Adipose tissue metabolism and cardiometabolic

health in obesity
Citation for published version (APA):

Stinkens, R. M. E. (2017). Adipose tissue metabolism and cardiometabolic health in obesity: effects of
pharmacological and lifestyle interventions. [Doctoral Thesis, Maastricht University]. Maastricht University.
https://doi.org/10.26481/dis.20171005rs

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Published: 01/01/2017

DOI:
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 Adipose tissue metabolism and
cardiometabolic health in obesity
Effects of pharmacological and lifestyle interventions
The research described in this thesis was performed within NUTRIM School of
Nutrition and Translational Research in Metabolism and was supported by Novartis
Pharma AG, Unilever and by grants from the Dutch Diabetes Research Foundation
(grant NL2009.60.003) and the Research Foundation Flanders (KAN 1507217N).

Layout & cover design: Rudi Stinkens

Printed by: Gildeprint – www.gildeprint.nl

ISBN: 978-94-6233-699-5

© Rudi Stinkens, 2017, Maastricht, The Netherlands

For articles published or accepted for publication, the copyright has been
transferred to the respective publisher. No parts of this thesis may be reproduced,
stored in a retrieval system, or transmitted in any form or by any means without the
permission of the author, or, when appropriate, from the publishers of the
manuscript.
 Adipose tissue metabolism and
cardiometabolic health in obesity
Effects of pharmacological and lifestyle interventions

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit Maastricht, op gezag
van de Rector Magnificus, Prof. dr. Rianne M. Letschert volgens het besluit van het
College van Decanen, in het openbaar te verdedigen
op Donderdag 5 Oktober 2017 om 12.00 uur.

door

Rudi Michel Elisabeth Stinkens

PROMOTOR
Prof. dr. E.E. Blaak

CO-PROMOTOR
Dr. G.H. Goossens

BEOORDELINGSCOMMISSIE
Prof. dr. C.D.A. Stehouwer (Chair)
Prof. dr. J.F.C. Glatz
Prof. dr. A.H. Kersten (Wageningen University & Research)
Prof. dr. L.J.C. van Loon
Prof. dr. B.M. Stallknecht (University of Copenhagen)
Voor papa en mama
TABLE OF CONTENTS
10 Chapter 1
General introduction

38 Chapter 2
Targeting fatty acid metabolism to improve glucose
metabolism

116 Chapter 3
Improved insulin sensitivity with angiotensin receptor
neprilysin inhibition in individuals with obesity and
hypertension

142 Chapter 4
Effect of sacubitril/valsartan on exercise induced lipid
metabolism in individuals with obesity and hypertension

162 Chapter 5
The effects of angiotensin receptor neprilysin inhibition
by sacubitril/valsartan on adipose tissue transcriptome
and protein expression in obese hypertensive patients

180 Chapter 6
Exercise training-induced effects on the abdominal
subcutaneous adipose tissue phenotype in obese
humans

202 Chapter 7
Coordinated regulation of adipose tissue adrenergic-
and non-adrenergic-mediated lipolysis during exercise
in lean and obese individuals: the effect of exercise
training

234 Chapter 8
General discussion

254 Chapter 9
Addenda
CHAPTER 1
GENERAL INTRODUCTION
Chapter 1

OBESITY – THE BURDEN OF A BIG PLAYER

Over the last decades, the worldwide prevalence of obesity has increased
enormously and reached pandemic proportions. According to the World Health
Organization, 39% of adults over 18 years of age (38% of men and 40% of women)
were overweight, while 13% of the world’s adult population (11% of men and 15%
of women) was obese in 2014 [1]. Furthermore, globally, approximately 6.3% of the
children under the age of 5 years were overweight [2] and on their way to become
obese. Clearly, not only the Western society is affected by obesity, also many low-
and middle-income countries have increased morbidity and mortality rates due to
the increasing prevalence of obesity [1]. Nowadays, the amount of people suffering
from obesity exceeds the number of people suffering from malnutrition [3], making
th
obesity the 5 leading cause of death worldwide [1].
Obesity is associated with an increased risk for developing insulin resistance [4]
and type 2 diabetes [5]. Peripheral insulin resistance is defined as a reduced ability
of insulin to stimulate glucose uptake in peripheral tissues, mainly skeletal muscle
[6], while type 2 diabetes is characterized by insulin resistance accompanied with
pancreatic beta-cell failure (inability of the pancreatic beta-cells to adequately
secrete insulin) [7]. Furthermore, obesity is also related to the development of
hypertension and cardiovascular diseases [8], fatty liver disease [9], mental
disorders (e.g. depression) [10], musculoskeletal disorders (e.g. osteoarthritis) [11]
and certain types of cancer [12, 13].
Since obesity is a major public health issue and one of the most important risk
factors for the development of cardiometabolic diseases, it is needless to say that
the increasing obesity prevalence has major socioeconomic consequences [3]. The
World Health Organization defines overweight as a body mass index (BMI)
2 2
between 25-30 kg/m and obesity as a BMI of 30 kg/m or greater [1]. However, the
use of BMI gives an underestimation of the amount of people suffering from obesity
and the related health complications globally [14, 15] since it does not take into
account the body composition (i.e. skeletal muscle mass) and body fat distribution
(i.e. abdominal and gluteofemoral fat depot) [16]. Also, the use of universal BMI
cut-off points does not consistently reflect adiposity in different ethnic populations.
South Asian populations, displaying a greater proportion of body fat for a given BMI
than Caucasians [17], have a higher susceptibility to develop type 2 diabetes and
coronary artery disease, despite lower BMI values [18]. Therefore, BMI is only an
approximation and should be considered as a rough indicator to determine obesity
and cardiometabolic disease risk [19]. Nowadays, more sensitive techniques are
available and assessment of a patient’s metabolic phenotype is needed to enhance
diagnosis, prognosis and optimization of interventions [20]. Metabolic phenotyping
generates detailed information regarding a patient’s (patho)physiological state and
makes it possible to stratify subjects into different subgroups. This stratification at
baseline may improve the effectiveness of a particular intervention in a specific
subgroup of the population [21]. Therefore, detailed metabolic phenotyping is
necessary to identify individuals or subgroups of a population that have an
increased risk for developing metabolic diseases and to optimize prevention and
treatment strategies.

10
 Chapter 1

OBESITY AND CARDIOMETABOLIC HEALTH

The etiology of obesity is complex and multifactorial. However, the fundamental
cause of overweight and obesity is an imbalance between energy intake (i.e.
energy-dense diet) and energy expenditure (i.e. physical inactivity), which depends
on the interaction between biological, genetic, behavioral, social and environmental
factors [22].
The development of obesity is accompanied by a substantial increase in adipose
tissue mass. While adipose tissue used to be considered a passive organ, only
involved in the storage and release of energy, it has become clear over the last two
decades that adipose tissue is an active metabolic and endocrine organ. Moreover,
adipose tissue mass per se may not be the most important contributor to the
development of obesity-related disorders. For example, abdominal liposuction,
which is the surgical removal of abdominal subcutaneous adipose tissue, did not
significantly improve obesity-related disturbances like insulin resistance [23].
Furthermore, pharmacological treatment with insulin-sensitizing thiazolidinediones
(PPARγ agonists) is accompanied by a significant increase in subcutaneous
adipose tissue mass [24]. Additionally, patients with lipodystrophy, who are
characterized by a partial or complete lack of adipose tissue, have pronounced
insulin resistance and a high incidence of type 2 diabetes [25]. Collectively, these
findings indicate that total adipose tissue mass per se is not the predominant
contributor to the development of obesity-related complications. Body fat
distribution and adipose tissue (dys)function seem to play a more prominent role in
cardiometabolic health [26, 27]. Adipose tissue accumulation in the upper body
(abdominal region) is associated with the development of obesity-related
comorbidities and all-cause mortality, while fat accumulation in the lower body
(gluteofemoral region) is considered to be even protective against metabolic and
cardiovascular disturbances [28, 29]. Apparently, the functional properties of these
adipose tissue depots, seem to play an important role in the disease risk [16].
Adipose tissue dysfunction, which is characterized by an impaired capacity to store
lipids in combination with low-grade inflammation, seems to contribute to the
development of insulin resistance and impaired glucose metabolism by promoting
excessive fat storage in non-adipose tissues (ectopic fat deposition), such as the
liver, skeletal muscle, pancreas and heart [26, 30-33]. Furthermore, functional
impairments in either of these organs may further contribute to the development of
impaired glucose metabolism, type 2 diabetes and cardiovascular disease, as
extensively described in chapter 2 of this thesis [34].
Interestingly, around 10-30% of the obese population seems to be relatively
protected against the development of cardiometabolic complications and are
therefore referred to as ‘metabolically healthy obese’ (MHO) [35, 36]. The reason
why these obese individuals are protected from cardiometabolic complications is
currently not completely understood, but may be caused by several mechanisms,
including a beneficial body fat distribution (low visceral and ectopic fat storage
compared to subcutaneous fat depots), normal adipose tissue function (a normal
adipose tissue inflammatory phenotype and adipokine secretion pattern) as well as
high physical activity and fitness levels, together contributing to preserved insulin
sensitivity [37-39]. Importantly, at a given BMI, there are obese individuals who are
either very insulin sensitive or extremely insulin resistant. Thus, there is no clear

11
Chapter 1

BMI-cutoff separating these distinct obese subgroups [35]. It is also important to

acknowledge that the criteria to define MHO have only been established recently
[36, 40] and studies performed thus far characterized MHO as the absence of
metabolic disorders, such as dyslipidemia, insulin resistance, impaired glucose
metabolism and type 2 diabetes. Interestingly, the concept of MHO may be
misleading, since several meta-analyses of prospective cohort studies showed an
increased risk of developing type 2 diabetes [41] and cardiovascular disease [42,
43] over time when compared to healthy normal weight subjects. The duration of a
MHO phenotype for 5.5-10.3 years of follow-up was not associated with an
increased risk to develop diabetes and cardiovascular disease, compared to
metabolically healthy normal weight subjects, but in about one-third of the MHO
subjects, the healthy phenotype was transient [44]. However, a 24% increased
mortality and cardiovascular risk was observed in the MHO group compared to
healthy normal weight subjects, when the MHO phenotype persisted over 10 years
of follow up [42]. Clearly, the MHO individuals may develop a metabolically
‘unhealthy’ phenotype [45, 46] over time and therefore MHO should not be
considered a harmless condition [47, 48]. Major efforts should be made to prevent
obesity and obesity-related comorbidities to maintain or reach a metabolically
healthy phenotype.

There is substantial evidence to suggest that targeting the renin-angiotensin

system and the natriuretic peptide system, either via pharmacological treatment or
lifestyle intervention, may beneficially affect cardiometabolic health. In the next
paragraphs, these two systems and their metabolic effects will be described in
more detail. Thereafter, the potential of physical exercise interventions to enhance
metabolic health will be addressed.

THE RENIN-ANGIOTENSIN SYSTEM

The renin-angiotensin system (RAS) is known as an important regulator of
systemic blood pressure and electrolyte homeostasis [49]. It has also been shown
that several RAS components are increased in obesity and insulin resistance and
are present in different tissues, including the adipose tissue [50-53]. Furthermore,
an increased RAS activity has been linked to the development of type 2 diabetes
and cardiovascular disease [54, 55].
In the classical (systemic) RAS, renal-derived renin converts circulating
angiotensinogen (AGT), mainly produced in the liver, into angiotensin I (ANG I),
which is then converted to angiotensin II (ANG II) by the action of angiotensin-
converting enzyme (ACE) found in the lung capillaries. ANG II is the main effector
hormone of the RAS and binds to the ANG II type 1 (AT1) and ANG II type 2 (AT2)
receptors to exert its biological functions. Most of the (patho)physiological effects,
such as vasoconstriction, aldosterone production, sodium reabsorption and
nervous system activation are induced by binding of ANG II to AT 1. In contrast,
binding of ANG II to AT2 counteracts the AT1-mediated effects and promotes
vasodilation, natriuresis and anti-inflammatory responses. Clearly, AT1 and AT2
receptors induce opposite physiological and metabolic effects [54].

12
 Chapter 1

Metabolic effects of the renin-angiotensin system

Different RAS components have been identified in several tissues, such as the
adipose tissue [50, 51], skeletal muscle and pancreas [52, 53] and a role for the
local RAS in obesity-related metabolic disturbances has been suggested [54, 56].
The metabolic effects of RAS on adipose tissue, skeletal muscle and pancreatic
function or metabolism will be addressed below.

The renin-angiotensin system and adipose tissue function

The adipose tissue of obese, insulin resistant and type 2 diabetic patients is
characterized by enlarged (hypertrophic) adipocytes, which can be attributable to a
prolonged positive energy balance and impaired adipocyte differentiation. On the
one hand, ANG II has been shown to promote adipocyte growth and differentiation
via several mechanisms [57, 58], but on the other hand it has also been shown to
inhibit differentiation of cultured human preadipocytes, mediated via the AT1
receptor [59] and to reduce insulin-induced adipocyte differentiation in human
preadipocytes [60]. Long-term RAS blockade, with the angiotensin receptor blocker
(ARB) valsartan, reduced abdominal subcutaneous adipocyte size and increased
the amount of small adipocytes in obese individuals with impaired glucose
metabolism [61].
Furthermore, ANG II has been shown to increase lipid synthesis and storage in
both rodent and human adipocytes [58] and to reduce adipose tissue lipolysis [62-
64], while blockade of the RAS reversed these effects [65]. However, conflicting
results have also been reported, with ANG II increasing adipose tissue lipolysis in
lean, but not in obese men [66] and RAS blockade not affecting adipose tissue
lipolysis in obese subjects [67].
Adipocyte size plays an important role in adipokine expression and secretion, with
a more pro-inflammatory phenotype of hypertrophic adipocytes. It has been shown
that ANG II increased, while RAS blockade decreased adipose tissue gene
expression of pro-inflammatory markers in rodents, as reviewed [50, 51]. In human
adipose tissue, long-term RAS blockade with valsartan reduced the expression of
macrophage infiltration markers [61] and several studies observed that ARB
treatment altered circulating adipokine concentrations, although conflicting results
have been reported [50].
Another important contributor to adipose tissue lipid handling is adipose tissue
blood flow (ATBF), which is reduced in obese, insulin resistant and type 2 diabetic
individuals, in both the fasting and postprandial state, as reviewed elsewhere [68].
While ANG II reduces ATBF [62, 69], local RAS blockade with losartan increased
human abdominal subcutaneous ATBF [69]. In line, long-term RAS blockade with
valsartan increased both fasting and postprandial ATBF [61] and may have
contributed to the previously observed valsartan-induced increase in insulin
sensitivity [70].
Taken together, the RAS may affect adipose tissue function by altering adipocyte
size and lipid metabolism, inflammation and adipose tissue blood flow, thereby
contributing to the development of both insulin resistance and type 2 diabetes.
However, the metabolic effects of the RAS go beyond the adipose tissue and also
affect skeletal muscle and the pancreas.

13
Chapter 1

The renin-angiotensin system and skeletal muscle metabolism

An increased RAS activity may cause impairments in skeletal muscle metabolism
by affecting tissue perfusion, insulin signaling and mitochondrial function. The
vascular system delivers nutrients and hormones to the skeletal muscle and local
tissue perfusion may regulate skeletal muscle metabolism and contractile
performance. Local infusion of ANG II in the gastrocnemius muscle reduced
skeletal muscle blood flow in both lean and obese individuals [62], while chronic
RAS blockade, with the ACE inhibitor (ACEi) captopril, increased postprandial
forearm blood flow in subjects with type 2 diabetes [71]. However, an increased
basal and insulin-stimulated total forearm blood flow was not observed after 2
weeks of ACEi treatment in obese insulin resistant subjects [72]. Thus, it is not
completely clear whether the RAS plays a major role in the regulation of total
skeletal muscle blood flow and/or microvascular function under physiological
conditions.
As reviewed elsewhere [50, 52], evidence suggest that the RAS may contribute to
impaired insulin signaling in skeletal muscle, either directly or indirectly via the
induction of oxidative stress. Importantly, most results are obtained from rodent
studies and need further investigation in humans.
An impaired mitochondrial function in skeletal muscle has been suggested to
contribute to the development of insulin resistance and type 2 diabetes, but
whether reduced mitochondrial function is a cause or consequence of these
disturbances remains to be established [73]. Nevertheless, chronic ANG II infusion
in mice increased mitochondrial reactive oxygen species, decreased expression of
genes involved in mitochondrial biogenesis and reduced mitochondrial content in
C2C12 myocytes [74]. Furthermore, ANG II reduced muscle mitochondrial content,
increased intramuscular triacylglycerol concentrations and reduced glycemic
control in mice, while RAS blockade partially reversed these effects, leading to an
increased fat oxidation, a decreased intramuscular triacylglycerol concentration
and an improved glucose tolerance [74]. In contrast, treatment with the ACEi
ramipril for two weeks had no significant effects on whole-body substrate oxidation,
intramuscular triacylglycerol content and insulin sensitivity in obese insulin resistant
men [72].

The renin-angiotensin system and pancreatic beta-cell function

Evidence suggests that elevated RAS activity may also contribute to an impaired
insulin secretion [50]. In rodents, RAS activation reduced pancreatic islet blood
flow, induced pancreatic islet fibrosis, oxidative stress, inflammation and impaired
insulin secretion, whereas RAS blockade, with ACEi or ARB, improved pancreatic
islet functionality and morphology and increased glucose tolerance, as reviewed
[50]. Furthermore, in patients with impaired glucose metabolism, long-term RAS
blockade, with valsartan, showed beneficial effects on pancreatic insulin secretion
[70].
Taken together, the RAS may affect adipose tissue, skeletal muscle and pancreatic
function and metabolism via different mechanisms, thereby contributing to the
development of insulin resistance, an impaired insulin secretion and type 2
diabetes. Therefore, blockade of the RAS may exert protective effects against the
development of metabolic disturbances.

14
 Chapter 1

Pharmacological modulation of the renin-angiotensin system

Meta-analyses of comparative outcome trials have shown that RAS blockade, with
either ACEi or ARB, reduced the incidence of new-onset type 2 diabetes by 20-
30% in populations at high risk for developing type 2 diabetes [75, 76]. More
recently, the prospective NAVIGATOR trial also showed that ARB treatment with
valsartan, in addition to lifestyle modification, reduced the incidence of type 2
diabetes by 14% in subjects with impaired glucose homeostasis after a median
follow-up of 5.3 years [77]. However, less pronounced improvements in glucose
metabolism have been shown by the prospective DREAM trial [78]. In this trial,
ACE inhibition, compared to placebo, non-significantly reduced the incidence of
type 2 diabetes by 9% after a median follow-up of 3 years in subjects with impaired
glucose homeostasis but without cardiovascular disease. Nevertheless, ACEi
treatment significantly reduced 2-hour glucose concentrations and increased
regression to normoglycemia [78]. Differences in study design, population and
treatment duration may underlie the somewhat different outcomes in the DREAM
trial and NAVIGATOR trials. Taken together, most randomized clinical trials
indicate that RAS blockade may protect against the development of type 2
diabetes [75, 76].

THE NATRIURETIC PEPTIDE SYSTEM

Although data are not entirely consistent [79, 80], several community-based cohort
studies have shown a lower activity of the natriuretic peptide (NP) system in obese
subjects, which has been shown to be related the development of type 2 diabetes
and chronic metabolic diseases [81-83]. As described below, modulation of the NP
system can be protective against the development of insulin resistance and type 2
diabetes via multiple mechanisms, making it an interesting target to combat
metabolic diseases.
The natriuretic peptide system contains several hormones of which Atrial NP
(ANP), Brain-type NP (BNP) and C-type NP (CNP) are the most abundant ones.
ANP and BNP are secreted from the cardiac atria [84] and ventricles [79],
respectively, in response to cardiac wall stress, but also via other factors, such as
weight loss [85], exercise [86], cold exposure [87] and hypoxia [88]. They bind to
guanylyl cyclase-coupled receptors: type A (NPRA) and type B (NPRB), which are
expressed in several tissues, including the adipose tissue [89]. While NPRA
mediates the majority of the metabolic effects of ANP and BNP, type-C NP
receptor (NPRC) is known as a membrane-bound clearance receptor that binds
and incorporates circulating NP into cytoplasm where NP are inactivated [90, 91].
In addition to the NPRC-mediated degradation, NP are also degraded via
extracellular proteases, such as the insulin-degrading enzyme [92] and neprilysin
(NEP: neutral endopeptidase 24.11) [93] of which the latter is mainly expressed in
the kidneys, but also present in adipocytes [94].
The lower NP effects in obesity and type 2 diabetes can be explained by a reduced
cardiac NP secretion, a reduced tissue NPRA signaling and/or an increased
systemic and tissue clearance, leading to reduced circulating NP concentrations.
These reduced NP concentrations have been implicated in the development of
insulin resistance and type 2 diabetes [83, 95-98], while higher NP concentrations

15
Chapter 1

are associated with a lower prevalence of new-onset type 2 diabetes [99, 100].
Beside changes in circulating NP concentrations, changes in the NP-receptors
have also been observed. A decreased NPRA receptor expression at the level of
abdominal subcutaneous adipose tissue, together with an increased NPRC
receptor expression at both abdominal subcutaneous and omental adipose tissue
has been observed in obese subjects with or without type 2 diabetes as compared
to lean individuals, thereby altering the NPRA/NPRC ratio [80, 101-104]. This
altered receptor expression, in combination with an increased NEP expression in
adipose tissue of obese and insulin resistant subjects [94], may cause an
increased NP clearance, leading to reduced NP bioavailability, thereby contributing
to the development of cardiometabolic disturbances.

Metabolic effects of the natriuretic peptide system

The NP system was primarily known for the regulation of blood pressure and its
physiological effects on the cardiovascular system, body fluid and electrolyte
homeostasis [105, 106]. Nowadays, it is established that the NP system also exert
effects on several key metabolic organs such as the adipose tissue and skeletal
muscle, as reviewed elsewhere [107-109].

The natriuretic peptide system and adipose tissue function

The natriuretic peptides have been shown to stimulate lipolysis in human
adipocytes, with ANP as the strongest effector, followed by BNP and CNP [110].
ANP and BNP stimulated in vitro lipolysis as much as the non-selective β-
adrenergic receptor agonist isoproterenol, while in situ microdialysis experiments
confirmed these NP-mediated lipolytic effects in abdominal subcutaneous adipose
tissue of healthy young men [110]. Even under local α2- and β1/2-adrenergic
blockade, a substantial non-adrenergic-mediated lipolysis was observed in
subcutaneous adipose tissue of healthy young lean [111] and overweight men
[112]. Intravenous infusion of ANP also acutely increased plasma concentrations of
glycerol and free fatty acids (FFA) in young healthy lean and obese men,
independently of the activation of the sympathetic nervous system [113]. Beside
lipid mobilization, intravenous infusion of ANP also rapidly increased lipid oxidation
in healthy normal weight men, both in the fasted [114, 115] and postprandial state
[116]. When directly infused into the human subcutaneous adipose tissue of young
lean men, ANP increased extracellular glycerol concentration and enhanced
adipose tissue blood flow [110], together contributing to an increased lipid
mobilization. This NP-mediated lipolysis revealed to be a cyclic guanosine
monophosphate/protein kinase G (cGMP/PKG) dependent pathway, which induces
the phosphorylation of perilipin 1 and hormone sensitive lipase [117].
ANP-treated human adipocytes also showed an increased activation of AMP-
protein kinase (AMPK), a major metabolic energy sensor and master regulator of
metabolic homeostasis [118]. Indeed, ANP increased energy expenditure and
oxidative capacity and also increased mitochondrial biogenesis and function in
differentiated human adipocytes [87, 118].
Beside effects on adipose tissue lipid mobilization and oxidation, NP have also
been shown to possess anti-inflammatory properties [119, 120], to induce
expression and secretion of the insulin-sensitizing factor adiponectin in both

16
 Chapter 1

chronic heart failure patients [121] and healthy men [122], and to reduce systemic
leptin concentrations in healthy men [123].

The natriuretic peptide system and skeletal muscle metabolism

In skeletal muscle, a NP-induced increase in peroxisome proliferator-activated
receptor-gamma coactivator-1 alpha (PGC-1α) and proliferator-activated receptor-
delta (PPAR-δ) mRNA expression as well as an increase in mitochondrial mass
has been observed in C2C12 myocytes [124]. In line, in human myotubes, ANP
caused an increase in PGC-1α mRNA expression, which was paralleled by an
upregulation of several genes and proteins involved in oxidative phosphorylation
(OXPHOS), although mitochondrial proliferation and mitochondrial mass were not
affected by ANP treatment [125].
Taken together, these findings suggest that NP may enhance the oxidative
capacity of skeletal muscle.

The natriuretic peptide system and pancreatic beta-cell function

At the level of the pancreas, intravenous infusion of ANP increased insulin
secretion in human subjects [114, 116, 126] and improved pancreatic β-cell
function in rodents [127]. ANP directly enhanced glucose-stimulated insulin
secretion in cultured pancreatic islets of Langerhans [127] and induced β-cell
growth in isolated rat pancreatic islets [128].
Interestingly, NP treatment may also induce changes at the level of the
gastrointestinal tract. Intravenous BNP infusion, in young healthy lean men,
showed a fasting-induced increase in total and acylated ghrelin concentrations,
decreased the subjective rating of hunger and increased the feeling of satiety,
without significantly changing circulating peptide YY, glucagon-like peptide 1,
oxyntomodulin, pancreatic polypeptide, leptin and adiponectin concentrations
[129]. Furthermore, intravenous infusion of BNP showed a decreased gastric
emptying in a rodent model [130]. Also a novel gut-heart crosstalk has been
described in rodents in which ANP mediates the blood pressure-lowering effects of
glucagon-like peptide-1 (GLP-1) agonists [131]. The GLP-1 agonist, liraglutide,
induced ANP secretion from the cardiac atrium and the blood pressure lowering
effect of liraglutide was abrogated in Nppa (ANP) knockout mice.
Taken together, these data clearly show the involvement of the NP system in
metabolic homeostasis and indicate that modulation of the NP system can be
protective against the development of insulin resistance and type 2 diabetes via
multiple mechanisms. Targeting the NP system may therefore serve as an effective
strategy to combat these metabolic diseases.

17
Chapter 1

PHARMACOLOGICAL MODULATION OF THE RENIN-ANGIOTENSIN SYSTEM

AND THE NATRIURETIC PEPTIDE SYSTEM
As discussed above, obesity, type 2 diabetes and cardiovascular diseases are
interrelated and may be characterized by a reduced NP system activity, an
increased RAS activity as well as similar metabolic impairments. Recently, a novel
dual-acting drug (sacubitril/valsartan) has been developed that combines ARB and
NEP inhibition and facilitates beneficial effects of NP and other neprilysin
substrates, while inhibiting the detrimental effects of the RAS [132]. In this respect,
the latter cardiovascular medication, targeting the RAS and NP system, may also
have positive metabolic effects. Moreover, the dual-acting mechanism of
sacubitril/valsartan may have synergistic beneficial effects on several metabolic
parameters. As described earlier in this thesis, the ARB valsartan improved insulin
sensitivity [70] and beneficially affects adipocyte size and lipid metabolism,
inflammation and adipose tissue blood flow [61]. The additional effects of the
neprilysin inhibitor, sacubitril, may further enhance metabolic health (i.e. insulin
sensitivity) via the increased NP concentrations, which also beneficially affect lipid
mobilization from adipose tissue [114], postprandial lipid oxidation [116],
adiponectin release and muscular oxidative capacity [125, 133]. Combined,
sacubitril/valsartan facilitates beneficial effects of NP and other neprilysin
substrates, while inhibiting the detrimental effects of the RAS. However, until
recently, the metabolic effects of sacubitril/valsartan have not been investigated. In
chapters 3, 4 and 5 of this thesis, the effects of sacubitril/valsartan on metabolic
parameters, such as peripheral insulin sensitivity, whole-body and local
subcutaneous adipose tissue lipolysis as well as energy expenditure and substrate
oxidation were investigated in obese hypertensive patients [134].

LIFESTYLE INTERVENTION
The management and treatment of obesity and obesity-related disorders is more
complex than only achieving weight loss. The rational should not only focus on the
maintenance of fat mass loss, but also on prolonged cardiometabolic risk reduction
and health improvement. Lifestyle characteristics such as diet, physical inactivity,
smoking, alcohol consumption and stress are important factors that influence the
development of obesity and related comorbidities, and guidelines recommend
changes in these lifestyle characteristics for both prevention and management of
metabolic disease [135].
Manipulation of diet and physical activity levels are the first-choice interventions to
reverse metabolic disturbances. Even though several large lifestyle intervention
trials did not show significant beneficial effects of intensive exercise or combined
diet and exercise lifestyle interventions on reducing the risk for cardiovascular
outcomes [136, 137], other trials did observe beneficial effects on modifiable risk
factors for cardiovascular disease [138], type 2 diabetes incidence and the
metabolic syndrome [139]. This indicates that lifestyle interventions do have
beneficial effects. Several important large lifestyle intervention trials that used
dietary advice, exercise strategies and/or combinations have been performed to
prevent type 2 diabetes. In this respect, the Malmö-study [140, 141], the Chinese

18
 Chapter 1

Da Qing IGT and Diabetes study [142], the Finnish Diabetes Prevention Study
(DPS) [143], the Diabetes Prevention Program in the USA (DPP) [144] and the
Study on Lifestyle intervention in Impaired glucose tolerance Maastricht (SLIM)
[145] indicated that the incidence of type 2 diabetes was reduced by 40-60%
following intensive (combined) diet and exercise intervention program. Importantly,
long-term follow-up of these trials revealed that diabetes risk reduction still existed
after 3 to 14 years after cessation of the intervention program [146-149]. Thus,
lifestyle interventions clearly show improvements in a variety of health outcomes
related to cardiometabolic health and can reduce the incidence of diseases [150].
However, the exact effects of the different components of the lifestyle program are
not fully elucidated. While one study revealed that the combination of diet and
exercise was more beneficial in reducing insulin resistance [151], another study
showed that diet and exercise were equally effective compared to exercise alone to
prevent the progression towards type 2 diabetes [142]. Moreover, another study
found that the combination of diet and exercise was more effective than either
treatment alone [145]. Nevertheless, combined dietary and exercise interventions
have been shown to be most effective in reducing body weight [152].
Clearly, both prevention and management of disease progression can be achieved
by strategies such as increased physical activity as well as dietary manipulation via
a hypocaloric diet or an improved nutritional composition [153]. Interestingly,
several dietary components, such as fatty acids, polyphenols and fibers may
modulate fatty acid metabolism in tissues like skeletal muscle, liver and pancreas,
both acute (i.e. postprandial phase) and more long-term, ultimately improving
glucose metabolism, as extensively reviewed in chapter 2 [34]. Physical activity
strategies include increased habitual physical activity (e.g. sitting less) and physical
exercise levels (e.g. cycling) which lead to increased energy expenditure, improved
physical fitness [154, 155] and contribute to an improved metabolic health. The
exercise-induced improvements in metabolic risk profile have largely been
attributed to changes in skeletal muscle metabolism and function, but physical
exercise is likely to induce alterations in almost all metabolically active tissues,
including the adipose tissue, as will be discussed below.

Physical activity-induced effects on adipose tissue metabolism

While physical activity is defined as any movement exerted by the skeletal muscle
to increase resting energy expenditure, exercise is usually defined as a part of
physical activity that is planned and/or structured [156]. When other aspects of
energy expenditure or energy intake are not changed, increased physical activity
and exercise induce a negative energy balance, resulting in a reduced fat mass
[157]. This reduced fat mass depends on the accumulated effect of each bout of
exercise and is caused when adipose tissue lipolysis and oxidation exceeds fat
storage. Even though, physical activity and exercise-induced effects on adipose
tissue metabolism are still not completely understood, several rodent studies
suggest that exercise training may improve adipose tissue metabolism and function
[158]. However, human studies that investigated the effects of exercise training on
the adipose tissue function are scarce and need further investigation [157-159].
The exercise-induced fatty acid mobilization from adipose tissue is influenced by
fatty acid re-esterification, adipose tissue lipolysis and adipose tissue blood flow

19
Chapter 1

(ATBF). A part of the exercise-induced increase of fatty acid mobilization can be

attributed to a decreased rate of fatty acid re-esterification [160, 161] and an
increased ATBF [162]. The exact mechanism for the increased ATBF is currently
unknown and could be explained by an increased cardiac output or by other factors
such as increases in circulating catecholamines and natriuretic peptides
concentrations [163-165]. This increased ATBF contributes to an increased supply
of hormones and signaling molecules (e.g. catecholamines, myokines) to the
adipose tissue as well as the supply of mobilized FFA and adipokines to other
tissues, such as skeletal muscle [166].
Adipose tissue lipolysis is mostly stimulated by low-intensity exercise and does not
increase further at higher exercise intensities [167-169]. This process depends on
both the adrenergic pathway, in which catecholamines (adrenalin and
noradrenalin) bind to the adrenergic receptors, and the non-adrenergic pathways,
that consist of the natriuretic peptide system, insulin, growth hormone and cortisol.
In obesity, a blunted catecholamine-mediated lipolysis has been observed in
subcutaneous adipose tissue [170, 171], in which the anti-lipolytic α2-adrenergic
receptor becomes more predominant compared to the lipolytic β1,2-adrenergic
receptors [172, 173]. Also, as described earlier in this thesis, the natriuretic peptide
concentrations are reduced in obesity and may contribute to an altered exercise-
induced lipolysis. Interestingly, after blocking the β-adrenergic receptor in
subcutaneous adipose tissue during exercise in overweight men, it has been
shown that adrenalin is not the primary lipolytic stimulus and that ANP might be of
more importance in overweight men [112]. Noteworthy, several cross-sectional
studies and exercise intervention studies have shown an increased lipolytic
response in isolated adipocytes of active individuals compared to their sedentary
counterparts [174-176] or as result of exercise training [177-179], reflecting an
increased capacity for lipolysis at the cellular level. Furthermore, there is some
evidence that exercise-induced adipose tissue lipolysis is increased with training
[157], which is reflected by a similar or even greater lipolysis in spite of lower
circulating concentrations of lipolytic hormones during exercise [180-182]. In line,
exercise training has been shown to improve the β-adrenergic responsiveness in
overweight [164] and obese men [183] and to improve the natriuretic peptide
sensitivity in overweight men [164], which ultimately leads to an increased lipolysis.
However, results are not always consistent and are complicated by confounding
factors such as recent energy balance [157]. Nevertheless, in general, there seems
to be an improved adipose tissue lipolytic sensitivity after training, which causes an
increased fat mobilization and may reduce fat mass when the mobilized FFA are
oxidized.
Several rodent studies have shown that exercise training may increase adipose
tissue mitochondrial biogenesis [184, 185] and function [186-188] and induce
browning of white adipose tissue [184, 185, 187, 189, 190], leading to an increased
energy expenditure. In line, exercise training in human studies has been shown to
increase white adipose tissue gene expression of PGC-1α [191] and oxidative
metabolism markers [192], suggesting a higher mitochondrial capacity to oxidize
fatty acids.
This increased mitochondrial oxidation can contribute to a reduced fat mass and
reduced adipocyte size, which has beneficial effects on disease progression [193]
and may lead to an altered adipokine expression [194]. Several rodent studies

20
 Chapter 1

have shown that exercise training may beneficially alter adipokine expression [195,
196], but human data are conflicting. Although there is consistent evidence that
exercise increases adipose tissue interleukin-6 (IL-6) expression and secretion
after a single bout of exercise [197-199], results of other adipokines, such as
adiponectin, leptin, TNF-α after a single bout of exercise or after exercise training
are currently limited and conflicting [181, 200-206].
Collectively, these data may suggest that exercise improves adipose tissue
metabolism and function and can ultimately contribute to a reduced disease
progression and improved peripheral insulin sensitivity. Indeed, transplantation of
white adipose tissue from trained animals to untrained recipients markedly
improved skeletal muscle glucose uptake [187], suggesting that improvement of
adipose tissue function may contribute to the increased peripheral insulin sensitivity
after exercise training.
In chapters 6 and 7 of this thesis, the exercise training-induced effects on
abdominal subcutaneous adipose tissue metabolism and lipolysis were
investigated in obese and lean subjects.

THESIS OUTLINE
This thesis describes the effects of a pharmacological intervention as well as
physical exercise interventions to improve metabolic health in obese individuals,
with a focus on adipose tissue metabolism.
As described earlier in this thesis, adipose tissue dysfunction contributes to the
development of insulin resistance and impaired glucose metabolism. By targeting
fatty acid metabolism in the adipose tissue, liver, skeletal muscle or even the
pancreas and the intestine, insulin sensitivity and glucose homeostasis may be
improved. In Chapter 2, an extensive overview is provided of the fatty acid
metabolism-related pathways in several metabolically active organs that can be
targeted by dietary interventions, thereby improving whole-body glucose
metabolism and insulin sensitivity.
Targeting the renin angiotensin system and the natriuretic peptide system by
cardiovascular medication may improve adipose tissue and metabolic dysfunction,
next to its effect on the cardiovascular system and hypertension. To investigate
this, we conducted in Chapter 3, a multicenter, randomized, double-blind, double-
dummy, parallel-group study to examine the metabolic effects of
sacubitril/valsartan, which is a first-in-class angiotensin receptor neprilysin inhibitor,
in obese hypertensive patients. By means of a hyperinsulinemic-euglycemic
glucose clamp, we investigated the effects on peripheral insulin sensitivity, while
2
whole-body lipolysis was determined using a stable isotope tracer ([1,1,2,3,3- H]-
glycerol) and abdominal subcutaneous adipose tissue lipolysis was measured with
the microdialysis technique. In addition to the collection of adipose tissue biopsies,
we also performed indirect calorimetry measurements to assess energy
expenditure and substrate utilization in these patients.
Chapter 4 extends the outcomes from the previous chapter and describes the
effects of the 8-weeks treatment with sacubitril/valsartan compared to amlodipine
on whole-body and adipose tissue lipolysis and lipid oxidation during a single bout
of exercise.

21
Chapter 1

To obtain more detailed insight into possible mechanisms underlying the findings
described in chapters 3 and 4, we assessed adipose tissue gene expression
patterns using microarray analysis and protein expression profiles of enzymes
involved in lipolysis, the natriuretic peptide signaling pathway and mitochondrial
oxidative phosphorylation complexes. The results from these analyses are
provided in Chapter 5.
There is some evidence that exercise training may improve adipose tissue function,
which may contribute to the reduced risk for developing obesity-related insulin
resistance and other comorbidities. However, human studies that investigated the
effects of exercise training on the adipose tissue function are limited. Chapter 6
addresses the results of a supervised, progressive, combined endurance and
resistance exercise training intervention for 12 weeks in well-phenotyped, obese
subjects. In this study, we investigated exercise training-induced effects on adipose
tissue by measuring abdominal subcutaneous adipocyte morphology, gene and
protein expression of markers related to adipose tissue function. Moreover, we
determined the exercise training-induced effects on ex vivo adipocyte lipolysis.
The aim of the study, described in Chapter 7, was to elucidate the physiological
role of ANP-mediated lipolysis in abdominal subcutaneous adipose tissue of
middle-aged obese insulin sensitive, obese insulin resistant and age-matched lean
insulin sensitive men. By means of local combined blockade of the α- and β-
adrenergic receptors (using a microdialysis approach), abdominal subcutaneous
adipose tissue lipolysis was investigated during a single bout of low-intensity
endurance exercise. In addition, we examined whether a combined endurance and
resistance exercise training intervention for 12 weeks could improve abdominal
subcutaneous adipose tissue lipolysis in obese insulin resistant individuals.
The main conclusions from the studies described in this thesis are discussed in
Chapter 8 and placed into a broader perspective, accompanied by suggestions for
further research.

22
 Chapter 1

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adiponectin receptors in human visceral and subcutaneous adipose tissue is related

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Kraus I, Langin D, Stich V. Effect of aerobic training on plasma levels and
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2002;283(4):E861-5.

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CHAPTER 2
Targeting fatty acid metabolism to
improve glucose metabolism

Stinkens R., Goossens G.H., Jocken J.W., Blaak E.E.

Obesity Reviews, 2015; 16(9):715-57

Chapter 2

ABSTRACT
Disturbances in fatty acid metabolism in adipose tissue, liver, skeletal muscle, gut
and pancreas play an important role in the development of insulin resistance,
impaired glucose metabolism and type 2 diabetes mellitus. Alterations in diet
composition may contribute to prevent and/or reverse these disturbances through
modulation of fatty acid metabolism.
Besides an increased fat mass, adipose tissue dysfunction, characterized by an
altered capacity to store lipids and an altered secretion of adipokines, may result in
lipid overflow, systemic inflammation and excessive lipid accumulation in non-
adipose tissues like liver, skeletal muscle and the pancreas. These impairments
together promote the development of impaired glucose metabolism, insulin
resistance and type 2 diabetes mellitus. Furthermore, intrinsic functional
impairments in either of these organs may contribute to lipotoxicity and insulin
resistance. The present review provides an overview of fatty acid metabolism-
related pathways in adipose tissue, liver, skeletal muscle, pancreas and gut, which
can be targeted by diet or food components, thereby improving glucose
metabolism.

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 Chapter 2

INTRODUCTION
Obesity is considered a global health problem, since it is closely associated with
the development of chronic metabolic diseases, including cardiovascular disease,
type 2 diabetes mellitus (T2D) and certain types of cancer [1]. The pathogenesis of
T2D is characterized by the development of both insulin resistance in peripheral
tissues and pancreatic β-cell failure [2, 3]. Disturbances in fatty acid metabolism
play a crucial role in the development of an impaired glucose metabolism and
diabetes. Combined dietary and physical activity intervention may reduce the
incidence of T2D by 30-60% [4, 5], which may for a considerable part be explained
through modulation of fatty acid metabolism [6]. A better understanding of the
interaction between diet, fatty acid metabolism, insulin resistance and β-cell
dysfunction is needed to develop novel strategies to prevent impairments in
glucose metabolism and, consequently, the development of T2D.
A tight interplay between adipose tissue, skeletal muscle, liver, pancreas and the
gut regulate fatty acid metabolism in the human body. Besides an increased fat
mass, adipose tissue dysfunction, characterized by an altered capacity to store
lipids and low-grade inflammation, plays a major role in the development of insulin
resistance and impaired glucose metabolism by promoting excessive fat storage in
non-adipose tissues like liver, skeletal muscle, pancreas, the heart and kidneys [7-
10] (Figure 1).
Furthermore, intrinsic functional impairments in either of these organs may
contribute to lipotoxicity and insulin resistance. Skeletal muscle is generally
considered the most important organ in peripheral insulin resistance [11]. Beside
an increased fatty acid (FA) supply [12], a reduced skeletal muscle oxidative
capacity [13, 14] may contribute to the accumulation of triacylglycerol (TAG) and
bioactive lipid metabolites [15-17]. An increased lipid supply to the liver may result
in an increased hepatic TAG content and very low-density lipoprotein (VLDL-TAG)
output [18], a higher glucose production and output [19-21] and a reduced insulin
clearance by the liver [22-24], leading to hyperlipidemia, insulin resistance and
glucose intolerance [25]. Furthermore, hyperglycemia may, together with
hyperlipidemia-related lipotoxicity in the pancreas, result in decreased glucose-
stimulated insulin secretion by the pancreatic β-cells [26].
Recent evidence also indicates that the gut microbiota and its products may
contribute to the development of insulin resistance and a disturbed glucose
metabolism (Figure 1). There is accumulating evidence that alterations in the gut
microbiota composition and function may affect adipose tissue, liver and skeletal
muscle lipid and glucose metabolism, at least partly through effects on bile acid
metabolism [27-29].
Energy intake and diet composition (i.e. dietary fatty acids, polyphenols, fibers)
may have a significant impact on many aspects of fatty acid metabolism in different
tissues. These effects can be both acute (i.e. postprandial phase) and more long-
term, ultimately affecting health status. The aim of the present review is to provide
an overview of fatty acid metabolism related pathways in adipose tissue, liver,
skeletal muscle, pancreas and gut that can be targeted by diet or food components
and, as a consequence, improve whole-body glucose metabolism and insulin
sensitivity. The focus of the present review will be on the major tissues involved in
inter-organ substrate metabolism and lipid-induced insulin resistance. Associations

39
Chapter 2

between an increased myocardial triglyceride content or epicardial fat volume with

insulin resistance, T2D and cardiac dysfunction [30-32], and the beneficial effects
of diet on these deleterious parameters have been described elsewehere [33-35].
In addition, recent evidence points out that lipid accumulation in the kidneys [36,
37] and bones [38-40] may contribute to disturbances in whole-body glucose
metabolism. These findings may be promising areas of future research but are
beyond the scope of the present review.

40
 Chapter 2

Figure 1. Inter-organ crosstalk in fatty acid metabolism and insulin resistance

An impaired adipose tissue lipid metabolism, as observed in obesity, is characterized by a decreased

lipid storage capacity, which contributes to lipid overflow in the circulation (1), resulting in excessive fat
storage in peripheral tissues such as skeletal muscle, liver, pancreas, kidney and heart (ectopic fat
storage) (2). Furthermore, adipose tissue dysfunction is characterized by an altered expression and
secretion of adipokines, inducing a state of chronic low-grade inflammation (3). This inflammatory state
may on the one hand affect local adipose tissue lipid metabolism and on the other hand contribute to
systemic inflammation (4), which together may affect lipid handling in peripheral tissues such as liver
and skeletal muscle and promote insulin resistance through interference with insulin signaling. An
increased lipid supply to the liver may result in a higher glucose production, an increased hepatic TAG
content and VLDL-TAG output and a reduced insulin clearance by the liver (5). The increased insulin
concentration stimulates de novo lipogenesis and, together with an increased VLDL-TAG output, results
in hypertriglyceridemia, ultimately leading to insulin resistance and glucose intolerance. In skeletal
muscle, besides an increased fatty acid supply and uptake, an impaired muscle lipid turnover may
contribute to accumulation of TAG and bioactive lipid metabolites (DAG, LCFA-CoA and ceramides),
which may interfere with insulin signaling (6). Hyperglycemia may, together with the formation of
hyperlipidemia-related toxic metabolites and lipid accumulation in the pancreas, result in decreased
glucose-stimulated insulin secretion by the pancreatic β-cells (7). Finally, alterations in gut microbiota
composition and function (8) may affect adipose tissue, liver and skeletal muscle lipid and glucose
metabolism, possibly through effects on SCFA production and bile acid metabolism. Ectopic fat storage
is associated with impaired function of the liver, skeletal muscle and pancreas, leading to derangements
in whole-body glucose homeostasis and, consequently, type 2 diabetes (9).
Symbols: (): Altered; (): Increased; (): Decreased

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Chapter 2

ADIPOSE TISSUE DYSFUNCTION AND IMPAIRED GLUCOSE METABOLISM

Adipose tissue is highly important in buffering the daily influx of dietary lipids. The
buffering capacity of adipose tissue refers both to its ability to suppress the release
of free fatty acids (FFA) into the circulation and to increase lipid uptake, which is
most pronounced in the postprandial state [41].
Firstly, disturbances in the adipose tissue lipid buffering capacity, like impairments
in lipid uptake, lipolysis and fatty acid storage, adipocyte differentiation, adipose
tissue expandability and adipose tissue mitochondrial function will be discussed.
Subsequently, the browning of white adipose tissue (WAT) in relation to
thermogenesis will be adressed. Thirdly, lipid-induced inflammation in relation to
impaired glucose metabolism will be described, as depicted in Figure 2. Finally,
putative nutritional targets that may improve adipose tissue dysfunction will be
addressed.

Lipid uptake in adipose tissue

In the postprandial period, VLDL- and chylomicron-TAG are hydrolyzed in the
process of intravascular lipolysis by lipoprotein lipase (LPL) [42]. The FFA that are
liberated by this process can be taken up by the adipose tissue but a significant
proportion will also spillover in the circulation and enter the plasma FFA pool [43].
LPL is regulated in a tissue-specific manner by nutrients and hormones [44]. In
adipose tissue, the major activator of LPL activity is insulin [45, 46]. Therefore,
adipose tissue LPL activity is high in the fed state and low when fasted [47, 48].
Furthermore, research has shown that dexamethasone (a synthetic glucocorticoid)
decreased in vitro and in vivo LPL mRNA expression and activity in isolated rat and
mice adipose cells and tissue, resulting in increased circulating TAG
concentrations [49, 50]. In contrast, dexamethasone increased LPL mRNA and
activity in human adipose tissue cultures [51, 52]. Whether these differences in LPL
response reflect species-specific differences remains unclear. Another feeding-
related hormone that could be responsible for changes in LPL expression and
activity is glucose-dependent insulinotropic polypeptide (GIP), which increases LPL
activity and mRNA in isolated human and rodent adipocytes [53, 54], although the
regulation of adipose LPL activity by GIP in vivo remains to be established.
Recent studies also showed that LPL activity is altered by
glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1
(GPIHBP1), which binds LPL [55] in the interstitial space and transports it to the
capillary lumen across endothelial cells [56, 57]. Fasting and peroxisome
proliferator-activated receptor (PPAR)-γ agonists increased GPIHBP1 expression
in rodents, while PPAR-α and PPAR-δ agonists had little or no effect [58].
Furthermore, hyperglycemia altered GPIHBP1 expression and activity in rodent
and bovine endothelial cells [59]. Whether nutritional status or PPAR-γ agonists
alter GPIHBP1 expression in humans is currently unknown, but several GPIHBP1
mutations have been discovered in humans [60] and result in severe
hypertriglyceridemia [55, 56, 61]. Therefore, modulation of GPIHBP1 expression or
activity might provide a strategy to treat hypertriglyceridemia, cardiovascular
disease and T2D.

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 Chapter 2

A considerable part of the variation in LPL activity during fasting, exercise and in
the postprandial state may be explained by post-translational regulation by
apolipoproteins (APO-C1, -2, -3, -5, APO-E) and the angiopoietin-like proteins
(ANGPTL-3, -4, -8), as reviewed in detail elsewhere [62]. ANGPTL4, in particular,
has been identified as an inhibitor of LPL activity and may be regulated by diet and
fasting, since its expression and secretion changes in parallel with circulating FFA
concentrations [62-66].
In obese conditions, basal LPL activity has been reported to be increased, whereas
FA spillover from LPL-mediated TAG hydrolysis across adipose tissue during
hyperinsulinemia is less suppressed as compared to lean healthy individuals [67,
68]. Indeed, the removal of TAG across adipose tissue was found to be impaired in
obesity, insulin resistance and T2D, due to a reduced insulin-mediated stimulation
of LPL activity [68-73], suggesting less efficient removal of dietary lipids by adipose
tissue in these subjects. In line, a recent study has demonstrated that the relative
quantity of meal fat stored in adipose tissue after the intake of subsequent meals
was significantly reduced in (abdominally) obese versus lean individuals [69], which
in turn may contribute to increased lipid spillover and ectopic fat deposition. To
define nutritional targets affecting the regulation of LPL activity, which may
subsequently reduce lipid spillover, ectopic fat deposition and improve insulin
sensitivity, additional studies to determine the role of different LPL modulators in
the regulation of its activity in vivo are clearly needed.
In addition to impaired LPL action in obese and insulin resistant conditions,
disturbances in the uptake of the liberated FFAs (lipid spillover) may contribute to
impaired lipid buffering. FAs are able to cross the endothelial barrier via passive
diffusion, which is dependent on the concentration gradient of FAs across the
membrane of the adipocyte, or via active transport [41, 43, 74, 75]. Active FA
transport is facilitated by several enzymes and proteins, of which numerous FA-
transport proteins have been identified in adipocytes [76], including fatty acid
translocase/CD36 (CD36), membrane-bound and cytosolic fatty acid binding
protein (FABPpm and FABPc, respectively) and fatty acid transporter protein
(FATP) [77]. It has been shown that the translocation of CD36 from the intracellular
stores to the plasma membrane is regulated by various stimuli, including insulin
and increased activation of AMP-activated protein kinase (AMPK) [78-81]. Although
CD36 protein expression is higher in the subcutaneous adipose tissue of obese,
overweight and T2D compared to lean subjects [82], the net removal of TAG and
FAs by adipose tissue is reduced in the former groups, suggestive of impairments
in intracellular CD36 trafficking. Taken together, strategies to reduce spillover and
increase FFA trapping in adipose tissue, mainly in the postprandial phase, will lead
to a reduced lipid spillover and may consequently prevent or at least reduce
ectopic fat deposition. On the one hand, this may be achieved by an increased
FFA concentration gradient across the adipocyte membrane and a subsequent
decreased intracellular FFA concentration. This might be accomplished by
increasing FA reesterification and/or oxidation, which will be discussed later in this
section. On the other hand, an increased facilitated diffusion by fatty acid
transporters like CD36 may possibly increase fatty acid trapping, thereby reducing
FFA spillover. Indeed, it has been shown that tissue-specific CD36 transcripts
differentially influence fatty acid homeostasis and insulin sensitivity [83]. Adipocyte
CD36 appears to be metabolically protective, and its selective upregulation might

43
Chapter 2

have therapeutic potential in insulin resistance. Nevertheless, it has been shown

that CD36 only facilitates fatty acid transport in adipose tissue and muscle when
extracellular concentrations are low, suggesting no major regulatory role when
circulating FFA concentrations are equal or above those seen in the overnight
postabsorptive state [84].

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 Chapter 2

Figure 2. Disturbances in adipose tissue fatty acid metabolism

Adipose tissue lipid uptake (1) is impaired in obesity, insulin resistance and T2D, due to a reduced
insulin-mediated stimulation of LPL activity and, possibly, decreased facilitated FFA uptake via CD36 in
adipocytes, which contributes to lipid overflow in the circulation. TAG and DAG are broken down by the
lipases ATGL and HSL. Fasting lipolysis, expressed per unit fat mass, may be decreased in obese
subjects (2). Nevertheless, insulin-mediated suppression of adipose tissue lipolysis per unit fat mass is
attenuated in obese individuals, resulting in an increase in whole-body lipolysis during postprandial
conditions. A decreased adipose tissue mitochondrial function (3) may lead to impaired scavenging of
fatty acids, resulting in increased intracellular FFA concentrations, reducing FFA trapping and an
enhanced release of FFA into the circulation. Furthermore, dynamic changes occur in the adipose
tissue immune cell populations during the development of obesity, causing a shift from an anti-
inflammatory towards a more pro-inflammatory phenotype, resulting in a state of low-grade inflammation
(4), affecting insulin sensitivity through different mechanisms. Finally, a reduced adipose tissue
differentiation and expandability (5), leading to a reduced lipid storage capacity (6), contributes to lipid
overflow into the circulation.
Abbreviations: LPL: Lipoprotein lipase; FFA: Free fatty acid; CD36: Fatty acid translocase CD36; TAG:
Triacylglycerol; DAG: Diacylglycerol; MAG: monoacylglycerol; ATGL: Adipose triglyceride lipase; HSL:
Hormone sensitive lipase; ANGPTL4: Angiopoietin-like protein 4; PUFA: Poly unsaturated fatty acid;
SCFA: Short chain fatty acid; FGF21: Fibroblast growth factor 21.
Dashed lines indicate inhibition. Solid lines indicate stimulation. Green lines indicate beneficial effects.
Symbols: (=): unchanged; (): Increased; (): Decreased.

45
Chapter 2

Fatty acid release from adipose tissue

When energy is required (e.g. during exercise and fasting), the lipids that have
been stored in intracellular lipid droplets (LD’s) as TAG, are hydrolyzed via
activation of the intracellular lipolytic pathway, which involves different lipases and
lipid droplet-associated proteins that are under hormonal control [85].
Catecholamines and natriuretic peptides (NPs) act as lipolytic hormones, while
insulin acts as the major anti-lipolytic hormone in human adipose tissue. The net
lipolytic effect of catecholamines is determined by the ratio between lipolytic beta
(1,2 and 3)- and anti-lipolytic alpha (2)-receptors, and subsequent protein kinase
A (PKA) activation. NPs act through natriuretic peptide receptor type A, B and C
(NPRA, NPRB, NPRC) to increase protein kinase G (PKG) activity [86-88].
Stimulation of intracellular lipolysis is dependent on PKA and PKG-mediated
phosphorylation of LD-associated proteins, including perilipin 1 (PLIN-1), hormone-
sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), as extensively
reviewed elsewhere [89, 90].
Whole-body lipolysis under fasting conditions (basal lipolysis) may be increased in
obesity because of the increased total adipose tissue mass. However, if adipose
tissue would release FFA at the same rate in obese and lean subjects, then
circulating FFA would be much higher than observed in obesity (only 20-30%
higher), suggesting that FFA concentrations are not elevated in proportion to fat
mass in obese individuals. Indeed, others and we have demonstrated that fasting
lipolysis expressed per unit fat mass is rather reduced in obesity [91-93]. This was
accompanied by downregulation of the expression of the key lipolytic enzymes
HSL and ATGL [91, 94, 95]. Data on PLIN-1 are inconclusive, being reduced [96-
98] or even elevated [99] in obese adipose tissue. Since humans are in the
postprandial state most of the day, insulin-mediated inhibition of adipose tissue
lipolysis is a major regulator of lipolytic rate. Insulin-mediated suppression of
adipose tissue lipolysis per unit fat mass is attenuated in obese individuals,
suggesting that chronic hyperinsulinemia cannot overcome the increase in whole-
body lipolysis. Therefore, inhibition of adipose tissue lipolysis might be a
therapeutic strategy to limit excess FFA release, thereby alleviating the
development of insulin resistance and obesity-associated metabolic abnormalities.
Although data are scarce, reduced plasma FFA and glucose levels have been
demonstrated in diabetic rats treated with a selective HSL inhibitor for 3-8 hours
[100]. Recently, Girousse et al. [101] have shown that mice treated with the same
+/-
pharmacological HSL inhibitor for 7 days and haploinsufficient HSL mice were
paradoxically resistant to diet-induced obesity due to a reduction in FFA uptake
and reesterification, suggestive of reshaping the FFA flux in peripheral tissues
[101]. Furthermore, in that study, systemic glucose tolerance was improved
through induction of de novo lipogenesis in murine and human adipocytes. In
addition to selective inhibition of HSL, recent data report about the development of
a selective inhibitor of ATGL, Atglistatin [102], and about the mechanisms of
inhibition of ATGL by long-chain acyl-coenzyme A [103], highlighting the increasing
interest in selective lipase inhibition to correct defects in lipid metabolism for the
treatment and prevention of obesity and obesity-associated metabolic diseases.
In contrast to an elevated basal lipolysis, others and we have clearly shown that in
vitro and in vivo catecholamine-induced lipolysis is blunted in subcutaneous

46
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adipose tissue of obese subjects, which persists after significant weight loss [89,
94, 95]. These data suggest that impaired cathecholamine-induced lipolysis may
be an important primary factor in the development of obesity. Data are limited with
respect to NP-induced lipolysis. However, reduced circulating NP levels [104] and
a defective ANP-mediated lipolytic response in subcutaneous adipocytes and
adipose tissue from obese subjects have been observed [105]. Therefore, in
contrast to the anti-lipolytic approach with selective lipase inhibition, improving
catecholamine- and NP-sensitivity has been extensively investigated for the
treatment of obesity and obesity-related complications but has, so far, not shown
promising results due to cardiovascular side effects and receptor desensitization
[106-108].
In addition to classical lipolysis, three recent studies have implicated autophagy, a
homeostatic mechanism functioning as a ‘self-digestion’ system, in selective lipid
hydrolysis under basal and catecholamine-stimulated conditions in adipocytes,
termed lipophagy [109-111]. These data indicate that activation of cytosolic lipases
(i.e. ATGL and HSL) is no longer the sole molecular mechanism to liberate FA from
adipocyte TAG stores. Since ATGL and HSL are expressed at much higher level in
adipocytes than other cell types it is plausible that under normal physiological
conditions adipocytes rely mainly on classical cytosolic lipolysis, while the
alternative pathway for lipid breakdown, lipophagy, may become more important in
pathophysiological conditions, with a reduced ATGL and HSL activity, to maintain
lipid homeostasis. In line, autophagy markers and fluxes appear to be elevated in
adipose tissue of obese insulin resistant and T2D subjects [112-115]. In addition,
nutritional and hormonal regulation of adipose tissue autophagy is impaired in
obese rodents [116].
To summarize, obesity is characterized by an increased basal and a blunted
catecholamine and NP-stimulated lipolysis in subcutaneous adipocytes. This
altered lipid turnover may be an early factor in the development of increased fat
stores and obesity-associated metabolic complications. Modulation of classical
lipolysis recently regained interest in the treatment of obesity-related insulin
resistance, indicated by the development of selective ATGL and HSL inhibitors.
However, to prevent excessive gain in body weight, tissue FFA turnover (uptake,
esterification and oxidation) should be adapted accordingly. Furthermore, lipohagy
might be increased in adipose tissue of obese subjects as compensatory
mechanism to deal with the increased lipid availability due to an attenuated
classical lipolysis. Importantly, before considering manipulation of the classical
and/or alternative pathway of adipose tissue lipolysis for therapeutic purposes, a
better insight into its role in pathophysiology as well as hormonal and nutritional
regulation is warranted.

Lipid droplet formation and fatty acid storage in the adipose tissue
In humans, adipocytes, the major cell type in white and brown adipose tissue
(BAT), are specialized for storing lipids in LD. Several proteins bind the LD surface
and regulate LD size, fusion and number. They include PAT proteins (i.e. perilipin
1, perilipin 2/adipophilin/ADRP, perilipin 3/TIP47 and perilipin 4/S3-12), CIDE (Cell
Death inducing DNA Fragmentation Factor) proteins, Soluble NSF Attachment

47
Chapter 2

Protein Receptor (SNARE), caveolins and several lipases. LDs are dynamic
organelles, constantly forming, growing or shrinking. In recent years, our
knowledge of LD dynamics and biogenesis has increased, as reviewed extensively
elsewhere [117-123]. Impaired LD expansion and TAG storage capacity may play a
role in lipid storage diseases, including obesity and T2D. In obese conditions, the
storage capacity of the LD in adipocytes is exceeded. CIDE-C/FSP27 (fat-specific
protein 27) and PLIN-1 largely regulate TAG storage in human adipocytes by
facilitating lipid transfer from smaller to larger LDs and by regulating intracellular
lipolysis. Studies examining the expression of CIDE proteins and PLIN-1 as a
function of insulin sensitivity found that mRNA levels of these LD-associated
proteins correlate positively with insulin sensitivity in subjects with similar body
mass index [124, 125]. These findings in humans contrast with findings in mice, in
which the lack of CIDEC/FSP27 or PLIN-1 protected against high fat diet-induced
obesity and insulin resistance [126-128], highlighting the difficulties in extrapolating
results from mice to human pathologies.
In summary, although human data are limited, high levels of protein that promote
TAG storage, including CIDEC/FSP27 and PLIN-1, might help to sequester lipids in
the adipose tissue and to protect against insulin resistance. Furthermore, impaired
LD expandability may prevent the recruitment of new adipocytes by either initiating
a pro-inflammatory response or by preventing the secretion of yet unidentified
factors that promotes recruitment of adipocyte progenitors.

Adipocyte differentiation and adipose tissue expandability

A unique property of adipose tissue is its capacity to change its dimensions. This
can be achieved by fat cell enlargement (hypertrophy) or by recruitment of new
adipocytes from the resident pool of progenitor cells (hyperplasia).
During periods of chronic excessive energy intake, the adipose tissue mass
expands first by hypertrophy to the point that the maximal fat cell expandability is
achieved. Then, signals are released by the adipocyte to stimulate proliferation
and/or differentiation of preadipocytes (adipogenesis) [129], leading to an
increased number of mature adipocytes. This process is necessary to store the
excessively available FAs and to protect cells from detrimental effects of high
concentrations of circulating FAs [130]. Interestingly, there seems to be adipose
tissue depot-specific patterns in adipose tissue expansion. Overfeeding evokes
adipocyte hypertrophy in abdominal subcutaneous adipose tissue, whereas
adipocyte hyperplasia, but not hypertrophy, occurs in the femoral subcutaneous fat
[131]. Adipogenesis is also accompanied by specific changes in the adipocyte
extracellular matrix (ECM), as reviewed elsewhere [132-134], and this ECM
remodeling seems essential in adipose tissue expansion. Research showed that
ECM processes are disrupted in obesity and impair metabolic function and fat
mass expansion [135, 136]. Furthermore, diet induced weight gain by overfeeding,
resulted in fat mass expansion and upregulation of genes involved in lipid
metabolism and storage, angiogenesis and ECM remoddeling [137].
The inability of the adipose tissue to store excess lipids in newly differentiated
adipocytes results in enlargement of existing adipocytes [138]. These hypertrophic
adipocytes are less sensitive to the action of insulin and have impaired lipid

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buffering capacity [139]. The adipogenic potential of adipose-derived stem cells

(ASCs) depends on their depot specific origin and host characteristics such as age,
sex and metabolic status [140]. ASCs from subcutaneous fat have been reported to
differentiate better into mature adipocytes than those from visceral fat [141]. In
contrast to what was thought in the past, the expandability of the adipose tissue is
not an unlimited process. Importantly, impaired adipose tissue expandability has
been linked to metabolic derangements. For example, lipodystrophic patients with
severely reduced adipose tissue mass, show a substantial reduction in fat storage
capacity. Hypertrophic adipocytes in obese individuals, on the other hand, are
overloaded with stored TAG. In both conditions, the storage capacity of adipose
tissue seems insufficient, resulting in ectopic fat storage and, consequently, insulin
resistance [7].
Kim et al. [138] have shown that ectopic fat storage and insulin resistance can be
ameliorated when adipose tissue mass can properly expand to accommodate
excess calories. Involvement of ASCs in the impaired adipose tissue expandability
is only beginning to be explored. It has been reported that the number of mature
adipocytes is set during childhood and stays constant throughout adulthood
regardless of fat mass changes in humans [142], but it is not yet clear how adipose
progenitor cells contribute to this process. Thus, strategies to increase adipose
tissue expandability may reduce ectopic fat storage, thereby improving insulin
sensitivity, despite possible body weight gain.

Adipose tissue mitochondrial function and browning

Mitochondria play a central role in the catabolism of nutrients to provide energy that
is required for numerous cell functions. During periods of energy requirement,
FFAs are liberated to fuel mitochondrial β-oxidation. There is substantial evidence
that adipose tissue mitochondrial mass and function determine metabolic health.
Rodent studies have suggested that mitochondria in white adipose tissue
contribute to overall fat oxidation [143]. Impaired mitochondrial oxygen
consumption in adipose tissue is present in mouse models of obesity and T2D
[144], and mitochondrial oxidative pathways are downregulated in human adipose
tissue [145]. Importantly, adequate mitochondrial function is essential to maintain
adipose tissue function, glucose homeostasis [146] and protects against insulin
resistance and T2D [144]. This is further highlighted by a recently large-scale
microarray analysis using >1000 human abdominal subcutaneous adipose tissue
biopsies, revealing that mitochondrial oxidative pathways were markedly
downregulated, whereas inflammatory pathways were upregulated, in morbidly
obese insulin resistant as compared to morbidly obese insulin sensitive patients
[147]. In accordance, mitochondrial mass and electron transport chain genes in
adipose tissue are lower in obese and T2D subjects [148, 149]. A recent study has
demonstrated that both human adipocyte mitochondrial content and ex vivo
respiration (oxygen consumption) were significantly reduced in obese as compared
to lean individuals, independent of adipocyte size [150]. A decreased adipose
tissue mitochondrial function may lead to an incorrect scavenging of fatty acids,
resulting, on the one hand, in increased intracellular FFA concentrations reducing
FFA trapping and, on the other hand an enhanced release of FFA, liberated by

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intravascular or intracellular lipolysis into the circulation, contributing to lipid

overflow [151]. Interestingly, the ‘browning’ of white adipocytes, which will be
further discussed below, likely involves lipase-generated PPAR ligands, whereby
lipolysis may be an initiating factor (e.g. recruiting adult adipocyte progenitor cells
into a brown adipocyte lineage) [152].
Traditionally, fat cells have been divided into unilocular white and multilocular
brown adipocytes. Brown adipocytes are specialized heat producing cells, and
transfer energy from lipid and carbohydrate substrates into heat through the
actions of uncoupling protein-1 (UCP-1). In contrast to infants, who have
substantial amounts of BAT to maintain body temperature, it was thought for many
years that healthy adult humans living under normal environmental temperatures
18
lack BAT. However, F-fluoro-D-2-deoxy-D-glucose (FDG) positron emission
tomography (PET)-positive areas have recently been identified in lean healthy
adults, indicating that substantial amounts of BAT are present in humans [153-
155]. Furthermore, it has been demonstrated that BAT is inversely related to body
fat percentage and BMI [153], and BAT activity was increased 1 year after bariatric
surgery-induced weight loss in morbidly obese subjects [156]. Recently, BAT has
been implicated to improve whole body glucose homeostasis and insulin sensitivity
in humans [157]. Moreover, a third category of fat cells has been introduced,
namely “beige/brite” adipocytes, which have a brown fat-like morphology within
white fat depots [158-161]. Beige adipocytes seem to be programmed to be
bifunctional, meaning they are suitable for energy storage in the absence of
thermogenic stimuli but capable of switching on heat production [158]. It is not
entirely clear yet what distinguishes brown from beige adipocytes, and the relative
importance of BAT in energy homeostasis also requires further investigation.
Nevertheless, it has been estimated that BAT may contribute to 3-5% of basal
metabolic rate in humans [162]. Therefore, an intriguing question is how BAT can
be activated and whether this may induce weight loss in humans.
In addition to cold-induced BAT activity, several factors have been studied that
affect activity and recruitment of brown adipocytes. Briefly, these include
adrenergic stimulation, natriuretic peptides, irisin, capsinoids, and insulin in
humans [162, 163]. In rodents, it has been suggested that, in addition to the above-
mentioned factors, thyroid hormone, bile acids, fibroblast growth factor 21 (FGF21)
[164, 165] and bone morphogenetic proteins (BMP) [166] may also play a role.
Future studies will likely elucidate whether strategies to increase browning of white
adipose tissue hold promise to prevent and/or treat obesity and related
impairments in lipid and glucose metabolism.

Lipid-induced inflammation and impaired glucose metabolism

Adipose tissue used to be considered as a passive fat storage organ. However, it is
clear for more than two decades that adipose tissue is an active endocrine organ
by the release of a variety of lipids (lipokines) [167] and signaling molecules
(adipokines). These factors can both act locally (autocrine and/or paracrine) and at
the whole-body level (endocrine function) to exert a wide range of biological effects
and to regulate systemic metabolic homeostasis [7]. The endocrine function of
adipose tissue is impaired in obese, insulin resistant individuals, which may

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contribute to chronic low-grade inflammation and, consequently, obesity-related

insulin resistance and other complications [7]. In addition to adipocytes, adipose
tissue contains a wide variety of stromal vascular cells, including endothelial cells,
fibroblasts, preadipocytes and immune cells. Although mature adipocytes make up
>90% of adipose tissue volume, they account for only 20-40% of the cellular
content. Interestingly, immune cells present in adipose tissue have recently been
shown to play an important role in adipose tissue biology [164]. The low-grade
inflammatory state in obesity is the result of an imbalance between the production
of pro-inflammatory and anti-inflammatory factors. It was shown about 10 years
ago that many proinflammatory factors are produced by macrophages that infiltrate
hypertrophic adipose tissue [168, 169]. More recent evidence indicates that also
other innate and adaptive immune cells are present in adipose tissue, as reviewed
elsewhere [170]. Furthermore, it seems that dynamic changes occur in the adipose
tissue immune cell populations during the development of obesity, causing a shift
from an anti-inflammatory towards a more pro-inflammatory phenotype. It has been
demonstrated that there is a relative abundance of M1 polarized macrophages,
Treg cells and eosinophils in lean visceral adipose tissue, whereas the proportion
of M2 polarized macrophages, Th1 cells, mast cells, dendritic cell, neutrophils and
natural killer T cells is increased in obesity. Intriguingly, many of the latter
proinflammatory immune cells have been linked to impairments in glucose
homeostasis [171, 172] and decreasing the inflammatory status may improve
insulin resistance in rodents and humans [173].
An important but still unanswered question is what triggers adipose tissue
inflammation in obesity? Lipids act as important signal moieties regulating both
metabolism and immune responses. This is exemplified by the induction of hepatic
and peripheral insulin resistance [174] and pro-inflammatory responses in adipose
tissue [175] by lipids. Various mechanisms seem to be involved in the induction
and progression of inflammatory responses in adipose tissue and consequently
insulin resistance in obesity. Briefly, lipid-induced activation of innate receptors
(Toll-Like Receptors (TLR)) and the inflammasome, death of hypertrophic
adipocytes and resulting macrophage infiltration, lipid-induced endoplasmic
reticulum stress (ER stress) and the unfolded protein response and, more recently,
the gut microbiota have been linked to adipose inflammation and consequently
insulin resistance in obesity [176]. On the other hand, activation of PPAR-γ by
polyunsaturated fatty acids (PUFA) [177] may reduce adipose tissue inflammation
[178]. However, it remains to be established to what extent the above-mentioned
mechanisms contribute to systemic inflammation and whole-body insulin resistance
in humans. Over the last decade, evidence has emerged that ER stress in several
organs, including adipose tissue, plays a direct (i.e. as a negative modulator of the
insulin signaling pathway) and indirect role (i.e. by promoting lipid accumulation) in
the onset of insulin resistance, as reviewed elsewhere [179].
Adipose tissue inflammation may in turn exert detrimental effects on insulin
sensitivity through different mechanisms. For example, tumor necrosis factor alpha
(TNF-α) and Interleukin 6 (IL-6) inhibit adipocyte differentiation [180-182] and TNF-
α induces adipocyte apoptosis in pre- and mature adipocytes [183], which could
lead to the enlargement of the remaining fat cells and consequently, reduced
adipose tissue lipid buffering. Furthermore, TNF-α and IL-6 both stimulate
adipocyte lipolysis [184-187], thereby contributing to systemic FA release, which

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may in turn lead to ectopic fat storage and insulin resistance [188]. In addition,
lipid-induced impairments in adipokine secretion may also exert direct effects on
peripheral insulin sensitivity [189]. It has been demonstrated that adiponectin
increases skeletal muscle fat oxidation and therefore, the decreased adiponectin
concentrations in obese conditions may impact fat oxidation, and as such, affect
lipid accumulation and insulin sensitivity [190, 191].
As reviewed by Ohashi et al [192], several anti-inflammatory adipokines, such as
adiponectin, the family of the C1q/TNF-related Proteins (CTRP3, -6, -9), adipolin
and omentin-1 may exert beneficial effects on obesity-related complications.
CTRP3 is a novel adipokine, expressed at the AT and found in circulating plasma
[193, 194], which regulates hepatic glucose output [195], suppresses chemokine
production in response to lauric acid, LPS or TLR stimulation in macrophages and
adipocytes [196] and stimulates the expression of adiponectin in primary human
adipocytes and cultured 3T3-L1 adipocytes [197]. CTRP6 has been found to
increase the expression of the anti-inflammatory cytokine IL-10 in human
monocyte-derived macrophages [198], to stimulate activation of AMPK and
enhance fatty acid oxidation in skeletal muscle cells [199]. In cultured myocytes,
CTRP9 activates phosphorylation of AMPK and protein kinase B (PKB) and
promotes insulin stimulated glucose uptake [194]. It also reduced diet-induced
weight gain, decreased insulin resistance and hepatic steatosis, with enhanced
AMPK activation and fat oxidation in skeletal muscle in CTRP9 transgenic mice
[200]. CTRP12, also known as Adipolin (adipose-derived insulin-sensitizing factor)
[201], has been reported to activate insulin signaling in the liver and adipose tissue
of obese mice [202]. Furthermore, omentin-1, also known as intelectin-1, is
abundantly expressed in visceral fat tissue [203], is decreased in obese individuals
[204] and increased insulin stimulated glucose uptake in cultured adipocytes in
vitro [203].
In addition, dipeptidyl peptidase-4 (DPP4) is released by mature adipocytes and
inhibits skeletal muscle insulin signaling [205, 206]. Beside lipolysis and insulin
signaling, pro-inflammatory cytokines may also regulate adipocyte mitochondrial
function [207]. Of note, not only adipose tissue but also other organs such as the
liver, skeletal muscle, heart and pancreas may contribute to lipid-induced systemic
inflammation in obesity via secretion of cytokines [208, 209].
Taken together, lipids are important triggers for adipose tissue inflammation and
consequently insulin resistance in obesity. Therefore, interventions aimed at
improving lipid metabolism may improve glucose metabolism via reduction of
adipose tissue and systemic inflammation.

Putative nutritional targets to improve adipose tissue function

The balance between lipolysis, adipocyte differentiation and mitochondrial function
within adipose tissue is important to maintain adequate lipid storage capacity of
adipose tissue, thereby preventing lipid overflow in the circulation and ectopic fat
deposition. Therefore, improving the lipid buffering capacity of adipose tissue has
high potential to increase glucose tolerance and insulin sensitivity.

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Presently, there is a clear need for additional studies to determine the regulators of
LPL activity, including the involvement of proteins affecting post-translational
mechanisms, like ANGPTL4, which may be regulated by diet [63].
Secondly, as discussed above partial HSL or ATGL inhibition may reduce lipid
overflow and possibly improve metabolic profile. Interestingly, intravenous acetate
infusion [210] or colonic acetate administration may reduce systemic FFA
concentrations, through an effect on HSL phosphorylation [211]. Dietary
manipulation of colonic acetate and short chain fatty acid (SCFA) concentration
through pre- and probiotics may be a promising target in this respect.
Thirdly, modulation of the adipose tissue lipophagic pathway might be a potential
target pathway as well. Recently, we have shown that dietary polyphenols including
resveratrol and epigallocatechin-gallate (EGCG), found naturally in red wine and
green tea have caloric restriction-like effects in overweight humans [212].
Interestingly, our microarray data showed that an improved adipose tissue lipolysis
and function, is associated with selectively targeting of the master regulator of
lipophagy, TFEB, in human adipose tissue following resveratrol supplementation
[213]. However, it remains to be determined whether lipophagy-mediated lipid
catabolism in adipose tissue is directly involved in the potential beneficial effects of
polyphenols.
As indicated above, nutritional strategies to improve mitochondrial function, like
specific polyphenols or a combination of polyphenols, may be effective in balancing
lipid supply to utilization [212, 213], improving thereby adipose tissue function.
Moreover, recent data from our group also suggest that a dysbalance between
oxygen supply and oxygen utilization leading to an increased adipose tissue
oxygen tension may induced adipose tissue dysfunction [214, 215], again
illustrating the importance of a normal mitochondrial mass and function.
Factors affecting the activity and recruitment of BAT, may have positive effect with
respect to adipose tissue function, body weight control and insulin sensitivity.
Currently, most promising may be dietary components affecting our microbial bile
acid metabolism, leading to increased circulating FGF21, a mediator of lipid and
carbohydrate metabolism, also inducing recruitment of brown adipocytes [216].
The effect of dietary quality on adipose tissue function and ectopic fat accumulation
remains an area of particular interest in composing optimal diets that minimize
ectopic and abdominal fat accumulation. There is strong evidence that avoidance
of high saturated fatty acids (SFA) diet contributes to lower health risks among
obese, metabolic syndrome and diabetic patients [217]. There are indications that
the monounsaturated fatty acids (MUFA) and/or PUFA have more beneficial effects
compared to SFA on the action of insulin [218-221]. Research showed that n-3
PUFA (eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and
docosahexaenoic acid (DHA)) reduced LD formation in 3T3-L1 cells compared to
SFA [222]. Further, long chain n-3 PUFA may increase fatty acid oxidation and
mitochondrial biogenesis in adipose tissue [223, 224], may inhibit fat cell
proliferation [225] and may limit fat cell hypertrophy and hyperplasia [226].
Additionally, positive effect have been ascribed to specifically n-6 fatty acids in
reducing abdominal fat area, improving insulin sensitivity and in reducing visceral
fat/subcutaneous fat ratio compared with a SFA diet [227, 228].
Dietary fat quality not only modulates lipid metabolism, it may also affect low-grade
inflammation, as for instance seen by exposure of myotubes or adipocytes to SFA

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which increased IL-6 mRNA and protein expression, possibly via activation of
nuclear factor-κB (NF-κB) [229, 230]. A human intervention study with a SFA rich
diet for 5 weeks showed higher concentrations of C-reactive protein (CRP),
fibrinogen and IL-6 compared to a diet enriched in MUFA [231]. Substantial
increase in the PUFA intake can also decrease low-grade inflammation as
indicated by circulating CRP, IL-6 or soluble adhesion molecules [232, 233].
In summary, polyphenols, specific dietary fatty acids and pre- and probiotics may
be promising nutritional components in improving the balance between lipolysis,
autophagy and mitochondrial function and in stimulation of adipose tissue
browning, thereby improving adipose tissue function and whole-body glucose
homeostasis. For an overview of putative nutritional targets see Table 1.

ALTERED LIVER FUNCTION AND IMPAIRED GLUCOSE METABOLISM

Obesity is recognized as a major cause of the promotion of metabolic diseases
including non-alcoholic fatty liver disease (NAFLD), which is not only linked to an
impaired glucose metabolism and diabetes, but also evokes more severe liver
diseases like non-alcoholic steatohepatitis (NASH), hepatic cirrhosis and eventually
liver cancer.
Indeed, hepatic insulin resistance, which is defined as an impaired suppression of
hepatic glucose production [234], may originate from the accumulation of lipid
metabolites that interfere with insulin signaling [235]. This accumulation of lipids
and lipid metabolites may be caused by multiple factors [236]. Besides that, liver
lipid accumulation is also associated with the progression of ER stress,
mitochondrial stress and an impaired autophagy, resulting in lipotoxicity [237]. An
increased FFA release from visceral adipose tissue (the ‘portal hypothesis’) or
increased intake of dietary fat, followed by a reduction in postprandial lipid
clearance and an increased spillover of FFA in the circulation, due to a reduced
lipid storage capacity, can explain this phenomenon [238-241]. Furthermore,
mitochondrial dysfunction, associated with insulin resistance may precede liver fat
accumulation by impairing fatty acid β-oxidation [242]. Additionally, an increased
de novo lipogenesis highly contributes to liver lipid accumulation, ultimately leading
to steatosis [243] (Figure 3).
When FA supply to the liver is increased, VLDL-TAG secretion by the liver is also
increased, but it appears that this secretion of VLDL-TAG is not sufficient to
compensate for the increased uptake, resulting in a net lipid accumulation [244],
even though conditions of insulin resistance are marked by an increased
production of VLDL-TAG [245]. Below, the mechanisms contributing to liver fat
accumulation are briefly delineated.

Hepatic lipid uptake

In obesity, insulin resistance and T2D, circulating levels of FFA and lipoprotein
remnants are increased [246-248] and this causes an increased storage of TAG
and lipid metabolites in hepatocytes and may lead to hepatic insulin resistance.
This may further increase circulating VLDL-TAG concentrations due to a decreased
insulin-mediated suppression of VLDL production by the liver [249-252].

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In the liver, beside LPL, hepatic lipase (HL) plays an important role in fatty acid
metabolism, as it is both a phospholipase and a TAG lipase [253]. An increased HL
activity promotes FFA uptake into hepatocytes and has been shown to be
increased in obese humans [254-256], insulin resistant rodents [257], type 1
diabetic patients [258] and in NAFLD patients [259].
The rate of plasma FFA uptake by the liver is determined by both the plasma FA
concentration and the hepatocellular capacity for FA uptake [260], which depends
on the number and activity of transporter proteins on the plasma membrane of the
hepatocyte. The main proteins in the hepatic transmembrane FA transport are
FATP-2 [261], FATP-5 [262], caveolins, and to a lower extent fatty acid translocase
CD36 [263, 264]. FATP-5 knockout mice show resistance to diet-induced obesity
and hepatic lipid accumulation, but there is no evidence for involvement of this
isoform in human obesity [265]. Also, silencing of hepatic FATP-5 in hyperglycemic
mice showed a rapid (2-3 weeks) reduction in liver FFA uptake and a reduction in
serum glucose levels, reaching normal glycaemia after 5 weeks [262]. Additionally,
expression of FABP-4 and -5 correlated with hepatic fatty acid infiltration in NAFLD
patients [266]. Interestingly, it was shown that specific bile acids (BAs)
(ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA)) inhibit liver-specific
FATP-5 in mice [267], suggesting that specific bile acids can impact hepatic lipid
metabolism. Caveolin proteins consisting out of caveolin-1, -2 and -3, play an
important role in protein trafficking and the formation of lipid droplets. Caveolin-1
knockout mice have lower TAG accumulation in the liver and are resistant to the
development of diet-induced obesity, suggesting that this protein may be of
importance in liver fat accumulation and TAG synthesis [268]. Furthermore, rodent
in vivo and in vitro data show that overexpression of caveolin-3 in liver resulted in
an improved insulin receptor signaling, insulin sensitivity and glucose metabolism
[269]. Normally, CD36 is not highly expressed in liver, but its expression was
positively correlated with hepatic TAG content in NAFLD patients, underscoring the
importance of this protein in lipid accumulation [270]. Moreover, CD36 deficiency
increased insulin sensitivity in muscle, but resulted in an insulin resistant state in
the liver of mice [271]. It is evident that further research is required to elucidate the
role of these FA transporters in normal physiology as well as pathological
conditions.

Hepatic lipolysis
Rodent in vivo and in vitro data showed that hepatic ATGL (also known as PNPLA2
and desnutrin) knockdown enhances glucose tolerance by increasing hepatic
glucose utilization and improved insulin action from hepatic TAG accumulation
[272]. Furthermore, HSL-knockout mice also showed reduced hepatic TAG stores
and increased hepatic insulin sensitivity [273], but results are not consistent [274].
In adipose tissue, ATGL and HSL are highly expressed and active [275], but in the
liver, the contribution of other lipases may also play a role in the catabolism of
stored lipids. Several members of the carboxylesterase/lipase family and the
patatin-like phospholipase domain-containing protein (PNPLA) family have been
suggested as potential TAG hydrolases [276]. One of them, carboxyl esterase-
3/triglyceride hydrolase-1 (Ces-3/Tgh-1, ortholog of human Ces-1), has gained

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major interest because the recent characterization of Ces-3/Tgh-1-deficient mice

provided compelling evidence that the enzyme participates in the assembly and
secretion of hepatic VLDL [277]. PNPLA-4 and -5 exhibit TAG-hydrolase, DAG-
transacylase and retinylester hydrolase activity in vitro [278], but whether these
activities are also relevant in vivo remains to be determined [90]. The
adiponutrin/PNPLA-3 I148M polymorphism was reported to be associated with
insulin resistance and NAFLD, suggesting a role of importance in hepatic TAG
accumulation [279-281]. Furthermore, LD-covering proteins also play a role in the
pathophysiology in fatty liver disease, which is characterized by hepatocytes
containing LD with excessive neutral lipids [282]. Studies showed that LD proteins
such as PLIN-1 and PLIN-2 are highly expressed in liver steatosis [283, 284]. A
high fat diet increases expression of PLIN-2 in a PPAR-γ dependent manner,
resulting in the development of fatty liver [285-287]. Data from a rodent study
showed that PLIN-2 knockout enhanced insulin action in the liver, whereas muscle
and adipose tissue were not affected [288]. Furthermore, other LD-associated
proteins such as FSP27 and CIDEC have also been characterized. FSP27 was
expressed in the steatoic liver of a T2D mouse model and the expression was
markedly decreased in livers lacking PPAR-γ. Forced expression of FSP27 in
hepatocytes in vitro or in vivo led to an increase of LD through increased TAG
levels, as reviewed elsewhere [289].
In addition to the classical lipolysis, recent evidence suggests a role for autophagy
and lysosomal lipid degradation in liver LD (also termed lipophagy) [109, 290], via
the action of lysosomal lipase (LAL) [109]. Even though this lipophagy accounts for
a high percentage of the lipolysis that takes place in response to lipid challenges
and during prolonged starvation in liver [109], also a certain percentage of
degradation of lipids in lysosomes may occur continuously in many cell types (e.g.
adipose tissue and skeletal muscle) [109]. Further research is warranted to
determine the reasons behind the coexistence and possible co-regulation of the
two different lipolytic pathways, the one mediated by the cytosolic lipases (i.e.
ATGL and HSL) and the other mediated by the autophagic-lysosomal degradation
system (i.e. LAL). It is possible that activation of one lipolytic pathway or the other
may be related to total capacity (i.e. lipophagy may be able to degrade larger
amounts of triglycerides in shorter time). However, it is also possible that the
quality and type of the resulting lipolytic products differs between cytosolic and
lysosomal lipases, but data on this are largely lacking. Lastly, in light of the growing
evidence in support of the heterogeneity of the cellular LD, it is also plausible that
the two lipolytic systems target different subpopulations of LD’s [291].
In the presence of insulin resistance and hyperinsulinemia, hepatic autophagy has
been reported to be reduced [292] and it has been shown that defective autophagy
is causal to impaired hepatic insulin sensitivity and glucose homeostasis in obese
mice [293]. Since most evidence of autophagy comes from rodent studies, more
research is warranted to elucidate the exact mechanism of action of hepatic
autophagy and possible lysosomal dysfunction in human insulin resistant subjects.

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Figure 3. Disturbances in liver fatty acid metabolism

When circulating levels of FFA and lipoprotein remnants are increased, uptake and storage (1) of TAG
and lipid metabolites in hepatocytes occur via increased LPL, HL activity and number and activity of
transporter proteins (such as FATP2-5, FABP4) leading to hepatic insulin resistance. Increased insulin-
stimulated de novo lipogenesis (2) contributes to hepatic lipid accumulation. In addition, under insulin
resistant conditions hepatic mitochondrial function is decreased (3) and may inhibit FA oxidation,
contributing to hepatic lipid accumulation, increased VLDL production (4) and an inflammatory state (5).
During conditions of hepatic lipid accumulation, an overproduction of VLDL occurs and together with an
increased de novo lipogenesis results in hypertriglyceridemia, a condition often seen in insulin
resistance and T2D. Finally, circulating bile acid composition (6) is altered in insulin resistant and T2D
patients and might affect liver lipid metabolism via membrane (e.g. TGR5) and nuclear (e.g. RXR)
receptor signaling. Interestingly, plant sterols have been shown to be effective nuclear receptor
activators. Abbreviations: FFA: Free fatty acids; TAG: Triacylglycerol; DAG: Diacylglycerol; LPL:
Lipoprotein lipase; HL: Hepatic lipase; FATP2-5: Fatty acid transport proteins 2-5; FABP4: Fatty acid
binding protein 4; VLDL: Very-low-density lipoprotein; TGR5: G-protein coupled receptor TGR5; FXR:
Farnesoid X receptor; LXR: Liver X receptor; RXR: Retinoid X receptor.
Solid lines indicate stimulation. Green lines indicate beneficial effects. Symbols: (): Altered; ():
Increased; (): Decreased

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De novo lipogenesis
On the other hand, increased insulin concentrations stimulate de novo lipogenesis
(DNL), a process that is characterized by converting carbohydrates towards FAs
and generating “new lipids” [294, 295]. The overproduction of VLDL together with
an increased DNL, leads to hypertriglyceridemia, a condition often seen in insulin
resistance and T2D [245]. During DNL, the conversion of glucose to fatty acids
includes a coordinated series of enzymatic reactions of which fatty acid synthase
(FAS) is the key rate-limiting enzyme that regulates the conversion of malonyl-CoA
into palmitate, which is, thereafter, converted into complex fatty acids [296].
Knockout of FAS has been shown in mice to provoke a fatty liver phenotype upon
high carbohydrate feeding, perhaps due to an increase in hepatic malonyl-CoA
[297]. Another key enzyme is stearoyl-CoA desaturase 1 (SCD1), which plays a
role in the desaturation of fatty acids and is predominantly expressed in liver.
Depletion of SCD1 in mice showed a decreased lipogenesis and an increased β-
oxidation [298]. During DNL, the final step of TAG synthesis is catalyzed by
diacylglyceroltransferase (DGAT), which consists of DGAT1 and DGAT2. DGAT1
deficient mice are resistant to diet-induced obesity [299], have decreased levels of
tissue TAG and are more insulin and leptin sensitive [300]. Furthermore, glucose
and insulin regulate the expression of lipogenic enzymes via carbohydrate
responsive element binding protein (ChREBP) [301] and sterol regulatory element
binding protein-1 (SREBP1c) [302, 303], respectively. Both SREBP1c and
ChREBP are involved in the transcriptional regulation of lipogenic genes and have
been associated with increased DNL in NAFLD [304]. DNL is a highly regulated
process and can lead to adverse metabolic consequences when dysregulated.
Therapeutic targeting of this pathway may open a new window of opportunity for
combating various lipogenesis-driven pathological conditions like obesity and
insulin resistance.

Hepatic mitochondrial function and fatty acid oxidation

The majority of FAs are oxidized within mitochondria and lower amounts of
oxidation takes place in peroxisomes [305], which are dynamic, multifunctional
organelles that contribute to several anabolic and catabolic processes and are
essential for human health and development (for review, see: [306, 307]). While
medium- and short-chain FAs are thought to enter the mitochondrial matrix directly
[308], long-chain FAs require carnitine palmitoyltransferase I (CPT-1) to enter the
mitochondrial matrix. Insulin regulates CPT-1 by inhibiting transcription of the CPT-
1 gene [309] and this could lead to increased concentrations of TAG and lipid
metabolites inside hepatocytes. In contrast, the peroxisomal β-oxidation system is
CPT-1 independent, but complete FA oxidation does not take place [310] and the
chain-shortened acyl-CoAs are consequently exported to the mitochondria where
they undergo complete β-oxidation [311-313].
Research showed that during insulin resistant conditions, hepatic mitochondrial
function is altered [314-316]. During insulin resistant conditions, an increased
circulating glucose concentration may downregulate PPAR-α [317], which inhibits
FA-oxidation in men [318] and contributes to hepatic lipid accumulation and
increased VLDL production [319]. Furthermore, as reviewed by Koliaki et al. [320],

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an up-regulation of hepatic mitochondrial β-oxidation may occur as a result of lipid

oversupply. This would stimulate hepatic ATP and reactive oxygen species
production, possibly leading to local lipotoxicity, inflammation and oxidative stress
and resulting in impairments in mitochondrial function and morphology. Since non-
diabetic obese humans show normal or even greater hepatic mitochondrial function
compared to lean humans, the authors suggest that hepatic energy metabolism
transitionally adapts to chronic lipid overload by up-regulation of oxidative capacity
in states of obesity and steatosis, which could be followed by a progressive decline
in liver mitochondrial function during prolonged chronic insulin resistance,
associated with T2D and NASH [320].

Bile acid metabolism

Once synthesized in the liver, the free bile acids (BAs) are conjugated with the
amino acids taurine or glycine, secreted and stored in the gallbladder. After being
released into the intestinal tract to facilitate the absorption of dietary fats and fat-
soluble vitamins, about 95% of BAs are reabsorbed in the lateral ileum and
transported back to the liver via the entero-hepatic circulation with only 5% of BAs
excreted into the feces. The reabsorption of bile decreases the need for de novo
bile acid synthesis [321, 322] and the loss of BAs in the feces represents the
principal means of eliminating cholesterol from the body [321, 323, 324]. The
synthesis of BAs by oxidation of cholesterol occurs exclusively in the liver and
involves two different pathways. Firstly, the classical pathway converts cholesterol
to 7α-hydroxyl-cholesterol to ultimately form 2 major primary BA products:
chenodeoxycholic acid (CDCA) and cholic acid (CA) [325]. Secondly, the
alternative pathway (the “acidic pathway”) converts cholesterol to 27-hydroxy-
cholesterol and produces mainly CDCA [326]. The intestinal microbiota metabolize
primary BAs by dehydroxylation and deconjugation to form the secondary BAs
lithocholic acid (LCA) and deoxycholic acid (DCA) [321, 323, 324]. The gut
microbiota may not only regulate the conversion of BA, also the BA may regulate
the microbiota composition [327]. Complementary to the aid of absorbing lipids and
fat-soluble vitamins in the intestine, accumulating data also show that BAs have a
signaling function in regulating biological processes by binding to the nuclear
receptor farnesoid X receptor (FXR) and to the G-protein coupled receptor TGR5
(also known as GPR19) in gut and other tissues affecting glucose, lipid and energy
metabolism [321, 323, 324], as discussed more extensively below.

FXR-receptor
The FXR-receptor is highly expressed in the liver, intestine, kidney and adrenal
gland [321, 323, 324, 328] and is also shown to play a role in liver regeneration,
inflammation and tumorgenesis [321, 324]. Both conjugated and unconjugated BAs
can activate FXR and the order of potency is CDCA > LCA = DCA > CA. CA
feeding increased hepatic expression of ApoC-2, a LPL activator, specifically
-/-
through FXR as this effect was not observed in FXR mice [329]. In addition, FXR
activation increased the expression of VLDL receptor [330] and syndecan-1 [331],
which are responsible for increased clearance of TAG-rich lipoprotein and remnant
particles, respectively. Furthermore, BA sequestrants, such as cholestyramine,

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were found to increase plasma TAG, mediated at least in part, through FXR [332],
which increased plasma lipoprotein clearance. However, other mechanisms may
also be involved in FXR-mediated lipid lowering effects. For example, PPAR-α was
induced by CDCA and GW4064 (a synthetic FXR agonist) treatment in HepG2
cells and primary hepatocytes [333]. GW4064 treatment also increased the mRNA
expression of PDK4, a PPAR-α target gene involved in the regulation of substrate
metabolism, in both rat hepatoma cells, human primary hepatocytes and also
resulted in a reduced plasma TAG concentration in vivo in mice [334]. BA or
GW4064 induced activation of FXR also increased the expression and secretion of
FGF21 [335], a cytokine modulating systematic carbohydrate and lipid metabolism
and reducing hepatic TAG levels [336-338]. FGF21 has been reported to inhibit
lipogenesis through suppressing the transcriptional activity of SREBP-1c [339] and
therefore, the FGF21 pathway may play an important role in FXR-mediated
decrease in hepatic TAG levels, which may be of importance in decreasing hepatic
insulin resistance [340, 341]. Besides the nuclear receptor FXR, the liver X
receptor (LXR-α and LXR-β) and retinoid x receptor (RXR) play an important role in
regulating carbohydrate and lipid metabolism in humans [342, 343]. Interestingly,
sitosterol, campesterol and certain oxidized derivatives of phytosterols
(oxyphytosterols) are effective LXR activators [344]. A rodent study showed that
LXR-α and LXR-α/β knockout mice remained glucose tolerant and insulin sensitive,
while LXR-β knockout mice became highly insulin resistant after a high-fat diet
[345]. Treatment of lean and ob/ob mice with the pharmacological LXR activator
(GW3695) resulted in lower blood glucose levels and significantly improved whole
body insulin sensitivity in the ob/ob mice, but no changes were found in the lean
mice [346]. Furthermore, the synthetic LXR-α/β activator (T0901317) augmented
diet-induced hyperlipidemia, normalized glucose tolerance and improved insulin-
stimulated glucose uptake in isolated soleus muscle and completely restored
glucose transporter 4 (GLUT4) expression and insulin-stimulated AS160
phosphorylation in rat muscle [347]. Although the vast majority of studies have
been performed in non-adipose cells/tissues, results in recent years suggest that
LXRs may have important modulatory roles on adipose lipid and glucose
metabolism [348].
In addition, PPAR-γ and RXR agonists have complementary effects on glucose
and lipid metabolism in human skeletal muscle [349], as well as in diabetic and
obese rodent models [350]. It has also been shown that a RXR ligand (LG100754)
improved insulin resistance in vivo in db/db mice [351] and a recent study showed
that a novel RXR partial agonist (CBt-PMN (11b)) has a glucose-lowering effect
y
and improved insulin secretion and glucose tolerance in the liver of KK-A mice
[352].

TGR5-receptor
TGR5 is a family member of the G-protein coupled receptors (also known as
GPR19) and is highly expressed in gallbladder, ileum and colon and in lower
concentrations in BAT, liver, muscle and the central nervous system [336]. The
activation of TGR5 is highest with the bile acid LCA, followed by DCA, CDCA and
CA [323, 336] and results in the activation of PKA [336]. Furthermore, TGR5
activation regulates the expression of genes involved in inflammation [353],

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increases energy expenditure in skeletal muscle and BAT [354] through stimulation
of mitochondrial function [355-357] and modulates plasma glucose and lipid
concentrations [336, 355, 356, 358]. BA mediated activation of TGR5 has
beneficial metabolic effects and the BA composition is altered in patients with
insulin resistance [359] and T2D [360]. Since gram-positive bacteria have a more
pronounced effect on the transformation of primary to secondary BAs [361, 362],
compared to most of the gram-negative bacteria [363], an alteration in the gut
microbiota might have a distinct effect on bile acid metabolism and might be an
effective strategy to improve insulin sensitivity. Indeed, in the study of Vrieze et al.,
a vancomycin-induced decrease in gram-positive bacteria was associated with a
reduced conversion of primary to secondary BAs and a tendency towards a
reduced peripheral insulin sensitivity [364]. Also, transgenic liver overexpression of
cholesterol 7α-hydroxylase, protected mice against high-fat diet induced obesity,
fatty liver and insulin resistance [365]. Furthermore, glucose metabolism can also
be improved in diabetic patients by administration of BA sequestrants, such as
colesevelam [366, 367]. Thus, a promising nutritional strategy may be to alter the
gut microbiota by modifying the diet by either prebiotics or probiotics and
modulating thereby BA metabolism [368, 369].

Hepatic inflammation
Since insulin resistance and obesity are characterized by a low-grade inflammatory
status [370], inflammation induced by the increased lipid accumulation, could also
be an underlying cause for the development of hepatic insulin resistance. Of
interest, ER stress has been shown to be involved in the development of hepatic
inflammation and insulin resistance. First, ER stress can directly modify key hepatic
enzymes involved in gluconeogenesis and lipogenesis, and stimulate stress
kinases that interfere with insulin signaling. Secondly, ER stress may indirectly
induce inflammation and lipotoxicity by promoting fat accumulation in hepatocytes
[179, 371, 372].
Furthermore, hepatic peroxisomal FA oxidation causes an increased concentration
of reactive oxygen species [373], which in excess cause a decrease in the natural
antioxidant concentrations, leading to oxidative stress in hepatocytes [374, 375]. In
turn, this oxidative stress causes hepatocyte degeneration and death [374] evoking
an inflammatory response [376]. Specialized liver macrophages, called Kupffer
cells, play a central role in this inflammatory process since in vitro stimulation of
these cells by endotoxin (e.g. lipopolysaccharides (LPS)) or specific FAs (e.g. SFA)
and their metabolites, lead to toll-like receptor signal transduction and the
production of inflammatory cytokines, including TNF-α and IL-6 [377].
Furthermore, in analogy to the adipokines, the liver-derived proteins (known as
hepatokines) [378] such as leukocyte cell-derived chemotaxin 2 (LECT2) regulate
cross talk with other tissues and link obesity with skeletal muscle insulin resistance
[379].
Taken together, the oversupply of lipids to the liver, together with impaired
clearance of circulating remnants, increased de novo lipogenesis and decreased
FA oxidation contribute to lipid accumulation in the liver, referred to as hepatic
steatosis, which can progress to NAFLD, NASH and liver cancer [380].

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Putative nutritional targets to reduce liver fat accumulation

All factors that improve adipose tissue function may reduce lipid overflow and
spillover to the liver and will, as such, likely reduce hepatic steatosis.
Furthermore, it has been shown that BAs influence energy expenditure and
glucose homeostasis via their effects on gluconeogenesis, insulin secretion and
insulin sensitivity in both mouse and human studies [355, 358, 381]. BA-mediated
activation of TGR5 has beneficial metabolic effects and the BA composition is
altered in patients with insulin resistance. Secondary BAs can also affect host
metabolism via binding to several nuclear receptors (FXR, LXR, RXR). Therefore,
manipulating BA metabolism to increase concentrations of secondary BAs could be
an attractive target to tackle obesity and insulin resistance [358, 381]. An alteration
of the gut microbiota might have distinct effects on BA metabolism and might
therefore be an effective strategy to improve insulin sensitivity. Beside that,
modulating gut microbiota may affect fermentation products from dietary fibers like
SCFA and monosaccharides affecting thereby liver gluconeogenesis and
lipogenesis, which might directly or indirectly affect glucose homeostasis [382].
The nuclear receptor FXR and LXR are important in regulating liver, muscle and
adipose tissue lipid and glucose metabolism in humans and it has been shown that
sitosterol, campesterol and certain oxidized derivatives of phytosterols
(oxyphytosterols) are effective LXR activators [344].
In summary, in particular nutritional factors targeting BA metabolism, gut microbiota
like pre and probiotics and factors affecting the nuclear receptors FXR and LXR
like plant sterols seem promising targets in improving liver fat metabolism thereby
improving glucose homeostasis (Table 1).

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IMPAIRMENTS IN SKELETAL MUSCLE METABOLISM

Accumulation of intramuscular TAG (IMTAG) has been associated with skeletal
muscle insulin resistance in humans and is already present in young lean offspring
of type 2 diabetic parents [12]. Both high fat diets and acute intravenous intralipid
infusions result in increased IMTAG stores and a concomitant development of
insulin resistance [383-386], highlighting an important role of lipid supply in fatty-
acid induced insulin resistance. Nevertheless, also several studies report similar
IMTAG concentrations in obese insulin sensitive and obese T2D subjects [387,
388] indicating that intramuscular lipids are not directly linked to insulin resistance.
Further evidence against a direct role of IMTAG in insulin resistance originates
from the observation that endurance trained athletes also have high IMTAG levels
and are highly insulin sensitive [389-391]. This so called ‘athletes paradox’ may be
explained by a higher muscle oxidative capacity [389], a higher antioxidant
capacity in the athletic group [392] or a higher concentration of intramuscular lipid
droplets around the mitochondria after endurance exercise [393]. On the contrary,
in obese, insulin resistant and T2D patients, an increased IMTAG concentration
has been linked with a reduced oxidative capacity [394-398], which could lead to
FA storage, rather than oxidation and thereby provide a mechanism for lipid
accumulation within skeletal muscle (Figure 4). By now, it is obvious that the
relationship between IMTAG accumulation and insulin sensitivity is not as
straightforward as originally thought, but rather the accumulation of bioactive lipid
metabolites like diacylglycerol (DAG), long chain fatty acyl-CoA and ceramides are
involved in FA-induced insulin resistance [399-403]. Furthermore, others and we
recently highlighted that, besides the amount and type of lipid metabolite, also the
cellular localization is of major importance for the development of insulin resistance
[404-406]. Although results are not consistent [407-411], it was recently described
that skeletal muscle inflammation and macrophage markers are increased and
associated with insulin resistance in rodents as well as humans and that treatment
of high-fat-diet fed mice with the PPAR-γ agonist rosiglitazone decreased muscle
inflammation and improved local insulin signaling [412]. Also, several studies
showed that muscle insulin resistance is associated with ER stress activation, as
reviewed elsewhere [179, 413].
Taken together, it is obvious that the relationship between TAG or lipid metabolites
is more complex as originally proposed and that understanding lipid turnover within
skeletal muscle in addition to the role of increased lipid supply and uptake is crucial
for elucidating the relationship between ectopic fat accumulation and insulin
resistance. Therefore, the major components of skeletal muscle lipid turnover and
intrinsic disturbances are discussed below.

Skeletal muscle lipid uptake

As mentioned before, there is mixed evidence for the notion that obesity is
associated with increased fasting, postprandial, diurnal or nocturnal FFA
concentrations. Instead, elevated circulating TAG concentrations in both the fasting
and postprandial state may be far more striking associated with insulin resistance
as compared to increased FFA concentrations. TAG concentrations have been
reported to be increased in the obese insulin resistant state, which may be

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ascribed to an increased liver VLDL production [92] or to impairments in adipose

tissue TAG clearance from the circulation [68-73].
In human studies, postprandial systemic TAG concentrations and TAG extraction
across forearm muscle were significantly elevated in subjects with impaired
glucose metabolism (either subjects with impaired fasting glucose (IFG) or
impaired glucose tolerance (IGT)) versus normal glucose tolerant controls [414].
Another study comparing insulin resistant versus control subjects with the same
stable isotope methodology could not confirm an increased postprandial muscle
TAG extraction, despite elevated TAG concentrations [92]. Although the mixed
results remain to be elucidated, this study also confirms the apparent importance of
TAG metabolism in insulin resistance, showing that the insulin resistant state was
closely related to increased TAG rather than increased FFA concentrations, most
likely due to a partitioning towards TAG synthesis in the liver.
The expression and activation of muscle LPL plays a major role in skeletal muscle
TAG extraction. It was shown that in mice LPL deletion reduces lipid storage and
increases insulin signaling in skeletal muscle [415] and that muscle specific
overexpression of LPL causes muscle-specific insulin resistance by causing
defects in muscle signaling and action [416], whilst skeletal muscle LPL knockout
mice show opposite effects. Whether these effects are translational to humans is
currently unknown. Most studies indicate that fasting raises total LPL activity in
human skeletal muscle [417, 418], but results are not consistent [44]. Furthermore,
a single bout of physical exercise also leads to a marked increase in LPL activity,
protein, and mRNA in the exercising muscle [417, 419-422]. In contrast to adipose
tissue and liver, insulin infusion decreased skeletal muscle LPL activity in humans
[423]. Additionally, muscle LPL activity is inhibited at posttranslational level by
ANGPTL4 [424] but little is known about the physiological and molecular
mechanisms involved in the regulation of muscle LPL expression. It has been
shown that T2D patients have significantly lower plasma ANGPTL4 levels as
compared to healthy subjects, thereby suggesting a role for ANGPTL4 in diabetes
[63]. Furthermore, skeletal muscle ANGPTL4 expression is increased in human
muscle following fasting via elevation of plasma FFA. However, the functional
implications of fasting and lipid-induced ANGPTL4 expression in muscle as well as
possible impairments in insulin resistant conditions remain to be elucidated. In
rodents, ANGPTL4 overexpression and treatment markedly decreased blood
glucose concentration, improved glucose tolerance and hyperinsulinemia, but
induced hyperlipidemia, fatty liver and hepatomegaly in mice [425]. The FA that are
liberated after LPL-mediated lipolysis and those from the plasma FFA pool can be
taken up in skeletal muscle via passive diffusion, depending on the concentration
gradient over the muscle membrane and via membrane associated carrier proteins
[426] like CD36, FABPpm and a family of fatty acid transport proteins (FATP 1-6)
[427-430]. Of these FA transporters CD36 has been best characterized [46]. CD36
deficiency has been associated with a functionally important impairment in FA
transport in muscle and adipose tissue [427, 431]. In humans, it has been shown
that muscle CD36 protein expression may be acutely upregulated by insulin [80,
432] and that this upregulation may be more pronounced in insulin resistant
conditions [80, 433]. Furthermore, it has been shown that CD36 translocation to the
plasma membrane may be increased in muscle strips of obese subjects with T2D
and that FA uptake, as measured with the giant vesicle model, was increased 4-

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fold as compared to overweight and lean controls [434]. Nevertheless, several

human in vivo studies showed that the FFA concentrations were comparable
between groups and no difference was observed in skeletal muscle FA uptake
during fasting or insulin-stimulated conditions in obese versus lean subjects [434],
in patients with impaired glucose metabolism versus normal glucose tolerant
subjects [414, 435] or in insulin resistant men versus controls [436]. However,
results are contradicting, since 2 other studies showed a small increase in muscle
FFA uptake during fasting in obese versus lean [437] or insulin resistant versus
control subjects [92], despite comparable FFA concentrations. In summary, there
may be more human evidence for an elevation of plasma TAG concentrations as
compared to FFA in the obese insulin resistant or pre-diabetic state. Although
mixed evidence is available for increased muscle TAG extraction, not much
evidence is available from in vivo human studies for the notion that skeletal muscle
FFA uptake may be largely increased in the insulin resistant state.

Skeletal muscle fatty acid storage

The FAs that enter the myocyte, bind to cytoplasmic FABPc for transport through
the cell [438] and can either be directed towards storage in lipid droplets or towards
the mitochondria for oxidation. The synthesis of TAG in the myocyte involves the
activity of several glycerol-3-phosphates (GPAT1-4), lipin 1 and DGAT1-2 [439]
whilst the activity of SCD1 or ∆-9-desaturase is particularly important for the further
metabolism of SFA. Increased IMTAG synthesis via upregulation of lipogenic
enzymes (DGAT1, GPAT an SCD1) has been linked to protection against FA-
induced insulin resistance in rodents and humans [416]. Recently, Timmers et al.
[440] showed that unilateral overexpression of DGAT-1 (involved in the conversion
of TAG to DAG) in rat skeletal muscle, could rescue insulin sensitivity despite
increased DAG and TAG concentrations, possibly by increasing DAG and TAG
turnover [440]. Nevertheless, data on DGAT1 expression in human skeletal muscle
are mixed with several studies showing no differences in DGAT1 expression
between obese insulin resistant subjects and normal weight sedentary volunteers,
endurance trained athletes [441] or in obese volunteers after weight loss [442].
One of the putative mechanisms regulating turnover of in particular SFAs may be
related to the activity of SCD1 or ∆-9-desaturase, converting SFAs to MUFAs.
Overexpression of SCD1 has been reported to protect L6 myotubes from FA-
induced insulin resistance and increased TAG reesterification [443]. Nevertheless,
evidence is mixed with another study showing increased SCD1 expression in
skeletal muscle of extremely obese humans with severe muscle insulin resistance
[444]. Less information is available on the synthesis rate of muscle TAG in vivo in
humans. Bergman and coworkers [445] showed that IMTAG concentration and its
fractional synthetic rate were not related to insulin action in smokers compared to
non-smokers [445]. Additionally, the same group showed that obese prediabetic
men had higher muscle TAG concentrations, a lower TAG fractional synthetic rate
and a lower oxidative capacity in parallel to a reduced insulin action as compared
to obese normal glucose tolerant men [446]. These disturbances in IMTAG
metabolism were not found in women. In line, we recently showed that a more
pronounced degree of insulin resistance in subjects with IGT (either isolated or in

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combination with IFG) as compared to isolated IFG was accompanied by a

reduced fractional synthesis of TAG from dietary palmitate, an increased saturation
of the intramuscular FFA pool, a reduced saturation of the DAG and TAG pool and
a reduced expression of genes involved in oxidative metabolism, confirming that a
reduced muscle lipid synthesis and turnover may be an important characteristic of
the insulin resistant muscle [447]. Finally, a reduced incorporation of FFA into TAG
in primary myotubes from obese individuals with T2D has been recently observed.
The data indicate that the ability to incorporate FAs into TAG is an intrinsic feature
of human muscle cells that is reduced in individuals with T2D [448]. In summary,
there is evidence that partitioning of FFA towards TAG synthesis may be beneficial
for insulin sensitivity and that insulin resistance may be associated with a reduced
fractional TAG synthesis in skeletal muscle.

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Figure 4. Disturbances in skeletal muscle fatty acid metabolism

Skeletal muscle fatty acid uptake, which is regulated via LPL and fatty acid transport proteins (FATP1-6,
CD36), might be higher in subjects with impaired glucose metabolism compared to control subjects (1).
In addition, a reduced mitochondrial mass and/or mitochondrial function (2) have been proposed as
underlying mechanisms for reduced muscle fat oxidation, contributing to the accumulation of TAG and
lipid-intermediates (LCFA-CoA, DAG, ceramides, acylcarnitines) in myocytes (3), interfering with insulin
signaling (4) in the obese insulin resistant state. Finally, the capacity to increase intramyocellular fat
oxidation during conditions of high FA supply has shown to be impaired in obese, T2D subjects.
PUFA’s, polyphenols, plant sterols and bile acids may have beneficial effects on skeletal muscle lipid
metabolism.
Abbreviations: LPL: Lipoprotein lipase; FATP1-6: Fatty acid transport protein 1-6; CD36: Fatty acid
translocase CD36; TAG: Triacylglycerol; DAG: Diacylglycerol; LCFA-CoA: long chain fatty acyl co-
enzyme A; PUFA: Poly unsaturated fatty acids. Dashed lines indicate inhibition. Solid lines indicate
stimulation. Green lines indicate beneficial effects. Symbols: (): Increased; (): Decreased

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Skeletal muscle lipolysis

Besides FA uptake and incorporation into DAG and TAG, also intrinsic
disturbances in skeletal muscle lipolysis may contribute to the accumulation of
lipids and lipid metabolites in skeletal muscle of obese insulin resistant subjects. As
in adipose tissue, the intramuscular lipid stores are hydrolyzed by phosphorylation
and translocation of ATGL and HSL, which are under hormonal control of
catecholamines, NPs and insulin. HSL deficiency in mice has been shown to
increase DAG storage and signs of impaired skeletal muscle insulin sensitivity
[274], while ATGL deficient mice have increased TAG accumulation in skeletal
muscle and show improved glucose tolerance and insulin sensitivity [449]. In line,
in vitro data of Badin et al. [450] showed that overexpression of ATGL in human
myotubes promotes DAG and ceramide accumulation and disrupts insulin signaling
and action. These data suggest that a dysbalance in lipase expression and activity
contributes to an increased accumulation of lipotoxic metabolites, which might
interfere with insulin signaling. As far as we know, there is no human in vivo
evidence for a significant improvement in insulin sensitivity following inhibition of
muscle ATGL activity. However, it has been shown that ATGL deficiency in
humans with neutral lipid storage disease with myopathy (NLSDM), when
compared to healthy controls, show impaired insulin response to glucose,
preserved whole-body insulin sensitivity and a shift toward glucose metabolism in
the heart [451].
Furthermore, we showed that skeletal muscle of obese subjects exhibited a blunted
β-adrenergic mediated lipolysis as compared to lean controls in vivo [452].
Additionally, under fasting conditions total glycerol release was reduced across
forearm muscle of obese compared to lean men, which was accompanied by a
reduced HSL protein content and an increased ATGL protein content without
changes in CGI-58, a co-activator of ATGL [453]. This difference in lipase content
was accompanied by a 60% lower ratio of DAG to TAG hydrolase activity implying
incomplete muscle lipolysis. Nevertheless, the incomplete lipolysis was not
accompanied by DAG accumulation, but total DAG content was rather decreased
in obese subjects, again, as already indicated above not supporting an important
role of total DAG content in lipid-induced insulin resistance. Furthermore, we
recently showed that insulin-mediated suppression of skeletal muscle lipolysis is
blunted in T2D compared to normal glucose tolerant subjects and that this is
associated with increased accumulation of membrane saturated DAG and protein
kinase C (PKC) activation [404]. These data are supported by several other
observations that both cellular localization and composition of DAG influence the
relationship to insulin sensitivity [405, 406]. It remains to be elucidated whether
intracellular lipases directly contribute to membrane DAG accumulation in human
skeletal muscle. However, there are indications that in vitro ATGL mainly generates
specific DAG isoforms (1,3-DAGs and 2,3-DAGs) but not 1,2-DAGs, of which the
latter is found in the plasma membrane and able to activate PKC, suggesting that
muscle lipases are not directly involved in membrane DAG accumulation [90].
Nevertheless, targeting skeletal muscle lipases may constitute an interesting
strategy to improve insulin sensitivity in obesity and T2D.
Finally, accumulating evidence suggests that LD covering proteins play an
important role in skeletal muscle lipid turnover and the development of insulin

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resistance. The best-characterized group of LD coat proteins is the PLIN family. As

mentioned above, PLIN-1 is adipose tissue specific, although PLIN1 mRNA
expression is observed in human skeletal muscle [454], while PLIN-2 and PLIN-5
mRNA and protein are abundantly expressed in skeletal muscle [455, 456]. PLIN-3
and PLIN-4 are also present in skeletal muscle, but little is known to date about
their function in muscle lipid metabolism. It was recently reported that PLIN-5
protein is highly expressed in skeletal muscle of endurance athletes, corresponding
to their higher LD volume and their higher insulin sensitivity [456]. Nevertheless,
another study in humans showed no correlation between PLIN-5 protein content in
skeletal muscle and insulin sensitivity in obese T2D subjects and BMI-matched
control subjects [457]. PLIN-5 and PLIN-2 are co-localized with the LD [458] and
recent research using in vitro and in vivo methods showed that PLIN-2 is an
important facilitator of IMCL storage and that by improving IMCL storage, PLIN-2
protects against lipotoxicity improving thereby insulin sensitivity [455]. The role of
LD dynamics in myocellular insulin resistance is beyond the scope of this review
and has been reviewed in more detail elsewhere [399]. Taken together, although
the field is still in its infancy, mounting evidence suggests a role for LD covering
proteins in the protection against muscle lipotoxicity and insulin resistance. One
could hypothesize that storage of lipids in LD is not as harmful as opposed to
accumulating lipids elsewhere in the cell (e.g. the plasma membrane).

Skeletal muscle fat oxidation

The dynamics of fat oxidation and fine tuning with FA uptake and intramyocellular
TAG turnover may be very important to prevent accumulation of bioactive lipid
metabolites. To meet the complexity of changes in fuel oxidation, the concept of
metabolic flexibility has been introduced, defined as the capacity to increase fat
oxidation upon increased FA availability and to switch between fat and glucose as
the primary fuel source after a meal [459]. The insulin resistant muscle may be
characterized by a metabolic inflexibility to regulate substrate oxidation. Indeed, the
capacity to increase intramyocellular fat oxidation during conditions of increased
FA supply such as fasting has shown to be impaired in obese, T2D subjects [396,
460-462] and also during exercise and β-adrenergic stimulation in obese and T2D
subjects [463, 464]. Additionally, the postprandial suppression of muscle fat
oxidation has shown to be impaired in T2D and obesity [396, 435]. Together these
data suggest that an impaired metabolic flexibility may contribute to the
accumulation of lipid metabolites and may be driven by lipid supply from either
extracellular or intracellular sources (lipid turnover) as well as intrinsic impairments
in mitochondrial function.
Interestingly, a study in human myotubes indicated that elevated extracellular FAs
increase their own oxidation, which may in turn inhibit the oxidation of
intramyocellular lipids [465]. Also, inhibition of intracellular adipose tissue lipolysis
with acipimox decreased adipose tissue derived FA availability and increased the
oxidation of IMTAG derived FA during rest and during exercise [466]. Nevertheless,
since circulating FFA concentrations may only be slightly elevated or not increased
at all in insulin resistant conditions (see above), an increased lipid overflow from
FFA is probably not a major contributor to an impaired utilization of IMTAG in the

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obese insulin resistance state. Since there is increasing support for the notion that
lipid overflow in insulin resistance under physiological conditions is rather
characterized by increased circulating TAG concentrations than by increased FFA
concentrations, it is a plausible option that increased TAG-derived FA oxidation
may impair IMTAG utilization in the obese insulin resistant state. However, this
option remains to be investigated.
Furthermore, differences in IMTAG oxidation may be caused by differences in
IMTAG lipolysis. Interestingly, lipolysis from IMTAG might be necessary for the
maintenance of oxidative gene expression and FA oxidation. Recent findings
indicate that in particular ATGL-mediated lipolysis generates lipid ligands for PPAR
activation and the subsequent transcription of oxidative genes [90]. It was shown
that a cycle of FA esterification and rehydrolysis is required for activation of PPAR-
α and normal mitochondrial and oxidative phosphorylation, at leasat in
cardiomyocytes [467]. In vivo tracer data show that in patients with NLSDM
(caused by mutations in the ATGL gene) fat oxidative capacity is blunted and can
be partly reversed by PPAR-α treatment [468, 469]. These data highlight the
importance of ATGL activity in regulating muscle fat oxidation. Furthermore, it was
shown recently that activation of NP signaling in human skeletal muscle enhances
mitochondrial oxidative metabolism and fat oxidation [470]. However, further
studies are required linking lipolysis and NP signaling to muscle oxidative
metabolism and insulin resistance in human.
Finally, a reduced FA transport across the mitochondrial membrane and a reduced
mitochondrial mass and/or mitochondrial function have been proposed as
underlying causes of a reduced muscle fat oxidation in insulin resistance. In rodent
models of obesity and insulin resistance an increased content of malonyl-CoA, an
allosteric inhibitor of mitochondrial FA transport, has been shown in skeletal muscle
in combination with hyperglycaemia and hyperinsulinaemia and a reduced lipid
oxidation [471]. Also, in human skeletal muscle it has been shown that a
combination of hyperglycaemia and hyperinsulinaemia increases malonyl-CoA,
inhibits functional carnitine palmitoyl transferase 1 (CPT1b, muscle isoform) activity
and shunts long chain FA away from oxidation and towards storage in human
muscle [472]. Furthermore, two studies have shown that an improvement in fat
oxidation by exercise or by lifestyle intervention was accompanied by decreased
acetyl-CoA carboxylase (ACC) mRNA expression, involved in the formation of
malonyl-CoA, suggesting that a reduced inhibition of mitochondrial FA transport
through malonyl-CoA may have contributed to the improved fat oxidative capacity
[473, 474]. Furthermore, a lowered mitochondrial transport through CPT1 may
contribute to the reduced fat oxidation. In line, heterozygous CPT1b deficiency
results in mitochondrial abnormalities and lipid accumulation with elevated TAG
and ceramide content at least in mice cardiomyocytes [475]. Moreover, it has been
shown that an increased CPT1b activity is sufficient to improve high-fat diet
induced insulin resistance [476]. Peroxisome proliferator-activated receptor gamma
coactivator-1alpha (PGC-1α) is involved in mitochondrial biogenesis by supporting
the transcriptional activity of nuclear respiration factor 1 (NRF1), thereby regulating
the transcription of genes involved in oxidative metabolism. The expression of
genes responsible for oxidative phosphorylation is coordinately downregulated in
muscle of T2D individuals [477, 478] and muscle specific overexpression of PGC-
1α in rodents has been shown to improve lipid utilization, insulin signaling and

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glucose transport [479]. In vivo mitochondrial capacity and function have been
reported to be reduced in T2D, independent of mitochondrial content [388, 480,
481], although data are not consistent [482, 483]. Nevertheless, a reduced
mitochondrial capacity and a reduced activity of TCA cycle [484] and citrate
synthase (-14%) [485] have also been reported in primary myotubes from T2D
subjects, suggesting that skeletal muscle mitochondrial function is intrinsically
impaired in insulin resistance. Moreover, a marked reduction in mitochondrial
oxidative and phosphorylation activities, together with intramyocellular lipid
accumulation, were found in skeletal muscle in insulin resistant offspring of T2D
subjects [486, 487]. Furthermore, the definition of mitochondrial dysfunction may
also include a dysbalance balance between β-oxidation and TCA cycle activity
resulting in an accumulation of incompletely oxidized, fatty acid products (i.e.
acylcarnitines), which can impair skeletal muscle insulin sensitivity, as extensively
reviewed elsewhere [488].
In summary, skeletal muscle fat oxidation is reduced in the obese insulin resistant
state. This impaired fat oxidation may contribute to the accumulation of lipid
metabolites and may be driven by lipid supply from either extracellular or
intracellular sources as well as intrinsic impairments in mitochondrial function.
However, whether reduced mitochondrial function is causally related to insulin
resistance or rather a consequence of the sedentary lifestyle needs to be
investigated in more detail in future research.

Putative nutrional targets to improve skeletal muscle lipid metabolism

An improvement in adipose tissue and liver function may reduce lipid overflow and
low-grade systemic inflammation, thereby reducing the supply of lipids and
inflammatory mediators to skeletal muscle, which likely improves skeletal muscle
function.
As indicated above, targeting skeletal muscle lipases, LD covering proteins and
skeletal muscle fat oxidation/mitochondrial function to improve metabolic flexibility
may constitute an interesting strategy to improve insulin sensitivity in obesity and
T2D by limiting the accumulation of bioactive lipid metabolites interfering with
insulin sensitivity.
As discussed earlier in this review, specific polyphenols or a combination of
polyphenols may affect human lipid metabolism. The polyphenolic compound
resveratrol, which is abundantly present in grape skin, red wine and peanuts, has
been shown to improve in vitro FA oxidation and insulin sensitivity in murine
adipocytes [489], as well as in C2C12 myotubes [490] and improved skeletal
muscle mitochondrial function in rodents by activating Sirtuin-1 (SIRT-1) and PGC-
1α [491]. In humans, on the other hand, resveratrol supplementation has recently
been shown to improve metabolic profile and flexibility, skeletal muscle
mitochondrial function, energy expenditure, glucose control and insulin sensitivity
especially in patients with T2D [492, 493]. We have recently also shown that short-
term supplementation of the combination of EGCG and resveratrol may increase
resting energy expenditure and improve metabolic flexibility [212]. Nevertheless,
results of clinical trials are contradictory, as recently discussed [494].
Additionally, dietary fat quality may differentially affect (skeletal muscle) lipid

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.
metabolism and insulin sensitivity [218, 495] Indeed, we recently showed that a
PUFA rich meal acutely improved insulin sensitivity in obese insulin resistant
subjects as compared to a high-SFA meal, which was accompanied by a reduced
TAG-derived skeletal muscle FA uptake, an altered intramyocellular lipid
partitioning and a more oxidative transcriptional phenotype [496]. Additionally,
studies in human myotubes showed that palmitate induced a lower lipolysis rate
[497] and follows a different metabolic pathway as compared to oleic acid
[497,498]. Also, a lower palmitate oxidation has been shown in myotubes of T2D
subjects as compared to control, whilst oleate oxidation was not significantly
different [499]. These studies indicate that dietary fat quality may modulate the
nature of lipid-induced insulin resistance through affecting pathways of fatty acid
partitioning. Further studies are required to investigate the impact of gender and
dietary fat quality on FA dynamics and to investigate the causative role of an
impaired TAG synthesis capacity in the etiology of insulin resistance. In summary,
polyphenols and modulation of dietary fat quality may affect storage of bioactive
lipid metabolites in skeletal muscle, thereby improving insulin sensitivity and
glucose homeostasis. Beside that, manipulation the nuclear FXR and LXR
receptors by modulating bile acid metabolism or by ingestion of plant sterols may
also have positive effects on muscle lipid metabolism and insulin sensitivity, but
apparently more human evidence is required. For a summary see Table 1.

72
 Tabel 1: Summary of pathways, possible targets and (dietary) components in adipose tissue, liver and gut and skeletal muscle

Evidence for Strength of evidence

Tissue Pathway Target Dietary intervention or component improved glucose for improved
metabolism glucose metabolism

Adipose tissue Fatty acid trapping Stimulation of LPL Dietary modulation of ANGPTL4 [63] [425] Rodent - in vivo/ex vivo

Selective HSL and/or ATGL Pre- and probiotics affecting SCFA metabolism Rodent - in vitro/in vivo
Lipolysis [100, 101]
inhibition [210], [211] Human - in vitro/in vivo

Modulation of alternative pathway

Lipolysis Polyphenols [212, 213] Currently unknown Currently unknown
of lipid breakdown (lipophagy)

Mitochondrial function Dietary fat quality, PUFA quantity

Increase mitochondrial function [146] Rodent - in vivo/in vitro
and browning [223, 224]

Polyphenols [212] [492] [492] Human – in vivo

Increase BAT activity Capsinoids [163] [157] Human – in vivo

Increase FGF21 via BA metabolism / gut

[619] Rodent - in vivo
microbiota [165] [335]

Dietary fat quality, PUFA quantity

Inflammation Decrease low-grade inflammation [173] Rodent/human - in vivo
[229-233]

Modulation bile acid metabolism by pre- and

FXR and TGR5 receptors, probiotics, FXR and TGR5 agonists
Liver & Gut Bile acid manipulation [356] Rodent - in vivo
Increase FGF21 [333-335, 337-341]
[356, 357, 359, 360]

Rodent - in vivo
Bile acid manipulation Activate LXR Plant sterols [344] [346, 347]
Rodent - in vivo/ex vivo

Gut microbiota Alter gut microbiota (to alter

Pre-, probiotics [366] [369] [366, 367] Human - in vivo
manipulation secondary BA and SCFA)

Human - ex vivo/in vitro

Skeletal muscle Lipolysis Selective HSL, ATGL inhibition To be identified [450, 451]
Human - in vivo

TAG extraction Reduce TAG-derived- FA uptake PUFA [496] [496] Human - in vivo

Increase SIRT-1, PGC-1α, Polyphenols, PUFA Rodent - in vitro

Mitochondrial function [479] [490]
fat oxidation [212] [491, 492] [496] Rodent - in vitro/in vivo

Rodent - in vivo
Gut microbiota Plant sterols, Pre-, probiotics
Activate LXR, TGR5 [346, 347] [356] Rodent - in vivo/ex vivo
manipulation [344] [369]
Rodent - in vivo
Chapter 2

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Chapter 2

PANCREATIC BETA-CELL FUNCTION

T2D is a multifactorial disease that develops when insulin resistance is
accompanied by pancreatic β-cell failure [2, 3], which is defined as a deterioration
of β-cell function as well as loss of functional β-cell mass. In obese conditions, lipid
accumulation of TAG in pancreatic islets occurs [500], which is aggravated by the
simultaneous presence of hyperglycemia [501-503]. Elevated lipid accumulation
can damage the β-cell functionality (Figure 5) and evidence for this mainly comes
from studies with isolated islets exposed to high concentrations of FFA for periods
of 24-48 h [504, 505], while evidence from in vivo studies mainly comes from
studies with ZDF rats, since suitable pancreas specimens from individuals with
increasing degrees of β-cell dysfunction are scarce [235]. This decreased β-cell
functionality may lead to a decreased insulin secretion even when demands for
secretion are increased. However, in order to maintain normal glucose
homeostasis, a compensatory increased insulin secretion occurs. Prediabetic
patients however, in the long run, fail to compensate adequately for the greater
insulin requirements [2, 506-508] and will ultimately develop T2D. In this type 2
diabetic state, a decreased β-cell mass [509] and an increased level of β-cell
apoptosis [510-514] occurs. However, the rate of β-cell replication and neogenesis
was not different between a diabetic and a control group [510].

Putative targets to improve insulin secretion

The increased rate of β-cell apoptosis in T2D can originate from several
mechanisms [515], including oxidative stress [13, 509, 516, 517], inflammation
[518, 519], ER-stress [520-523], mitochondrial overload [524, 525] and lipotoxicity
[526]. As extensively reviewed elsewhere, human and animal data suggest that
there might be direct effects of bioactive compounds (including polyphenols,
vitamins and carotenoids) on enhancing insulin secretion and preventing β-cell
apoptosis, and some compounds might modulate β-cell proliferation [527].
Targeting these processes within the pancreatic islets, as already discussed in the
above paragraphs, may result in decreased β-cell apoptosis and maintain the
insulin secretory capacity, and as such could be a potential therapy to reduce the
incidence of T2D.

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Figure 5. Disturbances in pancreatic fatty acid metabolism

Lipid accumulation in pancreatic islets can result in inflammation, ER-stress, mitochondrial overload and
lipotoxicity. These mechanisms damage the β-cell function and lower β-cell mass, resulting in a
decreased insulin secretion and ultimately leading to the development of T2D. Polyphenols, vitamins
and carotenoids may counteract these negative effects.
Abbreviations: TAG: Triacylglycerol; DAG: Diacylglycerol; MAG: monoacylglycerol; FFA: Free fatty
acids; ER-stress: Endoplasmic reticulum stress. Dashed lines indicate inhibition. Solid lines indicate
stimulation. Green lines indicate beneficial effects. Symbols: (): Increased; (): Decreased

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Chapter 2

THE INTESTINE AND GUT MICROBIOTA IN INSULIN RESISTANCE

The intestinal tract has traditionally been seen as a passive organ, but currently it is
seen as an active organ that may affect substrate metabolism via the secretion of
several factors such as free fatty acids, incretins and inflammatory factors [528]
and may contribute to the development of obesity and insulin resistance [427, 529,
530]. The intestine plays an important role in lipid homeostasis and contributes
significantly to the systemic plasma cholesterol and lipid concentrations [531]. Fat
absorption by the small intestine is a very efficient process, which requires several
transport mechanisms, as reviewed elsewhere [532]. Fatty acids taken up in the
enterocyte can either be oxidized by the enterocyte, [533-535], stored as
intracellular LD’s [536] or excreted as chylomicrons, which is related to
hypertriglyceridemia [537, 538] contributing to T2D [539], metabolic syndrome
[540] and coronary artery disease [541, 542]. Targeting these processes can
significantly affect postprandial lipid concentrations and insulin sensitivity.
Improving intestinal fatty acid oxidation, for example by PPAR-α activation [543]
might contribute to reduced lipid accumulation in other organs, such as liver and
skeletal muscle. Furthermore, also PUFAs [544, 545] and DAG [546] appear to be
effective inducers of enterocyte fatty acid oxidation and may be beneficial in the
anti-diabetic and anti-obesity effects in various tissues. Besides fatty acid oxidation,
also the absorption and excretion of fatty acids might be altered. As reviewed
elsewhere, there are several components that might influence intestinal lipid
metabolism [547]. For example, JTT-130, a novel intestine-specific inhibitor of
microsomal triglyceride transfer protein (MTP), which is involved in the mobilization
and secretion of TAG rich lipoproteins from enterocytes and hepatocytes [548], has
been shown to suppress the absorption of dietary fat and cholesterol in the
intestine, decreased plasma TAG and total cholesterol levels [549] and improved
glucose and lipid metabolism in rodents [550-552].
Furthermore, recent evidence showed that a high-fat diet induced intestinal
inflammation [553, 554], which is related to obesity and insulin resistance [555-
557]. Interestingly, it has recently been shown in beta7 integrin-deficient mice
(Beta7(null)) that suppression of the gut immune system decreases HFD-induced
insulin resistance [558]. Furthermore, intestinal ER stress has been found in
obesity [559] and might contribute to intestinal and systemic inflammation [560],
leading to insulin resistance [561]. Taken together, this makes the intestine an
attractive therapeutic target.
Importantly, not only the intestine itself, but also its microbiota play an important
role in the intestinal lipid metabolism, obesity and insulin resistance. The human
14
adult intestines contain more than 10 bacteria from over 1000 species and the
genetic material of the intestinal microbes is collectively called the microbiome
[562]. The vast majority of all gut microbes include the phyla Gram-negative
Bacteroidetes, Proteobacteria and Verrucomicrobia as well as the Gram-positive
Firmicutes and Actinobacteria [562-566]. Growing evidence indicates that also our
gut and its microbiota play a crucial role in substrate metabolism and the
development of obesity, cardiometabolic diseases and T2D, as reviewed
extensively elsewhere [567, 568] (Figure 6).
Obese and T2D subjects seem to be characterized by an altered composition of
gut microbiota compared to lean and normal glucose tolerant individuals,

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respectively [569-572], although conflicting results have also been reported [570,
573-575]. Interestingly, germ-free mice infected with the gut microbiota of
conventionally raised mice demonstrated increased body weight, insulin resistance
and glucose intolerance together with reduced food intake and increased oxygen
consumption [576]. The body fat gain was related to suppressed gut expression of
the LPL inhibitor ANGPTL4, leading to increased fat accumulation in adipocytes
and increased energy harvest [570, 577-579].
In obese men, seven days treatment with the antibiotic vancomycin modulated their
gut microbiota and decreased peripheral insulin sensitivity [364]. Finally, the
transfer of intestinal microbiota from human lean donors to individuals with the
metabolic syndrome increased insulin sensitivity, along with increase of intestinal
butyrate-producing microbiota [580].
Several mechanisms link our gut microbiota to obesity and diabetes (diabesity), as
extensively reviewed elsewhere [581]. Firstly, alterations in gut microbiota
composition can increase the amount of LPS through increased production,
reduced breakdown or increased translocation across the gut wall, thereby
inducing a strong immune response to protect the organism from bacterial infection
[582]. This LPS-induced inflammatory state is referred to as ‘metabolic
endotoxemia’ and is accompanied by body weight gain and insulin resistance in
animal models [583]. In addition, major microbial products from the fermentation of
indigestible carbohydrates, like the SCFAs acetate, butyrate and propionate may
affect energy and fat metabolism through various mechanisms [584-587]. The type
of substrate is the primary determinant of the SCFA production rate and SCFA
ratio, compared to the composition of the intestinal microbiota [584] and the level of
substrate fermentability varies with fiber solubility. Soluble fibers are fermented
more completely than insoluble fibers [588] and highly fermentable substrates
produce higher amounts of SCFA [589], which have physiological effects in
different tissues [590]. At first, it has been proposed that they may play a role in
harvesting extra energy from the diet, which would be a promoting factor for body
weight gain. However, evidence that this largely contributes to human energy
balance is not yet convincing [591]. On the other hand, increased SCFA
concentrations have rather been associated with distinct generally positive,
metabolic effects, affecting hormonal release of glucagon-like peptide 1 (GLP1)
and peptide YY (PYY), cell proliferation and differentiation [584, 592-595], regulate
adipocyte lipolysis [210, 211], modulate inflammation, affecting adipose tissue fat
storage and ectopic fat storage, as has been extensively reviewed elsewhere
[596]. Effects of SCFAs may be related to the activation of their related G-protein
coupled receptors GPR41 (also known as free fatty acid receptor 3 (FFAR3)) and
GPR43 (FFAR2) [596], which are present in the gut epithelium but also in
peripheral tissues like adipose tissue and skeletal muscle [595, 597]. Thus,
manipulation of SCFA production by prebiotics and probiotics (or a combination of
both) may, among other processes, differentially affect human fat and glucose
metabolism.
Furthermore, besides converting non-digestible fibers, the microbiota are also
closely linked to BA metabolism. On the one hand, microbiota can transform
primary to the so-called secondary BAs [598], while on the other hand BAs by
themselves exert an anti-microbial effect on the microbiota [599, 600]. As
discussed above, the secondary BAs can affect host metabolism via binding to

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several nuclear receptors and GPRs and altering the intestinal microbiota could be
a potential therapeutic target to reduce obesity and insulin resistance.

Putative nutritional targets to modulate gut microbiota composition and function

Altering the diet, either via specific dietary FA or combinations of pre- and
probiotics, can rapidly change the human gut microbiota [601-603] and can
improve metabolic status [604, 605]. By fermenting dietary fibers that humans
cannot (fully) digest themselves, the human microflora produces SCFA [565, 570,
588, 606], which may play an important role in glucose homeostasis via modulation
of human substrate metabolism, as briefly discussed above. Besides endogenous
SCFA production, exogenous administration of SCFA has been studied in the
context of interest. Thus, an alteration of the SCFA production by microbiota, i.e.
via intake of pre- and probiotics, may be a useful tool to prevent obesity and
obesity-related insulin resistance. Additionally, prebiotics may also directly affect
postprandial glycemic and insulinemic responses by slower glucose absorption
[607].
Research has shown that FAs exert their effect on bacterial growth and survival, in
part, via altering the microbial cell membrane fluidity and disrupting cell
membranes of certain bacteria [608, 609]. As reviewed by Alcock et al. [610],
unsaturated fatty acids had a higher anti-microbial effect compared to SFA and
within the group of SFA, short chain saturated FA (4 to 12 carbons in length) had a
higher bactericidal effect, compared to long chain saturated FA (more than 12
carbons). Furthermore, while MUFA had a lower anti-microbial effect then PUFA of
the same chain length, the n-3 PUFA had a greater effect compared to n-6 PUFA,
but results are not consistent [610]. Although human evidence is scarce, research
showed that the type of dietary fat has an effect on the faecal microbiota. Fava et
al. [611] showed that a diet high in SFA increased Faecalibacterium prausnitzii and
that MUFA-rich diets reduced the total bacterial numbers, but not the specific
bacterial groups. In contrast, Simões et al. [612] showed that high habitual intake
of MUFA was associated with lower numbers of bifidobacteria and slightly higher
numbers of Bacteroides spp., that habitual n-3 PUFA intake had a significant
positive association with Lactobacillus group abundance and that a higher habitual
n-6 PUFA intake was associated with a decreased number of bifidobacteria.
Santacruz et al. [613] showed that the numbers of Lactobacilli remained the same,
eventhough the total PUFA consumption was greatly reduced.
Furthermore, it was shown that a high-fat diet significantly reduced the
bifidobacteria [614]. In addition, high-fat diets seem to increase metabolic
endotoxemia, which is related to an inflammatory state [583, 615, 616].
The altered gut microbiota composition also affects BA metabolism in rodents [617,
618] as well as in humans [364]. As such, it might be a possible target to combat
the development towards obesity and insulin resistance (Table 1), as discussed
above. However, further human studies are necessary to understand the exact
mechanisms of action of the human microbiome.

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Figure 6. Disturbances in gut microbiota composition and lipid metabolism in the

gut affect host metabolism

An altered gut microbiota composition, as seen in obesity and T2D, may increase local and systemic
lipopolysaccharide (LPS) concentrations, referred to as metabolic endotoxemia (1) that can lead to
systemic inflammation. Besides LPS, also bile acid composition and SCFA concentrations are altered.
Pre- and probiotics, plant sterols and PUFAs exert a beneficial effect on gut microbiota composition (2),
leading to an altered SCFA production, bile acid composition and a decreased LPS concentration,
thereby affecting gut inflammation (3) and gut lipid metabolism (4). In addition, changes in systemic
SCFA, bile acids and LPS affect host lipid metabolism and inflammation in adipose tissue, liver and
skeletal muscle (5).
Abbreviations: PUFA: Poly unsaturated fatty acids; SCFA: short chain fatty acid; LPS:
Lipopolysaccharide. Dashed lines indicate inhibition. Solid lines indicate stimulation. Green lines
indicate beneficial effects. Symbols: (): Altered; (): Increased; (): Decreased

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SUMMARY
This review focuses on the disturbances in fatty acid metabolism at both the tissue
and whole-body level that play a role in the etiology of insulin resistance, β-cell
dysfunction and an impaired glucose metabolism. The most important pathways
related to lipid metabolism in adipose tissue, liver, skeletal muscle, pancreas and
gut have been discussed, and targets to improve lipid metabolism and glycaemia,
which may be modulated by diet, have been described. A detailed overview of
pathways and potential targets to improve lipid metabolism and glycaemia in
adipose tissue, liver, skeletal muscle, pancreas and gut, which can be modulated
by dietary interventions and food components, is provided in Table 1, and will be
explained in more detail below.
Within adipose tissue, the balance between fatty acid extraction, lipolysis,
adipocyte differentiation and mitochondrial function is important to maintain
adequate lipid storage capacity. Improving the lipid storage capacity of adipose
tissue prevents lipid overflow in the circulation and subsequent ectopic fat
deposition, and therefore has high potential to improve glucose tolerance and
insulin sensitivity. Increasing adipose tissue fatty acid uptake, possibly via
stimulation of LPL, might reduce lipid overflow. On the other hand, a reduced lipid
overflow might also be achieved by partial inhibition of lipolysis (partial lipase
inhibition) and/or by modulation of the alternative pathway of lipid breakdown
(lipophagy). There is some evidence that partial lipolysis inhibition may be
achieved by modulation of SCFA metabolism through pro- and prebiotics and that
the lipophagic pathway may be modulated by specific polyphenols, like resveratrol.
Furthermore, adipose tissue mitochondrial function seems important in balancing
lipid supply and utilization, and thereby affects adipose tissue function. Improved
adipose tissue mitochondrial function may be achieved by nutritional strategies
such as supplementation with specific polyphenols (or a combination of
polyphenols) and altering dietary fatty acid composition. Dietary fat quality not only
modulates lipid metabolism but may also affect low-grade inflammation, which in
turn may lower the risk of developing insulin resistance and T2D.
An attractive target to tackle obesity and insulin resistance could be manipulation of
the bile acid metabolism and/or gut microbiota composition. On the one hand, bile
acids influence energy expenditure and glucose homeostasis via their effects on
gluconeogenesis, insulin secretion and insulin sensitivity, and an altered
concentrations of secondary bile acids can affect host metabolism via binding to
several nuclear receptors (e.g. FXR, LXR, RXR), some of which can be activated
by sitosterol, campesterol and certain oxidized derivatives of phytosterols
(oxyphytosterols). On the other hand, an alteration of the gut microbiota by
modulation of the dietary fat quality or pre- and probiotics might have a distinct
effect on bile acid composition and metabolism and may also affect fermentation
products from dietary fibers (e.g. SCFA and monosaccharides), affecting thereby
liver gluconeogenesis and lipogenesis, pathways directly or indirectly affecting
glucose homeostasis.
Targeting skeletal muscle lipid turnover and the balance between lipolysis, FA
uptake and mitochondrial function/fat oxidation may constitute an interesting
strategy to improve insulin sensitivity in obesity and T2D by limiting the
accumulation of bioactive lipid-intermediates interfering with insulin sensitivity and

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glucose homeostasis. The most promising dietary intervention to reduce FA uptake

and thereby muscle fat accumulation might be an improvement of the adipose
tissue lipid buffering capacity through the manipulation of dietary fat quality. On the
other hand, an increased activation of SIRT-1 and PGC-1α may result in an
enhanced skeletal muscle mitochondrial biogenesis/function, fat oxidation and
metabolic flexibility. Putative dietary strategies for the latter pathways may be
modulation of the dietary polyphenol content or the modulation of dietary fat
quality. In addition, stimulation of the nuclear FXR and LXR receptors by
modulation of bile acid metabolism or by ingestion of plant sterols may have
positive effects on skeletal muscle lipid metabolism and insulin sensitivity.
Finally, preventing β-cell apoptosis, and modulation of β-cell proliferation by
bioactive compounds (including polyphenols, vitamins and carotenoids) may
maintain the insulin secretory capacity, and as such could be a potential target to
reduce the incidence of T2D.
To conclude, the main most promising dietary interventions or components to
target fatty acid metabolism in order to improve glucose metabolism would be
components to alter the gut microbiota composition, like pre- and probiotics,
thereby affecting SCFA- and bile acid metabolism. Furthermore, the use of
(specific) polyphenol(s) to increase adipose tissue function possibly by affecting the
lipolytic pathway or the alternative pathway for lipid breakdown (lipophagy) and/or
adipose tissue and skeletal muscle mitochondrial function may be promising
strategies to improve glucose metabolism. Lastly, manipulation of the dietary fat
quantity and quality is of particular interest to improve adipose tissue function and
skeletal muscle lipid turnover and mitochondrial function. Further human
intervention studies are required targeting specific fatty acid related pathways to
translate the above indications into relevance for the control of glucose
homeostasis and insulin sensitivity.

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Improved insulin sensitivity with
angiotensin receptor neprilysin inhibition
in individuals with obesity and
hypertension

Jordan J., Stinkens R.*, Jax T.*, Engeli S., Blaak E.E., May M.,
Havekes B., Schindler C., Pal P., Heise T., Goossens G.H.,
Langenickel T.H.

* Shared authorship

Clinical Pharmacology and Therapeutics, 2017; 101(2):254-263

Chapter 3

ABSTRACT
Natriuretic peptide (NP) deficiency and sustained renin-angiotensin system
activation are associated with impaired oxidative metabolism and predispose to
type-2 diabetes. We hypothesized that sacubitril/valsartan (LCZ696), which
augments NP through neprilysin inhibition while blocking angiotensin II type-1
(AT1)-receptors, improves insulin sensitivity, lipid mobilization, and oxidation. After
8 weeks of treatment of obese patients with hypertension, sacubitril/valsartan 400
mg q.d., but not amlodipine 10 mg q.d., was associated with a significant increase
from baseline in the insulin sensitivity index (hyperinsulinemic-euglycemic clamp),
and tended to be higher in patients treated with sacubitril/valsartan compared to
amlodipine. Abdominal adipose tissue interstitial glycerol concentrations increased
with sacubitril/valsartan, but decreased with amlodipine. Whole-body lipolysis and
substrate oxidation did not change with either treatment. Results confirm that
sacubitril/valsartan treatment leads to a metabolic benefit in the study population
and supports the relevance of neprilysin inhibition along with AT1-receptor
blockade in the regulation of human glucose and lipid metabolism.

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INTRODUCTION
Obesity, type-2 diabetes mellitus (T2DM), and cardiovascular diseases are
interrelated. Patients with obesity and T2DM are at increased risk for arterial
hypertension and heart failure [1, 2], whereas patients with arterial hypertension or
heart ,failure frequently exhibit impaired muscular oxidative capacity predisposing
them to insulin resistance through accumulation of toxic lipid intermediates [3, 4].
The endopeptidase neprilysin is ubiquitously expressed, including in human
adipocytes [5]. Its plasma activity positively correlates with measures of obesity
and insulin resistance in humans, implicating neprilysin in the regulation of
cardiometabolic crosstalk presumably through cleavage of neprilysin substrates
with cardiometabolic effects, such as natriuretic peptides (NPs) and angiotensin II
[6]. Indeed, patients with obesity [7] or heart failure exhibit absolute or relative
natriuretic peptide deficiency, respectively. Circulating atrial natriuretic peptide
levels are inversely related to plasma glucose and insulin concentrations, and low
atrial natriuretic peptide levels predict the development of both arterial hypertension
and diabetes mellitus in humans [8]. Furthermore, polymorphisms in the genes
encoding atrial natriuretic peptide and B-type natriuretic peptide contribute to the
variability in the risk for type 2 diabetes after adjustment for age, gender, and body
mass index [9, 10]. NP promotes lipid mobilization from adipose tissue [11],
increases postprandial lipid oxidation [12], elicits adiponectin release, and
enhances muscular oxidative capacity [13, 14]. In contrast to atrial natriuretic
peptide, angiotensin II promotes insulin resistance, and angiotensin II type-1 (AT1)-
receptor blockade modestly improved insulin sensitivity and pancreatic beta-cell
function in humans [15].
Sacubitril/valsartan (LCZ696), a novel angiotensin receptor neprilysin inhibitor, was
recently approved in the United States, Europe, and many other countries to
reduce the risk of cardiovascular death and hospitalizations in patients with chronic
heart failure (New York Heart Association classes II–IV) and reduced ejection
fraction [16]. Upon oral administration, sacubitril/valsartan provides exposure to
sacubitril (AHU377), a prodrug that is rapidly metabolized to the biologically active
neprilysin inhibitor, sacubitrilat (LBQ657), and to the AT1-receptor blocker
valsartan. This active drug augments neprilysin substrates, such as natriuretic
peptides, while inhibiting AT 1-receptor mediated responses [17].
We hypothesized that sacubitril/valsartan, through its combined mechanism of
action, improves peripheral insulin sensitivity and increases lipid mobilization from
subcutaneous abdominal adipose tissue in patients with obesity and arterial
hypertension.
We conducted a mechanistic metabolic profiling study to investigate the effects of
8-week treatment with sacubitril/valsartan and amlodipine on peripheral insulin
sensitivity, abdominal subcutaneous adipose tissue lipolysis, whole-body lipolysis,
and energy expenditure and substrate oxidation in patients with obesity and
elevated blood pressure. In this study, the patient population served as an
exemplary human model for relative natriuretic peptide deficiency and impaired
insulin sensitivity, and was specifically selected to support the comparison of
sacubitril/valsartan with a metabolically neutral comparator, amlodipine.

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RESULTS

Subjects
Of the 98 patients enrolled, (sacubitril/valsartan=50; amlodipine=48), 92 patients
(93.9%) completed the study. All patients were included in the safety analysis set
(Figure 1). Patients with missing baseline or post-treatment assessments were
excluded from the pharmacodynamic analysis set, resulting in a maximum of 96
patients (sacubitril/valsartan=50; amlodipine=46) included in the pharmacodynamic
analysis set.
Patient demographics and baseline characteristics are summarized in Table 1 and
were comparable between the groups.

Figure 1. Study design

*Indicates those who had to discontinue antihypertensive medication. # Indicates those who had to
discontinue before the first dose. § Indicates subcutaneous adipose tissue microdialysis.
‡ Indicates indirect calorimetry by Spirometry. EOS, end of study; HEGC, hyperinsulinemic-euglycemic
glucose clamp.

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 Table 1. Patient demographics and baseline characteristics
Parameter LCZ696 (400 mg) N = 50 Amlodipine (10 mg) N = 48

Age, y 51.9 (9.6) 50.5 (9.4)

Weight, kg 101.2 (17.3) 104.3 (14.1)

Waist circumference, cm 112.1 (10.5) 113.9 (10.0)

2
BMI, kg/m 32.6 (4.6) 33.3 (4.4)

Gender, male, no. (%) 41 (82) 35 (72.9)

Race, white, no. (%) 50 (100) 48 (100)

msSBP, mmHg 141.7 (11.1) 139.7 (12.6)

msDBP, mmHg 90.2 (6.1) 91.9 (6.0)

Mean sitting pulse rate, bpm 70.6 (9.7) 70.3 (9.5)

Insulin SI (µg/kg*min/(mmol/L*pmol/L) 1.6 (1.0) 1.6 (0.9)*

† ‡
GIR (mg/min) 364.7 (192.5) 401.8 (189.6)
† ‡
Metabolic clearance rate (mg/(min*kg)) 64.4 (33.3) 71.1 (31.2)

Data are mean (SD) unless specified otherwise. BMI, body mass index; bpm, beats per minute; GIR, glucose infusion rate; msSBP, mean seated
Chapter 3

119
systolic blood pressure; msDBP, mean seated diastolic blood pressure; SI, sensitivity index. * N = 45; † N = 48; ‡ N = 42.
Chapter 3

Peripheral insulin sensitivity

Sacubitril/valsartan, but not amlodipine, was associated with a significant increase
from baseline in mean insulin sensitivity index (SI), as evidenced by the 95%
confidence interval (CI; mean change from baseline (95% CI); sacubitril/valsartan
0.192 (0.025–0.359) µg/kg*min/(mmol/L*pmol/L); and amlodipine 0.065 (–0.116 to
0.246) µg/kg*min/(mmol/L*pmol/L); Figure 2a). At week 8, the SI trended to be
higher after treatment with sacubitril/valsartan vs. amlodipine (sacubitril/valsartan
1.87 µg/kg*min/(mmol/L*pmol/L); amlodipine 1.76 µg/kg*min/(mmol/L*pmol/L));
however, the treatment difference did not reach statistical significance as
evidenced by the 97.5% CI (estimated treatment difference (97.5% CI), 0.128 (–
0.155 to 0.410) µg/kg*min/(mmol/L*pmol/L)). Importantly, 8 weeks of treatment with
sacubitril/valsartan, but not amlodipine, showed a significant increase from
baseline and a significant treatment difference in favor of sacubitril/valsartan in
glucose infusion rate (GIR; adjusted mean (95% CI) at week 8: LCZ696 445.3
(412.3–478.3) mg/min; amlodipine 390.5 (355.2–425.8) mg/min; estimated change
from baseline: sacubitril/valsartan 58.4 (25.4–91.4) mg/min; and amlodipine 3.6 (–
31.7 to 38.9) mg/min; adjusted treatment difference 54.8 (6.4–103.3) mg/min,
p=0.027; Figure 2b), the GIR normalized by body weight (adjusted mean (95% CI)
at week 8: sacubitril/valsartan 4.49 (4.15–4.83) mg/(min*kg); amlodipine 3.88
(3.51–4.24) mg/(min*kg); estimated change from baseline: sacubitril/valsartan 0.64
(0.31–0.98) mg/(min*kg); amlodipine 0.03 (–0.33 to 0.39) mg/(min*kg); adjusted
treatment difference 0.61 (0.12–1.11) mg/(min*kg), p=0.016; Figure 2c), and
metabolic clearance rate of glucose (adjusted mean (95% CI) at week 8:
sacubitril/valsartan 79.1 (73.1–85.0) (L*mg)/(min*mmol); amlodipine 69.4 (63.0–
75.8) (L*mg)/(min*mmol); estimated change from baseline: sacubitril/valsartan 10.7
(4.8–16.7) (L*mg)/(min*mmol); amlodipine 1.1 (–5.3 to 7.5) (L*mg)/(min*mmol); and
adjusted treatment difference 9.7 (0.9–18.4) (L*mg)/(min*mmol), p=0.031; Figure
2d).
To assess any significant effect of body weight and waist circumference, the
treatment difference for GIR was obtained from a similar analysis of covariance
model with additional covariates: change in body weight at week 8 and waist
circumference at screening. There was no significant covariate effect in either case
(body weight, estimated treatment difference (95% CI): 57.57 (8.98–106.15),
p=0.0208; waist circumference 50.86 (2.28–99.44), p=0.0404) compared to the
actual treatment difference for GIR (54.85 (6.39–103.30), p=0.0270). Scatter plot of
GIR vs. body weight and change in GIR vs. change in body weight did not display
any apparent trend of change at day 56 from baseline (Supplementary Figure S1a
and S1b, respectively).
Plasma insulin had reached steady-state values over the last 30 minutes of the
clamp procedure (Figure 3).

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Figure 2. Mean changes from baseline in insulin sensitivity variables after 8-week
treatment with sacubitril/valsartan vs. amlodipine.

Insulin sensitivity index (A); glucose infusion rate (GIR) (B); GIR by body weight (C); and metabolic
clearance rate (D). CI, confidence interval.

Figure 3. Insulin profiles during the hyperinsulinemic-euglycemic glucose clamp.

BAS, baseline.

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Abdominal subcutaneous adipose tissue lipolysis

The change in ethanol ratio from baseline was significant in the sacubitril/valsartan
group at the 45-minute (p=0.027) and 30 + 45-minute (p=0.042) time points.
However, there was no significant difference between treatment groups at any of
the time points (Figure 4a). An increase in interstitial glycerol from baseline to week
8 was observed at all time points in the sacubitril/valsartan group (adjusted
geometric mean ratio to baseline (95% CI) at 30 + 45-minute time points 1.05
(0.93–1.18)), but the change did not reach statistical significance. Compared with
the amlodipine group, the change from baseline in glycerol levels was significantly
higher with sacubitril/valsartan (adjusted geometric mean ratio between treatments
(95% CI) at 30 + 45-minute time points 1.22 (1.03–1.45), p=0.026; Figure 4b).
Glucose and lactate levels did not differ significantly from baseline in any of the
treatment groups and there was no difference between the treatment groups after
8 weeks of treatment (Figure 4c and 4d).

Figure 4. Adjusted geometric mean ratios of local adipose tissue lipolysis variables
after 8-week treatment with sacubitril/valsartan and amlodipine.

Ethanol ratio (A); glycerol (B); glucose (C); and lactate (D). Data were analyzed for repeated
measurements on log transformed values with treatment, visit, time and treatment*visit*time interaction
as fixed effect assuming an unstructured covariance. Subjects with missing data were excluded from
the analysis. The number of missing values ranged from 3 to 6 and 3 to 7 across all variables in the
sacubitril/valsartan and amlodipine groups, respectively. CI, confidence interval.

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Whole-body lipolysis
The rate of appearance (95% CI) of endogenous plasma glycerol at baseline on
-1
day 1 was 172.8 (158.0–189.0) and 200.6 (182.4–220.6) µmol/kg.min in the
sacubitril/valsartan and amlodipine groups, respectively. Whole-body glycerol rate
of appearance was not significantly affected by amlodipine treatment, but was
significantly lower at week 8 compared to baseline in the sacubitril/valsartan group
at the 30 + 45-minute time point (geometric mean ratio (95% CI), 0.93 (0.87–1.00),
p=0.045; Figure 5). There was no change from baseline in the sacubitril/valsartan
group at the 45-minute time point. There were no differences between treatment
groups in whole-body lipolysis after 8 weeks of treatment.

Figure 5. Whole-body lipolysis (rate of glycerol appearance).

The p value compared with baseline. CI, confidence interval.

Energy expenditure and substrate oxidation

At baseline, the mean respiratory quotient and resting energy expenditure were
comparable between groups (mean (SD); respiratory quotient: LCZ696 0.773
(0.063), amlodipine 0.768 (0.051); resting energy expenditure: sacubitril/valsartan
2,134.81 (397.98) kcal; and amlodipine 2,180.30 (548.78) kcal). At week 8,
respiratory quotient and resting energy expenditure were not significantly different
from baseline in either of the treatment groups or between the treatment groups
(mean treatment difference at week 8 (95% CI); respiratory quotient, 0.012 (–0.028
to 0.051), p=0.557; resting energy expenditure, 231.26 (–149.75 to 87.24) kcal,
p=0.601).

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Circulating metabolites, insulin and catecholamines in the fasted state

At week 8, there were no significant differences from baseline in either treatment
group or between treatment groups in fasting plasma glucose, insulin, glycerol, or
serum free fatty acids (Supplementary Table S1). However, sacubitril/valsartan, but
not amlodipine, was associated with a significant decrease from baseline in plasma
epinephrine (ratio to baseline (95% CI) for sacubitril/valsartan, 0.81 (0.72–0.91),
p<0.001), resulting in a significant treatment difference (sacubitril/valsartan vs.
amlodipine treatment ratio (95% CI), 0.77 (0.66–0.91), p=0.003). Conversely,
amlodipine was associated with a significant increase from baseline in
norepinephrine (ratio to baseline (95% CI) for amlodipine, 1.20 (1.07–1.33),
p=0.001); however, the difference between treatments at week 8 was not
statistically significant.

Blood pressure
Baseline data for mean seated blood pressure is presented in Table 1. At week 8,
mean seated blood pressure had decreased more in the sacubitril/valsartan group
compared with the amlodipine group (systolic blood pressure: -21.0 ± 16.1 mmHg
vs. -12.4 ± 14.7 mmHg; diastolic blood pressure: -12.4 ± 9.1 mmHg vs. -10.0 ± 7.8
mmHg; Figure 6).

Safety Assessments
Of the 98 patients, 67 patients (68.4%) experienced at least one adverse effect
(AE). Most AEs were mild or moderate in intensity. The overall AE incidence was
lower in the sacubitril/valsartan compared with the amlodipine group (60.0% vs.
77.1%; Supplementary Table S2). Five patients discontinued due to AEs. Two
patients discontinued due to a serious adverse event, which was unrelated to the
study drug (ruptured cerebral aneurysm in one patient in the sacubitril/valsartan
group and nephrolithiasis in one patient in the amlodipine group). Three patients
discontinued due to AEs (pruritus in one patient in the sacubitril/valsartan group,
and a single case of hypertension and peripheral edema in the amlodipine group).
The most commonly occurring AEs were nasopharyngitis, peripheral edema, and
headache, each of which had an overall incidence of >10% (Supplementary Table
S2). AEs suspected to be related to the study drug by the investigators occurred
more frequently in the amlodipine group than in patients receiving
sacubitril/valsartan (46% vs. 24%). Peripheral edema was more common in the
amlodipine group, whereas pruritus occurred only in the sacubitril/valsartan group
(Supplementary Table S2). Two patients in the sacubitril/valsartan group reported
mild orthostatic hypertension, both of which resolved by the end of the study. Both
of these events were suspected to be related to the study drug. No deaths were
reported in the study.

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Figure 6. Arithmetic mean (SD) of mean seated systolic blood pressure (SBP) (A);
and mean seated diastolic blood pressure (DBP) (B) after 8-week treatment with
sacubitril/valsartan and amlodipine.

BAS, baseline; EOS, end of study; SCR, screening; W-O, washout.

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DISCUSSION
Treatment with sacubitril/valsartan compared with amlodipine for 8 weeks improved
peripheral insulin sensitivity in obese patients with elevated blood pressure. This
improvement was not explained by changes in body weight or waist circumference.
Furthermore, sacubitril/valsartan significantly increased abdominal subcutaneous
adipose tissue lipolysis without changing whole-body lipolysis and plasma free fatty
acid concentrations. Therefore, this study is the first to show that simultaneous
neprilysin inhibition and AT 1-receptor blockade regulates glucose and lipid
metabolism in humans, supporting the concept that neprilysin substrates and AT 1-
receptor blockade have an important role in the crosstalk between the
cardiovascular system and metabolism in humans. These findings imply that
sacubitril/valsartan may improve cardiovascular and metabolic health in patients
with cardiovascular disease.
The metabolic effects of sacubitril/valsartan observed here could be mediated
through increased availability of neprilysin substrates, AT 1-receptor inhibition, or
both mechanisms combined. Indeed, individuals with obesity exhibit increased
renin angiotensin system (RAS) activity [18, 19] both systemically and at the tissue
level. Although genetically modified animals overexpressing RAS components are
characterized by insulin resistance that improved with pharmacological RAS
inhibition [20], the improvement in insulin resistance after RAS inhibition in humans
has been less consistent [16]. Among patients with impaired glucose tolerance and
cardiovascular disease or risk factors, the use of valsartan for 5 years, along with
lifestyle modification, led to a 14% relative reduction in T2DM incidence [21].
However, ramipril compared with placebo did not reduce the risk of new onset
T2DM in patients with impaired fasting glucose levels [22]. Overall, the
improvement of glucose metabolism after RAS inhibition seems to be modest and
may not solely explain the improvement in insulin sensitivity observed with
sacubitril/valsartan treatment. Moreover, AT1-receptor blockade does not increase
abdominal subcutaneous adipose tissue lipolysis, which was observed with
sacubitril/valsartan treatment [23, 24].
The present study suggests that neprilysin inhibition contributed to the metabolic
effect of sacubitril/valsartan treatment. Neprilysin degrades multiple peptides that
have the potential to modulate lipid and glucose metabolism, such as natriuretic
peptides, bradykinin, endothelin-1, and glucagon-like peptide 1. Therefore, we
cannot distinguish the contribution of individual neprilysin substrates to the
observed metabolic response. Eventually, the pharmacological effect of neprilysin
inhibition will depend on the net effect on all biologically relevant neprilysin
substrates. For example, bradykinin has been suggested to improve insulin
sensitivity and attenuate lipolysis, whereas endothelin-1 promotes insulin
resistance and increases lipolysis [25, 26]. Given the minor contribution of
neprilysin relative to angiotensin converting enzyme and aminopeptidase [27] and
the proven clinical safety of sacubitril/valsartan [16], it seems unlikely that an
increase in bradykinin explains the observed metabolic response. Likewise,
sacubitril/valsartan was shown to decrease, not increase, endothelin-1 plasma
concentrations in patients with heart failure and reduced ejection fraction. Indeed,
neprilysin contributes to both endothelin-1 formation from its precursors and
endothelin-1 degradation [28]. Hence, an involvement of endothelin-1 in mediation

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of metabolic effects of sacubitril/valsartan is also unlikely. Although the effect of

sacubitril/valsartan on multiple other metabolically relevant neprilysin substrates
has not been evaluated, its effect on the NP system, including increased NP and
cyclic guanosine monophosphate levels, has been well established in nonclinical
and clinical studies [17, 28, 29]. Given the strong evidence for an effect of NPs on
lipolysis and glucose metabolism in conjunction with the demonstrated increase in
NP and cyclic guanosine monophosphate availability with sacubitril/valsartan [17],
we propose that increased NP availability may have contributed, at least in part, to
metabolic improvements observed in our study.
NPs are more potent in stimulating human adipose tissue lipolysis than the
prototypical β-adrenoreceptor agonist isoproterenol [9]. Due to high NP clearance
receptor expression in adipose tissue of various animal species, NP-induced
lipolysis is only observed in primates [30], thus limiting the utility of standard
preclinical animal models. The present study, for the first time, examined the long-
term effects of simultaneous neprilysin inhibition and AT1-receptor blockade on
abdominal subcutaneous and whole-body lipolysis in humans. The sustained
increase in subcutaneous adipose tissue lipolysis is consistent with the observation
that ex vivo adipose tissue lipolysis is not desensitized in patients with heart failure
despite increased NP levels [30]. The observation that whole-body lipolysis does
not increase with sacubitril/valsartan treatment suggests that increased adipose
tissue lipolysis may be balanced by reduced lipolysis elsewhere in the body,
however, further studies are required to
elucidate the mechanism.
In the present study, patients treated with sacubitril/valsartan tended to exhibit
lower norepinephrine and epinephrine plasma concentrations compared with
patients treated with amlodipine. Given the central role of catecholamines in
regulation of lipid mobilization, the expected response is reduced whole-body and
adipose tissue lipolysis. Instead, sacubitril/valsartan-treated patients showed
increased adipose tissue lipolysis with sustained whole-body lipolysis, presumably
through increased availability of neprilysin substrates.
Unopposed increases in adipose tissue lipolysis could promote ectopic fat storage
and insulin resistance through accumulation of toxic lipid intermediates, including
diacylglycerol [3]. However, NPs acutely increase postprandial fatty acid oxidation
in humans, and circulating levels of the anti-inflammatory and insulin sensitizing
adipokine adiponectin [13]. Thus far, data on metabolic effects of sustained NP
activation were limited to nonclinical experiments. Nonclinical and in vitro studies
have demonstrated that NP activation and transgenic overexpression enhanced
mitochondrial oxidative capacity and lipid oxidation in human skeletal myotubes
and in adipocytes [14], and protected mice from high-fat diet induced obesity and
insulin resistance [31], respectively. Additionally, chronic NP infusion upregulated
muscular oxidative capacity in obese and diabetic mice, thereby ameliorating
lipotoxicity and insulin resistance [32]. The present clinical study did not indicate
alterations in whole-body fat oxidation by sacubitril/valsartan treatment. We
speculate that, after treatment with sacubitril/valsartan, NP-mediated changes in
oxidative metabolism at the organ level that cannot be captured by indirect
calorimetry may have attenuated accumulation of toxic lipid intermediates and
subsequent impairment of insulin signaling [3]. It is also possible that augmented
NP availability increased peripheral glucose and insulin supply through vascular

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actions. Augmenting cyclic guanosine monophosphate signaling through

phosphodiesterase 5 inhibition with sildenafil also improved insulin sensitivity in
overweight individuals with prediabetes [33].
Combination of state-of-the-art methodologies in a comparatively large sample size
is a particular strength of our study. In fact, hyperinsulinemic euglycemic clamp
2
testing, [1,1,2,3,3- H]-glycerol tracer kinetics, and microdialysis technics are
considered gold-standards in assessing insulin sensitivity, whole-body lipolysis,
and subcutaneous adipose tissue lipolysis, respectively. The total daily dose of
sacubitril/valsartan used in this study (400 mg) is known to provide superior blood
pressure control in patients with arterial hypertension when administered once daily
[34] and to reduce cardiovascular mortality and hospitalizations due to heart failure
in patients with systolic dysfunction when administered as sacubitril/valsartan 200
mg twice daily compared with standard-of-care RAS inhibition. Therefore, the
findings of this study are relevant for the future clinical use of sacubitril/valsartan.
We studied patients representing a human model for impaired insulin sensitivity.
Given the close association between adiposity and risk for T2DM, and the epidemic
increase in the incidence of T2DM in recent years, cardiovascular medications with
added benefits on insulin sensitivity are highly desirable. Neprilysin activity seems
to be upregulated in obese and insulin-resistant individuals [6], such that
sacubitril/valsartan directly targets a mechanism mediating relative natriuretic
peptide deficiency. Furthermore, heart failure, for which sacubitril/valsartan is
approved for, is also associated with insulin resistance and muscular metabolic
abnormalities. Patients with heart failure exhibit a reduction in oxidative slow twitch
(type I) relative to glycolytic fast twitch (type II) muscle fibers along with reduced
mitochondrial density [35]. This intrinsic impairment in skeletal muscle metabolism
likely limits exercise capacity and may contribute to associated insulin resistance in
patients with heart failure [36]. Decreased skeletal muscle oxidative capacity in
heart failure is prevalent and clinically relevant because poor exercise tolerance
and impaired insulin sensitivity predict mortality risk in these patients [37].
Therefore, metabolic improvements with sacubitril/valsartan may also be beneficial
in patients with heart failure, thereby differentiating sacubitril/valsartan from
currently available cardiovascular drugs.
The main limitation of our study was the fact that the change in the primary
endpoint SI between interventions was not statistically significant. To account for
an interim analysis conducted for SI, the p value for equality of treatments was
reported at 2.5% level of significance for SI, whereas it was reported at 5% level of
significance for all other glucose-clamp derived measurements. Nonetheless, there
was a clear trend for improvement of SI with sacubitril/valsartan compared to
amlodipine. Furthermore, GIR with or without adjustment for body weight, an
established marker for insulin sensitivity, was significantly improved with
sacubitril/valsartan, as were all other glucose-clamp derived measurements of
insulin sensitivity, supporting the overall conclusion. Moreover, the fact that we
used amlodipine as the metabolically neutral comparator does not allow
distinguishing individual contributions of neprilysin inhibition and angiotensin
receptor blockade to the effects of sacubitril/valsartan observed in this study.
Furthermore, we cannot completely rule out a carryover effect of metabolic testing,
particularly insulin infusion, on lipolysis measurements obtained on the following
study day. However, all patients underwent all study assessments in the same

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sequence suggesting that an impact on the comparison of investigational

treatments is unlikely. Finally, our study was conducted in a relatively homogenous
white population. Although there is no ethnic sensitivity with regard to exposure to
LCZ696 analytes (sacubitril, sacubitrilat, and valsartan), extrapolation to other
ethnic groups requires careful consideration.

CONCLUSION
In conclusion, our study demonstrated that sacubitril/valsartan treatment compared
with amlodipine resulted in improved peripheral insulin sensitivity and increased
abdominal subcutaneous adipose tissue lipid mobilization, but did not show
significant effects on whole-body lipolysis, energy expenditure, and substrate
oxidation. Moreover, our study highlights the utility of mechanistic profiling studies
in discerning metabolic drug actions that could go undetected in clinical
development.

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METHODS

Study design and participants

This was a multicenter, randomized, double-blind, double-dummy, active-
controlled, and parallel-group study. Eligible subjects were ≥18 years of age with
abdominal obesity (waist circumference ≥102 cm for men and ≥88 cm for women)
and elevated blood pressure either untreated (mean seated systolic blood pressure
≥130 mmHg and <180 mmHg at screening) or treated with up to two classes of
antihypertensive therapy (mean seated systolic blood pressure ≤160 mmHg at
screening and <180 mmHg at the end of the 4-week washout period). Women had
to be of nonchild-bearing potential. Key exclusion criteria were severe hypertension
(mean seated diastolic blood pressure ≥100 mmHg and/or mean seated systolic
blood pressure ≥180 mmHg at screening or at the end of the washout period), type
1 or type 2 diabetes mellitus, fasting plasma glucose ≥126 mg/dL or HbA1c
≥6.5%), dyslipidemia requiring pharmacological therapy with fibrates or nicotinic
acid, concomitant use of antihypertensive medications, antidiabetic medications or
drugs that may affect glucose or lipid metabolism, previous or current diagnosis of
cardiac structural and functional abnormalities, history or current diagnosis of heart
failure (New York Heart Association classes II-IV), history of myocardial infarction,
coronary bypass surgery or percutaneous coronary intervention during the 6
months before screening, history of angioedema, or known hypersensitivity to the
study drugs.
The study included a screening period of up to 4 weeks followed by a 4-week
washout period and an 8-week randomized, double-blind, and double-dummy
treatment phase (Figure 1). Patients receiving antihypertensive medications at the
time of screening discontinued the therapy during the washout period.
In the double-blind treatment period, patients were randomized to receive
sacubitril/valsartan 400 mg q.d. or amlodipine 10 mg q.d. along with matching
placebos for 8 weeks. Patients were stratified into four groups based on the
baseline Homeostasis Model Assessment of Insulin Resistance (<2.5 and ≥2.5)
and use of statin therapy within 8 weeks before randomization. Stratification was
intended to ensure that within each stratum the number of subjects receiving either
LCZ696 or amlodipine is comparable, thereby avoiding any impact of a potential
imbalance in baseline characteristics on study outcome.
All participants provided written informed consent before screening. The study
protocol was reviewed by the independent ethics committee or institutional review
board for each center, conducted in accordance with the Declaration of Helsinki,
and registered at clinicaltrials.gov under the identifier NCT01631864.

Peripheral insulin sensitivity

Changes in insulin sensitivity were measured from baseline to week 8 for
sacubitril/valsartan 400 mg q.d. vs. amlodipine 10 mg q.d. by a hyperinsulinemic-
euglycemic glucose clamp. Assessments were performed on day -1 (baseline) and
on day 56 under fasting conditions. A dorsal hand vein was catheterized and kept
warm for arterialized blood sampling and the contralateral arm was catheterized for
glucose and insulin infusions. The procedure consisted of a 2-hour primed infusion

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2
of insulin (a priming dose over the first 10 minutes (103 mU/m /min at 0–5 minutes
2
and 57 mU/m /min at 5–10 minutes) followed by a continuous infusion at 40
2
mU/m /min thereafter until 2 hours) and a variable glucose infusion to achieve
steady-state plasma insulin levels while maintaining blood glucose levels at 90.1
mg/dL (5.0 mmol/L). Blood samples were collected continuously (automated
clamp) or at approximately 5 minute intervals (manual clamp) to determine glucose
levels during hyperinsulinemic- euglycemic clamp testing. The last 30 minutes
(minutes 90 - 120) of the clamp were considered as the steady-state period and the
mean GIR was calculated for this period.
Insulin SI was calculated from steady-state GIRs and plasma insulin and glucose
concentrations (SI: glucose infusion rate/(plasma glucose x plasma insulin),
µg/kg*min/(mmol/L*pmol/L)). Whole-body glucose disposal rate (M, mg/min) was
calculated from mean GIRs at steady state to assess peripheral insulin sensitivity.
Finally, the metabolic clearance rate was calculated from the M-value and the
mean blood glucose concentration at steady state (metabolic clearance rate:
M/blood glucose, min*mmol). The glucose disposal rate was expressed per unit of
insulin at steady state, calculated from M-value and plasma insulin concentrations
(M/I, U/min).

Lipolysis
Lipolysis was assessed on day 1 before the first dosage and on day 57. One
microdialysis probe was placed in the subcutaneous adipose tissue 6–8 cm lateral
from the umbilicus with the patient under local anesthesia and sterile conditions, as
described previously [38]. The process comprised a recovery phase of 60 minutes
during which the probes were infused with perfusion solution (50 mM ethanol + T1
perfusion solution; CMA Microdialysis AB, Stockholm, Sweden) at a flow rate of 0.3
µL/min followed by a flow calibration phase of 60 minutes. During the flow
calibration phase, the perfusion rate was maintained at 0.3 µL/min for the first 30
minutes and increased thereafter to 2.0 µL/min for the remaining 30 minutes.
Microdialysate samples were collected at 30 minutes and 45 minutes in the flow
calibration phase.
Glycerol (an indicator of local lipolysis), glucose, and lactate concentrations were
measured in microdialysates. The ethanol ratio (ratio of ethanol concentration in
dialysate to ethanol concentration in perfusate) was measured as an indicator of
adipose tissue blood flow.
2
Whole-body lipolysis was assessed using [1,1,2,3,3- H]-glycerol tracer kinetics. A
glycerol tracer bolus (3.0 µg/kg) was injected at the start of the microdialysis flow
calibration phase, followed by a continuous infusion (0.1 µg/kg/min) until the end of
the sampling period. Blood samples were collected at 0, 30, 40, 50, and 60
minutes during the flow calibration phase and at 15, 30, and 45 minutes thereafter.
The rate of appearance of endogenous glycerol was calculated as the ratio of the
glycerol tracer infusion rate to the plasma glycerol tracer enrichment. At steady
state, glycerol rate of appearance was calculated from glycerol tracer enrichment
using Steele’s equation.

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Statistical analysis
Based on 80% power to detect a difference of 0.1 in SI at week 8 between the two
treatment groups at 2.5% significance level with two-sided alternative, 90 subjects
(45 in each group) were needed to complete the study. SI at 8 weeks was analyzed
using the analysis of covariance model with treatment as a fixed effect and
baseline insulin sensitivity as a covariate for assessing the difference in the mean
effect of sacubitril/valsartan vs. amlodipine. For the purpose of a two-sided
hypothesis of equality of treatment effects at 2.5% level of significance after the
completion of the study, the point estimate and 97.5% CI for the difference along
with the p-value for equality of the two treatments were reported. The smaller level
of significance (2.5%) was chosen to account for the conduct of an interim analysis.
Differences between treatments for change in SI from baseline were analyzed as
described above. Similar analyses at 5% level of significance were performed for
all other clamp-derived measures of insulin sensitivity.
For abdominal subcutaneous adipose tissue microdialysate data (ethanol ratio,
dialysate lactate, dialysate glucose, and plasma glycerol, glycerol, NEFA, glucose,
insulin, adrenalin, and noradrenalin) at rest, data were analyzed using repeated
measures analysis on log transformed values with treatment, visit, time and
treatment*visit*time interaction as fixed effects. Ratio to day 1 (day 57 vs. day 1) at
each treatment and ratio of sacubitril/valsartan vs. amlodipine for ratio to day 1
along with the corresponding 95% CIs and p-values are presented for 30 and 45-
minute microdialysis time points combined. Comparison of 30 and 45-minute
microdialysis time points demonstrated steady state of the microdialysis
experiment.
Oxidative metabolism was analyzed using the analysis of covariance with
treatment as fixed effect and baseline as covariate and the treatment mean
difference with 95% CIs and p-values are reported.
For biomarkers, data were analyzed using repeated measures of analysis on log
transformed values with treatment, visit, time and treatment*visit*time interaction as
fixed effects. Ratio to day 1 (day 57 vs. day 1) at each treatment and ratio of
sacubitril/valsartan vs. amlodipine for ratio to day 1 along with the corresponding
95% CIs and p-values are presented.

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controlled study. Hypertension. 2014;63(4):698-705.
35. Drexler H, Riede U, Munzel T, Konig H, Funke E, Just H. Alterations of skeletal
muscle in chronic heart failure. Circulation. 1992;85(5):1751-9.
36. Kemppainen J, Tsuchida H, Stolen K, Karlsson H, Bjornholm M, Heinonen OJ, et al.
Insulin signalling and resistance in patients with chronic heart failure. J Physiol.
2003;550(Pt 1):305-15.
37. Doehner W, Rauchhaus M, Ponikowski P, Godsland IF, von Haehling S, Okonko DO,
et al. Impaired insulin sensitivity as an independent risk factor for mortality in patients
with stable chronic heart failure. J Am Coll Cardiol. 2005;46(6):1019-26.
38. Goossens GH, Blaak EE, Saris WH, van Baak MA. Angiotensin II-induced effects on
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SUPPLEMENTARY MATERIAL

Energy Expenditure and Substrate Oxidation

Energy expenditure and substrate oxidation were assessed in parallel by indirect
calorimetry using a ventilated hood system. The ventilated hood measurements
were recorded for 30 minutes in the resting state (during the flow calibration phase)
and averaged for further analysis. Resting energy expenditure (REE) was
calculated using the abbreviated Weir equation (REE = [3.9 (VO2) + 1.1 (VCO2)]
1.44) (with VO2 and VCO2 expressed as mL/min).

Circulating metabolites, insulin, and catecholamines

Fasting plasma concentrations of free fatty acids, glycerol, glucose, insulin,
epinephrine, and norepinephrine were evaluated at baseline (Day 1) and on Day
57 at rest and concurrently with the microdialysis experiment. Blood samples were
taken at 30 minutes and 45 minutes following the calibration flow phase.

Blood Pressure
Office BP was measured at screening, during washout and throughout the study at
baseline, week 4, week 8 and end of study using the same arm and the same
automated equipment with an appropriate cuff size. Measurements were performed
in triplicates at 2-minute intervals after patients have been sitting for 15 minutes
with the back supported and both feet on the floor. During the home stay period,
patients were given a home measurement device and instructed to monitor BP
twice weekly at approximately the same time each morning (7–9 AM).

Safety Assessments
Safety assessments included adverse events (AEs) and serious adverse events
(SAEs) and were regularly monitored throughout the study based on hematology,
blood chemistry and urine analysis as well as assessments of vital signs (BP and
pulse measurements), ECG, physical condition and body weight.

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Supplementary Figure 1A. Comparison of glucose infusion rate with body weight
at baseline and Day 56.

Supplementary Figure 1B. Change from baseline in glucose infusion rate vs.
body weight following 8-week treatment with LCZ696 vs. amlodipine

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Supplementary Table 1. Mean Change in Biomarkers from Baseline to Day 57

Biomarker LCZ696 Amlodipine Treatment difference

Adjusted means (400 mg q.d.) (10 mg q.d.) (LCZ696 vs.
(95% CIs) (n=50) (n=46) Amlodipine)
Glucose (mmol/L)
Baseline 5.64 (5.51, 5.78) 5.52 (5.39, 5.65) -

Day 57 5.66 (5.55, 5.78) 5.50 (5.38, 5.62) -

Ratio to baseline 1.00 (0.99, 1.02) 1.00 (0.98, 1.01) 1.01 (0.98, 1.03)

Insulin (pmol/L)
Baseline 41.09 (34.68, 48.67) 37.54 (31.45, 44.79)
Day 57 41.07 (34.57; 48.79) 37.91 (31.61, 45.47)
Ratio to baseline 1.00 (0.88, 1.14) 1.01 (0.88; 1.16) 0.99 (0.82, 1.20)
Glycerol (µmol/L)
Baseline 85.39 (77.97; 93.52) 91.61 (83.32, 100.73)
Day 57 84.48 (76.85, 92.87) 87.90 (79.51, 97.18)
Ratio to baseline 0.99 (0.91, 1.08) 0.96 (0.87; 1.05) 1.03 (0.91, 1.17)
Non-esterified fatty acids (mmol/L)
Baseline, adjusted
0.63 (0.56, 0.70) 1.03 (0.91, 1.17)
mean
Day 57 0.60 (0.53, 0.68) 1.03 (0.91, 1.17)
Ratio to baseline 0.96 (0.86, 1.06) 0.92 (0.82, 1.03) 1.04 (0.89, 1.21)
Epinephrine (pg/mL)
Baseline 25.23 (21.20, 30.03) 20.98 (17.49, 25.16)
Day 57 20.42 (17.19, 24.25) 21.91 (18.28, 26.26)
a b
Ratio to baseline 0.81 (0.72, 0.91) 1.04 (0.93, 1.18) 0.77 (0.65, 0.91)
Norepinephrine (pg/mL)
Baseline, 217.3 (188.3, 250.7) 230.8 (198.8; 267.9)
Day 57 236.8 (204.2, 274.5) 276.2 (236.4; 322.9)
c
Ratio to baseline 1.09 (0.98, 1.21) 1.20 (1.07, 1.33) 0.91 (0.78, 1.06)

a
p<0.001; b p=0.002; c p=0.001
Data was analyzed using repeated measurements on log transformed values with treatment, visit, time
and treatment*visit*time interaction as fixed effect assuming an Unstructured covariance. Values are
then log back transformed.

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Supplementary Table 2. Incidence of AE occurring in more than one patient -

safety analysis set

LCZ696 400 mg Amlodipine 10 mg

N = 50, n (%) N = 38, n (%)

Subjects with AEs, n (%) 30 (60.0) 37 (77.1)

Nasopharyngitis 9 (18.0) 8 (16.7)

Peripheral edema 1 (2.0) 16 (33.3)

Headache 4 (8.0) 6 (12.5)

Pruritus 5 (10.0) -

Diarrhea 3 (6.0) 1 (2.1)

Dizziness 3 (6.0) 1 (2.1)

Pollakisuria 2 (4.0) 1 (2.1)

Circulatory collapse* - 2 (4.2)

Fatigue 1 (2.0) 1 (2.1)

Gastroenteritis 2 (4.0) -

Orthostatic hypotension 2 (4.0) -

Pruritus generalized 2 (4.0) -

Toothache 1 (2.0) 1 (2.1)

AE, adverse event

* Vasovagal reaction coded as circulatory collapse according to MedDRA

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Effect of sacubitril/valsartan on exercise
induced lipid metabolism in individuals
with obesity and hypertension

Engeli S., Stinkens R., Heise T., May M., Goossens G.H., Blaak E.E.,
Jax T., Albrecht D., Pal P., Tegtbur U., Haufe S., Langenickel T.H.,
Jordan J.

Submitted
Chapter 4

ABSTRACT
Background: sacubitril/valsartan (LCZ696), a novel angiotensin receptor-neprilysin
inhibitor, was recently approved for the treatment of heart failure with reduced
ejection fraction. Neprilysin degrades several peptides that modulate lipid
metabolism, including natriuretic peptides. In this study, we investigated the effects
of 8 weeks’ treatment with sacubitril/valsartan on whole body and adipose tissue
lipolysis and lipid oxidation during defined physical exercise compared with the
metabolically neutral comparator amlodipine.
Methods: This was a multicenter, randomized, double-blind, active-controlled,
parallel-group study enrolling subjects with abdominal obesity and moderate
hypertension (mean sitting systolic blood pressure [msSBP] ≥130-180 mmHg).
Lipolysis during rest and exercise was assessed by microdialysis and [1,1,2,3,3-
2
H]-glycerol tracer kinetics. Energy expenditure and substrate oxidation were
measured simultaneously using indirect calorimetry. Plasma non-esterified fatty
acids, glycerol, insulin, glucose, adrenaline and noradrenaline concentrations,
blood pressure and heart rate were also determined.
Results: Exercise elevated plasma glycerol, free fatty acids and interstitial glycerol
concentrations and increased the rate of glycerol appearance. However, exercise-
induced stimulation of lipolysis was not augmented upon sacubitril/valsartan
treatment compared with amlodipine treatment. Furthermore, sacubitril/valsartan
did not alter energy expenditure and substrate oxidation during exercise compared
with amlodipine treatment.
Conclusion: Sacubitril/valsartan treatment for 8 weeks did not elicit clinically
relevant changes in exercise-induced lipolysis or substrate oxidation in obese
patients with hypertension, implying that its beneficial cardiovascular effects cannot
be explained by changes in lipid metabolism during exercise.

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INTRODUCTION
Fatty acids are stored in the form of triglycerides in the adipose tissue and are
released during lipolysis to fuel lipid oxidation in energy consuming tissues.
Lipolysis and skeletal muscle lipid oxidation decrease following carbohydrate
ingestion and increase in the fasting state or during physical exercise [1]. An
imbalance between fatty acid mobilization and utilization may adversely affect
cardiovascular and metabolic health. Acute experimental increases in circulating
fatty acids in humans worsened hepatic [2] and skeletal muscle [3] insulin
sensitivity and endothelium-mediated vasodilation [4]. Chronic increase in fatty acid
availability promotes hepatic, skeletal muscle and myocardial lipotoxicity,
dyslipidemia, insulin resistance, and type 2 diabetes mellitus [5, 6]. Conversely,
interventions that reduce fatty acid levels, improve metabolic health [5]. These
observations are highly relevant for cardiovascular medications with the potential to
affect lipid turnover. Sacubitril/valsartan, comprising a novel neprilysin inhibitor pro-
drug sacubitril and angiotensin receptor blocker (valsartan) has been approved for
the treatment of chronic heart failure (NYHA Class II-IV) with reduced ejection
fraction [7]. The endopeptidase neprilysin is ubiquitously expressed, including in
human adipocytes, and degrades multiple peptides such as natriuretic peptides
(NPs), angiotensin II, bradykinin and endothelin that may modulate lipid
metabolism [8, 9]. Notably, NPs potently augment human adipose tissue lipolysis,
postprandial lipid oxidation, and skeletal muscle oxidative capacity [9], whereas,
angiotensin II elicits more subtle changes in fatty acid turnover [10].
Given the role and association of aberrant NP- and renin-angiotensin-aldosterone
signaling in cardiovascular diseases and metabolic dysfunction, we hypothesized
that simultaneous blockade of angiotensin receptor and neprilysin with
sacubitril/valsartan can potentially ameliorate metabolic dysfunction, especially
lipid-turnover, compared with amlodipine. In the present study, we investigated the
effects of 8-weeks treatment with sacubitril/valsartan compared with the
metabolically neutral comparator amlodipine on whole-body and adipose tissue
lipolysis, energy expenditure and substrate oxidation during defined physical
exercise, which is known to stimulate NP release and induces lipolysis and lipid
oxidation.

METHODS

Study design
The study design, key inclusion and exclusion criteria of the patients and the
primary results of this study have been described earlier [11]. Briefly, this was a
multicenter, randomized, double-blind, double-dummy, active-controlled, parallel-
group study enrolling adult subjects with abdominal obesity (waist circumference
≥102 cm for men and ≥88 cm for women) and moderate hypertension (mean sitting
systolic blood pressure [msSBP] ≥130 and<180 mmHg). Key exclusion criteria
were severe hypertension (msSBP>180 mmHg), type 1 or 2 diabetes (fasting
plasma glucose ≥126 mg/dL or HbA1c ≥6.5%), dyslipidemia requiring therapy with
fibrates or nicotinic acid, concomitant use of antihypertensive, anti-diabetic or other

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medications that affect glucose and/or lipid metabolism, and a history or current
diagnosis of heart failure (NYHA class II-IV).
The study included a screening period of up to 4-weeks followed by a 4-week
washout period and an 8-week, randomized, double-blind, double-dummy
treatment phase. Patients receiving antihypertensive medications at the time of
screening discontinued the therapy during the washout period. During the
treatment period, patients were randomized to receive either sacubitril/valsartan
400 mg every day (QD) or amlodipine 10 mg QD along with a matching placebo for
8 weeks. Patients were stratified into 4 groups based on baseline homeostatic
model assessment of insulin resistance (HOMA-IR) and statin use.
All patients provided written informed consent before screening. The clinical study
protocol was reviewed and approved by the Independent Ethics Committee or
Institutional Review Board at each center and, conducted in accordance with
declaration of Helsinki and registered at clinicaltrials.gov under the identifier
NCT01631864.

Exercise test
An incremental exercise test on a bicycle ergometer was conducted before the
start of the intervention (Day -14) to determine the maximal aerobic capacity
(VO2peak) at volitional exhaustion by measuring the individual maximum workload
prior to stopping for exhaustion or until pre-defined heart rate or blood pressure
(BP) criteria were met. At baseline (Day 1) and after 8 weeks (Day 57), subjects
exercised at 50% of VO2peak (as determined on Day -14) for a period of 60 min.

Measurement of lipolysis
Local adipose tissue and whole body lipolysis were assessed at baseline and after
8 weeks of treatment, as described previously [11]. Local adipose tissue lipolysis
was measured by microdialysis and assessed during a 45 min interval at rest,
followed by a 60 min interval during which the patients exercised at 50% of their
individual VO2peak. Dialysates were collected from abdominal subcutaneous
adipose tissue at the lower right abdominal quadrant at rest and at 15-min intervals
during exercise. Concentrations of glycerol (as an indicator of lipolysis), glucose
and lactic acid in dialysates were measured. The ethanol outflow/inflow ratio (ratio
of ethanol concentration in the dialysate and the perfusate) was measured as an
indicator of adipose tissue blood flow.
2
Whole body lipolysis was estimated using [1,1,2,3,3- H]-glycerol tracer kinetics
-1
following an intravenous glycerol bolus (2 µmol·kg ) after insertion of the
microdialysis catheter, approximately 60-min before the baseline measurements
-1 -1
started, and subsequent infusion at an infusion rate of 0.1 µmol·kg min at rest
-1 -1
and 0.2 µmol·kg min during exercise. Blood samples were collected at 15-min
intervals at rest and during exercise. The rate of appearance (Ra) of endogenous
glycerol was calculated as the ratio of glycerol tracer infusion rate to plasma
glycerol tracer enrichment. At steady state, glycerol Ra was calculated from
glycerol enrichment using Steele’s equation.

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Energy expenditure and substrate oxidation

Energy expenditure and substrate oxidation during rest and exercise were
assessed by indirect calorimetry using a ventilated hood system. The ventilated
hood measurements were recorded for 30 minutes in the resting phase with the
patient in supine position and during the last 10 minutes of the 60-min exercise
period.

Circulating metabolites and hormones

Samples for fasting plasma biomarkers (non-esterified fatty acid [NEFA], glycerol,
glucose, insulin, adrenaline and noradrenaline) were collected at baseline (Day 1)
and on Day 57 at rest and during exercise concurrently with microdialysis
measurements.

Blood pressure
Office BP was measured at screening, during washout and throughout the study at
baseline, Week 4, Week 8 and at the end of study using the same automated
equipment with an appropriate cuff size. Measurements were performed in
triplicate at 2-min intervals after patients had been sitting for 15 minutes with the
back supported and both feet on the floor. BP was also measured during the
exercise phase. During the home stay period, patients were given a home
measurement device and instructed to monitor BP twice weekly at approximately
the same time each morning.

Statistical analysis
Following 8 weeks of treatment with sacubitril/valsartan or amlodipine,
assessments of local adipose tissue lipolysis, whole body lipolysis, oxidative
metabolism, BP and biomarkers during exercise were performed as pre-specified
study objectives.
For abdominal subcutaneous adipose tissue microdialysate data (ethanol ratio,
dialysate lactate, dialysate glucose, dialysate glycerol), plasma biomarkers
(glycerol, NEFA, glucose, insulin, adrenaline and noradrenaline) and whole-body
lipolysis (rate of glycerol appearance) data for 45 minutes at rest and 4 time-points
during exercise (15, 30, 45 and 60 minutes) were analyzed using repeated-
measures analysis on log-transformed values with treatment, visit, time and
treatment*visit*time interaction as fixed effects. Geometric mean ratios of each
exercise time-point to 45 minutes resting for each day and treatment, ratios of Day
57 to Day 1 for each treatment and each exercise time-point, and the ratio between
sacubitril/valsartan and amlodipine for Day 57 to Day 1 were calculated.
Oxidative metabolism was analyzed using analysis of covariance (ANCOVA) with
treatment as the fixed effect and baseline as the covariate. Oxidative metabolism
during exercise was analyzed using analysis of variance (ANOVA) for repeated
measurements with treatment, visit and treatment*visit interaction as fixed effects.
Mean difference to Day1 (Day 57 vs. Day 1) for each treatment along with the
corresponding 95% CIs and p-values are presented. Data for exercise and resting
phase were analyzed for each day and treatment with a mixed-effects linear model
with phase (exercise or resting) as the fixed effect and subject as the random effect

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to obtain the mean difference estimate and 95% CI for exercise vs. rest
comparison. Respiratory quotient (CO2/O2 ratio) was calculated at each of the Days
1 and 57 at rest and during exercise. A statistical comparison of the quotients was
made between rest and exercise within each day.

RESULTS

Exercise testing
On Day 1, 39 patients in the sacubitril/valsartan group and 24 patients randomized
to the amlodipine group completed the constant workload exercise for 60 minutes.
On Day 57, 36 patients treated with sacubitril/valsartan and 23 treated with
amlodipine completed the exercise for 60 minutes. Similar observations were made
in patients completing only 45 and 60 minutes of exercise, suggesting that 8 weeks
of treatment of patients with obesity and hypertension with sacubitril/valsartan or
amlodipine did not have any clinically relevant impact on the exercise duration.
Oxygen consumption and workload were comparable between Day 1 and Day 57
in both treatment groups (Supplementary Table 1).

Plasma glucose and insulin concentrations

With exercise, plasma glucose concentrations increased in the amlodipine group
for all time-points and for 30 (p=0.017), 45 (p=0.002) and 60 (p<0.001) minutes in
the sacubitril/valsartan group on Day 1. On Day 57, the increase was significant
during 60 min of exercise in the sacubitril/valsartan group (p=0.031) but the
difference was not significant at any time point in the amlodipine group. A decrease
in glucose concentration was noticed on Day 57 in both the treatment groups as
compared to baseline (Day 1), with the difference being significant only in the
amlodipine group at 30 (p=0.017) and 45 (p<0.001) minutes of exercise. However,
no statistically significant differences in glucose concentrations were observed
between the treatment groups at any time-point.
A decrease in insulin concentrations with increasing exercise duration was
observed in both treatment groups. When compared with resting insulin
concentrations, a significant decrease was observed at 45 min (p=0.015) and 60
min (p<0.001) on Day 1 and at 45 min (p=0.044) on Day 57 in the
sacubitril/valsartan group. However, exercise-induced decreases in insulin
concentrations were not statistically significant in the amlodipine group, either on
Day 1 or Day 57. After 8 weeks of treatment, compared with baseline, insulin
concentrations were significantly lower in the amlodipine group at all time-points
except 60 minutes, while the change was not significant at any time-point in the
sacubitril/valsartan group. Significant differences in insulin concentrations were
observed at 30 min (p=0.017) and 45 min (p=0.027) between treatment groups on
Day 57 compared to baseline.

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Subcutaneous adipose tissue lipolysis during exercise

Compared with resting measurements, microdialysate glycerol concentrations
increased during exercise, indicating increased subcutaneous adipose tissue
lipolysis in both the amlodipine and sacubitril/valsartan groups on Day 1 as well as
on Day 57. Compared with baseline, microdialysate glycerol concentrations during
exercise were numerically lower in the amlodipine group on Day 57. In the
sacubitril/valsartan group, microdialysate glycerol concentrations increased
similarly at the beginning and at the end of treatment, but this increase was not
statistically significant (Figure 1). Microdialysate glucose concentrations were
comparable between sacubitril/valsartan and amlodipine at baseline
[sacubitril/valsartan vs. amlodipine: 15 min (1.07 vs. 0.94 mmol/L); 30 min (1.06 vs.
1.02 mmol/L); 45min (1.05 vs 0.99 mmol/L); 60 min (1.03 vs. 0.91 mmol/L)] and on
Day 57 [15 min (1.12 vs. 0.95 mmol/L); 30 min (1.08 vs. 0.94 mmol/L); 45min (1.07
vs 1.02 mmol/L); 60 min (1.06 vs. 1.01 mmol/L)]. No statistically significant
differences in glucose levels from baseline to Week 8 were observed between the
two treatment groups. A similar trend was observed with lactate levels.
Ethanol ratios were comparable between sacubitril/valsartan and amlodipine on
Day 1 [sacubitril/valsartan vs. amlodipine: 15 min (0.42 vs. 0.43); 30 min (0.42 vs.
0.44); 45 min (0.43 vs 0.45); 60 min (0.47 vs. 0.46)]. Ethanol ratios increased on
Day 57 in both the sacubitril/valsartan and amlodipine groups, but remained
comparable between treatment groups [15 min (0.49 vs. 0.49); 30 min (0.51 vs.
0.49); 45 min (0.53 vs. 0.51); 60 min (0.55 vs. 0.53)]. These data suggest that there
was no relevant change in blood flow that needs to be accounted for when
interpreting glycerol measurements.

† p=0.003 vs. baseline

Figure 1. Comparison of local adipose tissue lipolysis (dialysate glycerol) variable

during exercise following 8-weeks of treatment with sacubitril/valsartan and
amlodipine
Error bars indicate 95% CI

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Whole-body lipolysis
Plasma glycerol concentrations increased with exercise in both treatment groups,
both on Day 1 and Day 57 [amlodipine group, Day 1 vs. Day 57: resting (89.77 vs.
88.04 µmol/L); 15 min (141.12 vs. 119.56 µmol/L); 30 min (184.78 vs. 156.03
µmol/L); 45 min (216.04 vs. 179.27 µmol/L); 60 min (224.85 vs. 191.95 µmol/L) and
sacubitril/valsartan, Day 1 vs. Day 57: resting (85.64 vs. 83.93 µmol/L); 15 min
(139.3 vs. 126.92 µmol/L); 30 min (177.65 vs. 157.29 µmol/L); 45 min (205.68 vs.
189.84 µmol/L); 60 min (225.62 vs. 205.26 µmol/L)]. Compared to baseline, plasma
glycerol levels were lower in both the treatment groups on Day 57. While the
change from baseline to Day 57 was significant at all time-points in the amlodipine
group (p<0.05), it was significant at 30 minutes in the sacubitril/valsartan group
(p=0.012). The differences in plasma glycerol levels between treatment groups
were not significant.
As compared with glycerol Ra following 45 minutes rest, a significant increase was
observed during exercise at all time-points in both treatment groups on Day 1 and
Day 57 (p<0.001). The change from baseline to Day 57 was statistically significant
in the sacubitril/valsartan group at 15 min (p=0.026), 30 min (p=0.012) and 45 min
(p=0.035), but was not significant at any time-point in the amlodipine group (Figure
2A). However, there was no significant difference between treatment groups at any
time-point.
Plasma NEFA concentrations decreased on Day 57 at 15 minutes in the
sacubitril/valsartan group (p=0.018) and at 15 min and 30 min (p<0.05) in the
amlodipine group. No significant differences were observed between treatment
groups. When compared with NEFA levels at rest (45 minutes), the levels were
lower during the initial phases of exercise, but increased gradually with increasing
exercise duration in both treatment groups (Figure 2B).

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† p=0.026,* p=0.012, ‡ p=0.035 vs. baseline

*** p<0.001, ** p<0.002 vs. 45 min rest

Figure 2. Whole body lipolysis at exercise: comparison of rate of glycerol

appearance between treatments (A) and plasma concentration of NEFA (B).

Error bars indicate 95% CI

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Oxidative metabolism during exercise

Oxygen consumption was comparable between the sacubitril/valsartan and
amlodipine groups at baseline (O2 consumption: amlodipine, 1.31 ± 0.45 L/min;
sacubitril/valsartan, 1.40 ± 0.41 L/min) and on Day 57 (amlodipine, 1.27 ± 0.39
L/min; sacubitril/valsartan, 1.37 ± 0.44 L/min) and no differences were found
between treatment groups.
The respiratory quotient significantly increased during exercise in both the
treatment groups, on Day 1 and Day 57 (Figure 3). The respiratory quotient was
comparable between treatments at baseline and on Day 57.

*** p<0.001, exercise vs. rest

Figure 3. Oxidative metabolism-comparison of respiratory quotient between resting

and exercise status- carbon dioxide to oxygen ratio.

Error bars indicate 95% CI

Plasma catecholamine concentrations

When compared with resting levels, adrenaline concentrations increased
significantly during exercise at all time-points in both treatment groups on Day 1
and 57 (Figure 4A). Compared with baseline, a significant reduction in adrenaline
concentrations were observed on Day 57 in the amlodipine groups at all time-
points while the decrease was not statistically significant in the sacubitril/valsartan
group. However, no significant differences were observed in the adrenaline
concentrations between treatment groups at any time-point, except at 30 minutes
(p=0.012).
Plasma noradrenaline levels were significantly increased during exercise in both
the treatment groups on Day 1 and Day 57 (p<0.001) when compared with resting

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levels (Figure 4B). Noradrenaline levels increased incrementally during exercise on

Day 1 and Day 57 in both the treatment groups, with no significant differences
between treatments.

† p=0.044, *p=0.022, **p=0.019, ***p<0.001 vs. baseline. a p=0.012 vs. amlodipine

p<0.001 vs. rest

Figure 4. Analysis of plasma biomarkers during exercise: adrenaline (A) and

noradrenaline (B)

Error bars indicate 95% CI.

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Blood pressure
After 8-weeks of treatment, systolic BP, diastolic BP and pulse pressure decreased
from baseline in both treatment groups at rest. Systolic and diastolic BP and pulse
rate values increased during exercise in both treatment groups on both Day 1 and
Day 57 without clinically relevant differences between treatment groups (Table 1).

Table 1. Comparison of BP and pulse rate between treatments during

exercise and rest
Amlodipine
LCZ696 (400 mg
Parameter Day Rest/Exercise (10 mg QD)
QD) N=50
N=48
Rest 137.9 ± 14.7 135.8 ± 12.8
Day 1
Exercise 157.5 ± 23.8 146.5 ± 23.3
SBP (mmHg)
Rest 119.9 ± 14.3 125.9 ±10.2
Day 57
Exercise 146.8 ±21.9 137.6 ± 20.5

Rest 88.0 ± 10.6 86.8 ± 8.9

Day 1
Exercise 82.8 ±12.6 84.3 ± 12.4
DBP (mmHg)
Rest 77.2 ± 8.5 81.2 ± 7.2
Day 57
Exercise 78.4 ± 12.3 78.8 ± 11.9

Rest 67.4 ± 8.6 66.7 ± 9.3

Day1
Pulse rate Exercise 121.2 ± 15.4 111.4 ± 25.6

(BPM) Rest 65.2 ± 8.7 69.2 ± 10.5

Day 57
Exercise 123.5 ± 18.6 115.6 ± 16.5

Data are expressed as mean ± SD.

SBP, systolic blood pressure; DBP, diastolic blood pressure, BPM, beats per minute

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DISCUSSION
The present study demonstrated that treatment with sacubitril/valsartan compared
with amlodipine for 8 weeks did not elicit relevant changes in exercise-induced
lipolysis and substrate oxidation in obese patients with hypertension. The exercise-
induced increase in abdominal subcutaneous adipose tissue and whole-body
lipolysis was not augmented following sacubitril/valsartan treatment compared with
amlodipine treatment. Moreover, the shift in substrate oxidation towards
carbohydrate catabolism during exercise was comparable in both treatment
groups, implying that sacubitril/valsartan did not significantly affect lipid utilization
during acute exercise. We have previously observed significantly improved whole-
body insulin sensitivity and a modest increase in resting abdominal subcutaneous
adipose tissue lipolysis with no marked changes in whole-body lipolysis with
sacubitril/valsartan compared with amlodipine treatment [11]. Overall, these
findings imply that the beneficial cardiometabolic effects of sacubitril/valsartan may
not be explained by changes in lipid mobilization or oxidation.
2
In this study, we used state-of-the-art methodology including [1,1,2,3,3- H]-glycerol
tracer kinetics and abdominal subcutaneous adipose tissue microdialysis to assess
whole-body and local lipolysis, respectively, in a large patient sample. Furthermore,
we treated patients with a total daily dose of sacubitril/valsartan which provided
superior BP control in patients with arterial hypertension (400 mg QD) [12] and
reduced cardiovascular mortality and heart failure hospitalizations in patients with
heart failure and reduced ejection fraction (HFrEF) (200 mg twice daily) compared
with standard-of-care renin-angiotensin system (RAS) inhibition [7]. This study,
therefore, was appropriately designed to study the effect of sacubitril/valsartan on
lipid turnover.
Our study extends previous investigations on the role of neprilysin substrates and
angiotensin II type-1 (AT1)-receptors in the regulation of lipid turnover. All
components of the RAS are expressed in adipose tissue, and AT 1-receptors have
been implicated in the regulation of adipose tissue differentiation, inflammation,
and metabolism [10]. Conflicting findings have been reported with respect to the
effects of angiotensin II on adipose tissue lipolysis. More specifically, both
increased [13, 14] and decreased [15] subcutaneous adipose tissue lipolysis have
been demonstrated [14]. Moreover, intravenous angiotensin II infusions and
angiotensin converting enzyme inhibition did not elicit major changes in whole-body
lipolysis as determined by glycerol tracer kinetics [16]. AT1-receptor blockade in
human subjects did not increase lipolytic gene expression or lipolysis in abdominal
subcutaneous adipose tissue [17, 18]. However, long-term AT1-receptor blockade
altered intramuscular lipid partitioning, manifested by decreased saturation of
skeletal muscle triacylglycerol and diacylglycerol stores, reduced postprandial fatty
acid spillover and lipolysis [19]. Overall, angiotensin II actions on AT 1-receptors
appear to have modest effects on lipid turnover. Although postprandial fatty acid
handling has not been examined in this study, the present findings suggest that
AT1-receptor blockade in the context of neprilysin inhibition by sacubitril/valsartan
does not have clinically relevant effects on lipid mobilization or utilization.
Neprilysin degrades multiple peptides potentially modulating lipid metabolism such
as natriuretic peptides (NPs), bradykinin, endothelin-1, and glucagon-like peptide 1
(GLP-1) [20]. We cannot discern contributions of individual neprilysin substrates to

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the observed metabolic response. While bradykinin has been suggested to

attenuate lipolysis, endothelin-1 may increase lipolysis. However, endothelin-1 was
significantly decreased following treatment of patients with HFrEF with
sacubitril/valsartan for 21 days, and no changes in lipolysis for GLP-1 at high
concentrations have been reported [21-25]. Among neprilysin substrates, lipolytic
effects of NPs are particularly striking. NPs are more potent in stimulating human
adipose tissue lipolysis than the prototypical β-adrenoreceptor agonist
isoproterenol [26]. Since NP-induced lipolysis is observed only in primates, the
utility of many preclinical animal models is limited [27]. NPs are released during
physical exercise and have been suggested to provide lipid fuel to the working
skeletal muscle. Excess NP-mediated lipid mobilization has been suggested as a
potential limitation of therapeutic neprilysin inhibition. In fact, ex vivo lipolysis of
subcutaneous adipose tissue was not desensitized in patients with heart failure
despite increased circulating NP levels [28]. However, the lack of changes in
exercise-induced lipolysis by sacubitril/valsartan observed in the present study is
clinically reassuring.
Given the potent acute effect of NPs on human lipolysis, our findings are somewhat
unexpected. Plasma noradrenaline and adrenaline increased to a similar extent at
baseline and following treatment with amlodipine and sacubitril/valsartan,
suggesting that opposing changes in sympatho-adrenal activity did not mask a
direct treatment effect on lipolysis. The reduction in catecholamine concentrations
observed with sacubitril/valsartan in this study is consistent with our previous
observation [11]. Conflicting observations have been reported with respect to the
effect of amlodipine therapy on noradrenaline concentrations [29-31] while
valsartan treatment has been demonstrated to attenuate increases in plasma
noradrenalin concentrations with larger reductions from baseline associated with
lower risk of mortality and morbidity [32, 33]. While sacubitril/valsartan improved
insulin-mediated glucose disposal compared with amlodipine [11], potential anti-
lipolytic effects of insulin in adipose tissue following sacubitril/valsartan have not
been investigated before. We cannot completely rule out that improved insulin
action in adipose tissue confounded our analysis. However, an alternative and
more likely explanation is that NP actions in adipose tissue are not or to a lesser
degree dependent on neprilysin activity. Indeed, a study in isolated human
adipocytes suggests that clearance via the natriuretic peptide type-C receptor, the
so-called scavenger receptor, may be more important than neprilysin activity to
control local NP availability [34]. Indeed, completely abolishing neprilysin activity
using thiorphan did not modify atrial natriuretic peptide (ANP)-mediated lipolysis
[34].
Noteworthy, we conducted our study in obese patients with hypertension. Given
the differences in neurohormonal activity between patients with hypertension and
heart failure, the extent to which our findings can be extrapolated to patients with
HFrEF remains to be elucidated. However, a recent post-hoc analysis of the
PARADIGM-HF trial showed that in patients with heart failure and type 2 diabetes
mellitus, treatment with sacubitril/valsartan resulted in greater reductions in HbA1 c
concentrations compared to those treated with enalapril. Moreover,
sacubitril/valsartan treated patients with type 2 diabetes were less likely to require
initiation of insulin treatment during the trial suggesting potential metabolic benefits
of sacubitril/valsartan therapy in HF patients [35]. Finally, the selection of

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amlodipine as a metabolically neutral comparator does not allow distinguishing the

contributions of neprilysin inhibition from those of AT1-receptor blockade to the
effects of sacubitril/valsartan observed in this study.
In conclusion, our study demonstrated that sacubitril/valsartan treatment did not
result in clinically relevant changes in exercise-induced abdominal subcutaneous
adipose tissue and whole-body lipolysis, energy expenditure and substrate
oxidation compared with amlodipine. This finding is relevant because neprilysin
substrates, particularly NPs, have been implicated in lipolysis and the pathogenesis
of cardiac cachexia. Our findings further support the idea that neprilysin is of lesser
importance in regulating NP availability in the vicinity of adipocytes.

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P, Ayalasomayajula S, Prescott MF, Pal P, Langenickel TH, Jordaan P, Rajman I.
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Trial (Val-HeFT). Circulation. 2003;107(9):1278-83.
34. Moro C, Klimcakova E, Lafontan M, Berlan M, Galitzky J. Phosphodiesterase-5A and
neutral endopeptidase activities in human adipocytes do not control atrial natriuretic
peptide-mediated lipolysis. Br J Pharmacol. 2007;152(7):1102-10.
35. Seferovic JP, Claggett B, Seidelmann SB, Seely EW, Packer M, Zile MR, Rouleau
JL, Swedberg K, Lefkowitz M, Shi VC, Desai AS, McMurray JJV, Solomon SD. Effect
of sacubitril/valsartan versus enalapril on glycaemic control in patients with heart
failure and diabetes: a post-hoc analysis from the PARADIGM-HF trial. Lancet
Diabetes Endocrinol. 2017;5(5):333-40.

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The effects of angiotensin receptor
neprilysin inhibition by sacubitril/valsartan
on adipose tissue transcriptome and
protein expression in obese hypertensive
patients

Stinkens R., van der Kolk B.W., Jordan J., Jax T., Engeli S.,
Heise T., Jocken J.W., May M., Schindler C., Havekes B.,
Schaper N., Albrecht D., Kaiser S., Hartmann N., Letzkus M.,
Langenickel T.H., Goossens G.H., Blaak E.E.

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Chapter 5

ABSTRACT
Increased activation of the renin-angiotensin system is involved in the onset and
progression of cardiometabolic diseases, while natriuretic peptides (NP) may exert
protective effects. We have recently demonstrated that sacubitril/valsartan
(LCZ696), a first-in-class angiotensin receptor neprilysin inhibitor, which blocks the
angiotensin II type-1 receptor and augments natriuretic peptide levels, improved
peripheral insulin sensitivity in obese hypertensive patients. Here, we investigated
the effects of sacubitril/valsartan (400mg QD) treatment for 8 weeks on the
abdominal subcutaneous adipose tissue (AT) phenotype compared to the
metabolically neutral comparator amlodipine (10mg QD) in 70 obese hypertensive
patients. Abdominal subcutaneous AT biopsies were collected before and after
intervention to determine the AT transcriptome and expression of proteins involved
in lipolysis, NP signaling and mitochondrial oxidative metabolism. Both
sacubitril/valsartan and amlodipine treatment did not significantly induce AT
transcriptional changes in pathways related to lipolysis, NP signaling and oxidative
metabolism. Furthermore, protein expression of adipose triglyceride lipase (ATGL)
(Ptime*group=0.195), hormone-sensitive lipase (HSL) (Ptime*group=0.458), HSL-ser660
phosphorylation (Ptime*group=0.340), NP receptor-A (NPRA) (Ptime*group=0.829) and
OXPHOS complexes (Ptime*group=0.964) remained unchanged. In conclusion,
sacubitril/valsartan treatment for 8 weeks did not alter the abdominal subcutaneous
AT transcriptome and expression of proteins involved in lipolysis, NP signaling and
oxidative metabolism in obese hypertensive patients.

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INTRODUCTION
Obesity is strongly associated with cardiometabolic risk factors [1], which is
reflected by an increased risk for arterial hypertension, heart failure and type 2
diabetes mellitus (T2DM) [2]. An impaired adipose tissue function and excessive fat
mass in obesity represent key factors in the development of insulin resistance and
related chronic diseases, including cardiovascular disease and T2DM [3]. Evidence
suggests that impaired insulin sensitivity in obesity might be related to an altered
renin-angiotensin system (RAS) and natriuretic peptide (NP) signaling in adipose
tissue. Blockade of the RAS using angiotensin-converting enzyme (ACE) inhibitors
or angiotensin type-1 receptor blockers (ARB) has been shown to improve insulin
sensitivity and beta-cell function [4] and reduces the incidence of T2DM [5] as
reviewed elsewhere [6]. However, results are not consistent [7]. In addition, NPs
are positively associated with insulin sensitivity and low atrial natriuretic peptide
(ANP) concentrations are associated with an increased risk of developing arterial
hypertension and T2DM [8]. In accordance, neprilysin (NEP), which is involved in
the degradation and inactivation of NP, is linked to insulin resistance, increased
blood pressure and impaired lipid metabolism [9]. Therefore, combined RAS
blockade and NEP inhibition might have synergistic beneficial effects on blood
pressure and peripheral insulin sensitivity. We recently demonstrated that
combined ARB and NEP inhibition, using sacubitril/valsartan (LCZ696), improved
peripheral insulin sensitivity following 8 weeks of treatment compared to amlodipine
(AMLO) in obese hypertensive patients [10]. However, the mechanisms underlying
these beneficial effects remain to be established.
Evidence suggests that both the RAS and ANP affect adipose tissue metabolism,
thereby determining insulin sensitivity [6, 11]. It has been shown that valsartan
(ARB) reduced adipocyte size, increased adipose tissue blood flow and decreased
gene expression of angiogenesis, adipogenesis and macrophage infiltration
markers [12], which may have contributed to the valsartan-induced increased
insulin sensitivity [4]. Furthermore, angiotensin II inhibited lipolysis in vitro in mature
human adipocytes [13], although conflicting findings on adipose tissue lipolysis in
vivo in humans have been reported [14, 15]. ANP has been shown to increase
adipose tissue lipid mobilization and oxidation [11] and we and others have
recently demonstrated that ANP-mediated lipolysis is impaired in subcutaneous
mature adipocytes from obese men with and without T2DM [16, 17] .
Therefore, it is hypothesized that ARB and NEP inhibition with sacubitril/valsartan
may affect adipose tissue function, thereby contributing to the observed improved
peripheral insulin sensitivity in obese individuals [10]. The present study
investigated the effects of sacubitril/valsartan compared to amlodipine treatment for
8 weeks on the abdominal subcutaneous adipose tissue transcriptome and protein
expression profiles in obese hypertensive individuals.

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METHODS

Study design
Ninety-eight obese hypertensive patients participated in a multicenter, randomized,
double-blind, parallel-group study to investigate the effects of sacubitril/valsartan
(400mg QD) compared with amlodipine (10mg QD) treatment for 8 weeks. A
detailed description of the inclusion and exclusion criteria is described elsewhere
(clinicaltrials.gov - NCT01631864). Before and after treatment, abdominal
subcutaneous adipose tissue biopsies were collected by needle aspiration under
local anesthesia after an overnight fast. We determined adipose tissue gene
expression profiles using microarray analysis in a subgroup of 70 patients who had
a RNA Integrity Number (RIN) ≥5.0, as described in detail below. Next, in a
subgroup (N=12-13), we determined the expression of proteins involved in the
lipolytic pathway, the natriuretic peptide signaling pathway and mitochondrial
oxidative phosphorylation. Subjects showing a treatment-induced increase in
adipose tissue lipolysis in vivo following sacubitril/valsartan and unchanged
lipolysis after amlodipine treatment were included in the latter analyses.
All patients gave written informed consent before participation. The Independent
Ethical Committee or Institutional Review Board of each center reviewed the study
protocol. The study was performed in accordance with the Declaration of Helsinki.

Adipose tissue transcriptomics

First, the integrity of abdominal subcutaneous adipose tissue total RNA was
determined using the sample RNA Integrity Number (RIN), generated using a
Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Foster City, CA, USA). RIN
values <5.0 indicated high level of sample total RNA degradation and were
excluded from analyses [18]. Biopsies of 70 patients (N sacubitril/valsartan=36, N
amlodipine=34) were eligible for transcriptional analyses (RIN ≥5.0).
Whole-transcriptome analysis was carried-out using Affymetrix HG-U133plus2.0
oligonucleotide microarrays (Affymetrix Inc., Santa Clara, CA, USA) and the
microarray files were pre-processed using the Robust Multi-array Average
algorithm (RMA). The Chip Definition File (CDF) used for the RMA procedure
corresponded to the public domain Michigan University Entrez CDF version 17.0.
Transcripts showing median expression higher than 6 (log2-value) were combined
and considered for statistical analyses. Evaluations comprised unsupervised
(hypothesis-free) and supervised (targeted) assessments of which the latter
explored the longitudinal treatment-induced effects on specific transcripts linked to
lipolysis, fatty acid oxidation, mitochondrial biogenesis and adipokines.

Adipose tissue protein expression

A detailed description of the analysis can be found in the Supplementary Material.
Briefly, abdominal subcutaneous adipose tissue was ground to a fine powder under
liquid nitrogen and homogenized in RIPA buffer. The homogenate was lysed,
vortexed and centrifuged and the supernatant was collected and stored at -80°C.
The protein concentration was determined by the Bradford-based protein assay.
Next, solubilized proteins (15 µg) were separated on a precast gel and transferred

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onto a nitrocellulose membrane and quantitative Western Blot analysis was

performed to determine the levels of proteins involved in the lipolytic pathway
(adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL) and HSL
serine 660 phosphorylation), the natriuretic peptide signaling pathway (natriuretic
peptide receptor A (NPRA)) and mitochondrial oxidative phosphorylation
(OXPHOS).

Statistics
Statistical analyses of gene expression data was performed with R v.3.2.2 (R
Development Core Team, Vienna, http://www.R-project.org) and the Bioconductor
limma R package v.3.26.8. Statistical significance was set at nominal P≤0.05 and
P-values were corrected for multiple testing using the Bonferroni method. A total of
8319 transcripts (out of assessed 18898 transcripts/microarray) fulfilled the
expression level filtering criteria and the Bonferroni adjusted threshold was
-6
therefore defined as P<6.01·10 . Longitudinal treatment-induced transcriptional
changes were expressed as Ratio Change from baseline (RC; (post-treatment
-1
level)·(pre-treatment level) ). Thresholds for minimum relevant treatment-induced
effects were defined as 0.66≥RC≥1.50 (i.e. RC range equivalent to absolute fold
changes≥1.5).
Protein expression data are expressed as mean±SEM. All variables were checked
for normal distribution by Shapiro-Wilk test and variables were Ln-transformed to
satisfy conditions of normality (HSL, HSL S660 phosphorylation, NPRA and
OXPHOS). Data was analyzed using two-way repeated measures ANOVA, with
time (pre, post) and treatment (sacubitril/valsartan, amlodipine) as factors.
Bonferroni post-hoc correction was applied when a significant time*treatment
interaction was found. Calculations were performed using SPSS v.21 for Mac OSX
(IBM, Chicago, IL, USA) and P≤0.05 was considered statistically significant.

RESULTS

Subject characteristics
Baseline characteristics of patients contributing to the present study are shown in
Table 1. Importantly, the two groups were well matched and there were no major
differences in clinical characteristics between groups.

Transcriptomics
At baseline, no significant differences in the abdominal subcutaneous adipose
tissue transcriptome were found between treatment arms. Unsupervised
assessments revealed 1443 longitudinally modulated transcripts (nominal P<0.05),
but none of these fulfilled both thresholds for Bonferroni adjusted P-value and
minimum treatment-induced RC effect. A set of selected transcripts is shown in
Supplementary Table S1.
Supervised analyses identified no significant treatment-induced changes (defined
based on the two thresholds stated above) in gene expression levels of transcripts
involved in lipolysis, NP signaling, oxidative metabolism and adipokines (Figure 1).

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More specifically, gene expression of ATGL (PNPLA2), MGL (MGLL), lipoprotein

lipase (LPL), perilipin-1 (PLIN1) and fatty acid binding protein 4 (FABP4) was not
significantly altered by sacubitril/valsartan treatment (Figure 1). In addition, gene
expression of natriuretic peptide receptors (NPR1, NPR3), adipokines (adiponectin
(ADIPOQ), leptin (LEP)) and oxidative metabolism markers (peroxisome
proliferator-activated receptor gamma co-activator-related 1 (PPRC1), peroxisome
proliferator-activated receptor gamma co-activator 1 alpha (PPARGC1A), nuclear
respiratory factor 1 (NRF1), acyl-coenzyme A oxidase 1 (ACOX1) and uncoupling
protein 2 (UCP2)) were not significantly affected (Figure 1).

Figure 1. Targeted assessment of selected treatment-induced transcriptional

changes in abdominal subcutaneous adipose tissue.

The heatmap plot shows per patient longitudinal treatment-induced gene expression changes [defined
as log2(post treatment expression) - log2(pre-treatment expression)] for selected transcripts related to
natriuretic peptide signaling, lipolytic pathway, oxidative pathway and adipokines in abdominal
subcutaneous adipose tissue (n=70 patients; grouped per treatment arm). Color in the heatmap reflects
transcript change from baseline per subject. Blue: downregulated transcripts; yellow: upregulated
transcripts. The adjacent table depicts the corresponding transcript ratio change from baseline (RC) and
P-value per treatment arm.

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 Table 1. Baseline clinical characteristics of patients involved in transcriptomic and protein expression analyses

Transcriptomic analyses

Sacubitril/valsartan Amlodipine Total

Parameter
(N = 36) (N = 34) (N = 70)

Age (yrs.) 52.4 (8.56) 51.6 (9.21) 52.0 (8.83)

Gender (N) Male 28 25 53
Female 8 9 17
Weight (kg) 100 (18.2) 104 (15.8) 102 (17.1)
Waist circumference (cm) 111.0 (10.53) 113.4 (10.81) 112.2 (10.66)
2 -1
BMI (kg·(m ) ) 32.4 (4.77) 33.6 (4.59) 33.0 (4.69)
Mean sitting SBP (mm Hg) 143.2 (13.26) 138.8 (11.61) 141.0 (12.60)
Mean sitting DBP (mm Hg) 89.6 (7.79) 90.4 (5.87) 90.0 (6.89)
-1 -1
Glucose infusion rate (μmol·kg bw ·min ) 344.5 (161.96) 409.1 (192.69) 375.9 (179.22)

Protein expression analyses

Sacubitril/valsartan Amlodipine Total
Parameter
(N = 15) (N = 12) (N = 27)
Age (yrs.) 53.6 (6.92) 54.9 (7.04) 54.2 (6.87)
Gender (N) Male 13 8 21
Female 2 4 6
Weight (kg) 108 (19.2) 102 (14.4) 105 (17.1)
Waist circumference (cm) 114.6 (12.98) 111.2 (12.89) 113.1 (12.80)
2 -1
BMI (kg·(m ) ) 34.3 (5.89) 33.1 (3.93) 33.8 (5.06)
Mean sitting SBP (mm Hg) 147.2 (10.62) 137.8 (12.59) 143.0 (12.27)
Mean sitting DBP (mm Hg) 92.0 (6.10) 87.5 (5.63) 90.0 (6.22)

Glucose infusion rate (μmol·kg bw-1·min-1) 362.6 (141.71) 401.1 (104.46) 379.7 (125.74)
Chapter 5

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Data are expressed as mean (SD).
Chapter 5

Protein expression
Sacubitril/valsartan treatment did not significantly change protein expression of
ATGL (LCZ696: 1.00±0.21 vs. 0.85±0.19 AU; AMLO: 1.00±0.26 vs. 1.36±0.30 AU;
Ptime=0.583, Ptime*treatment=0.195; Figure 2A), HSL (LCZ696: 1.00±0.23 vs. 1.38±0.24
AU; AMLO: 1.00±0.26 vs. 1.00±0.18 AU; Ptime=0.141, Ptime*treatment=0.458; Figure
2B), HSL serine 660 phosphorylation (LCZ696: 1.00±0.16 vs. 1.04±0.14 AU;
AMLO: 1.00±0.25 vs. 0.71±0.19 AU; Ptime=0.551, Ptime*treatment=0.340; Figure 2C) or
NPRA (LCZ696: 1.00±0.24 vs. 0.99±0.29 AU; AMLO: 1.00±0.36 vs. 0.96±0.30 AU;
Ptime=0.775, Ptime*treatment=0.829; Figure 2D). Furthermore, total OXPHOS protein
expression (LCZ696: 1.00±0.22 vs. 1.76±0.48 AU; AMLO: 1.00±0.15 vs. 1.74±0.50
AU; Ptime=0.125, Ptime*treatment=0.964; Figure 2E) remained unchanged following
treatment. More specifically, OXPHOS complexes I-V were not affected (data not
shown). Representative Western Blots are presented in Supplementary Figure S1.

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Figure 2. Protein expression in abdominal subcutaneous adipose tissue.

Expression of proteins involved in the lipolytic pathway (ATGL, N=12, A; HSL, N=13, B; HSL serine 660
phosphorylation, N=13, C), the natriuretic peptide signaling pathway (NPR-A, N=12, D) and
mitochondrial oxidative metabolism (total OXPHOS, N=13, E) expressed as fold change relative to
baseline for each treatment arm (Sacubitril/valsartan: LCZ696 or Amlodipine: AMLO). Data are
expressed as mean ± SEM. White bars: baseline values; black bars: post-treatment values.
Representative Western Blots are presented in Supplementary Figure S2.

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DISCUSSION
Here, we investigated the effects of sacubitril/valsartan versus amlodipine
treatment for 8 weeks on the abdominal subcutaneous adipose tissue
transcriptome and protein expression in obese hypertensive patients. We
demonstrated that sacubitril/valsartan treatment did not significantly alter adipose
tissue gene and protein expression of factors related to lipolysis, natriuretic peptide
signaling and oxidative metabolism.
We recently demonstrated that in obese hypertensive patients sacubitril/valsartan
treatment, which provides simultaneous ARB blockade and NEP inhibition,
significantly increased peripheral insulin sensitivity [10]. Furthermore,
sacubitril/valsartan slightly but significantly increased abdominal subcutaneous
adipose tissue lipolysis, although no changes in whole-body lipolysis were
observed [10]. Therefore, we hypothesized that RAS blockade and NEP inhibition
has synergistic beneficial effects on abdominal subcutaneous adipose tissue
metabolism and might underlie the observed improvement in insulin sensitivity. We
showed that the abdominal adipose tissue phenotype was not significantly affected
by 8 weeks of sacubitril/valsartan treatment in obese hypertensive patients. First,
sacubitril/valsartan treatment did not reveal any transcriptional changes in
abdominal subcutaneous adipose tissue, as determined by an unsupervised
analysis. More specific, no treatment-induced changes in expression of genes
involved in lipolysis, the NP signaling pathway and mitochondrial oxidative pathway
were found. Secondly, the expression of proteins involved in these pathways, as
well as post-translational modification of HSL, remained unchanged after the
intervention.
Evidence suggests that both the RAS and NP system may affect adipose tissue
metabolism, thereby contributing to improved insulin sensitivity [6, 11]. It has
previously been shown that angiotensin II decreased adipose tissue lipolysis in vivo
in humans [14, 19, 20] and in human isolated adipocytes [13], which seems to be
mediated via the angiotensin II type-1 receptor. However, increased adipose tissue
lipolysis has also been reported [15]. In accordance with our results, long-term
ARB treatment with valsartan improved insulin sensitivity in subjects with impaired
glucose metabolism [4], but adipose tissue gene and protein expression of several
lipolytic enzymes remained unchanged [12].
Several studies have shown that ANP promotes adipose tissue lipid mobilization
and oxidation in healthy individuals [11] via cGMP-mediated phosphorylation of
HSL [21, 22]. Furthermore, ANP induced mitochondrial biogenesis and uncoupling
in human adipocytes from healthy, non-diabetic women [23]. Here, we did not find
significant changes in gene and protein expression of markers involved in lipolysis,
phosphorylation of HSL and mitochondrial oxidative metabolism, which may be
explained by reduced ANP-mediated signaling in the study population. Indeed, an
impaired ANP-mediated lipolysis has recently been described in situ in
subcutaneous adipose tissue and in vitro in subcutaneous adipocytes from obese
individuals [16, 17]. Moreover, it has been shown that NPRC (NP clearance
receptor) is increased in adipose tissue of obese hypertensive patients compared
to lean and normotensive individuals [24], together with increased NEP expression
in obesity [9]. These data suggest reduced NP signaling and increased NP
clearance in adipose tissue in obesity. This may explain the unaltered

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subcutaneous adipose tissue metabolic phenotype following sacubitril/valsartan

treatment in the present study.
Due to conservative multiple testing correction, we may have missed relevant
treatment-induced changes in gene expression. However, the observed relative
changes in gene expression were very modest, revealing only minor, potentially not
physiologically-relevant, changes in gene expression.

CONCLUSION
The present study demonstrated that simultaneous RAS blockade and NEP
inhibition following sacubitril/valsartan treatment for 8 weeks did not significantly
alter the adipose tissue metabolic phenotype in obese hypertensive patients. More
specifically, abdominal subcutaneous adipose tissue gene and protein expression
of factors involved in lipolysis, natriuretic peptide signaling and mitochondrial
oxidative metabolism remained unchanged. Therefore, alterations in the adipose
tissue metabolic phenotype do not seem to contribute to the improved peripheral
insulin sensitivity following 8 weeks of sacubitril/valsartan treatment [10].

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subcutaneous adipocytes of obese type 2 diabetic men. Clin Sci (Lond).
2016;130(13):1105-14.
17. Ryden M, Backdahl J, Petrus P, Thorell A, Gao H, Coue M, Langin D, Moro C, Arner
P. Impaired atrial natriuretic peptide-mediated lipolysis in obesity. Int J Obes (Lond).
2016;40(4):714-20.
18. Archer KJ, Mas VR, O'Brien TR, Pfeiffer R, Lum NL, Fisher RA. Quality assessment
of microarray data in a multicenter study. Diagn Mol Pathol. 2009;18(1):34-43.
19. Boschmann M, Ringel J, Klaus S, Sharma AM. Metabolic and hemodynamic
response of adipose tissue to angiotensin II. Obes Res. 2001;9(8):486-91.
20. Boschmann M, Rosenbaum M, Leibel RL, Segal KR. Metabolic and hemodynamic
responses to exercise in subcutaneous adipose tissue and skeletal muscle. Int J
Sports Med. 2002;23(8):537-43.
21. Moro C, Galitzky J, Sengenes C, Crampes F, Lafontan M, Berlan M. Functional and
pharmacological characterization of the natriuretic peptide-dependent lipolytic
pathway in human fat cells. J Pharmacol Exp Ther. 2004;308(3):984-92.
22. Sengenes C, Bouloumie A, Hauner H, Berlan M, Busse R, Lafontan M, Galitzky J.
Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated
hormone-sensitive lipase phosphorylation in human adipocytes. J Biol Chem.
2003;278(49):48617-26.
23. Bordicchia M, Liu D, Amri EZ, Ailhaud G, Dessi-Fulgheri P, Zhang C, Takahashi N,
Sarzani R, Collins S. Cardiac natriuretic peptides act via p38 MAPK to induce the
brown fat thermogenic program in mouse and human adipocytes. J Clin Invest.
2012;122(3):1022-36.
24. Dessi-Fulgheri P, Sarzani R, Tamburrini P, Moraca A, Espinosa E, Cola G,
Giantomassi L, Rappelli A. Plasma atrial natriuretic peptide and natriuretic peptide
receptor gene expression in adipose tissue of normotensive and hypertensive obese
patients. J Hypertens. 1997;15(12 Pt 2):1695-9.

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SUPPLEMENTARY MATERIAL

Protein expression analysis

Adipose tissue (~500 mg) was ground to a fine powder under liquid nitrogen and
homogenized in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris
(Calbiochem)-HCl (Merck, Darmstadt, Germany) buffered saline (Merck) with 0,1%
SDS (Bio-Rad Laboratories Inc, Hercules, CA, USA), 1% Na-Deoxycholate (Sigma-
Aldrich, St. Louis, MO, USA), 1% NP-40 (Fluka) and a protease/phosphatase
inhibitor cocktail (Cell Signaling Technology, Beverly, MA, USA). The homogenate
was lysed on iced and vortexed for 5 min and centrifuged at 20,000 g for 30 min at
10°C. The supernatant was carefully collected and aliquots were stored at -80°C.
The protein concentration was determined by the Bradford-based protein assay
(Santa Cruz Biotechnology, Dallas, TX, USA).
Next, solubilized proteins were separated on a precast gel (Criterion™ TGX any
kD, Bio-Rad Laboratories Inc, Hercules, CA, USA) and transferred onto a
nitrocellulose membrane (Trans Blot® Turbo™ transfer system; Bio-Rad).
Differences in loading were adjusted to total adipose tissue protein content (via
Ponceau S (Sigma-Aldrich, St. Louis, MO, USA) staining), and appropriate positive
controls (lysates of abdominal subcutaneous adipose tissue) were included.
Thereafter, quantitative Western Blot analysis was performed to determine the
levels of ATGL (Cell Signaling Technology, Beverly, MA, USA), HSL (kind gift from
Prof. Cecilia Holm, [Department of Cell and Molecular Biology, Lund University,
Sweden]), HSL serine 660 phosphorylation (Cell Signaling Technology, Beverly,
MA, USA) and NPRA (Abcam, Cambridge, MA, USA). The secondary antibody
was a horseradish peroxidase (HRP) swine-anti-rabbit antibody (DakoCytomation,
Glostrup, Denmark). Furthermore, OXPHOS blots were probed with Total
OXPHOS Antibody Cocktail (Abcam, Cambridge, MA, USA) and a secondary HRP-
conjugated Rabbit-anti-Mouse antibody (DakoCytomation, Glostrup, Denmark).
Antigen-antibody complexes were visualized using chemiluminescence by a
ChemiDoc™ XRS apparatus (Bio-Rad) and analyzed with Quantity One® software
(Bio-Rad), which calculated the optical density units that are expressed as average
intensity [average intensity = total intensity of the rows of pixels inside the band
boundary divided by the number of rows, minus the background intensity].

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SUPPLEMENTARY TABLE S1. Set of selected genes for the untargeted

assessment of transcriptional changes in abdominal subcutaneous adipose tissue.

A total of 1443 transcripts were modulated by either sacubitril/valsartan (n=916) or amlodipine (n=668)
treatment (nominal P≤0.05, no multiple correction applied). A set of selected genes with corresponding
ratio changes from baseline (RC) and P-value per treatment arm is shown.

BIOLOGICAL LCZ696 AMLO

GENES ID
PATHWAY RC P-value RC P-value
INFLAMMATION ALOX5AP 0.76 0.001 0.93 0.483
AOX1 0.93 0.083 0.89 0.025
BLNK 0.91 0.040 0.96 0.430
BTK 0.88 0.011 0.94 0.337
C3AR1 0.90 0.072 0.88 0.024
CCL16 1.14 0.009 1.01 0.838
CCL18 1.24 0.040 0.97 0.781
CD14 0.89 0.035 0.95 0.351
CD163 0.94 0.222 0.91 0.027
CD44 0.95 0.044 0.89 0.000
CD74 0.98 0.530 0.94 0.028
CLU 0.96 0.036 0.98 0.372
CTSS 0.85 0.009 0.88 0.118
CXCL9 1.20 0.042 1.15 0.134
CYBB 0.86 0.003 0.93 0.215
E2F3 0.95 0.020 1.00 0.887
FCER1G 0.87 0.023 0.87 0.042
FYB 0.85 0.050 1.01 0.944
FYN 0.98 0.075 0.96 0.036
GAB2 0.96 0.131 0.95 0.027
GBP2 0.91 0.005 0.96 0.236
HCLS1 0.90 0.022 0.98 0.687
HLA-DMA 0.99 0.660 0.93 0.033
IFI30 0.89 0.153 0.77 0.000
IFIT2 0.91 0.009 1.03 0.593
IFNGR1 0.99 0.649 0.95 0.021
IRF1 0.90 0.040 1.09 0.134
ITGB2 0.85 0.031 0.82 0.020
LCP1 0.85 0.016 0.82 0.019
LYN 0.87 0.009 0.98 0.823
MAP2K1 1.03 0.048 1.04 0.119
MAP2K4 0.94 0.004 0.98 0.414
MAP3K7 0.97 0.044 0.93 0.004
MAX 0.96 0.041 0.98 0.514
PAK2 0.97 0.023 0.99 0.447
PIK3CB 0.96 0.027 0.97 0.290
PLA2G7 1.01 0.917 0.71 0.001
PLCG1 1.07 0.010 0.99 0.748
PPP3CC 1.00 0.927 0.96 0.047
PTPN6 0.88 0.042 0.98 0.779
PTPRC 0.83 0.017 1.00 0.989
RAC2 0.85 0.034 0.96 0.652
RIPK1 0.98 0.249 0.95 0.021
STAT1 0.93 0.006 1.03 0.316
SYK 0.85 0.001 0.89 0.078

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BIOLOGICAL LCZ696 AMLO

GENES ID
PATHWAY RC P-value RC P-value
OXIDATIVE COX11 1.05 0.001 1.03 0.128
METABOLISM COX4I2 1.09 0.016 1.11 0.024
COX7A2 1.01 0.312 1.03 0.049
COX7A2L 0.97 0.028 0.98 0.288
IDH2 1.02 0.530 1.10 0.011
NDUFA5 1.01 0.831 1.06 0.049
NDUFB4 1.03 0.179 0.94 0.047
NDUFS1 1.00 0.939 1.08 0.010
PDP1 0.94 0.002 0.99 0.497
PPARGC1A 0.89 0.016 1.00 0.997
SLC25A11 1.01 0.804 1.10 0.009
SLC25A16 0.99 0.820 1.10 0.019
SLC25A18 1.08 0.041 1.07 0.117
SLC25A27 0.99 0.862 0.92 0.046
SLC25A33 1.04 0.337 1.10 0.038
SUCLG1 0.96 0.017 1.02 0.417

NATRIURETIC ADM 0.95 0.027 0.95 0.071

PEPTIDE EDN1 1.00 0.958 0.82 0.006
SIGNALING FAP 1.27 0.000 1.05 0.361
GUCY1A2 1.12 0.000 1.11 0.001
GUCY1A3 1.14 0.000 1.07 0.047
GUCY1B3 1.12 0.000 1.04 0.150
MME 1.05 0.177 1.11 0.001
PDE3A 1.06 0.048 1.04 0.295
PDE9A 1.09 0.016 0.99 0.797
RAPGEF5 1.09 0.031 1.04 0.362

LIPID ACACA 1.06 0.153 1.14 0.008

METABOLISM ADIPOR1 0.91 0.006 0.99 0.802
ANG 0.94 0.076 0.91 0.037
CIDEA 0.90 0.096 0.85 0.024
ELOVL5 0.99 0.522 1.03 0.048
ELOVL6 1.09 0.431 1.42 0.003
ELOVL7 0.93 0.095 0.90 0.047
GPAM 1.01 0.498 1.08 0.002
LPL 1.01 0.578 1.04 0.004
PPARGC1A 1.08 0.016 0.93 0.997
PLA2G4C 0.89 0.043 1.00 0.096

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SUPPLEMENTARY FIGURE S2. Representative Western Blots for proteins

involved in the lipolytic pathway, the natriuretic peptide signaling pathway and
mitochondrial oxidative metabolism.

Membranes were probed with antibodies directed against total ATGL, total HSL, phosphorylated HSL
(p-HSL) on Ser660, NPRA (A) and OXPHOS protein expression (B).

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Exercise training-induced effects on the
abdominal subcutaneous adipose tissue
phenotype in obese humans

Stinkens R.*, Brouwers B.*, Jocken J.W., Blaak E.E.,

Theunissen-Beekman K.F., Hesselink M. K., van Baak M.,
Schrauwen P., Goossens G.H.

* Shared authorship

To be submitted
Chapter 6

ABSTRACT
Aims/hypothesis: Rodent studies have indicated that physical exercise may
improve adipose tissue (AT) function. We investigated the effects of a 12-weeks
supervised, progressive exercise training program on adipocyte morphology and
abdominal subcutaneous AT function in well-phenotyped, obese subjects.
Methods: 21 obese men (14 metabolically compromised - non-alcoholic fatty liver
and/or type 2 diabetes - and 7 metabolically healthy controls) participated in a 12-
weeks supervised, progressive, combined exercise training program. At baseline
and after intervention, abdominal subcutaneous AT biopsies were collected to
determine 1) adipocyte morphology, 2) gene expression of markers for lipolysis,
inflammation, browning, adipokines and mitochondrial biogenesis/function, 3)
protein expression of mitochondrial oxidative phosphorylation (OXPHOS)
complexes and 4) ex vivo basal and β2-adrenergic stimulated lipolysis.
Results: At baseline, AT gene expression of HSL (P=0.005), CGI-58 (P<0.001)
and PGC-1α (P=0.037) were significantly lower in the metabolically compromised
as compared to metabolically healthy obese subjects. Mean adipocyte diameter
and total OXPHOS protein content in AT were comparable between groups. The
exercise training program, which increased maximal aerobic capacity (P time<0.001)
and muscle strength (Ptime<0.001), slightly reduced AT mass (~ 0.7 kg, Ptime=0.037)
but did not affect abdominal subcutaneous adipocyte size (Ptime=0.860), AT gene
expression of markers for mitochondrial biogenesis and function, browning,
lipolysis, inflammation and adipokines, total OXPHOS protein content (P time=0.826)
and β2-adrenergic sensitivity of lipolysis (Ptime=0.555), irrespective of baseline
metabolic status.
Conclusions/interpretation: A 12-weeks supervised, progressive exercise
training program did neither alter abdominal subcutaneous adipocyte morphology
and AT gene and protein expression of markers related to adipose tissue function,
nor β2-adrenergic sensitivity of lipolysis in obese subjects, irrespective of baseline
metabolic status.

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INTRODUCTION
The obesity epidemic is paralleled by a tremendous increase in the prevalence of
obesity-related diseases, including type 2 diabetes (T2DM), non-alcoholic fatty liver
(NAFL), cardiovascular disease and certain types of cancer [1]. A sedentary
lifestyle is a major contributor to obesity and related complications. In line,
increased habitual physical activity and exercise training may have beneficial
effects on insulin sensitivity and glucose homeostasis in obese, insulin resistant
and T2DM patients [2-4]. Therefore, increasing physical activity is a recommended
lifestyle modification in the prevention and treatment of obesity-related disorders,
including T2DM [5].
Since skeletal muscle is responsible for the majority of glucose disposal,
adaptations in skeletal muscle metabolism are thought to play a central role in the
exercise training-induced improvement of insulin sensitivity. Adipose tissue
dysfunction in obesity, however, represents a key step in the development of
obesity-related insulin resistance and chronic diseases [6, 7]. The reason for this is
that adipocyte hypertrophy in obesity promotes low-grade inflammation and
decreases the adipose tissue lipid buffering capacity. Consequently, lipids
accumulate in non-adipose tissues (e.g. skeletal muscle and liver) when lipid
supply exceeds fat oxidation, thereby accelerating the development and
progression of insulin resistance and chronic metabolic diseases [6-8].
Interestingly, there is evidence that exercise training may improve white adipose
tissue function [9]. Several rodent studies demonstrated that exercise training
increased adipose tissue mitochondrial biogenesis [10, 11] and function [12-14],
induced browning of white adipose tissue [10, 11, 13, 15, 16] and altered adipokine
expression [17, 18]. Furthermore, transplantation of white adipose tissue from
trained animals to untrained recipients markedly improved skeletal muscle glucose
uptake [13], suggesting that improvement of adipose tissue function may contribute
to the increased peripheral insulin sensitivity after exercise training. However,
human studies that investigated the effects of exercise training on the adipose
tissue phenotype are scarce. Exercise training has been shown to increase gene
expression of peroxisome proliferator-activated receptor-gamma coactivator-1
alpha (PGC-1α) [19] and oxidative metabolism markers [20], yet conflicting data
regarding the expression of adipokines and markers of lipolysis in human adipose
tissue have been reported [21-29]. Furthermore, the evidence that exercise training
enhances adipose tissue lipolysis, assessed either ex vivo in isolated adipocytes or
in vivo at rest and in response to a lipolytic stimulus, is inconsistent and
complicated by confounding factors such as recent energy balance, as reviewed
[30]. Importantly, most human studies that have investigated the exercise training-
induced effects on adipose tissue metabolism did not perform detailed metabolic
phenotyping. Therefore, it remains to be established whether the metabolic
phenotype at baseline determines study outcomes.
The aim of the present study was to investigate the effects of a 12-weeks
supervised, progressive, combined exercise training program on abdominal
subcutaneous adipocyte morphology, adipose tissue gene expression of markers
related to mitochondrial biogenesis/function, browning, lipolysis, inflammation and
adipokines and protein expression of mitochondrial oxidative phosphorylation
(OXPHOS) in obese, metabolically healthy and metabolically compromised

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individuals, matched for age and BMI. Furthermore, using isolated adipocytes from
these subjects, we determined the exercise training-induced effects on ex vivo
basal and β2-adrenergic stimulation of lipolysis.

METHODS

Study design
Twenty-one sedentary, middle-aged (40–70 yrs), overweight/obese men (14
NAFL/T2DM and 7 age and BMI-matched metabolically healthy control subjects)
participated in the present study, which was conducted within the framework of a
larger clinical trial designed to primarily investigate the effects of exercise training
on liver fat content, hepatic, adipose tissue and peripheral insulin sensitivity [31].
Control subjects (n=7) had low liver fat content (all ≤4%), as measured with proton
1
magnetic resonance spectroscopy ( H-MRS), in the absence of liver dysfunction
(defined as alanine aminotransferase (ALAT) >2.5 times normal values) and had to
be normoglycemic according to the WHO criteria. Subjects were defined as NAFL
1
patients (n=7) when having a liver fat content ≥5% as measured with H-MRS, in
addition to a fasting plasma glucose concentration <7.0 mmol/l. Furthermore, at
screening, T2DM patients (n=7) were allowed to be on sulphonyl urea, metformin,
dipeptidyl peptidase-4 inhibitors therapy (or a combination) for at least 6 months
with stable dosage for at least 2 months or on a dietary treatment for 6 months,
with fasting plasma glucose concentrations ≥7.0 and <10.0 mmol/l. Liver fat
content was not a selection criteria for T2DM patients. All subjects gave written
informed consent before participation in the study. The Medical Ethical Committee
+
of Maastricht University Medical Center approved the study protocol, which was
performed according the principles expressed in the Declaration of Helsinki.
All participants were asked not to change their habitual dietary intake during the
study period. General exclusion criteria were unstable body weight, cardiovascular
disease, impaired renal function, hemoglobin <7.5 mmol/l, blood pressure
>160/100 mmHg, participation in a weight-loss or exercise program, history of
substantial alcohol use (>3 units/day), history of drug abuse, use of beta-blockers,
anti-thrombotic medication, insulin therapy and use of medication known to
interfere with glucose homeostasis (except for T2DM patients).
At screening, routine laboratory analyses and physical examinations were
performed, medical history was checked and a resting electrocardiogram (ECG)
was taken. Maximal power output (W max) and maximal aerobic capacity (VO2max)
were assessed during a graded cycling test with concurrent ECG until exhaustion.
Body composition was determined using DEXA (Hologic Discovery A, Waltham,
MA, USA). Furthermore, a two-step hyperinsulinemic-euglycemic clamp with
2
primed D-[6,6- H2]-glucose was performed to assess peripheral, hepatic and
adipose tissue insulin sensitivity, as described elsewhere [31].

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Exercise training protocol

Subjects participated in a 12-weeks supervised, progressive exercise training
program. Aerobic exercise training was performed on a cycle ergometer twice a
week for 30 min at 70% W max, which was determined just before the start of the
intervention. Resistance exercise training, which focused on large muscle groups
(chest press, lat pull down, leg extension, shoulder press, horizontal row, leg press,
triceps extensions and biceps curls), was performed once a week and comprised
three series of ten repetitions at 60% of subjects’ previously determined one
repeated maximum (1RM). The 1RM test was preceded by a familiarization trial.
Warming-up and cooling-down sessions of 5 min were performed on a stationary
bike at 45% W max. Every 4 weeks, 1RM and VO2max were reassessed and training
loads were adjusted accordingly to assure that the training stimulus was
maintained. At baseline and after the 12-weeks training program, several
measurements were performed, as described below.

Adipose tissue biopsies

After an overnight fast, an abdominal subcutaneous adipose tissue biopsy (~1g)
was collected 6–8 cm lateral from the umbilicus, under local anesthesia (2%
lidocaine) by needle biopsy. The biopsy was washed with sterile saline and visible
blood vessels were removed. One part of the biopsy was snap frozen in liquid
nitrogen and stored at -80°C for gene and protein expression analyses, whereas
two parts were processed for determination of adipocyte morphology and
measurement of ex vivo lipolysis, as described below.

Adipocyte morphology
A part of the adipose tissue biopsy was fixed overnight in 4% paraformaldehyde
and embedded in paraffin for histological sections (8 μm). Sections were cut from
paraffin-embedded tissue, mounted on microscope glass slides and dried overnight
in an incubator at 37°C. The sections were stained with hematoxylin (VWR,
Radnor, PA, USA) and eosin (Klinipath BV, Duiven, The Netherlands). Digital
images were captured with a Leica DFC320 digital camera (Leica, Rijswijk, The
Netherlands) at x20 magnification (Leica DM3000 microscope, Leica, Rijswijk, The
Netherlands) and computerized morphometric analysis (Leica QWin V3,
Cambridge, UK) of individual adipocytes was performed in a blinded manner.
Approximately 400 adipocytes per sample were measured.

Gene expression
Total RNA was extracted from frozen adipose tissue biopsies (~500 mg) using
Trizol chloroform extraction (Invitrogen, Cergy Pontoise, France) and 300ng RNA
was reversed transcribed using iScript cDNA synthesis kit (BIO-RAD). Gene
expression for markers of lipolysis (ATGL [PNPLA2], HSL [LIPE], CGI-58 and
PLIN1 [perilipin 1]), inflammation (TNFα, IL-6, MCP-1 [CCL2], CD68), browning
(CIDEA, PRDM16), mitochondrial biogenesis (PGC-1α [PPARGC1A]) and
adipokine expression (ADIPOQ and LEP) (supplementary table 1 for primer
sequences) was determined in a total volume of 25 μL containing 12.5 ng cDNA
using SYBR-Green based qPCR (iCycler/MyIQ, BIO-RAD). Results were

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-ΔCT
calculated via the 2 method and normalized for 18S (housekeeping gene)
ribosomal RNA.

Protein expression
A detailed description can be found in the supplementary material. Briefly,
subcutaneous adipose tissue (~500mg) was ground to a fine powder under liquid
nitrogen and homogenized in radioimmunoprecipitation assay buffer. The
homogenate was lysed, vortexed and the supernatant was collected and stored at -
80°C. The protein concentration was determined by the Bradford-based protein
assay (Santa Cruz Biotechnology, Dallas, TX, USA).
Next, solubilized proteins (15 µg) were separated on a precast gel (Criterion™
TGX any kD, Bio-Rad Laboratories Inc, Hercules, CA, USA) and transferred onto a
nitrocellulose membrane (Trans Blot® Turbo™ transfer system; Bio-Rad).
Thereafter, quantitative Western Blot analysis was performed to determine the
levels of OXPHOS proteins. OXPHOS blots were probed with Total OXPHOS
Antibody Cocktail (Mitoscience/Abcam, Cambridge, MA, USA) and a secondary
horseradish peroxidase (HRP)-conjugated Rabbit-anti-Mouse antibody
(DakoCytomation, Glostrup, Denmark). Antigen-antibody complexes were
visualized using chemiluminescence by a ChemiDoc™ XRS apparatus (Bio-Rad)
®
and analyzed with Quantity One software (Bio-Rad), which calculated the optical
density units that are expressed as average intensity.

Ex vivo adipocyte lipolysis

Ex vivo adipocyte lipolysis was determined in 15 individuals and mature adipocytes
were isolated from the subcutaneous adipose tissue following collagenase
digestion. First, digestion was performed for 60 min at 37°C in a Krebs-Ringer
phosphate buffer, containing 100 mg glucose/100 ml and 4% bovine serum
albumin with 2 mg/ml collagenase (Sigma-Aldrich, Zwijndrecht, The Netherlands).
Secondly, adipocytes (~5,000-10,000 cells/incubation) were incubated with or
without increasing concentration of salbutamol (specific β 2-adrenergic receptor
-9 -4
agonist) (10 –10 M; GlaxoSmithKline, Zeist, The Netherlands) for 2 h at 37°C in
Krebs–Ringer phosphate buffer. Thereafter, incubation medium was collected and
stored at -80°C until analysis. Glycerol concentration in the medium, which is an
indicator of complete TAG hydrolysis (lipolysis), was determined using the EAPL-
200 EnzyChrom Adipolysis Assay Kit (Glentaur Europe BVBA, Kampenhout,
Belgium).

Biochemistry
Arterialized blood samples were collected and immediately centrifuged at 4°C for
10 min at 1000 g and plasma was snap frozen in liquid nitrogen and stored at -
80°C until further analysis. Plasma non-esterified fatty acid (NEFA; Wako NEFA C
test kit; Wako Chemicals, Neuss, Germany) and glucose (hexokinase method;
LaRoche, Basel, Switzerland) concentrations were measured with enzymatic
assays, whereas triacylglycerol (TAG) concentrations were measured
colorimetrically (Roche, Vienna, Austria), automated on a Cobas Fara/Mira.
Plasma insulin and serum liver function parameters (aspartate aminotransferase

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(ASAT), alanine aminotransferase (ALAT), γ-glutamyl transpeptidase (GGT)) were

routinely measured and analyzed at the clinical chemistry department in the
hospital.

Statistics
Student’s unpaired t-test was used for baseline comparisons between groups. The
effects of exercise training in metabolically healthy and metabolically compromised
obese subjects were compared by two-way repeated measures ANOVA, using
time (baseline and post-intervention) as within-subject factor and group as
between-subject factor. When a significant time*treatment interaction was
observed, post-hoc analysis with Bonferroni correction was applied to identify
significant within-group effects. All variables were checked for normal distribution
and were Ln-transformed to satisfy conditions of normality. All data are presented
as means ± SEM. Calculations were done using SPSS 21 for Mac OS X (IBM,
Chicago, IL, USA). P<0.05 was considered statistically significant.

RESULTS

Anthropometric and clinical characteristics

Subject characteristics before and after the 12-weeks supervised, progressive
exercise training program are summarized in Table 1. At baseline, fasting plasma
glucose (P=0.009) and HOMA-IR (P=0.016) were significantly higher, whereas
peripheral (P<0.001), adipose tissue (P=0.048) and hepatic insulin sensitivity
(P<0.001) were significantly lower in the obese metabolically compromised as
compared to age and BMI-matched obese metabolically healthy controls (Table 1).
As expected, VO2max (Ptime<0.001), W max (Ptime<0.001) and 1RM (Ptime<0.001) were
significantly increased following the exercise training intervention in both groups.
The training program significantly improved peripheral insulin sensitivity to a similar
extent in all subjects (Ptime=0.015; Ptime*group=0.106), but did not significantly affect
hepatic (Ptime=0.213) and adipose tissue insulin sensitivity (Ptime=0.943) (Table 1).
Furthermore, total fat mass (Ptime=0.037) and body fat percentage (Ptime=0.008)
were slightly but significantly decreased, whereas body weight (P time=0.866), BMI
(Ptime=0.890), fat free mass (Ptime=0.309) and plasma glucose, NEFA and TAG
concentrations remained unaltered after the training intervention (Table 1).

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Adipocyte morphology
At baseline, no differences in mean adipocyte size and adipocyte size distribution
were observed between groups (Figure 1A). The training intervention did not affect
mean adipocyte size, neither in the total group (62.9 ± 1.4 vs. 63.3 ± 1.4 μm,
P=0.860, Figure 1A) nor in both groups separately (Figure 1A). In line, adipocyte
size distribution was not affected by the exercise training in the total group (Figure
1B) and subgroups (Figure 1C).

Figure 1. Exercise training-induced effects on adipose tissue morphology.

Mean adipocyte size (A) and adipocyte size distribution in the total group (B); adipocyte size distribution
in the metabolically healthy and metabolically compromised subjects (C).
Panel A and B: white bars, baseline values; black bars, post-intervention values.
Panel C: white and grey bars, baseline values of the metabolically healthy and compromised subjects,
respectively; black and striped bars, post-intervention values of the metabolically healthy and
compromised subjects, respectively.

186
 Table 1. Anthropometric and clinical subject characteristics before and after the 12-weeks supervised, progressive exercise training program.

Total group Obese metabolically healthy Obese metabolically compromised

Post
Baseline Post Baseline Baseline Post Ptime Pgroup Ptime*group
intervention
intervention intervention
Age (years) 58.1 ± 1.6 - 60.1 ± 2.3 - 57.1 ± 2.2 - - - -

Body weight (kg) 95.4 ± 2.6 95.4 ± 2.6 93.6 ± 5.5 93.9 ± 5.6 96.3 ± 3.6 96.2 ± 3.8 0.866 0.676 0.707

-2
BMI (kg·m ) 30.0 ± 0.6 30.0 ± 0.6 29.7 ± 1.2 29.8 ± 1.2 30.1 ± 0.8 30.0 ± 0.8 0.890 0.849 0.684

Fat mass (kg) 27.9 ± 1.2 27.2 ± 1.3 27.4 ± 3.0 26.8 ± 3.0 28.3 ± 1.4 27.4 ± 1.6 0.037 0.806 0.838

Fat free mass (kg) 65.7 ± 1.5 66.0 ± 1.6 64.4 ± 3.0 64.8 ± 3.2 66.4 ± 2.2 66.6 ± 2.4 0.309 0.602 0.772

Body fat percentage (%) 28.8 ± 0.7 28.2 ± 0.7 28.6 ± 1.5 28.0 ± 1.6 28.9 ± 0.8 28.3 ± 0.9 0.008 0.888 0.907

-1 -1
VO2max (ml·min ·kg ) 26.9 ± 0.9 29.5 ± 1.0 26.9 ± 1.8 29.3 ± 2.0 27.0 ± 1.0 29.6 ± 1.1 <0.001 0.929 0.910

Wmax (W·kg-1) 2.0 ± 0.8 2.4 ± 1.0 2.1 ± 0.2 2.4 ± 0.2 2.0 ± 0.1 2.3 ± 0.1 <0.001 0.805 0.927

Strength (kg) 83.4 ± 3.8 97.1 ± 4.7 78.2 ± 6.8 91.2 ± 9.6 88.5 ± 5.2 101.8 ± 5.8 <0.001 0.355 0.822

-1
Fasting plasma glucose (mmol·l ) 6.6 ± 0.5 6.6 ± 0.5 5.2 ± 0.2 5.2 ± 0.1 7.2 ± 0.8 7.4 ± 0.9 0.959 0.040 0.982

-1
Fasting insulin (mU·l ) 13.1 ± 1.5 12.4 ± 1.4 9.8 ± 1.3 9.8 ± 1.1 13.7 ± 2.1 13.7 ± 2.0 0.353 0.188 0.748

HOMA-IR 3.7 ± 0.5 3.4 ± 0.4 2.3 ± 0.3 2.3 ± 0.2 4.1 ± 0.6 4.0 ± 0.6 0.399 0.048 0.751

Fasting plasma NEFA (μmol·l-1) 702 ± 31.7 648 ± 38.8 669 ± 46.6 633 ± 90.4 735 ± 44.3 670 ± 36.2 0.184 0.566 0.691

-1
Fasting plasma TAG (mmol·l ) 1.6 ± 0.1 1.6 ± 0.2 1.4 ± 0.2 1.4 ± 0.3 1.6 ± 0.2 1.7 ± 0.2 0.712 0.302 0.709

-1 -1
Rd (μmol·kg ·min ) 11.5 ± 2.2 14.2 ± 2.8 22.1 ± 2.9 27.8 ± 2.5 6.7 ± 1.1 8.0 ± 1.9 0.015 <0.001 0.106

EGP suppression (%) -48.6 ± 4.5 -53.4 ± 5.6 -71.0 ± 3.6 -78.3 ± 10.6 -38.4 ± 2.8 -42.0 ± 2.5 0.213 <0.001 0.663

NEFA suppression (%) -61.6 ± 3.2 -62.1 ± 3.2 -69.8 ± 2.4 -68.9 ± 3.0 -56.8 ± 4.4 -58.1 ± 4.4 0.943 0.036 0.736

Data are expressed as mean ± SEM (n=21). VO2max: maximal aerobic capacity; Wmax: maximal power output; TAG: triacylglycerol; Rd: glucose rate of disappearance (reflects peripheral insulin
sensitivity); EGP: endogenous glucose production (EGP suppression reflects hepatic insulin sensitivity); NEFA: non-esterified fatty acid (NEFA suppression reflects adipose tissue insulin
Chapter 6

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sensitivity)
Chapter 6

Gene expression
At baseline, gene expression of HSL (0.35 ± 0.08 vs. 0.45 ± 0.11, P=0.005), CGI-
58 (0.39 ± 0.04 vs. 0.75 ± 0.19, P<0.001) and PGC-1α (-0.35 ± 0.22 vs. 0.00 ±
0.13, P=0.037) was significantly lower in the obese metabolically compromised as
compared to obese metabolically healthy individuals. In the total group, the
exercise training did not alter adipose tissue gene expression of ATGL
(Ptime=0.332, Figure 2A), HSL (Ptime=0.862, Figure 2B), PLIN1 (Ptime=0.614, Figure
2C) and CGI-58 (Ptime=0.546, Figure 2D). Furthermore, the inflammatory markers
TNFα (Ptime=0.604, Figure 2E), IL-6 (Ptime=0.507, Figure 2F), MCP-1 (Ptime=0.222,
Figure 2G) and CD68 (Ptime=0.688, Figure 2H) were unchanged after the
intervention. Next, gene expression of the browning markers CIDEA (Ptime=0.943,
Figure 2I) and PRDM16 (Ptime=0.839, Figure 2J) and PGC-1α (Ptime=0.835, Figure
2K), a major regulator of mitochondrial biogenesis and function, remained
unchanged following the exercise training. Finally, gene expression of LEP
(Ptime=0.840, Figure 2L) and ADIPOQ (Ptime=0.413, Figure 2M) was also not
significantly altered after the 12-weeks training program. In line, no significant
differences in exercise-induced alterations in these parameters were observed
between groups, except for a slight but significant change in CGI-58
(Ptime*group=0.037).

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Figure 2. Exercise training-induced effects on adipose tissue gene expression.

Genes involved in lipolysis (A-D), inflammation (E-H), browning (I-J), mitochondrial biogenesis and
function (K) and adipokine expression (L-M) are expressed as fold change relative to the baseline
values of the total group and of the obese metabolically healthy control group.
* Ptime*group<0.05; #P<0.05 compared to baseline value of obese metabolically healthy control group.
White bars, baseline values; black bars, post-intervention values.

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Protein expression
At baseline, total OXPHOS protein content was not significantly different between
groups (P=0.176). In the total group, total OXPHOS protein expression remained
unchanged following the training program (27.0 ± 9.7 vs. 24.9 ± 6.9 AU,
Ptime=0.826, Figure 3A). More specific, OXPHOS complex I (2.9 ± 1.2 vs. 2.9 ± 1.0
AU, Ptime=0.857, Figure 3B), complex II (9.2 ± 2.4 vs. 8.9 ± 1.7 AU, P time=0.804,
Figure 3C), complex III (4.7 ± 2.7 vs. 2.1 ± 1.0 AU, Ptime=0.549, Figure 3D),
complex IV (0.8 ± 0.2 vs. 0.9 ± 0.3 AU, Ptime=0.870, Figure 3E) and complex V
(14.5 ± 6.8 vs. 13.3 ± 5.3 AU, Ptime=0.666, Figure 3F) were not affected. In line, no
significant differences in exercise-induced alterations in OXPHOS protein
complexes were observed between groups (Figure 3A-F).

Figure 3. Exercise training-induced effects on adipose tissue mitochondrial

oxidative phosphorylation (OXPHOS) protein expression.

Protein content of total OXPHOS (A), OXPHOS complex I (B), OXPHOS complex II (C), OXPHOS
complex III (D), OXPHOS complex IV (E) and OXPHOS complex V (F), expressed as fold change
relative to baseline values of the total group (time effect) and of the obese metabolically healthy group
(time*treatment interaction).
White bars, baseline values; black bars, post-intervention values.

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Ex vivo adipocyte lipolysis

The potency of salbutamol to stimulate lipolysis was determined by its EC 50, which
represents the concentration of agonist inducing 50% of its maximal lipolytic
response. The dose-response curve for salbutamol on ex vivo adipocyte lipolysis is
presented in supplementary Figure S1. In the total group, the training intervention
did not induce a significant change in basal lipolysis (7.0 ± 0.9 vs. 5.9 ± 0.6 µmol
7 -1 -1
glycerol·10 cells ·2h incubation , P=0.108), maximal lipolysis (18.1 ± 2.1 vs. 14.5
7 -1 -1
± 1.9 µmol glycerol·10 cells ·2h incubation , P=0.111) or the potency of
salbutamol (-logEC50) (6.0 ± 0.2 vs. 5.8 ± 0.3, P=0.555).

DISCUSSION
The aim of the present study was to investigate the effects of a 12-weeks
supervised, progressive exercise training program on the abdominal subcutaneous
adipose tissue phenotype in metabolically healthy and metabolically compromised,
well-phenotyped obese individuals. Here, we demonstrate that exercise training did
neither alter abdominal subcutaneous adipocyte morphology and adipose tissue
gene expression of markers for mitochondrial biogenesis/function, browning,
lipolysis, inflammation and adipokines, adipose tissue OXPHOS protein content,
nor β2-adrenergic stimulation of adipocyte lipolysis in obese subjects, irrespective
of baseline metabolic status. These data suggest that alterations in the phenotype
of abdominal subcutaneous adipose tissue do not significantly contribute to the
exercise-induced improvement in peripheral insulin sensitivity in obese men when
adipose tissue mass is only slightly reduced (~0.7 kg).
The training program induced a significant increase in aerobic capacity, maximal
power output and maximal muscle strength, indicating that the supervised,
progressive nature of the program was successful regarding enhancement of
physical fitness. This was accompanied by a slight but significant decrease in fat
mass and body fat percentage. In agreement with the findings in a larger study
population [31], we observed that peripheral insulin sensitivity was significantly
increased, whereas hepatic and adipose tissue insulin sensitivity remained
unchanged after the training program.
Exercise training interventions may affect adipocyte morphology in humans [30].
We demonstrated that a 12-weeks exercise training intervention did not
significantly alter mean adipocyte size or adipocyte size distribution, despite a 0.7
kg decrease in total fat mass. In contrast, Despres et al. [32] demonstrated that 20
weeks of endurance training decreased mean adipocyte size in young men, but not
in women. Importantly, they observed a more pronounced reduction in body weight
(~3.0 kg). Thus, a more prolonged intervention period, leading to a more
pronounced decrease in adipose tissue mass, seems necessary to induce
beneficial changes in adipocyte morphology.
Since an altered rate of lipolysis is one of the characteristics of adipose tissue
dysfunction and relates to peripheral insulin resistance [8], we determined adipose
tissue gene expression of lipolytic enzymes and genes encoding lipid droplet-
associated proteins. At baseline, gene expression of HSL and CGI-58 was lower in
obese metabolically compromised as compared to obese metabolically healthy
subjects. These findings are in line with previous studies from our group and

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others, showing a reduced expression of lipolytic genes in obese insulin resistant

[33] and patients with T2DM [34] as compared to obese insulin sensitive
individuals. Conflicting results on the impact of exercise training on basal and
stimulated adipose tissue lipolysis in humans has been reported, as extensively
reviewed [30]. Here, we show that exercise training did not alter the expression of
genes related to lipolysis under fasting conditions in obese individuals, irrespective
of baseline metabolic status, except for a slight change in CGI-58. It has previously
been demonstrated that β2-adrenergic stimulation of lipolysis is impaired in obese
as compared to lean subjects, whereas β 1-adrenergic receptor sensitivity was
comparable between groups [35]. We therefore determined if exercise training
altered β2-adrenergic sensitivity of lipolysis. In agreement with unchanged adipose
tissue gene expression of lipolytic markers, the potency of the β 2-adrenergic
receptor agonist salbutamol to stimulate abdominal subcutaneous adipocyte
lipolysis was comparable before and after the exercise training program.
Furthermore, basal and maximal β2-adrenergic receptor-mediated lipolysis
remained unchanged after exercise training. The present findings are in agreement
with a previous study showing no improvement of β 2-adrenergic stimulation of
lipolysis after 12 weeks of training in obese non-diabetic men [36]. However, in
contrast to the present findings, a decreased basal lipolysis was found after
exercise training in the latter study [36]. This may be explained by the modest loss
of fat mass in the present study, which did not result in a reduction in adipocyte
size. Indeed, it has previously been demonstrated that substantial weight loss,
which decreased adipocyte size, increased and normalized the sensitivity to
catecholamine-stimulated lipolysis in obese subjects [37].
In addition to impairments in lipolysis, a pro-inflammatory phenotype of adipose
tissue is associated with insulin resistance in obese subjects and T2DM patients [6-
8]. In the present study, no changes in macrophage infiltration and inflammatory
markers in adipose tissue were found following exercise training. The absence of
alterations in the inflammatory profile after exercise training is in agreement with
most previous studies in obese subjects [22, 23, 25, 26]. Furthermore, we found no
effects of exercise training on adipose tissue gene expression of leptin and
adiponectin, which is in accordance with observations in both lean and
overweight/obese humans [25, 26, 38]. Nevertheless, some studies have shown a
reduction in adipose tissue gene expression of MCP-1 [22] and an increase in
adiponectin expression [23] following exercise training in obese subjects. The
present findings further support the notion that a reduction in adipocyte size is
required to achieve beneficial changes in the adipose tissue phenotype.
Rodent studies demonstrated that increasing brown adipose tissue mass/activity or
inducing browning of white adipose tissue via cold exposure or other stimuli might
be a promising strategy for the treatment of obesity and obesity-related
impairments in glucose homeostasis [39]. Interestingly, physical exercise has been
shown to induce browning of white adipose tissue in rodents, possibly via the
secretion of the myokine irisin [15], although findings are controversial [40], or via
increased natriuretic peptide concentrations [41]. However, the expression of
browning markers in white adipose tissue of endurance-trained athletes was not
different from lean sedentary controls [42]. In the present study, the expression of
PRDM16 and CIDEA, markers of beiging/browning of white adipose tissue, did not
change following the intervention. In agreement with our findings, no exercise

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training-induced changes in browning markers in white adipose tissue were found

in lean [43, 44] and overweight [20, 44] subjects.
The oxidative phenotype of white adipose tissue is impaired in obesity and seems
related to an altered glucose homeostasis in rodents and humans [45-48]. Here,
we found that baseline gene expression of PGC-1α was significantly lower in
obese metabolically compromised as compared to metabolically healthy obese
subjects, although this did not translate into significant differences in OXPHOS
protein content between these BMI-matched groups. Furthermore, the exercise
training did not induce alterations in adipose tissue gene expression of PGC-1α,
which is in accordance with most [20, 38, 43], but not all previous studies [19, 49].
Finally, we observed no significant exercise-induced alterations in adipose tissue
OXPHOS protein content after the training intervention, which is in line with high-
intensity interval training in healthy lean [43] and overweight [50] subjects. In
contrast to human data, previous rodent studies showed that endurance training
had beneficial effects on intra-abdominal and epididymal white adipose tissue
mitochondrial activity in rats [12], indicating species differences in mitochondrial
protein expression and/or function in response to exercise.
In conclusion, the present study demonstrated that a 12-weeks supervised,
progressive exercise training intervention, which improved physical fitness and
peripheral insulin sensitivity, had no significant effects on abdominal subcutaneous
adipocyte morphology, adipose tissue gene and protein expression of markers
related to adipose tissue function, nor β2-adrenergic sensitivity in obese subjects,
irrespective of their baseline metabolic status. Noteworthy, we cannot exclude that
exercise training may induce beneficial alterations in the adipose tissue phenotype
after a more prolonged intervention period, leading to a more pronounced loss of
fat mass, or in other fat depots.

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SUPPLEMENTARY MATERIAL

Protein expression analysis

Firstly, subcutaneous adipose tissue (500mg) was ground to a fine powder under
liquid nitrogen and homogenized in radioimmunoprecipitation assay buffer (10mM
Tris (Calbiochem)-HCl (Merck, Darmstadt, Germany) buffered saline (Merck) with
0,1% sodium dodecyl sulfate (SDS) (Bio-Rad Laboratories Inc, Hercules, CA,
USA), 1% sodiumdeoxycholate (Sigma-Aldrich, St. Louis, MO, USA), 1% NP-40
(Fluka) and a protease/phosphatase inhibitor cocktail (Cell Signaling Technology,
Beverly, MA, USA). The homogenate was lysed on iced and vortexed for 5 min and
centrifuged at 20,000 g for 30 min at 10°C. The supernatant was carefully collected
and aliquots were stored at -80°C. The protein concentration was determined by
the Bradford-based protein assay (Santa Cruz Biotechnology, Dallas, TX, USA).
Next, solubilized proteins were separated on a precast gel (Criterion™ TGX any
kD, Bio-Rad Laboratories Inc, Hercules, CA, USA) and transferred onto a
nitrocellulose membrane (Trans Blot® Turbo™ transfer system; Bio-Rad).
Differences in loading were adjusted to total protein content (via Ponceau S
(Sigma-Aldrich, St. Louis, MO, USA) staining) and appropriate positive controls
were included.
Thereafter, quantitative western blot analysis was performed to determine the
levels of the OXPHOS proteins. OXPHOS blots were probed with Total OXPHOS
Antibody Cocktail (Mitoscience/Abcam, Cambridge, MA, USA) and a secondary
horseradish peroxidase (HRP)-conjugated Rabbit-anti-Mouse antibody
(DakoCytomation, Glostrup, Denmark). Antigen-antibody complexes were
visualized using chemiluminescence (ECL) by a ChemiDoc™ XRS apparatus (Bio-
Rad) and analyzed with Quantity One® software (Bio-Rad), which calculated the
optical density units that are expressed as average intensity ([average intensity =
total intensity of the rows of pixels inside the band boundary divided by the number
of rows, minus the background intensity]).

Supplementary Figure S1. The effects of salbutamol on ex vivo adipocyte

lipolysis before and after exercise training in the total group (n=15).

Glycerol release into the medium (µmol glycerol·107 cells-1·2h incubation-1) was used as an indicator of
lipolysis.
Open circles represent baseline values; closed circles represent post-intervention values.

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Supplementary Table S1: RT-qPCR Primer Sequences

The following RT-qPCR primer sequences were used for gene expression analysis.
Upper sequences represent forward primers (5' - 3'), while lower sequences represent reverse primer
(5' - 3').

Genes Sequences

GTGTCAGACGGCGAGAATG
ATGL
TGGAGGGAGGGAGGGATG

GCGGATCACACAGAACCTGGAC
HSL
AGCAGGCGGCTTACCCTCAC

CAGCATCCAGTCCTTACGACCA
CGI-58
GTTCAGTCCACAGTGTCGCAGA

CTCTCGATACACCGTGCAGA
PLIN1
TGGTCCTCATGATCCTCCTC

CCGAGTGACAAGCCTGTAGC
TNFα
GAGGACCTGGGAGTAGATGAG

AAATTCGGTACATCCTCGACGG
IL-6
GGAAGGTTCAGGTTGTTTTCTGC

CCCCAGTCACCTGCTGTTAT
MCP-1
TCCTGAACCCACTTCTGCTT

CCCTATGGACACCTCAGCTTT
CD68
GAAGGACACATTGTACTCCACC

TCAGACCTTGGGAGACAACACG
CIDEA
CGAAGGTGACTCTCGCTATTCC

CAGCCAATCTCACCAGACACCT
PRDM16
GTGGCACTTGAAAGGCTTCTCC

TCTGAGTCTGTATGGAGTGACAT
PGC-1α
CCAAGTCGTTCACATCTAGTTCA

TGGTGAGAAGGGTGAGAA
ADIPOQ
AGATCTTGGTAAAGCGAATG

GAACCCTGTGCGGATTCTTGT
LEP
TCCATCTTGGATAAGGTCAGGAT

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Coordinated regulation of adipose tissue
adrenergic- and non-adrenergic-
mediated lipolysis during exercise in lean
and obese individuals:
the effect of exercise training

Stinkens R.*, Verboven K.*, Hansen D., Wens I., Frederix I.,
Eijnde B.O., Jocken J.W., Goossens G.H.#, Blaak E.E.#

* Shared first authorship, # Shared last authorship

Submitted
Chapter 7

ABSTRACT
Background: Adipose tissue dysfunction, which includes impairments in (adipose
tissue) lipolysis, contributes to insulin resistance. Subcutaneous adipose tissue
(SCAT) lipolysis in obesity is characterized by catecholamine resistance and an
impaired ANP responsiveness. It remains to be established whether exercise
training improves non-adrenergically-mediated lipolysis, next to the adrenergic
pathway, in metabolically compromised conditions. The aim of the present study
was to investigate the effect of local combined α- and β-adrenergic receptor
blockade on SCAT lipolysis in obese insulin sensitive (IS), obese insulin resistant
(IR) and age-matched lean IS men. Moreover, obese individuals underwent
endurance and resistance exercise training to improve metabolic profile and (non-)
adrenergically-mediated SCAT lipolysis.
Methods: Abdominal SCAT lipolysis was investigated in 10 obese IS, 10 obese IR
and 10 age-matched lean IS men using microdialysis in the presence or absence
of local combined α- and β-adrenergic receptor blockade at rest, during 60 min of
low-intense (40% VO2max) endurance-type exercise and recovery. Systemic
responses were investigated using venous blood sampling. Obese individuals
participated in a supervised, endurance and resistance exercise training
intervention for 12 weeks (3 sessions/week) after which the microdialysis
measurements were repeated in obese IR men.
Results: Exercise-induced increase in abdominal SCAT lipolysis (expressed as
total area under the curve) was more pronounced in obese IS (81%) and IR (34%)
as compared to lean individuals (Pgroup=0.012). Abdominal SCAT lipolysis was
significantly reduced (~40%) following local combined α-/β-adrenoceptor blockade
in obese IS individuals only. Despite improvements in body composition, physical
fitness and exercise-induced changes in circulating free fatty acids, lactate and
adrenalin, exercise intervention did not significantly affect (non-)adrenergically-
mediated lipolysis in abdominal SCAT of obese IR individuals.
Conclusion: Our findings indicate a major contribution of non-adrenergically
mediated lipolysis during exercise in abdominal SCAT of lean and obese
individuals. Furthermore, a 12-week exercise training program improved metabolic
profile and body composition in obese individuals, but did not affect abdominal
SCAT lipolysis.

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INTRODUCTION
Adipose tissue (AT) dysfunction is commonly observed in human obesity and
contributes to insulin resistance (IR) and chronic metabolic diseases, including
cardiovascular disease, type 2 diabetes mellitus (T2D) and certain types of cancer
[1, 2]. Disturbances in AT lipid metabolism, including a decreased lipid uptake and
impairments in lipid mobilization are closely linked to ectopic fat deposition and
obesity-related IR [3]. An important function of the AT is to release fatty acids
through lipolysis [4, 5], especially during fasting and increased energy demanding
conditions such as exercise. Multiple endocrine factors affect the activity of lipid
droplet-associated proteins and lipases, thereby regulating the release of free fatty
acids (FFA) and glycerol [6]. However, impairments in the regulation of lipolysis
have been identified in subcutaneous AT (SCAT) of obese humans [7], including a
blunted catecholamine-mediated lipolysis [8, 9]. More specific, β-adrenergically-
mediated lipolysis is reduced [9] and inhibitory α2-adrenoceptors become
predominant on adipocytes in the obese insulin resistant state [10] [11], leading to
a blunted adrenergically-mediated lipolysis [9, 12]. Of interest, local β-adrenergic
blockade (alone or in combination with α2-adrenergic blockade) in SCAT, inhibits
exercise-induced lipolysis only to a minor extent at low-to-moderate intensities in
healthy lean [13-15] and overweight individuals [16]. In this respect, Moro et al. [16]
demonstrated that non-adrenergically-mediated lipolysis in SCAT substantially
contributes to lipid mobilization during exercise in healthy young lean men [15] and
healthy young overweight men [16]. Other key regulators of lipolysis are insulin [17]
and lactate [18], which both exert an inhibitory role in the physiological control of
AT during exercise [17-19].
More recently, evidence has emerged that natriuretic peptides (NP) not only affect
the cardiovascular system, but also have pronounced effects in several key
metabolic organs such as AT and skeletal muscle [20]. Interestingly, several
studies have indicated that the circulating NP concentrations are reduced in human
obesity and T2D [21-23]. The latter findings, together with evidence that reduced
systemic NP concentrations increase the risk of developing T2D [24, 25], highlight
the importance of NP in metabolic disease. Of the NP family, atrial natriuretic
peptide (ANP) has been shown to be the most potent stimulator of human AT
lipolysis [26], via guanylyl cyclase-coupled natriuretic peptide receptor type A
(NPRA)-mediated activation of hormone-sensitive lipase (HSL) [27, 28].
Interestingly, we have recently found that maximal ANP responsiveness is impaired
in isolated abdominal subcutaneous adipocytes of obese non-diabetic and T2D
men [29]. In line, Rydén and colleagues [30] have recently shown a blunted
lipolytic effect of ANP in isolated abdominal subcutaneous adipocytes of obese
women and in situ (microdialysis) in abdominal SCAT of overweight men under
resting conditions. Importantly, however, the physiological role of exercise-induced
ANP-mediated lipolysis in human obesity remains to be established.
It has been shown that endurance exercise training can partly improve β-
adrenoceptor activity, reduce anti-lipolytic α2-adrenoceptor sensitivity in human
SCAT [31-33], and alleviate ANP-mediated lipolysis in subcutaneous adipocytes in
young, metabolically healthy overweight individuals [28, 34]. However, to date, it
remains elusive if endurance and resistance exercise intervention improves ANP-
induced activation of lipolysis in metabolically compromised conditions.

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The aim of the present study was to compare the effect of local combined α- and β-
adrenoceptor blockade on local SCAT lipolysis at rest, during low-intensity
endurance-type exercise and during recovery from exercise in middle-aged obese
insulin sensitive (IS), obese insulin resistant (IR) and age-matched lean IS men. In
addition, we investigated whether a 12-week endurance and resistance exercise
training improved the metabolic profile in obese men and (non-)adrenergically-
mediated abdominal SCAT lipolysis in obese IR men.

METHODS

Subjects
Ten middle-aged healthy lean insulin sensitive (IS), 10 obese IS and 10 obese
insulin resistant (IR) men, matched for age and BMI (obese groups) participated in
the present study. Subjects were included when they had a stable body weight for
at least 3 months prior to the start of the intervention and had no contraindications
for participation in an exercise training intervention based on their medical history.
Major exclusion criteria were a history, or clinical symptoms, of heart, lung or
kidney disease, presence of endocrine anomalies and/or the use of beta-blockers,
glucose or lipid-lowering medication. Insulin sensitivity was assessed via
homeostasis assessment of insulin resistance (HOMA-IR) [35]. Subjects were
classified as insulin sensitive or insulin resistant when HOMA-IR was ≤ 2.3 [36] or ≥
3.8 [37], respectively. Height, weight, waist and hip circumference and blood
pressure were measured during screening. Body composition was measured using
a Dual Energy X-ray Absorptiometry scan (Hologic Series Delphi-A Fan Beam X-
ray Bone Densitometer). One week before the investigational protocol, peak
oxygen uptake (VO2peak) was determined during a maximal cardiopulmonary
exercise test performed on an electrical braked cycle ergometer (Gymna Ergofit
Cycle 400, Bilzen, Belgium) by using an incremental procedure (work rate
increased by 15W/min until volitional exhaustion). Heart rate (electrocardiography)
was monitored continuously and VO2peak was measured using a Metalyzer II
(Cortex Medical, Leipzig, Germany). The study was approved by the Medical
Ethical Committee of the Jessa Hospital and Hasselt University, Hasselt, Belgium,
and performed in accordance with the declaration of Helsinki (2008). All individuals
gave written informed consent prior to the start of the study.

Experimental protocol
Subjects arrived at the hospital at 07:30 AM after an overnight fast. They were
instructed to consume a standardized meal and snack the evening before the test
day (total energy: 2628 kJ (626 kcal); 23.4g fat (10.4g saturated fat); 73.8g
carbohydrates (of which 6.8g sugar); 28.8g protein; 2.9g salt; 2.3g fibres) and to
abstain from exhausting activities 48 hours prior to the experimental protocol. On
arrival, a catheter was inserted into the antecubital vein for blood sampling. Two
microdialysis catheters (CMA 63, CMA Microdialysis AB, Stockholm, Sweden)
®
were inserted percutaneously into SCAT after epidermal anesthesia (EMLA
crème: lidocaine 2.5% and prilocaine 2.5%, AstraZeneca AB) at a distance of 6-8

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cm from the umbilicus (one probe on the left side and one probe on the right side of
the umbilicus). The probes were connected to a microinfusion pump (Harvard
apparatus, Plato BV, Diemen, The Netherlands) and perfused with Ringer solution
(in mmol/l: 147 sodium, 4 potassium, 2.25 calcium and 156 chloride; Fresenius
Kabi BV, ‘s Hertogenbosch, The Netherlands) at a perfusion rate of 2.0 µl/min.
Ethanol (50 mmol/l) was added to the perfusate to semi-qualitatively estimate
changes in local adipose tissue blood flow (ATBF), using the ethanol outflow/inflow
(out/in) ratio [38]. A higher ethanol out/in ratio, corresponding to a lower ethanol
wash-out, reflects a lower regional ATBF.
One microdialysis catheter was perfused with Ringer solution (control), while the
contralateral catheter was perfused with Ringer, supplemented with 100 μmol/l
phentolamine (α1,2-adrenergic receptor antagonist) (Regitin 10 mg/ml; Novartis
Pharma BV, The Netherlands) and 100 μmol/l propranolol (nonselective β-
adrenergic receptor antagonist) (propranolol hydrochloride, Dociton 1 mg/ml, Mibe
GmbH, Germany), concentrations that completely suppress lipolysis [28, 39, 40].
After a 60-min equilibration period (recovery from insertion), two 30-min fraction of
dialysate were collected at a flow rate of 0.3 µL/min after which the perfusion rate
was increased to 2.0 µL/min for the remaining of the experiment. During the resting
phase, three 15-min fractions of the outgoing dialysate were collected from both
sites to determine the extracellular glycerol concentration (reflecting basal
lipolysis). Next, subjects performed a single bout of endurance exercise for 60 min
at 40% of their VO2max on a cycle ergometer while heart rate was monitored
continuously (Polar, Kempele, Finland). Exercise was followed by a 60-min
recovery period in supine position. During exercise and recovery, dialysate
samples were collected at 15 min intervals without disconnecting the microdialysis
probes from the microinfusion pumps.
Ethanol concentrations were determined both in the ingoing (perfusate) and
outgoing (dialysate) fluid to assess the ethanol out/in ratio as an indicator of local
nutritive blood flow. Ethanol concentrations were determined at the same day,
whereas dialysate samples for measurement of extracellular glycerol, glucose and
lactate concentrations were immediately frozen and stored at -80 °C until analysis.
Venous blood samples were taken at rest, during exercise and recovery in
prechilled 20 mL tubes at 15 min intervals throughout the study protocol.

Indirect calorimetry
Substrate utilization and energy expenditure were determined at rest and during
submaximal exercise via indirect calorimetry using a Metalyzer II (Cortex Medical,
Leipzig, Germany). Substrate oxidation rates (g/min) and energy expenditure were
calculated from VO2 and VCO2 [41, 42]. Water intake was allowed ad libitum during
the exercise and recovery period.

Exercise training protocol

Obese IS and IR subjects participated in a supervised, exercise training program
for 12 weeks (3 sessions per week) [43]. Subjects were asked not to change their
habitual diet during the intervention period. Each training session started with
cycling (Excite Bike, Technogym, Zaventem, Belgium) for 45min at 65% VO 2peak
(heart rate based) from which mean heart rate, mean workload and total energy

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expenditure (calories) were collected. Next, resistance exercises of 5 large muscle

groups were performed at 65-70% of 1 RM (leg press, leg curl, leg extension,
vertical traction, arm curl and chest press; Technogym). Training volume and load
were gradually increased during the intervention whereby resistance training was
increased every 3 weeks. Training sessions were supervised to assure compliance
and safety of the participants. After the 12 weeks of exercise training the
experimental protocol was repeated and venous blood samples were taken at rest,
during exercise and recovery at 15 min intervals. In addition, SCAT microdialysis
was performed in obese IR subjects, as described above.

Biochemical analysis
Microdialysate samples were analyzed for glycerol, glucose and lactate
concentrations by means of bioluminescence on an ISCUS clinical microdialysis
analyzer (M dialysis AB, Stockholm, Sweden). Ethanol concentrations in dialysate
(out) and perfusate (in) were measured spectrophotometrically using a COBAS
FARA semi-automatic analyzer (Roche Diagnostics, Basal, Switzerland) and using
a standard ethanol assay kit (Boehringer Mannheim, Germany).
Blood samples were centrifuged at 4°C for 10 min at 1200 g and plasma and
serum was stored at -80°C until further analysis. Plasma free glycerol was
TM
measured after precipitation with an enzymatic assay (Enzytec Glycerol, Roche
Biopharm, Switzerland), automated on a Cobas Fara spectrophotometric
autoanalyzer (Roche Diagnostics, Basel, Switzerland). Plasma FFA, glucose and
lactate concentrations were measured with enzymatic assays on an automated
spectrophotometer (ABX Pentra 400 autoanalyzer, Horiba ABX, Montpellier,
France). Plasma ANP concentrations were measured using an enzyme
immunoassay (RayBiotech, Norcross GA, USA). Catecholamine concentrations
(adrenalin and noradrenalin) were determined using high performance liquid
chromatography with electrochemical detection (ClinRep® Complete Kit for
Catecholamines in Plasma, RECIPE chemicals & Instruments GmbH, Munich,
Germany). Serum insulin concentrations were determined with radioimmunoassay
kits (Human Insulin specific RIA Kit, Millipore Corporation, MA, USA).

Statistical analysis
All data are expressed as means ± SEM. Normal distribution was tested by the
Kolmogorov-Smirnov test. Subjects were excluded from analyses when dialysate
samples of 2 subsequent time points were missing, in order to maintain paired
samples. Dialysate and systemic exercise responses were expressed as the area
under the curve (AUC) and the incremental area under the curve (iAUC),
calculated by the trapezoid method. Cross-sectional analyses (differences between
groups and conditions) for the microdialysis lipolysis data were analyzed with a
two-way repeated-measures ANOVA. In case of significance, post-hoc analyses
with Bonferroni correction were applied to identify significant within-group effects.
Differences in plasma concentrations and substrate metabolism between groups
were tested with a one-way ANOVA and differences within groups were analyzed
by means of paired t-test. Intervention effects in the obese groups were analyzed
with a two-way repeated-measures ANOVA (with pre- and post-intervention as
conditions), with Bonferroni post-hoc correction to detect within-group effects.

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Three subjects dropped out of the exercise intervention, due to medical (n=1) or
motivational reasons (n=2) and were therefore excluded from the intervention (pre
vs. post) analyses. SPSS 21 for Macintosh OS X was used to perform all
calculations (IBM Corporation, Armonk, NY, USA). The level of statistical
significance was set at p<0.05 (2-tailed), while p<0.10 was considered a tendency.

RESULTS

BASELINE
Anthropometric and clinical characteristics
Subjects’ characteristics are presented in Table 1. By design, there was a
significant difference between the lean and the obese IS and/or obese IR group
with respect to body weight, BMI, WH-ratio, whole-body fat percentage, android
(i.e. trunk region) and gynoid (including hip and leg regions) fat mass (all p<0.05).
Furthermore, HOMA-IR and fasting serum insulin concentrations were significantly
higher in obese IR compared to the lean and obese IS individuals (p<0.001 for both
parameters in both groups). Obese IS and obese IR individuals only differed in
android fat mass, which was higher in the obese IR group (p=0.017) (Table 1).
With respect to physical fitness, VO2peak/FFM and W peak/FFM were significantly lower in
the obese IS and obese IR group as compared to the lean group (p<0.01 and
p<0.001, respectively) (Table 1), while maximal heart rate and maximal respiratory
quotient (RQ) were comparable between groups.

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Table 1. Characteristics of obese insulin sensitive, obese insulin resistant individuals and healthy
lean controls
Obese insulin Obese insulin
Lean
sensitive resistant
(n=10) (n=10) (n=10) P ANOVA
Age, years 45 ± 2 47 ± 2 43 ± 1 0.527

Cardiometabolic risk
Fasting plasma glucose
5.5 ± 0.0 5.2 ± 0.1 5.8 ± 0.2 † 0.050
(mmol/L)
Fasting serum insulin (mU/L) 7.3 ± 0.6 9.2 ± 0.6 19.6 ± 1.6 *** † <0.001
HOMA-IR 1.8 ± 0.1 2.1 ± 0.1 5.0 ± 0.4 *** † <0.001
Systolic BP (mmHg) 122 ± 2 135 ± 6 143 ± 6 0.066
Diastolic BP (mmHg) 72 ± 1 81 ± 4 86 ± 5 0.108

Body composition
Body weight (kg) 79.9 ± 2.9 101.5 ± 3.2 ** 109.6 ± 4.7 *** <0.001
Body mass index (kg/m²) 23.7 ± 0.4 32.6 ± 0.4 *** 33.9 ± 0.7 *** <0.001
Waist-to-hip ratio 1.00 ± 0.00 1.04 ± 0.01 * 1.05 ± 0.01 * 0.010
Fat mass (kg) 16.4 ± 1.1 30.0 ± 1.7 *** 34.2 ± 1.8 *** <0.001
Fat percentage (%) 22.0 ± 0.8 31.4 ± 0.9 *** 33.2 ± 1.1 *** <0.001
Fat free mass (kg) 57.4 ± 1.8 65.0 ± 1.5 68.4 ± 2.8 ** 0.004

Exercise capacity
VO2 peak (ml*min-1*kg-1 (FFM)) 62 ± 3 48 ± 2 ** 48 ± 1 ** 0.002
Wmax (Watt*kg-1 (FFM)) 4.9 ± 0.1 3.7 ± 0.2 *** 3.4 ± 0.1 *** <0.001

Data are mean ± SE. * Significantly different from lean group p < 0.05; ** p < 0.01; *** p < 0.001.
† Significantly different from obese insulin sensitive group (p < 0.05). FFM: fat free mass; HR: heart
rate; RER: respiratory exchange ratio; VO2 peak: maximum oxygen uptake; Wmax: maximum power
output

Systemic responses during rest, exercise and recovery phase

Under resting conditions, plasma glycerol, FFA, glucose, lactate, ANP, adrenalin
and noradrenalin concentrations were comparable between groups, while fasting
serum insulin concentration was higher in obese IR compared to lean and obese IS
individuals (Figure 1 A-H). During exercise, plasma concentrations of glycerol,
FFA, glucose, ANP, adrenalin and noradrenalin increased to the same extent in all
groups (Figure 1). Exercise increased plasma lactate concentrations in all groups,
which was most pronounced in obese IR as compared to lean individuals
(pANOVA=0.044) (Figure 1 D). The exercise-induced increase in plasma ANP was
similar in all groups (pANOVA=0.300), but peak plasma ANP concentrations were
reached earlier during exercise in the lean compared to the obese groups
(p=0.034) (Figure 1 E). Serum insulin levels were significantly higher during
exercise in obese IR as compared to obese IS and lean individuals, with no
differences between the latter two groups (Figure 1 F). During exercise, the
increase in plasma adrenalin and noradrenalin concentrations was comparable
between groups (Figure 1 G-H). During recovery, plasma glycerol, glucose,
adrenalin, noradrenalin and ANP concentrations decreased back to baseline
concentrations (Figure 1), while plasma lactate concentrations tended to remain
elevated in the obese IR group (pANOVA=0.061) (Figure 1 D). Furthermore, plasma
FFA concentrations peaked in the first 15 minutes of the recovery period and

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remained significantly elevated in obese IR as compared to lean individuals

(pANOVA=0.020) (Figure 1 B). Serum insulin concentrations remained significantly
elevated during recovery in the obese IR group compared to the obese IS and lean
group (both pANOVA<0.001), with no differences between the latter two groups
(Figure 1 F). Detailed systemic plasma responses during baseline, exercise and
recovery are shown in Supplementary Table S1.

Figure 1. Plasma glycerol, FFA, glucose, lactate, ANP, adrenalin, noradrenalin and
serum insulin concentrations at rest, during exercise and recovery.

Systemic glycerol (A), FFA (B), glucose (C), lactate (D), ANP (E), insulin (F), adrenalin (G) and
noradrenalin (H) responses in lean (white circles), obese insulin sensitive (white squares) and obese
insulin resistant (black triangles) individuals. Data are presented as mean ± SEM. P ANOVA values
represent differences in exercise-induced systemic responses between groups.

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Substrate oxidation and energy expenditure

Substrate oxidation and energy expenditure were determined at rest and during
exercise. Whole-body energy expenditure (kJ/min), RQ, carbohydrate and fat
oxidation (as percentage of energy expenditure) were not different between groups
at rest, nor during exercise (Supplementary Figure 1 A-E).

Microdialysis

Abdominal subcutaneous adipose tissue blood flow

At rest, lean individuals had a significantly lower ethanol out/in ratio, reflecting a
higher ATBF, compared to both obese groups (p<0.01), whilst no significant
difference in ATBF was observed between both obese groups (Figure 2 A, C and
E). Local α-/β-adrenergic blockade induced a significant increase in ethanol out/in
ratio in the lean group (Figure 2 A), reflecting a reduced ATBF, while this effect
disappeared during exercise. Moreover, this adrenergic sensitivity of ATBF was not
observed in the obese IS or IR group (Figure 2 C and E). Exercise induced a
decrease in ethanol out/in ratio, reflecting an increase in ATBF, in all groups. This
exercise-induced increase in ATBF tended to be higher in lean as compared to
obese IS and obese IR individuals (p=0.093, p=0.087, respectively) (Figure 2 A, C
and E). During recovery, ATBF returned to resting levels, with a significantly higher
ATBF (i.e. a lower ethanol out/in ratio) in the lean group compared to both obese
groups (Figure 2 A, C and E). Details with respect to ethanol out/in ratio during
baseline, exercise and recovery are shown in Supplementary Table S2.

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Figure 2. Changes in subcutaneous adipose tissue extracellular glycerol

concentration and adipose tissue blood flow indices.
Subcutaneous adipose tissue ethanol ratio’s in lean (A), obese insulin sensitive (B) and obese insulin
resistant (C) individuals. Changes in extracellular glycerol concentration in lean (D), obese insulin
sensitive (E) and obese insulin resistant (F) individuals at rest, during exercise and recovery in control
probe (white circles) and the probe perfused with phentolamine and propranolol (black squares). Data
are presented as mean ± SEM. * Significantly different from the control probe (p<0.05).

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Abdominal subcutaneous adipose tissue lipolysis

In SCAT, resting extracellular glycerol concentrations were comparable between
groups (Figure 2 B, D and F). Local α-/β-adrenergic blockade had no significant
effects on resting extracellular SCAT glycerol concentration in either of the groups
(Figure 2 B, D and F). During exercise, extracellular glycerol concentration
significantly increased in all groups. In obese IS individuals, exercise-induced
increase in glycerol concentration (AUC0-60) was higher compared to lean
(p=0.011), but not obese IR individuals (p=0.816) ((Figure 2 B, D and F). Local α-
/β-adrenergic blockade induced a significant reduction in the exercise-induced
increase in extracellular glycerol in the obese IS group (p=0.020), but not in the
lean IS or obese IR group (Figure 3). During recovery, extracellular glycerol
concentrations decreased in all groups, but remained significantly elevated in the
obese IS as compared to the lean group, with no differences between both obese
groups (Figure 2 B, D and F). Additionally, there were no significant effects of α-/β-
adrenergic blockade on the extracellular glycerol concentration during recovery in
any group (Figure 2 B, D and F). Details with respect to dialysate glycerol
concentrations at rest, during exercise and recovery are shown in Supplementary
Table S2.
Abdominal SCAT extracellular glucose and lactate concentrations and responses
were comparable between groups at rest, during exercise and recovery
(Supplementary Table S3).

Figure 3. Exercise-induced increase in subcutaneous adipose tissue extracellular

glycerol concentration.

Mean changes in subcutaneous adipose tissue extracellular glycerol concentration during 1 h of low-
intense exercise (40% VO2peak). Extracellular glycerol concentrations are determined in control probe
(white bars) and the probe perfused with phentolamine and propranolol (black bars). Changes were
calculated by the difference between the mean glycerol concentrations during exercise and the baseline
concentration (pgroup=0.009, ptreatment=0.069, ptreatment*group=0.035). Data are presented as mean ± SEM.
* Significantly (p<0.05) different compared to the control probe from the lean group; N.S.: not significant.

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EXERCISE TRAINING INTERVENTION

Anthropometry, exercise capacity and systemic responses

In both obese groups, exercise training led to a significant reduction in body
weight, BMI, whole-body fat percentage as well as android and gynoid fat mass
(Table 2). Whole-body insulin sensitivity (HOMA-IR) was significantly improved in
the obese IR group (ptime=0.005), but not in the obese IS group. Furthermore,
physical fitness (VO2peak/FFM as well as W max/FFM) improved significantly following the
12-week exercise training (Table 2).
The training intervention induced a significant reduction in resting plasma FFA
(Figure 4 C and D) and tended to reduce fasting ANP concentrations in both obese
groups (Figure 4 K and L). Resting blood glucose concentration increased in the IS
group but not in the IR group (Figure 4 E and F). The training intervention did not
induce significant changes in plasma glycerol, insulin, lactate, adrenalin or
noradrenalin concentrations. However, resting insulin concentrations, as well as
insulin concentration during exercise and recovery remained elevated in the obese
IR compared to obese IS group (Figure 4 I-J).
The exercise-induced increase in plasma FFA (Figure 4 C and D), lactate (Figure 4
G and H) and adrenalin (Figure 4 M and N) concentrations were significantly
blunted after intervention. Peak blood ANP concentrations (Figure 4 K and L)
tended to be reduced in both obese groups. The increase in plasma glycerol,
insulin and glucose concentrations during exercise remained unchanged.
In the recovery period, beside reduced plasma FFA (Figure 4 C and D) and
adrenalin concentrations (Figure 4 M and N), no significant training-induced
changes were observed in plasma glycerol, glucose, lactate, ANP, noradrenalin or
serum insulin concentrations (Figure 4). In addition, the training intervention did not
induce changes in whole-body energy expenditure and substrate oxidation
(Supplemental Figure 1 A-E). Detailed post-intervention systemic plasma
responses at rest, during exercise and recovery are shown in Supplementary Table
S4.

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Figure 4. Exercise training-induced changes in systemic plasma glycerol, FFA,

glucose, lactate, ANP, adrenalin, noradrenalin and serum insulin in obese
individuals.

Systemic glycerol (A-B), FFA (C-D), glucose (E-F), lactate (G-H), insulin (I-J), ANP (K-L), adrenalin (M-
N) and noradrenalin (O-P) of obese insulin sensitive and obese insulin resistant individuals at baseline
(white circles) and after 12 weeks of exercise training intervention (black squares). Data are presented
as mean ± SEM.

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Abdominal subcutaneous adipose tissue blood flow and lipolysis

Following exercise intervention, SCAT lipolysis was investigated in the obese IR
group only. Ethanol out/in ratio (Figure 5 A) as well as resting, exercise-induced
and recovery-related extracellular glycerol concentration (Figure 5 B) were not
altered after exercise intervention. Additionally, α-/β-adrenergic blockade had no
significant effect on resting, exercise-induced or recovery-related extracellular
glycerol concentration in SCAT following the exercise training program (Figure 5 B;
Figure 3).

Figure 5. Exercise training-induced changes in subcutaneous adipose tissue

extracellular glycerol concentration and adipose tissue blood flow indices in obese
insulin resistant individuals.

Subcutaneous adipose tissue ethanol ratio (A) and extracellular glycerol concentration (B) in the obese
insulin resistant individuals at rest, during exercise and recovery after 12 weeks of exercise training in
control probe (white circles) and the probe perfused with phentolamine and propranolol (black squares).
Data are presented as mean ± SEM.

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 Table 2. Body composition and exercise capacity before and after 12 weeks of supervised endurance and
resistance training

Obese insulin sensitive Obese insulin resistant

P Time P Group P Time*Group
N=8 N=9

Body composition PRE POST PRE POST

Body weight (kg) 104.5 ± 3.2 102.8 ± 2.6 110.6 ± 5.1 108.3 ± 5.0 0.002 0.360 0.585

Body mass index (kg/m²) 32.7 ± 0.5 32.2 ± 0.3 34.1 ± 0.8 33.3 ± 0.8 0.002 0.225 0.550

Waist-to-hip ratio 108.8 ± 1.6 108.8 ± 1.5 117.6 ± 3.3 113.5 ± 1.3 0.039 0.004 0.076

Fat mass (kg) 31.22 ± 1.91 29.11 ± 1.69 35.00 ± 1.92 33.07 ± 2.02 <0.001 0.168 0.833

Fat percentage (%) 31.6 ± 1.1 30.0 ± 1.0 33.7 ± 1.1 32.8 ± 1.2 <0.001 0.150 0.267

Fat free mass (kg) 66.8 ± 1.4 67.2 ± 1.3 68.5 ± 3.1 67.5 ± 3.3 0.377 0.779 0.037

Exercise capacity

VO2 peak (ml*min-1*kg-1 (FFM)) 49 ± 2 53 ± 1 48 ± 1 55 ± 2 0.012 0.800 0.521

Wmax (Watt*kg-1 (FFM)) 3.8 ± 0.2 4.1 ± 0.1 3.4 ± 0.1 4.0 ± 0.1 <0.001 0.382 0.069

Data are mean ± SE. FFM: fat free mass; VO2 peak: maximum oxygen uptake; Wmax: maximum power output.
Chapter 7

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DISCUSSION
The present study is the first to investigate (non-)adrenergically-mediated lipolysis
during low-intensity endurance-type exercise in abdominal subcutaneous adipose
tissue (SCAT) in middle-aged obese IS and obese IR men as compared to age-
matched lean IS men. Furthermore, we examined whether a 12-week exercise
intervention altered resting and exercise-induced (non-)adrenergically-mediated
SCAT lipolysis in obese IR individuals. Here, we demonstrated that the exercise-
induced increase in abdominal SCAT extracellular glycerol concentration (reflecting
local lipolysis) was more pronounced in obese as compared to lean individuals,
which may at least partly be explained by the higher adipose tissue blood flow
(ATBF) in lean individuals. Exercise-induced SCAT lipolysis was substantially
reduced (~40%) following local combined α-/β-adrenergic blockade in obese IS
individuals, but not in obese IR or lean IS individuals. Finally, the exercise training
intervention improved body composition, physical fitness and exercise-induced
systemic responses in both obese groups, and insulin sensitivity in the obese IR
group. However, this was not accompanied by changes in adrenergically- and non-
adrenergically-mediated lipolysis in the SCAT of obese IR individuals. Collectively,
our findings indicate that exercise-induced lipolysis is predominantly mediated by
non-adrenergic factors, most likely mediated by natriuretic peptides (NP) in middle-
aged lean IS, obese IS and obese IR individuals.
The present study showed a more pronounced increase in exercise-mediated
SCAT extracellular glycerol concentration in obese IS as compared to lean
individuals. Although the lipolytic response in abdominal SCAT is often blunted in
human obesity [9], the higher exercise-induced extracellular glycerol levels in both
obese groups is likely explained by the substantially lower ATBF in the obese state,
which contributes to higher extracellular glycerol concentrations due to a lower
removal of glycerol from the AT [16, 44]. Local α-/β-adrenergic blockade
substantially reduced basal ATBF in lean but not in the obese individuals. This
might suggest that adrenergic sensitivity of ATBF is reduced in the obese state,
which is in line with previous findings [39, 45]. Thus, these differences in ATBF
between lean and obese individuals should be taken into account when interpreting
local SCAT lipolysis.
Interestingly, combined α-/β-adrenergic receptor blockade reduced exercise-
induced SCAT lipolysis in obese IS men but not in lean and obese IR individuals. In
obese IR individuals, the lipolytic activity of the β-adrenergic receptors is
attenuated [9, 11, 12], while an increase in anti-lipolytic α2-adrenergic receptors in
SCAT reduce exercise-mediated lipolysis [39]. In addition, obese IR individuals
often display lower plasma catecholamine (adrenaline, noradrenaline) responses to
physical exercise [46], although the latter was not observed in the current study.
Therefore, the reduced exercise-mediated lipolytic response upon combined α-/β-
adrenergic receptor blockade in the obese IS individuals, as opposed to the obese
IR group, might suggest differences in adrenergic receptor expression and
sensitivity. The blunted lipolytic response in obese IR individuals might also be
explained by the significantly higher fasting and exercise induced serum insulin
levels. Since the anti-lipolytic effects of insulin might be normal or only slightly
impaired in obese AT [47-49], the observed hyperinsulinemia in the obese IR group
might have contributed to the attenuated adrenergically-mediated SCAT lipolysis

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as compared to the obese IS group, as previously shown [50]. In addition,

increased plasma lactate concentrations in obese IR individuals, may have
contributed to the reduced lipolytic response, since lactate has been shown to
inhibit lipolysis in mice [51] and human primary adipocytes in vitro [18].
The present study implies that non-adrenergic regulators of lipolysis play a major
role during low-intensity endurance-type exercise. It has previously been
demonstrated that propranolol [28] and phentolamine [39, 40] fully inhibit
adrenergically-mediated SCAT lipolysis at the concentrations used in the present
study. The exercise-induced lipolytic response was only suppressed to a minor
extent in all groups, clearly indicating a major contribution of non-adrenergic
components to SCAT lipolysis during exercise. Importantly, other (anti-)lipolytic
factors, such as the parathyroid hormone, cortisol and growth hormone are less
important during the type and duration of exercise as applied in the current study
[7, 16]. Noteworthy, atrial natriuretic peptide (ANP) may be responsible for the
exercise-induced increase in SCAT lipolysis in the present study, especially since
ANP is one of the major lipolytic hormones produced upon exercise [26, 52], next
to sympathetic nervous system activation. In line with our findings, it has previously
been shown that non-adrenergically mechanisms are involved in SCAT lipolysis,
accounting for ~65% of the exercise-mediated lipolysis in young healthy lean and
overweight men [15, 16]. Previously, a lower in vivo ANP responsiveness in SCAT
of overweight (31) and obese (30) individuals was observed. In line, the non-
adrenergically regulation of SCAT lipolysis has been reported to be more
pronounced in young healthy overweight as compared to lean men [16],
suggesting that ANP-mediated SCAT lipolysis is particularly important in the
overweight and obese state. Interestingly, in contrast to obese IS individuals, we
found that SCAT lipolysis in obese IR men was not affected by local α-/β-
adrenergic blockade, which may propose an interaction between SCAT
adrenergically-mediated lipolysis and whole-body insulin resistance. These findings
support a catecholamine-resistance phenotype of the SCAT during exercise
especially in the obese IR state. Therefore, a major role for ANP in SCAT lipolysis
during exercise can be suggested, which is sustained in the obese IR state.
To ensure adequate fatty acid delivery to the working skeletal muscles during
exercise, the sustained ANP-mediated lipolytic response, the adrenergically-
mediated lipolysis and a blunted insulin- and lactate-mediated inhibition of lipolysis,
might have contributed to the more pronounced SCAT lipolysis as observed in the
obese IS men. Because most of the anti-lipolytic action of insulin is mediated
through stimulation and activation of cellular phosphodiesterase-3B [53, 54], which
degrades cAMP, attenuation of the ANP-related SCAT lipolysis by insulin is
unlikely, since the latter is cGMP-mediated [27]. Likewise, it has been shown that
NP receptor expression (both functional NPRA and scavenging NPRC) in SCAT
associates with whole-body insulin sensitivity [55].
The 12-week exercise intervention increased insulin sensitivity in the obese IR
group. While plasma glycerol, FFA, lactate, ANP and adrenalin concentrations
were significantly reduced after the intervention, only minor reductions were
observed for circulating glucose, insulin and noradrenalin concentrations. However,
local exercise-induced abdominal SCAT lipolysis was not improved following the
exercise intervention. Moreover, the efficiency of local α-/β-adrenergic blockade
was not affected by the exercise intervention. Together, these data suggest that

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even after a substantial improvement in metabolic profile (as indicated by our

systemic responses) and body composition after a 12-week exercise intervention,
lipolytic disturbances remain unaffected in SCAT of obese IR individuals.
Optimized therapies are warranted to achieve enhancements in the regulation of
SCAT lipolysis, especially in metabolically compromised individuals.
Although evidence suggests that exercise training induces beneficial changes in
SCAT insulin sensitivity [56], SCAT adrenergic sensitivity [56-58] as well as ANP-
mediated lipolysis [59], future mechanistic studies are needed to obtain a better
understanding of the hormonal (exercise-induced) lipolytic regulation in lean and
obese individuals with a different degree of insulin sensitivity. Unfortunately, a
NPRA receptor agonist/antagonist for use in humans is currently unavailable,
which hampers strong conclusions about the physiological role of ANP in human
AT lipolysis in vivo.
In conclusion, this study is the first to report the integrated physiological role of
adrenergically- and non-adrenergically-mediated SCAT lipolysis during low-
intensity endurance-type exercise in middle-aged lean IS, obese IS and obese IR
men, and the effects of a 12-week supervised exercise intervention on these
processes in obese individuals. The present data demonstrate a major role for non-
adrenergically regulated lipolysis in SCAT during low-intensity exercise, likely
involving ANP-mediated lipolysis. Furthermore, the exercise training intervention
was not accompanied by changes in SCAT lipolysis in obese IR individuals,
regardless of improvements in metabolic profile and body composition.

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SUPPLEMENTARY MATERIAL

SUPPLEMENTARY FIGURE S1. Fasting and exercise-induced whole-body energy

expenditure and substrate oxidation before and after exercise intervention.

Indirect calorimetry was performed during resting conditions and during an acute exercise bout of
moderate intensity. Mean O2-consumption and CO2-production over 10 min were used for calculations
of respiratory quotient (A), energy expenditure and substrate oxidation during fasting (B + D) and during
exercise (C + E). Post intervention data are added for both obese groups (A-E). CHO, carbohydrates;
EE, energy expenditure; IR, insulin resistant; IS, insulin sensitive. Data are presented as mean± SEM.

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226
 Supplementary Table S1. Mean plasma and serum concentrations of systemic glycerol, insulin, FFA, glucose, lactate,
ANP, adrenalin and noradrenalin at rest, during low-intense exercise and recovery in obese insulin sensitive, obese insulin
resistant and lean individuals
Obese insulin Obese insulin
Lean (n= 10) sensitive (n= 10) P (vs lean) resistant (n= 10) P (vs lean) P (vs OB IS) P (ANOVA)

GLYCEROL
Baseline (μmol/l) 70.5 ± 4.8 81.8 ± 7.5 0.580 77.5 ± 5.5 1.000 1.000 0.416
Exercise (AUC0-60) (μmol/l*60min) 136.73 ± 12.5 163.2 ± 19.4 0.978 168.48 ± 22.8 0.722 1.000 0.448
Recovery (AUC75-105) (μmol/l*45min) 107.2 ± 6.5 124.2 ± 10.4 1.000 157.0 ± 28.4 0.183 0.660 0.159
INSULIN
Baseline (μU/ml) 7.0 ± 0.6 9.4 ± 1.1 0.563 16.9 ± 1.8 <0.001 0.001 <0.001
Exercise (AUC0-60) (μU/ml*60min) 7.4±0.64 9.3±1.2 0.791 15.0±1.5 <0.001 0.005 <0.001
Recovery (AUC75-105) (μU/ml*45min) 8.5 ± 1.0 11.7 ± 1.2 0.209 21.43 ± 1.3 <0.001 <0.001 <0.001
FFA
Baseline (μmol/l) 451.1 ± 30.9 528.4 ± 58.3 0.658 556.7 ± 36.3 0.292 1.000 0.224
Exercise (AUC0-60) (μmol/l*60min) 466.4 ± 42.6 555.2 ± 56.0 0.628 530.9 ± 46.9 1.000 1.000 0.425
Recovery (AUC75-105) (μmol/l*45min) 600.7±37.2 777.6 ± 63.8 0.189 864.5±81.9 0.019 1.000 0.020
GLUCOSE
Baseline (mmol/l) 5.2 ± 0.1 5.0 ± 0.1 0.722 5.1 ± 0.1 1.000 1.000 0.488
Exercise (AUC0-60) (mmol/l*60min) 5.3 ± 0.1 5.1 ± 0.1 1.000 5.3 ± 0.2 1.000 1.000 0.679
Recovery (AUC75-105) (mmol/l*45min) 5.3 ± 0.1 5.3 ± 0.2 1.000 5.3 ± 0.1 1.000 1.000 0.975
LACTATE
Baseline (mmol/l) 0.9 ± 0.1 0.9 ± 0.0 1.000 1.0 ± 0.1 0.628 0.600 0.339

effects were tested with a one-way ANOVA with Bonferroni post-hoc.

Exercise (AUC0-60) (mmol/l*60min) 1.0 ± 0.7 1.1 ± 0.1 0.100 1.4 ± 0.1 0.044 0.313 0.044
Recovery (AUC75-105) (mmol/l*45min) 0.9 ± 0.1 1.0 ± 0.1 0.730 1.2 ± 0.1 0.057 0.679 0.061
ANP
Baseline (pg/ml) 43.8 ± 5.2 41.7 ± 3.2 1.000 36.2 ± 4.0 0.548 0.793 0.356
Exercise (AUC0-60) (pg/ml*60min) 47.2 ± 4.8 45.9 ± 3.5 1.000 39.2 ± 2.2 0.452 0.680 0.300
Recovery (AUC75-105) (pg/ml*45min) 42.7 ± 4.4 44.0 ± 2.3 1.000 37.5 ± 3.2 0.864 0.616 0.390
ADRENALIN
Baseline (pg/ml) 16.4 ± 3.8 26.7 ± 6.5 0.561 19.4 ± 4.0 1.000 1.000 0.376
Exercise (AUC0-60) (pg/ml*60min) 54.0 ± 5.7 76.2 ± 11.2 0.233 58.0 ± 8.1 1.000 0.429 0.171
Recovery (AUC75-105) (pg/ml*45min) 35.2 ± 3.2 48.2 ± 9.0 0.537 39.4 ± 5.9 1.000 1.000 0.380
NORADRENALIN

AUC75-105 (area under the curve during the recovery period (from timepoint 75 until 105min)). Group
Data are mean ± SEM. AUC0-60 (area under the curve during exercise (from timepoint 0 till 60min)),
Chapter 7

227
Baseline (pg/ml) 492.6 ± 84.7 437.1 ± 91.4 1.000 430.3 ± 42.2 1.000 1.000 0.816
Exercise (AUC0-60) (pg/ml*60min) 1107.1 ± 80.8 1095.6 ± 83.1 1.000 1007.3 ± 110.6 1.000 1.000 0.708
Recovery (AUC75-105) (pg/ml*45min) 610.1 ± 70.9 539.8 ± 48.3 1.000 580.2 ± 56.1 1.000 1.000 0.725
 Supplementary Table S3. Interstitial glucose and lactate concentrations in the control probe and the
probe containing phentolamine and propranolol in the obese insulin sensitive, obese insulin resistant
and lean individuals.
Obese Obese
Lean ANOVA
insulin sensitive insulin resistant
(n=10) P Group P Treatment P Treatment*Group
(n= 10) (n= 10)
GLUCOSE
Baseline (mmol/l) 0.081 0.622 0.877
Control 3.62 ± 0.31 2.88 ± 0.39 2.56 ± 0.48
α/β blocker 3.72 ± 0.45 2.86 ± 0.31 2.82 ± 0.23
Exercise (AUC0-60) 0.811 0.194 0.655
Control 4.30 ± 0.36 4.36 ± 0.73 4.60 ± 1.06
α/β blocker 4.24 ± 0.55 3.43 ± 0.38 3.93 ± 0.47
Recovery (AUC75-120) 0.925 0.624 0.482
Control 3.38 ± 0.30 3.45 ± 0.50 3.65 ± 0.92
α/β blocker 3.68 ± 0.41 3.32 ± 0.37 3.02 ± 0.16
LACTATE
Baseline (mmol/l) 0.121 0.091 0.709
Control 1.79 ± 0.35 0.98 ± 0.16 1.80 ± 0.60
α/β blocker 1.37 ± 0.37 0.78 ± 0.13 1.08 ± 0.14
Exercise (AUC0-60) 0.354 0.078 0.449
Control 2.63 ± 0.58 1.79 ± 0.41 3.16 ± 0.95
α/β blocker 2.36 ± 0.62 1.46 ± 0.34 1.94 ± 0.32
Recovery (AUC75-120) 0.349 0.234 0.558
Chapter 7

Control 2.70 ± 0.64 1.62 ± 0.45 2.42 ± 0.90

α/β blocker 2.65 ± 0.84 1.38 ± 0.36 1.52 ± 0.16

228
Data are mean ± SEM. Control (probe containing Ringer), α/β blocker (probe containing Phentolamine
+ Propranolol). P-values were calculated via a 2-way repeated ANOVA.
 Supplemental Table S2. Interstitial glycerol concentrations and ethanol ratio in the control probe and the probe containing
phentolamine and propranolol in obese insulin sensitive, obese insulin resistant and lean individuals
ANOVA
Obese Obese
Lean insulin sensitive insulin resistant
P P P
(n=10) (n= 10) (n= 10)
Group Treatment Treatment*Group

GLYCEROL

Baseline (μmol/l) 0.279 0.266 0.674

Control 311.4 ± 33.7 403.6 ± 41.1 338.9 ± 46.0

α/β blocker 345.8 ± 42.7 404.8 ± 49.2 407.8 ± 41.5

Exercise (AUC0-60) 0.012 0.883 0.335

Control 537.2 ± 71.0 973.7 ± 74.9 721.6 ± 79.4

α/β blocker 570.5 ± 110.5 851.1 ± 116.4 839.7 ± 123.5

Recovery (AUC75-120) 0.014 0.894 0.348

Control 466.8 ± 69.2 909.6 ± 77.4 624.3 ± 82.7

α/β blocker 519.5 ± 99.3 803.8 ± 111.1 699.0 ± 118.7

ETHANOL RATIO

Baseline 0.003 0.037 0.190

Control 0.44 ± 0.05 0.66 ± 0.03 0.64 ± 0.05

α/β blocker 0.56 ± 0.05 0.67 ± 0.05 0.69 ± 0.03

Exercise 0.042 0.365 0.501

Control 0.42 ± 0.05 0.60 ± 0.05 0.59 ± 0.05

α/β blocker 0.50 ± 0.05 0.59 ± 0.05 0.60 ± 0.05

Recovery 0.003 0.025 0.620

Control 0.44 ± 0.04 0.65 ± 0.05 0.66 ± 0.05

α/β blocker 0.53 ± 0.04 0.69 ± 0.05 0.71 ± 0.05

Data are expressed as mean ± SEM. Control (probe containing Ringer), α/β blocker (probe containing Phentolamine + Propranolol). P-values were calculated via a
2-way repeated ANOVA.
Chapter 7

229
Chapter 7

230
 Supplementary Table S4. Mean plasma and serum concentrations of circulating glycerol, insulin, FFA, glucose, lactate, ANP, adrenalin and noradrenalin
at rest, during exercise and recovery, before and after 12 weeks of exercise training in the obese insulin sensitive and obese insulin resistant subjectss

Obese insulin sensitive Obese insulin resistant ANOVA

Pre Post Pre Post P Time P Group P Time*Group

GLYCEROL
Baseline (μmol/l) 74.0 ± 5.0 64.2 ± 4.8 79.9 ± 5.5 74.8 ± 6.4 0.100 0.235 0.593
Exercise (AUC0-60) 157.3 ± 22.7 150.3 ± 12.7 175.7 ± 24.1 144.0 ± 23.6 0.026 0.730 0.500
(μmol/l*60min)
Recovery (AUC75-105) 118.9 ± 12.7 124.4 ± 6.6 164.3 ± 30.7 122.1 ± 19.3 0.316 0.405 0.198
(μmol/l*45min)
INSULIN
Baseline (μU/ml) 9.5 ± 1.4 9.4 ± 1.3 16.9 ± 2.0 14.3 ± 1.3 0.196 0.008 0.263
Exercise (AUC0-60) 9.2 ± 1.5 9.1 ± 1.2 14.8 ± 1.7 13.4 ± 1.3 0.313 0.021 0.393
(μU/ml*60min)
Recovery (AUC75-105) 11.6 ± 1.6 13.8 ± 1.5 21.6 ± 1.5 19.4 ± 2.5 0.981 0.003 0.206
(μU/ml*45min)
FFA
Baseline (μmol/l) 506.2 ± 48.5 382.2 ± 30.2 571.3 ± 37.1 476.5 ± 41.1 0.016 0.060 0.723
Exercise (AUC0-60) 533.0 ± 55.4 436.7 ± 42.6 540.5 ± 51.3 439.0 ± 47.7 0.040 0.930 0.954
(μmol/l*60min)
Recovery (AUC75-105) 759.0 ± 70.1 698.0 ± 38.5 892.5 ± 86.1 726.4 ± 76.1 0.034 0.401 0.296
(μmol/l*45min)
GLUCOSE
Baseline (mmol/l) 5.0 ± 0.1 5.3 ± 0.2 5.1 ± 0.1 5.1 ± 0.1 0.030 0.671 0.021
Exercise (AUC0-60) 5.2 ± 0.1 5.3 ± 0.1 5.3 ± 0.2 5.1 ± 0.1 0.973 0.790 0.202
(mmol/l*60min)
Recovery (AUC75-105) 5.4 ± 0.3 5.4 ± 0.1 5.3 ± 0.2 5.1 ± 0.1 0.564 0.437 0.564
(mmol/l*45min)
LACTATE
Baseline (mmol/l) 0.9 ± 0.0 0.8 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 0.335 0.322 0.824
Exercise (AUC0-60) 1.1 ± 0.1 0.9 ± 0.1 1.4 ± 0.1 1.0 ± 0.1 0.016 0.127 0.609

effects were tested with a one-way ANOVA with Bonferroni post-hoc.

(mmol/l*60min)
Recovery (AUC75-105) 1.0 ± 0.1 0.9 ± 0.1 1.2 ± 0.1 1.0 ± 0.1 0.078 0.392 0.606
(mmol/l*45min)
ANP
Baseline (pg/ml) 39.7 ± 3.5 33.8 ± 3.6 38.8 ± 3.5 37.5 ± 3.5 0.042 0.769 0.162
Exercise (AUC0-60) 44.6 ± 4.7 38.1 ± 5.2 41.1 ± 2.5 39.1 ± 3.5 0.043 0.819 0.256
(pg/ml*60min)
Recovery (AUC75-105) 39.9 ± 2.8 36.2 ± 5.0 39.2 ± 3.0 37.6 ± 3.8 0.105 0.946 0.530
(pg/ml*45min)
ADRENALIN
Baseline (pg/ml) 29.4 ± 6.8 15.8 ± 2.5 21.9 ± 5.6 19.7 ± 5.4 0.155 0.773 0.295
Exercise (AUC0-60) 85.6 ± 7.1 62.9 ± 10.4 58.0 ± 9.2 48.1 ± 7.2 0.029 0.057 0.354
(pg/ml*60min)
Recovery (AUC75-105) 55.0 ± 6.9 36.6 ± 5.0 36.2 ± 6.7 24.4 ± 3.6 0.007 0.045 0.483
(pg/ml*45min)
NORADRENALIN

AUC75-105 (area under the curve during the recovery period (from timepoint 75 until 105min)). Group
Chapter 7

231
Data are mean ± SEM. AUC0-60 (area under the curve during exercise (from timepoint 0 till 60min)),
Baseline (pg/ml) 452.1 ± 118.1 395.6 ± 28.2 442.5 ± 45.8 400.8 ± 41.7 0.468 0.973 0.912
Exercise (AUC0-60) 1078.5 ± 92.2 1002.6 ± 93.3 1043.1 ± 118.7 1045.6 ± 106.2 0.875 0.822 0.534
(pg/ml*60min)
Recovery (AUC75-105) 519.6 ± 50.7 552.8 ± 68.1 584.7 ± 63.4 608.1 ± 56.8 0.613 0.377 0.930
(pg/ml*45min)
CHAPTER 8
GENERAL DISCUSSION
Chapter 8

The prevalence of obesity has increased enormously over the last decades and is
associated with an increased risk for metabolic impairments and chronic diseases
such as insulin resistance [1], type 2 diabetes [2] and cardiovascular diseases [3].
Obesity results from a chronic positive energy balance that leads to an increased
amount of adipose tissue mass. However, adipose tissue mass per se does not
seem to be the most important contributor to the development of obesity-related
disorders. Body fat distribution and adipose tissue dysfunction play a more
prominent role in the determination of cardiometabolic health [4, 5]. Therapies to
reduce obesity and related comorbidities can include dietary manipulation (as
extensively discussed in Chapter 2), physical activity strategies and a
pharmacological approach.
There is evidence to suggest that both the renin-angiotensin system (RAS) and the
natriuretic peptide (NP) system can influence cardiometabolic risk. Indeed, several
RAS components, which are also present in different key metabolic organs such as
the adipose tissue, skeletal muscle and the liver, are increased in obesity and
insulin resistance [6-9]. Also, reduced circulating NP concentrations have been
observed in obesity and type 2 diabetes [10]. An increased RAS activity [6] and
reduced NP concentrations [10] have detrimental metabolic effects and may
increase disease progression [11-15]. Recently, a novel dual acting drug,
sacubitril/valsartan, has been developed that facilitates the beneficial effects of the
NP system, while inhibiting the detrimental effects of the RAS [16]. This
combination therapy may have beneficial synergistic effects [17, 18] with respect to
disease progression. Indeed, sacubitril/valsartan has been shown to be superior in
reducing the risks of cardiovascular death or hospitalization for heart failure as
compared to monotherapy with enalapril [19]. Due to the common
pathophysiological impairments in individuals with cardiovascular and several
metabolic diseases (e.g. reduced oxidative capacity, altered lipolysis, increased
inflammation, insulin resistance), treatment with sacubitril/valsartan could induce
clinical benefits in both patients with cardiovascular and/or metabolic diseases.
However, it remains to be established whether sacubitril/valsartan may also have
(superior) beneficial metabolic effects as compared to other blood pressure
lowering agents.
Beside a pharmacological approach, changes in lifestyle are effective in preventing
the development of type 2 diabetes and related cardiometabolic complications [20,
21]. Increasing physical activity levels lead to increased energy expenditure,
improved physical fitness and contribute to an improved metabolic health [22, 23],
which could be attributable to changes in adipose tissue metabolism. Although
several rodent studies suggest that exercise training may improve adipose tissue
metabolism and function [24], human data are limited and need further
investigation [24-26].
Therefore, in the present thesis, the metabolic effects of pharmacological treatment
with sacubitril/valsartan, as well as exercise-training induced effects on adipose
tissue metabolism and the metabolic profile were investigated. In this chapter, our
findings will be discussed and put into a broader perspective.

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 Chapter 8

PHARMACOLOGICAL TREATMENT WITH SACUBITRIL/VALSARTAN

Combination therapies that simultaneously target more than one biological pathway
or mechanism may be more effective in reducing disease progression because of
additional and/or synergistic effects as compared to monotherapies [17, 18].
Therefore, due to common involved pathways, it is tempting to postulate that
combination therapy using sacubitril/valsartan through its distinct mechanisms of
action, may target risk factors for both cardiovascular (e.g. hypertension) and
metabolic diseases such as an impaired lipid mobilization, lipid oxidation and
substrate utilization, as will be discussed below.

Effects on insulin sensitivity

In Chapter 3, we investigated the effects of 8 weeks treatment with
sacubitril/valsartan in obese hypertensive patients on peripheral insulin sensitivity.
Compared to the metabolically neutral comparator, the calcium antagonist
amlodipine, peripheral insulin sensitivity was significantly improved after
sacubitril/valsartan, independent of changes in body weight or waist circumference.
The increased peripheral insulin sensitivity may be due to valsartan-induced AT1-
receptor inhibition, increased availability of neprilysin (NEP) substrates or both
mechanisms combined. Unfortunately, the present study design does not allow any
conclusions regarding the separate effects of valsartan and sacubitril on peripheral
insulin sensitivity.
The improvement in peripheral insulin sensitivity following sacubitril/valsartan is in
line with most [27-31] but not all [32] previously performed human studies using
valsartan treatment. Importantly, however, the latter study [32] investigated the
effects of a lower dosage of valsartan (40 mg/day vs. 400 mg/day in our study)
after 4 weeks of treatment and used surrogate markers of insulin sensitivity rather
than the hyperinsulinemic-euglycemic clamp, which is the golden-standard. Taken
together, our data and previous studies collectively show that valsartan improves
peripheral insulin sensitivity in humans.
Although it has been shown that interference with the RAS improves glucose
metabolism and reduces the incidence of type 2 diabetes [6], the valsartan-induced
effects may not solely explain the observed improvement in peripheral insulin
sensitivity with sacubitril/valsartan treatment in the present study and most likely
also NEP inhibition is involved. The effects of NEP inhibition per se on insulin
sensitivity have not been investigated previously, although it is known that a
reduced NP system activity is associated with insulin resistance and type 2
diabetes and that modulation of the NP system can be protective against the
development of insulin resistance [10].
An increased adipose tissue lipolysis, accompanied by an increased fatty acid
oxidation has beneficial effects on insulin sensitivity [33, 34]. Therefore, we
hypothesized that enhanced lipolysis and whole-body fat oxidation contributed to
the improved peripheral insulin sensitivity following sacubitril/valsartan treatment,
as described in Chapter 3 and Chapter 4, since it has been shown that activation
of NP signaling induces lipolysis [35-39], promotes fatty acid oxidation [36, 37, 40]
and enhances mitochondrial oxidative metabolism [41-43].
While monotherapy with valsartan increased peripheral insulin sensitivity by
approximately 10% in subjects with impaired glucose metabolism [27, 44],

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treatment with sacubitril/valsartan showed an increase of ~20% in insulin

sensitivity, with no marked effects of amlodipine treatment. Although the previous
study [27] used a lower dosage of valsartan as compared to our study (320 mg/day
vs. 400 mg per day, respectively), this difference is not likely to explain the
additional 10% increase in insulin sensitivity after treatment with
sacubitril/valsartan. Therefore, these data suggest that the NEP inhibitor sacubitril
has significantly contributed to the effects on peripheral insulin sensitivity that were
found in the present study.

Effects on subcutaneous adipose tissue lipolysis and substrate oxidation

Since adipose tissue metabolism and function and substrate oxidation are
important factors that contribute to peripheral insulin sensitivity, we investigated
sacubitril/valsartan-mediated effects on abdominal subcutaneous adipose tissue
lipolysis, whole-body lipolysis and substrate oxidation, both at rest and during an
acute bout of exercise.
After 8 weeks of treatment, there was a slight, but significant increase in abdominal
subcutaneous adipose tissue lipolysis at rest in the sacubitril/valsartan as
compared to the amlodipine group (Chapter 3). Surprisingly, the increased
subcutaneous adipose tissue lipolysis at rest did not translate into significant
changes in whole-body lipolysis, as measured with the stable isotope [1,1,2,3,3-
2
H]-glycerol (Chapter 3). Together, these findings either suggest that the increased
subcutaneous adipose tissue lipolysis might be paralleled by a reduced lipolysis in
other adipose tissue depots or, more likely, that the effects on lipolysis may not be
physiologically relevant. An increased adipose tissue lipolysis has beneficial effects
on insulin sensitivity, only when the mobilized fatty acids are oxidized at an
increased rate [33, 34].
In Chapter 3, we found no significant changes in total energy expenditure and
substrate oxidation in resting conditions. In Chapter 4, we examined the effects of
sacubitril/valsartan on abdominal subcutaneous and whole-body lipolysis during an
acute bout of aerobic exercise, which is known to stimulate lipolysis [45, 46], and
observed no significant differences in abdominal subcutaneous adipose tissue
lipolysis, whole-body lipolysis, energy expenditure and substrate oxidation between
groups. Thus, there were no differences between resting (Chapter 3) and exercise
(Chapter 4) conditions, except for a slightly higher abdominal subcutaneous
adipose tissue lipolysis under resting conditions (Chapter 3). Therefore, an
increased fat oxidation does not seem to contribute to the improved peripheral
insulin sensitivity following sacubitril/valsartan treatment.
Next, we examined the effects of sacubitril/valsartan on adipose tissue gene and
protein expression, as described in Chapter 5, and we found no significant
changes in the expression of genes and proteins of factors involved in lipolysis,
natriuretic peptide signaling and mitochondrial oxidative metabolism.
These results are in line with a previous study that did not show an increased
adipose tissue lipolytic gene and protein expression after 26 weeks of valsartan
treatment [44]. Furthermore, AT1-receptor blockade did not increase abdominal
subcutaneous adipose tissue lipolysis [47] and, therefore, it seems unlikely that
AT1-receptor blockade with sacubitril/valsartan treatment contributed to the mild
alteration in lipid mobilization. Rather, a NEP inhibition-mediated increased NP

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availability could have contributed to the observed effects on subcutaneous

adipose tissue lipolysis, since NP have been shown to affect adipose tissue lipid
mobilization and oxidation in lean and overweight men and women [35-40].
Taken together, alterations in adipose tissue lipolysis or whole-body substrate
oxidation at rest and during exercise do not seem to contribute to the
sacubitril/valsartan-induced improvement in peripheral insulin sensitivity. The
underlying mechanisms remain to be elucidated, but seem to be explained by
metabolic effects in other tissues (e.g. visceral adipose tissue, the liver or skeletal
muscle).

EXERCISE TRAINING INTERVENTIONS TO IMPROVE METABOLIC HEALTH

Regular physical exercise has beneficial effects on cardiometabolic health and
improves glucose tolerance, insulin sensitivity and circulating lipid concentrations
[48, 49]. These exercise-induced improvements in cardiometabolic risk profile have
largely been attributed to changes in skeletal muscle metabolism and function, but
physical exercise may also induce alterations in other metabolically active tissues,
including the adipose tissue. Indeed, several rodent studies suggest that exercise
training may improve adipose tissue metabolism and function [24], although the
evidence for this in humans is limited [24-26].

Effects on insulin sensitivity

In Chapter 6 and Chapter 7 of this thesis, we investigated the metabolic effects of
a 12-week supervised, combined endurance and resistance exercise training
program in sedentary, middle-aged overweight/obese men with or without
metabolic impairments (Chapter 6) and sedentary, middle-aged obese insulin
sensitive, obese insulin resistant and age-matched lean insulin sensitive men
(Chapter 7).
Both exercise training programs improved aerobic capacity, maximal power output
and maximal muscle strength, indicating that the supervised, progressive nature of
both programs was successful with respect to enhancement of physical fitness.
This enhanced physical fitness was accompanied by improved peripheral insulin
sensitivity, as determined using either a hyperinsulinemic-euglycemic clamp
(Chapter 6) or HOMA-IR (Chapter 7). These findings are in line with previous
studies showing an improved insulin sensitivity after 12 weeks of exercise in obese
individuals [50, 51] and could, among other mechanisms, be explained by an
altered body composition [52]. In Chapter 6, we observed a modest but significant
reduction in body fat mass (~0.7 kg) and body fat percentage (~0.6 %) in both
obese groups, without significant changes in fat-free mass. In Chapter 7, we
observed a significantly reduced fat mass (~2.1 kg in the obese insulin sensitive
group and ~1.9 kg in the obese insulin resistant group), body fat percentage (~1.6
% in the obese insulin sensitive and ~0.9 % in the obese insulin resistant group),
BMI and body weight, while fat-free mass remained unchanged and was not
different between groups. Previous data showed that increased fat-free mass (e.g.
skeletal muscle cross-sectional area) and improved skeletal muscle function (e.g.
skeletal muscle capillarization, oxidative capacity) are associated with improved
insulin sensitivity [53-56], while reduced fat-free mass induced insulin resistance

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Chapter 8

[57, 58]. However, our data suggest that peripheral insulin sensitivity can improve
without significant changes in fat-free mass.
Although exercise training significantly increased peripheral insulin sensitivity it did
not induce changes in adipose tissue and hepatic insulin sensitivity, as described
in Chapter 6. These findings are in contrast with previous rodent [59, 60] and
human [61] studies. The unaltered adipose tissue and hepatic insulin sensitivity
may be explained by the rather minor changes in fat mass loss. Nevertheless, our
data showed that exercise training beneficially improved physical fitness, body
composition and peripheral insulin sensitivity, irrespective of the baseline metabolic
status.

Effects on subcutaneous adipose tissue metabolism and adipokine expression

Subcutaneous adipocyte size is closely associated with adipose tissue function
and insulin resistance [4] and adipocyte hypertrophy is correlated with impairments
in adipose tissue metabolism [62]. Since marked weight loss is associated with
decreased adipocyte size in subcutaneous adipose tissue, which is accompanied
by improved insulin sensitivity [63-66], we investigated if exercise training could
alter adipocyte morphology, thereby contributing to an improved subcutaneous
adipose tissue metabolism and whole-body insulin sensitivity.
In Chapter 6, we found that physical exercise training did not change abdominal
subcutaneous adipocyte morphology, neither mean adipocyte size nor the
proportion of small and large adipocytes, in metabolically healthy obese and
metabolically compromised obese subjects. In contrast, a previous study
demonstrated a decreased adipocyte size in young men, but not in women, after
20 weeks of endurance training [67]. Importantly, in the latter study, a more
pronounced reduction in body weight (~3.0 kg) was achieved. Since we observed a
weight reduction of only ~0.7kg in Chapter 6, it is likely that a more pronounced
decrease in adipose tissue mass is needed to induce significant changes in
adipocyte morphology and, consequently, adipose tissue metabolism. While
exercise training duration was comparable between the exercise training studies in
chapter 6 and 7, we observed a greater reduction in fat mass in Chapter 7, which
is likely due to the higher training volume. In Chapter 6, subjects performed three
training sessions per week, of which two sessions consisted of 30min cycling at
70% VO2max and one training session included resistance training at 60% of 1RM
(3 sets of 10 reps). In Chapter 7, the participants performed three training sessions
per week of which all consisted of 45min cycling at 65% VO 2max combined with
resistance training at 65% of 1RM (4 sets of 10 reps). Since a greater reduction in
body weight (fat mass) induces more favorable metabolic effects [68, 69], the
greater reduction in fat mass, as observed in Chapter 7, could have altered
adipocyte morphology and improved adipose tissue metabolism, although this was
not investigated.
However, it remains to be established how much weight loss is needed to induce a
significant reduction in adipocyte size and which exercise training modality is most
optimal [63, 68, 70].

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 Chapter 8

Since adipocyte hypertrophy is associated with impairments in adipose tissue

metabolism [4, 62], the minor reduction in fat mass and the lack of changes in
adipocyte morphology seems to explain the unaltered expression of genes and
proteins related to adipose tissue metabolism, as reported in Chapter 6. More
specific, there were no exercise training-induced changes in expression of genes
involved in inflammation (e.g. TNF-a, IL-6, MCP-1 and CD68), which have been
implicated to induce insulin resistance. Additionally, gene expression of the
adipokines leptin and adiponectin, which are related to adipocyte size [71, 72], also
remained unchanged after exercise training. These observations are in line with
most other exercise training studies in obese subjects [73-78]. Furthermore, an
adequate mitochondrial function is essential to maintain adipose tissue function,
glucose homeostasis [79] and protects against insulin resistance and type 2
diabetes [80]. Browning of white adipose tissue may increase cellular energy
expenditure [81, 82], thereby improving whole-body glucose homeostasis and
insulin sensitivity in humans [83]. We did not found changes in browning markers
after training, which is in line with studies in lean [84, 85] and overweight [85, 86]
subjects, but in contrast to rodent data [41, 87]. Furthermore, the exercise training
intervention did not induce changes in mitochondrial gene expression and did not
alter mitochondrial oxidative phosphorylation protein expression, which was in
accordance with most [78] [84, 86, 88], but not all previous studies [89, 90]. When
investigating adipose tissue lipolysis, which is one of the characteristics of adipose
tissue dysfunction and relates to peripheral insulin resistance [91], we observed no
major significant exercise training-induced effects on expression of genes related
to lipolysis. Furthermore, ex vivo basal and maximal lipolysis, as well as β 2-
adrenergic sensitivity of lipolysis in mature human adipocytes, were not affected by
the exercise training program. The latter observation is in agreement with a
previous study in obese non-diabetic men [92] and may be explained by the minor
change in fat mass loss, since it has previously been demonstrated that substantial
2
weight loss (BMI: -6.1 kg/m ) increased and normalized the sensitivity to
catecholamine-stimulated lipolysis in obese subjects [93].
Overall, Chapter 6 demonstrated that a 12-weeks supervised, progressive
exercise training intervention did not significantly affect abdominal subcutaneous
adipocyte morphology, adipose tissue gene and protein expression of markers
related to adipose tissue function, nor β2-adrenergic sensitivity in obese subjects,
irrespective of their baseline metabolic status.

Disturbances in subcutaneous adipose tissue (SCAT) lipolysis have been reported

in obese humans [46], including a reduced adrenergically-mediated lipolysis [94-
96]. On the other hand, non-adrenergically-mediated lipolysis in SCAT substantially
contributes to lipolysis during exercise in healthy young lean [38] and healthy
young overweight men [39]. Atrial natriuretic peptide (ANP) is the most potent
stimulator of human adipose tissue lipolysis [35] and one of the major lipolytic
hormones produced upon exercise [35, 97]. However, ANP responsiveness has
been shown to be impaired in middle-aged obese men with or without type 2
diabetes [98] and in middle-aged obese women and overweight men [99].
It has been shown that endurance exercise training can partly improve β-
adrenoceptor sensitivity and reduce anti-lipolytic α2-adrenoceptor sensitivity in
human SCAT [76, 100, 101], and can alleviate ANP-mediated lipolysis in

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Chapter 8

subcutaneous adipocytes in young, metabolically healthy overweight individuals

[102, 103]. However, it remained to be investigated if combined endurance and
resistance exercise training could improve ANP-mediated lipolysis in metabolically
compromised conditions.
In Chapter 7, we aimed to investigate the effects of local combined α- and β-
adrenergic blockade on SCAT lipolysis in obese insulin sensitive, obese insulin
resistant and age-matched lean insulin sensitive men. Therefore, we determined in
situ SCAT lipolysis at rest, during a single bout of low-intensity endurance exercise
and during recovery in age-matched obese insulin sensitive, obese insulin resistant
and lean insulin sensitive men in the presence or absence of combined α- and β-
adrenergic receptor blockade in abdominal SCAT. Next, the obese individuals
participated in a 12-week combined endurance and resistance exercise training
intervention to investigate whether exercise training was able to improve (non-)
adrenergic-mediated abdominal SCAT lipolysis in obese insulin resistant subjects.

Our findings indicated a major contribution of non-adrenergically mediated lipolysis

during exercise in abdominal SCAT of lean and obese individuals. Additionally, we
found a greater increase in extracellular glycerol concentration in SCAT during
exercise in obese insulin sensitive as compared to lean insulin sensitive
individuals. Although extracellular glycerol concentration is a reflection of lipolysis,
the higher exercise-induced lipolysis in the obese insulin sensitive group is likely
explained by the lower adipose tissue blood flow (ATBF) in the obese state, which
contributes to higher extracellular glycerol concentrations due to a lower removal of
glycerol from the adipose tissue [39, 104]. Local α-/β-adrenergic blockade
substantially reduced basal ATBF in lean but not in the obese individuals, which
suggests that adrenergic sensitivity of ATBF is reduced in the obese state, which
has been shown before [105, 106].
The exercise-induced SCAT lipolysis following combined α-/β-adrenergic blockade
was substantially reduced in obese insulin sensitive but not in lean insulin sensitive
or obese insulin resistant men. This might suggest differences in adrenergic
receptor expression and/or sensitivity. Previous data showed that the lipolytic
activity of the β-adrenergic receptors is attenuated in obese insulin resistant
individuals [95, 96, 107], while inhibitory α2-adrenoceptors become predominant on
adipocytes in the obese insulin resistant state [107, 108] and reduce exercise-
mediated SCAT lipolysis [105]. Additionally, obese insulin resistant individuals
often show lower plasma catecholamine (adrenaline, noradrenaline) responses to
physical exercise [109], although we did not find this in Chapter 7. Furthermore,
the blunted SCAT lipolysis in the obese insulin resistant individuals, as compared
to the obese insulin sensitive group, might also partly be explained by the
significantly higher insulin concentrations, as shown previously [110]. In addition,
increased plasma lactate concentrations, as observed in the obese insulin resistant
individuals, may have contributed to the reduced lipolytic response, since lactate
has been shown to inhibit lipolysis in mice [111] and human primary adipocytes in
vitro [112].
In all groups, we found a major contribution of non-adrenergic components in
SCAT lipolysis during low-intensity exercise, which is likely involving ANP-mediated
lipolysis. This observation is in line with previous studies, which showed that non-
adrenergically-mediated mechanisms are involved in SCAT lipolysis, accounting for

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~65% of the exercise-mediated lipolysis in young healthy lean and overweight men
[38, 39]. We found that SCAT lipolysis in obese insulin resistant men, compared to
the obese insulin sensitive group, was not affected by local α-/β-adrenergic
blockade, which may point toward an interaction between SCAT adrenergically-
mediated lipolysis and whole-body insulin resistance. These findings support a
catecholamine-resistant phenotype of the SCAT during exercise, especially in the
obese insulin resistant state.
The 12-week combined endurance and resistance exercise training intervention did
not improve exercise-induced abdominal SCAT lipolysis. Furthermore, the
efficiency of local α-/β-adrenergic blockade was not affected and these data
combined suggest that the lipolytic disturbances in SCAT of obese insulin resistant
individuals remain unaffected after 12 weeks of exercise training. This observation
is in contrast with previous studies that showed beneficial changes in SCAT insulin
sensitivity [61], SCAT adrenergic sensitivity [61, 92, 113] as well as ANP-mediated
lipolysis [114] after exercise training in obese subjects.
Taken together, the results described in Chapter 7 show a major role for non-
adrenergically-mediated lipolysis in SCAT during low-intensity exercise, likely
involving ANP-mediated lipolysis. Furthermore, our data suggest that even after a
substantial improvement in body composition, physical fitness and insulin
sensitivity, lipolytic disturbances remain unaffected in SCAT of obese insulin
resistant individuals after a 12-week exercise training intervention.

CONCLUSION AND FUTURE PERSPECTIVES

This thesis describes the effects of a pharmacological intervention as well as
physical exercise interventions to improve metabolic health in obese individuals,
with a focus on adipose tissue metabolism.

In Chapter 3, we found improved peripheral insulin sensitivity after 8 weeks of

combination therapy with sacubitril/valsartan in obese hypertensive subjects, which
was accompanied by an increased abdominal subcutaneous adipose tissue
lipolysis at rest. However, this finding did not translate into alterations in whole-
body lipolysis, energy expenditure and substrate oxidation. In Chapter 4, no
changes were found for these parameters during a single bout of moderate-
intensity endurance exercise. Therefore, it seems that sacubitril/valsartan had no
clinically relevant effects on adipose tissue and whole-body lipolysis and substrate
utilization. In line, in Chapter 5, no significant effects on gene and protein
expression of markers related to lipolysis, natriuretic peptide signaling and
mitochondrial oxidative metabolism were found. Therefore, we conclude that
treatment with sacubitril/valsartan improved peripheral insulin sensitivity, without
altering the abdominal subcutaneous adipose tissue metabolic phenotype and
substrate oxidation.
In Chapter 6, we showed that a 12-weeks supervised, progressive, combined
endurance and resistance exercise training program improved body composition,
physical fitness and peripheral insulin sensitivity in sedentary, middle-aged
overweight/obese men with or without metabolic impairments. However, no
significant effects on hepatic and adipose tissue insulin sensitivity, abdominal

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subcutaneous adipocyte morphology, adipose tissue gene and protein expression

of markers related to adipose tissue function, nor β 2-adrenergic sensitivity of
abdominal SCAT lipolysis were observed in obese subjects, irrespective of their
baseline metabolic status.
In Chapter 7, we showed a major contribution of non-adrenergically-mediated
lipolysis during exercise in SCAT of sedentary, middle-aged obese insulin
sensitive, obese insulin resistant and age-matched lean insulin sensitive men.
Furthermore, 12 weeks of supervised, combined endurance and resistance
exercise training did not significantly improve exercise-induced abdominal SCAT
lipolysis.

The outcomes of this thesis provide evidence for an improved metabolic health
after sacubitril/valsartan treatment and exercise training interventions, but several
questions and issues should be addressed in future research:

1. Since we used a combination drug, we cannot distinguish between the

individual effects of sacubitril (NEP inhibition) and valsartan (RAS blockade) on the
improved peripheral insulin sensitivity. It would be of interest to investigate the
effects of NEP inhibition compared to placebo, valsartan (at a similar dosage as
applied in this thesis), or a metabolically neutral blood pressure lowering agent
and/or sacubitril/valsartan, to elucidate whether the observed effects of
sacubitril/valsartan on peripheral insulin sensitivity are attributable to the slightly
higher valsartan concentration as compared to a previous study [27], or whether
the effects are attributable to the additional effects of increased neprilysin
substrates, such as NP concentrations.
2. We treated obese hypertensive patients with sacubitril/valsartan for 8
weeks, since literature reports show that 8 weeks of treatment is sufficiently long to
demonstrate metabolic effects of several drug classes used for the treatment of
hypertension and heart failure, including ACE inhibitors and ARBs [27, 115, 116].
However, it remains to be investigated whether metabolic effects would be more
pronounced after a more prolonged treatment period. Furthermore, the persistence
and durability of the metabolic effects after cessation of treatment also remains to
be established.
3. It remains to be established whether sacubitril/valsartan has effects on
other key metabolic organs (i.e. visceral adipose tissue, the liver and skeletal
muscle), which may underlie the observed improvement in peripheral insulin
sensitivity.
4. Future studies should investigate the exercise-mediated metabolic
responses in females, since there is a sex difference in metabolic regulation [117-
119], related to circulating sex hormones [120, 121]. Interestingly, there is a sex
difference with respect to circulating NP concentrations [122], and estrogen
administration in postmenopausal women increased circulating ANP
concentrations [123]. The sex dependent NP regulation might contribute to the
well-known differences in cardiovascular risk between men and women. Clearly,

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results obtained in a male population cannot simply be extrapolated to the female

population.
5. There is a large inter-individual variation in several metabolic health
outcomes following different interventions, including exercise training [124].
Metabolic phenotyping at baseline makes it possible to stratify subjects into
different subgroups and may improve the effectiveness of a particular intervention
in a specific subgroup of the population [125]. However, we do not find clear
evidence that obese individuals with a different metabolic phenotype at baseline,
differentially affected exercise training-induced study outcomes. Thus, based on
the findings described in this thesis, we cannot conclude that a subgroup-based
approach is more beneficial than a population-based approach. Therefore, studies
including more detailed metabolic phenotyping such as tissue-specific profiling are
needed not only to identify individuals or subgroups at increased risk of developing
metabolic diseases but also to design optimized prevention and treatment
strategies for specific subgroups of the population, and ultimately leading to
personalized exercise strategies.

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92. De Glisezinski I, Crampes F, Harant I, Berlan M, Hejnova J, Langin D, et al.
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123. Maffei S, Del Ry S, Prontera C, Clerico A. Increase in circulating levels of cardiac

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ADDENDA
Chapter 9

SUMMARY

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Obesity is associated with an increased risk for metabolic impairments and chronic
diseases, including insulin resistance, type 2 diabetes and cardiovascular
diseases. Strategies to reduce body weight and obesity-related comorbidities
include dietary (as discussed in Chapter 2), pharmacological and physical activity
interventions. This thesis describes the effects of a pharmacological intervention as
well as physical exercise interventions to improve metabolic health in obese
individuals, with a focus on adipose tissue metabolism .
An increased renin-angiotensin system activity and a lower activity of the natriuretic
peptide system have been linked to the development of type 2 diabetes and
cardiovascular disease. Combination therapy with sacubitril/valsartan, a combined
angiotensin receptor blocker (ARB) and neprilysin (NEP) inhibitor, facilitates the
beneficial effects of the natriuretic peptide system, while inhibiting the detrimental
effects of the renin-angiotensin system.

In Chapter 3, we performed a multi-centre, randomized, double-blind, double-

dummy, parallel-group study to assess the effects of 8 weeks treatment with
sacubitril/valsartan as compared to amlodipine on whole-body insulin sensitivity,
determined by a hyperinsulinemic-euglycemic clamp in 98 obese hypertensive
patients. We found that sacubitril/valsartan significantly improved peripheral insulin
sensitivity without affecting body weight or waist circumference. Furthermore,
abdominal subcutaneous adipose tissue lipolysis at rest was slightly but
significantly increased in the sacubitril/valsartan as compared to the amlodipine
group. Surprisingly, the increased subcutaneous adipose tissue lipolysis at rest did
not translate into significant changes in whole-body lipolysis, as measured using
2
the stable isotope [1,1,2,3,3- H]-glycerol. Moreover, we also found no significant
changes in total energy expenditure and substrate oxidation in resting conditions.

In Chapter 4, we extended the outcome of this multi-centre trial by investigating

the effects of sacubitril/valsartan on abdominal subcutaneous adipose tissue and
whole-body lipolysis as well as energy metabolism and substrate oxidation during a
single bout of moderate-intensity aerobic exercise, which is known to stimulate
lipolysis. We observed no significant effects of sacubitril/valsartan as compared to
amlodipine on abdominal subcutaneous adipose tissue lipolysis, whole-body
lipolysis, energy expenditure and substrate oxidation. Therefore, it seems that
sacubitril/valsartan had no physiological relevant effects on adipose tissue lipolysis,
whole-body lipolysis and substrate utilization.

To obtain more detailed insight into possible mechanisms underlying the findings
described in Chapters 3 and 4, we assessed the effects of sacubitril/valsartan on
abdominal subcutaneous adipose tissue gene expression patterns using
microarray analysis and determined adipose tissue protein expression profiles in
Chapter 5. We showed no significant changes in expression of genes and proteins
of factors involved in lipolysis, natriuretic peptide signalling and mitochondrial
oxidative metabolism.
Collectively, these data indicate that alterations in abdominal subcutaneous
adipose tissue lipolysis, whole-body lipolysis or whole-body substrate oxidation at
rest and during exercise, do not seem to contribute to the sacubitril/valsartan-

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induced improvement in peripheral insulin sensitivity. It remains to be established

whether sacubitril/valsartan has effects on other key metabolic organs, which may
underlie the observed improvement in peripheral insulin sensitivity.

Beside pharmacological therapy, changes in lifestyle are effective in preventing the

development of type 2 diabetes and related cardiometabolic complications. There
is some evidence, mainly from rodent studies, that exercise training may improve
adipose tissue function, thereby reducing obesity-related insulin resistance and
other comorbidities. However, human studies that investigated the effects of
exercise training on adipose tissue function are limited. Therefore, a second
objective of this thesis was to determine the effects of physical exercise training on
abdominal subcutaneous adipocyte morphology, adipose tissue gene and protein
expression of markers related to adipose tissue function and ex vivo adipocyte
lipolysis in metabolically healthy and metabolically compromised individuals.

In Chapter 6, we investigated the effects of a 12-weeks supervised, progressive,

combined endurance and resistance exercise training program on insulin sensitivity
and adipose tissue function in metabolically healthy and metabolically
compromised sedentary, middle-aged men. We found that 12 weeks of exercise
training improved body composition, physical fitness and peripheral insulin
sensitivity, as assessed by a two-step hyperinsulinemic-euglycemic clamp.
However, no significant effects on hepatic and adipose tissue insulin sensitivity,
abdominal subcutaneous adipocyte morphology, adipose tissue gene and protein
expression of markers related to adipose tissue function, nor β 2-adrenergic
sensitivity of abdominal subcutaneous adipose tissue lipolysis were observed in
obese subjects, irrespective of their baseline metabolic status. Importantly, since
we observed only a slight but significant decrease in fat mass, it is likely that a
more pronounced decrease in adipose tissue mass is needed to induce significant
changes in adipocyte morphology and, consequently, adipose tissue metabolism.

Also, since atrial natriuretic peptide (ANP) increases during exercise and plays an
important role in adipose tissue lipolysis, we investigated abdominal subcutaneous
adipose tissue (non-)adrenergically-mediated lipolysis before, during and after a
single bout of endurance exercise and after 12-weeks of exercise training in
metabolically healthy and metabolically compromised individuals in Chapter 7.
Therefore, we investigated the effect of local combined α- and β-adrenoceptor
blockade on local subcutaneous adipose tissue lipolysis at rest, during low-intensity
endurance-type exercise and during recovery from exercise in sedentary, middle-
aged obese insulin sensitive, obese insulin resistant and age-matched lean insulin
sensitive men. In addition, we investigated whether a 12-week supervised,
progressive, combined endurance and resistance exercise training improved the
metabolic profile in obese men and (non-)adrenergically-mediated abdominal
subcutaneous adipose tissue lipolysis in obese insulin resistant individuals. We
demonstrated a major contribution of non-adrenergically-mediated lipolysis during
exercise in all groups. Furthermore, we showed that the exercise training
intervention improved body composition, physical fitness and exercise-induced
changes in circulating free fatty acids, lactate and adrenalin concentrations in both
obese groups and insulin sensitivity in the obese insulin resistant group. However,

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this was not accompanied by changes in adrenergically- and non-adrenergically-

mediated lipolysis in the subcutaneous adipose tissue of obese insulin resistant
individuals. Together, these data suggest that even after a substantial improvement
in metabolic profile and body composition after a 12-week exercise intervention,
lipolytic disturbances remain unaffected in subcutaneous adipose tissue of obese
insulin resistant individuals. Optimized therapies are warranted to achieve
enhancements in the regulation of subcutaneous adipose tissue lipolysis,
especially in metabolically compromised individuals.

Combined, these data indicate that even after exercise-induced improvements in

body composition, physical fitness and peripheral insulin sensitivity, changes in
abdominal subcutaneous adipose tissue metabolism and function are lacking and
will most likely occur only after a more pronounced decrease in adipose tissue
mass. Currently, it remains to be established which exercise training duration and
modality is most optimal to induce beneficial effects in abdominal subcutaneous
adipose tissue.

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VALORIZATION

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SOCIAL RELEVANCE
The worldwide prevalence of obesity has increased enormously over the last
decades and numbers are still increasing every year. According to the World
Health Organization, 13% of the world’s adult population (11% of men and 15% of
women) was obese in 2014, while in the same year, obesity affected around 18,5%
of men and 19% of woman in The Netherlands [1]. If post-2000 trends continue,
this global prevalence of obesity is suggested to reach 18% in men and 21% in
women by 2025. Obesity is associated with an increased risk of developing chronic
diseases, including insulin resistance [2], type 2 diabetes [3], cardiovascular
diseases [4] and certain types of cancer [5, 6]. To reduce these obesity related
comorbidities, nowadays, millions of people are in need of medication, such as
glucose-, cholesterol- and/or blood pressure lowering medication, and surgical
treatments such as gastric bypass or cardiovascular surgery. Since obesity is a
major public health issue and one of the most important risk factors for the
development of metabolic diseases, it is clear that the increasing obesity
prevalence has major socioeconomic consequences [7]. National and international
guidelines recommend changes in modifiable lifestyle characteristics, such as diet
and physical activity for both prevention and management of metabolic diseases
[8]. While weight loss has been shown to be effective in reducing disease risk,
implementation of the recommended lifestyle in the long-term is often hard to
maintain by the majority of people. Therefore, to reduce the incidence of obesity
and thereby partly improving global health, it is important to obtain better insights in
the development and treatment of obesity and related metabolic diseases and to
implement new treatment strategies. The results described in this thesis contribute
to a better understanding of the role of adipose tissue metabolism in
cardiometabolic health and obesity, and provide leads for possible treatment
strategies to reduce or prevent obesity and cardiometabolic complications.

TARGET GROUPS

Scientific community
The results described in this thesis have and will become available to the scientific
community via publication of scientific articles in international peer-reviewed
journals. Additionally, results have been presented at (inter)national conferences to
scientists as well as physicians, healthcare professionals and dieticians, working in
the fields of obesity, diabetes and metabolism.

Industry
A part of this thesis was accomplished by the close collaboration between
academia and industry and research outcomes are of valuable information to the
academic community and both the nutritional and the pharmaceutical industry. The
industrial partners can translate the research outcomes to develop improved or
novel treatment strategies or products that help to prevent or reduce the
prevalence of obesity and obesity-related complications. More specific, the
nutritional industry can translate the results from chapter 2 in defining new

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nutritional targets. The pharmacological industry can use the results from this
thesis to develop and/or implement new or improved pharmacological therapies or
to expand the rationale for prescribing certain cardiovascular drugs in metabolically
compromised conditions, since in our studies combination therapy with
sacubitril/valsartan was shown to improve both cardiovascular and metabolic risk
factors.

Health care professionals

Healthcare professionals (e.g. dieticians, physiotherapists and physicians) play an
important role in stimulating a healthy lifestyle among patients and people at
increased risk of developing obesity and related complications. On the other hand,
physicians prescribe drugs to reverse for example cardiovascular risk (e.g.
hypertension) or to improve glucose metabolism and insulin sensitivity. Although
the results from this thesis do no provide direct guidelines for healthcare, they
provide better insight in metabolic parameters that can be targeted through certain
interventions, applied by physicians and other healthcare professionals.
Furthermore, this thesis might provide an indication for more targeted prevention
programs in high risk groups of the population (i.e. people with both cardiovascular
and metabolic risk factors) and it gives more insight in the mechanism of action of a
cardiovascular drug (i.e. sacubitril/valsartan) that also has effects on insulin
sensitivity and which therefore may alter the rationale for prescription.

ACTIVITIES AND PRODUCTS

In this thesis, we have put forward fatty acid metabolism-related pathways in
several metabolically active organs that can be targeted by dietary interventions,
thereby improving whole-body glucose metabolism and insulin sensitivity. The
results from the nutritional review can potentially lead to novel functional foods or
food supplements (e.g. pre- and probiotics, polyphenols, plant sterols, improved
dietary fat quality). However, before these novel products will become available on
the market, more scientific research is necessary to confirm mechanisms, safety
and health benefits in individuals at increased risk of developing cardiometabolic
diseases.

The results from the pharmacological intervention with sacubitril/valsartan, which

has recently been approved by the U.S. Food and Drug Administration (FDA) and
the European Medicines Agency (EMA) for the treatment of heart failure [9],
showed that the combination therapy has a positive effect on cardiovascular and
metabolic risk factors. We showed that treatment with sacubitril/valsartan, as
compared to the metabolically neutral calcium antagonist amlodipine, improved
peripheral insulin sensitivity in obese hypertensive patients. This knowledge will be
immediately used by the pharmaceutical industry. Moreover, sacubitril/valsartan
may be a promising alternative for the treatment of hypertension in patients who
are at increased risk for developing chronic metabolic diseases (e.g. hypertensive
individuals with impaired glucose metabolism).

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The exercise training intervention studies showed that exercise training beneficially
affects body composition and physical fitness and is effective to improve obesity-
related disturbances like whole-body insulin resistance. Although 12 weeks of
exercise training induced a slight but significant reduction in fat mass, no significant
changes in abdominal subcutaneous adipocyte morphology, adipose tissue
function, and abdominal subcutaneous adipose tissue lipolysis were observed in
obese subjects, irrespective of their baseline metabolic status. Furthermore, we
showed that 12 weeks of exercise training did not improve disturbances in
subcutaneous adipose tissue lipolysis in obese insulin resistant individuals. It
seems that a more pronounced decrease in adipose tissue mass is needed to
induce significant changes in adipose tissue metabolism. Currently, it remains to be
established which exercise training duration and modality is most optimal to induce
beneficial effects in abdominal subcutaneous adipose tissue.

INNOVATION & IMPLEMENTATION

All results described in this thesis are novel findings and have, partly, been
performed at the Department of Human Biology and Movement Sciences of
Maastricht University Medical Center+ and in close collaboration with other
universities and industrial partners within The Netherlands and Europe, using state-
of-the-art methodologies for both in vivo and ex vivo analyses.

An attractive approach, as described in this thesis, is the application of combination

therapy, which simultaneously targets more than one biological pathway or
mechanism and therefore may be more effective in reducing disease progression
because of additional and/or synergistic effects as compared to monotherapies [10]
[11]. With respect to the pharmacological treatment with the combination drug,
sacubitril/valsartan, this thesis is the first to describe the beneficial metabolic
effects (i.e. improved insulin sensitivity) in obese hypertensive patients. However,
to implement these results in treatment strategies for the hypertensive population,
more research is necessary to unravel the underlying mechanisms in different
metabolic tissues (e.g. skeletal muscle) and long-term health outcomes of therapy
with the combinational drug sacubitril/valsartan.

The innovative aspect of the exercise training interventions, was the investigation
of exercise training-induced effects on abdominal subcutaneous adipose tissue,
since these studies have mainly been performed in rodents whereas human
studies are scarce. Our results contribute to the knowledge and provide better
insight in exercise training-mediated metabolic changes in abdominal
subcutaneous adipose tissue.
Furthermore, metabolic phenotyping at baseline makes it possible to stratify
subjects into different subgroups and may improve the effectiveness of a particular
intervention in a specific subgroup of the population [12]. However, in this thesis we
did not find clear evidence that metabolic phenotype at baseline affected exercise
training-induced study outcomes. Therefore, before extrapolating our findings to a
larger population, more research is necessary in larger study populations following
different intervention strategies (e.g. nutritional, pharmacological as well as

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prolonged exercise training interventions). Studies including more detailed

metabolic phenotyping such as tissue-specific profiling are needed not only to
identify individuals or subgroups at increased risk of developing metabolic
diseases, but also to design optimized prevention and treatment strategies for
specific subgroups of the population. These promising strategies will be further
investigated by the current project team by performing human intervention trials
including in vivo and laboratory analyses, performed via state-of-the-art research
methodologies.

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REFERENCES
1. World Health Organization. Fact sheet: Obesity and overweight. Updated June 2016.
2016.
2. Despres JP, Lemieux I. Abdominal obesity and metabolic syndrome. Nature.
2006;444(7121):881-7.
3. Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin resistance
and type 2 diabetes. Nature. 2006;444(7121):840-6.
4. Van Gaal LF, Mertens IL, De Block CE. Mechanisms linking obesity with
cardiovascular disease. Nature. 2006;444(7121):875-80.
5. Bhaskaran K, Douglas I, Forbes H, dos-Santos-Silva I, Leon DA, Smeeth L. Body-
mass index and risk of 22 specific cancers: a population-based cohort study of 5.24
million UK adults. Lancet. 2014;384(9945):755-65.
6. Lauby-Secretan B, Scoccianti C, Loomis D, Grosse Y, Bianchini F, Straif K,
International Agency for Research on Cancer Handbook Working G. Body Fatness
and Cancer--Viewpoint of the IARC Working Group. N Engl J Med. 2016;375(8):794-
8.
7. Di Cesare M, Bentham J, Stevens GA, Zhou B, Danaei G, Lu Y, Bixby H, Cowan MJ,
Riley LM, Hajifathalian K, Fortunato L, Taddei C, Bennett JE, Ikeda N, Zhu D,
Zimmermann E, J. ZC. Trends in adult body-mass index in 200 countries from 1975
to 2014: a pooled analysis of 1698 population-based measurement studies with 19.2
million participants. Lancet. 2016;387(10026):1377-96.
8. American Diabetes Association. Standards of Medical Care in Diabetes. Diabetes
Care. 2016;39(Suppl 1).
9. US FDA. Entresto Prescribing Information 2015 [updated July 7, 2015. Available
from:
http://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/207620Orig1s000Lbl.pdf.
10. Tschop MH, Finan B, Clemmensen C, Gelfanov V, Perez-Tilve D, Muller TD,
DiMarchi RD. Unimolecular Polypharmacy for Treatment of Diabetes and Obesity.
Cell Metab. 2016;24(1):51-62.
11. Levin PA. Practical combination therapy based on pathophysiology of type 2
diabetes. Diabetes Metab Syndr Obes. 2016;9:355-69.
12. Stefan N, Fritsche A, Schick F, Haring HU. Phenotypes of prediabetes and
stratification of cardiometabolic risk. Lancet Diabetes Endocrinol. 2016;4(9):789-98.

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ACKNOWLEDGEMENTS

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Promoveren is iets dat je zeker niet alléén kunt doen en ik ben ervan overtuigd dat
een goede samenwerking onmisbaar is tijdens het promoveren. Er zijn een
heleboel mensen die een belangrijke bijdrage hebben geleverd aan het tot stand
komen van deze thesis en ik wil deze mensen dan ook heel graag bedanken.

Allereerst wil ik mijn promotieteam, Prof. Ellen Blaak en Dr. Gijs Goossens
bedanken voor het vertrouwen en de kans die ze mij ongeveer 5 jaar geleden
hebben gegeven om aan dit promotieonderzoek te starten.
Ellen, bedankt dat je altijd voor me klaar stond wanneer dat nodig was. Dankzij
jouw wetenschappelijk inzicht werd altijd de juiste breedte en diepgang aan de
manuscripten gegeven. Ik bewonder het enorm dat je, ondanks je drukke agenda,
steeds op korte termijn mijn papers na kon kijken en met constructieve
commentaar had voorzien.
Gijs, bedankt voor de goede begeleiding. Zowel tijdens het praktisch werk op de
testdagen en de planning daarvan als op wetenschappelijk gebied heb ik veel van
je geleerd. Ik heb het enorm gewaardeerd dat ik altijd bij je terecht kon wanneer
dat nodig was en dat je altijd bereid was om (uitgebreide ;) ) raad te geven. Jouw
kritische blik op de manuscripten en de grammaticale aanpassingen (al vond ik die
soms wat te uitgebreid :) ) hebben er steeds voor gezorgd dat de manuscripten
verbeterden. Ik vond het ook leuk dat we naast het werk gezellige tijden hebben
gehad, zoals bijvoorbeeld op de congressen in Barcelona, Götenborg of in
Denemarken.
Ellen en Gijs, bedankt voor de zeer prettige samenwerking gedurende de
afgelopen jaren!

Next, I would like to thank all members of the thesis assessment committee, Prof.
C. Stehouwer, Prof. J. Glatz, Prof. S. Kersten, Prof. L. van Loon and Prof. B.
Stallknecht for taking the time and effort to review my thesis and being present at
the official dissertation.

I would also like to thank all co-authors for the pleasant cooperation and for the
excellent contributions to improve the work that is presented in this thesis.

Zonder toegewijde proefpersonen had deze thesis nooit tot stand kunnen komen.
Mijn oprechte dank gaat daarom uit naar alle mensen die hebben deelgenomen
aan de experimentele onderzoeken; voor de moeite, flexibiliteit, gemaakte
kilometers en het afstaan van bloed, vet- en spierweefsel en vooral heel veel
zweet. Ik heb veel plezierige gesprekken en momenten gehad tijdens de
verschillende test- en trainingsdagen.

Mijn paranimfen wil ik even extra bedanken!

Max, bijna 4 jaar lang hebben we een kamer gedeeld en ik had me eerlijk gezegd
geen betere kamergenoot kunnen wensen! Wanneer er gewerkt moest worden,
werd er gewerkt en wanneer we even wouden pauzeren, kon dat gewoon en
konden we echt over vanalles praten. We hebben samen veel gelachen en een
hele leuke tijd gehad op de summer school in Lissabon. Naast het werk ben je ook
een hele gezellige persoon en ik kijk er naar uit om samen nog eens een whisky te
gaan proeven ;)

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Birgitta, ook wij hebben een aanzienlijke tijd een kamer gedeeld. We zijn ongeveer
tegelijkertijd begonnen op de universiteit en ik heb je leren kennen als een sociale,
harde werker die heel direct en eerlijk is. Naast het werk kunnen we het goed met
elkaar vinden en hebben we vele leuke gesprekken en tijden gehad, zoals
bijvoorbeeld op het congres in Barcelona en op verschillende feestjes.
Max en Birgitta, ik ben blij dat jullie mijn paranimfen willen zijn en het is voor mij
een hele geruststelling dat jullie aan mijn zijde staan!

Een groot deel van deze thesis gaat over een farmacologische interventie die
synergetische effecten kan hebben. Het is terecht om te zeggen dat ons
onderzoeksteam tijdens deze studie ook een synergetisch geheel was ;) Birgitta en
Laura, ik wil jullie alle twee heel erg bedanken voor de hulp tijdens deze toch wel
stressvolle periode, waarin we soms verschillende screeningen én VO2max testen
op één ochtend moesten combineren met een testdag. Zonder jullie hulp zou het
nooit gelukt zijn om deze studie binnen de deadline tot een goed einde te brengen!
Ook Bas, Kirsten, Bart, Irene, Nicolaas en iedereen die op één of andere manier
heeft geholpen, bedankt voor de flexibiliteit en bereidwilligheid!

Kenneth, ik wil jou ook even bedanken voor de aangename samenwerking tijdens
onze studie in Hasselt. Je hebt enorm veel werk geleverd, hele lange dagen
gemaakt (zeker wanneer er zowel in de ochtend als in de avond training sessies
plaatsvonden) en heel veel cupjes geplakt ;) Ik ben blij dat we de studie samen tot
een goed einde hebben gebracht, dat er mooie data zijn uitgekomen en dat we niet
nog één aflevering van “In de gloria” of de film “Intouchable” moeten zien ;)
Ik wens je nog veel succes met het uitbouwen van je verdere academische
carrière.

Ik wil ook alle (ex)collega’s binnen onze onderzoeksgroep, Johan, Nicole H,

Yvonne, Jasper, Emanuel, Dorien, Birgitta, Max, Kenneth, Laura, Mattea, Qing,
Nicole V, Ruth, Rens, Manuel, Adriyan en Kelly bedanken voor de
wetenschappelijke discussies en de leuke tijden die we samen hebben gehad
tijdens onze meetings of congressen. De collegialiteit binnen onze
onderzoeksgroep vind ik echt super! Wanneer er iemand hulp nodig heeft met
testdagen of als iemand met een vraag zit is iedereen wel bereid om te helpen
waar hij kan en niets is teveel gevraagd…echt top! Niet alleen professioneel, maar
ook naast het werk kunnen we het goed met elkaar vinden. De leuke tijden op
congressen, dagjes uit of (trouw)feesten ga ik zeker missen! Ik wil iedereen nog
heel veel succes wensen met zijn verdere carrière, maar ik hoop dat we elkaar nog
regelmatig kunnen zien. Nicole en Yvonne, bedankt voor de goede begeleiding en
de leuke samenwerking op het lab en natuurlijk de leuke praatjes tussendoor!

Alle collega’s van Humane Biologie en Bewegingswetenschappen (ik ga bewust

geen namen noemen, want het zijn er gewoon te veel en ik wil niemand vergeten),
bedankt voor de leuke momenten gedurende de afgelopen 5 jaar! Weekendjes in
de Ardennen, dagjes uit, feestjes, de gezellige lunches of diners, de koffiepauzes,
kerstdiners en de feestjes die daarop volgden en niet te vergeten de vrijdagmiddag
borrels...jullie hebben allemaal bijgedragen aan deze leuke tijden en ik hoop dat de
onderlinge sfeer binnen de afdeling zo goed blijft!

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Ik wil alle betrokken analisten bedanken voor het uitvoeren van de vele analyses.
Zonder jullie was dit alles nooit gelukt!
De secretaresses wil ik bedanken voor alle ondersteuning en het administratieve
werk.

Natuurlijk wil ik ook mijn vrienden bedanken voor de leuke en ontspannende

momenten! Het is altijd heel gezellig met jullie en ik hoop dat we nog lang en
regelmatig kunnen afspreken, zeker aangezien er nu bouwgronden worden
gekocht en over gezinsuitbreiding wordt nagedacht ;)

Mijn familie mag zeker niet ontbreken…Peter en Els (en Amber), Inge en Geert
(Stan en Sien), dank jullie voor de interesse, steun en vooral voor de gezellige en
leuke momenten die we altijd samen hebben!
Liefste mama en papa, bedankt voor alles wat jullie voor mij gedaan hebben!
Bedankt voor jullie onvoorwaardelijke liefde, om altijd voor ons klaar te staan
wanneer dat nodig was, voor jullie interesse, raad en steun en dat jullie mij de
mogelijkheden hebben gegeven om te komen waar ik nu ben! Ik hoop dat we nog
lang in goede gezondheid bij elkaar kunnen zijn! Ik kan jullie niet genoeg
bedanken, maar als teken van dank, wil ik deze thesis heel graag aan jullie
opdragen!

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CURRICULUM VITAE

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nd
Rudi Stinkens was born on June 2 1983 in Sittard, The Netherlands and raised in
Belgium. He studied Social and Technical Sciences and completed secondary
school at the Sint-Augustinus Insituut in Bree, Belgium in 2001. Consecutively, he
started a Bachelor of Science in Nutrition and Dietetics at the Katholiek
Hogeschool Kempen in Geel, Belgium, where he graduated in 2005. During the
following years, he specialised in sports nutrition at both the HAN University of
Applied Sciences in Nijmegen, The Netherlands and the Artesis & Plantijn
Hogeschool in Antwerpen, Belgium, while working in the food industry.
In 2010 he decided to make a switch in his career and started the Master of
Science program in Physical Activity and Health at Maastricht University, The
Netherlands, where he graduated in 2011. During this Master program, he
investigated astaxanthin supplementation in endurance trained athletes during a 6-
months internship at the department of Human Movement Sciences at Maastricht
University. Following his internship, he performed several months pro deo research
towards beet root juice supplementation, under the supervision of Dr. Cermak N.
and Prof. van Loon LJ.
In November 2012, he started as a Ph.D. candidate at the department of Human
Biology at Maastricht University (NUTRIM School of Nutrition and Translational
Research in Metabolism), under supervision of Prof. Dr. Ellen Blaak and Dr. Gijs
Goossens. His research is described in this thesis and is entitled “Adipose tissue
metabolism and cardiometabolic health in obesity - Effects of pharmacological and
lifestyle interventions”. During his Ph.D., Rudi was selected by the European
st
Association for the Study of Obesity (EASO) to attend the 1 Young Investigators
United summer school in Lisbon, Portugal (2015). He was also selected as one of
the 10 best candidates from the Netherlands to present his research findings at the
North European Young Diabetologists (NEYD) meeting in cooperation with the
Danish Diabetes Academy in Snekkersten, Denmark (2016). He presented his
research findings at several national and international conferences. Furthermore,
he received a travel grant from The Netherlands Association for the Study of
st
Obesity (NASO) to present his research at the 1 European Obesity Summit in
Göteborg, Sweden (2016) as well as a travel grant from the European Association
rd
for the Study of Diabetes (EASD) to present his research at the 53 Annual
Meeting of the European Association for the Study of Diabetes (EASD) in Lisbon,
Portugal (2017).

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Chapter 9

LIST OF PUBLICATIONS

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 Chapter 9

FULL PAPERS

Improved insulin sensitivity with angiotensin receptor neprilysin inhibition in

individuals with obesity and hypertension
Jordan J, Stinkens R, Jax T, Engeli S, Blaak EE, May M, Havekes B, Schindler C,
Albrecht D, Pal P, Heise T, Goossens GH, Langenickel TH.
Clin Pharmacol Ther. 2017 Feb;101(2):254 - 263

Targeting fatty acid metabolism to improve glucose metabolism

Stinkens R, Goossens GH, Jocken JW, Blaak EE.
Obes Rev. 2015 Sep;16(9):715-757

Astaxanthin supplementation does not augment fat use or improve endurance

performance
Res P, Cermak NM, Stinkens R, Tollakson TJ, Haenen GR, Bast A, van Loon
LJC.
Med Sci Sports Exerc. 2013 Jun;45(6):1158-65

No improvement in endurance performance after a single dose of beetroot juice

Cermak NM, Res P, Stinkens R, Lundberg JO, Gibala MJ, van Loon LJC.
Int J Sport Nutr Exerc Metab. 2012 Dec;22(6):470-8

Effect of sacubitril/valsartan on exercise induced lipid metabolism in individuals

with obesity and hypertension
Engeli S, Stinkens R, Heise T, May M, Goossens GH, Blaak EE, Jax T, Albrecht
D, Pal P, Tegtber U, Haufe S, Langenickel TH, Jordan J.
Submitted

The effects of angiotensin receptor neprilysin inhibition by sacubitril/valsartan on

adipose tissue transcriptome and protein expression in obese hypertensive
patients
Stinkens R, van der Kolk BW, Jordan J, Jax T, Engeli S, Heise T, Jocken JW, May
M, Schindler C, Havekes B, Schaper N, Albrecht D, Kaiser S, Hartmann N, Letzkus
M, Langenickel TH, Goossens GH, Blaak EE.
Submitted

Exercise training-induced effects on abdominal subcutaneous adipose tissue

phenotype in obese humans
Stinkens R*, Brouwers B*, Jocken JW, Blaak EE, Theunissen-Beekman KF,
Hesselink MK, van Baak M, Schrauwen P, Goossens GH.
To be submitted * Shared first authorship

Coordinated regulation of adipose tissue adrenergic- and non-adrenergic-mediated

lipolysis during exercise in lean and obese individuals: the effect of exercise
training
Stinkens R*, Verboven K*, Hansen D, Wens I, Frederix I, Eijnde BO, Jocken JW,
# #
Goossens GH , Blaak EE.
#
Submitted * Shared first authorship, Shared last authorship

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Chapter 9

ABSTRACTS

Coordinated regulation of adipose tissue adrenergic- and non-adrenergic-mediated

lipolysis during exercise in lean and obese individuals: the effect of exercise
training
Stinkens R, Verboven K, Hansen D, Wens I, Frederix I, Eijnde BO, Jocken JW,
Goossens GH, Blaak EE.
Diabetologia, 2017; 60(Suppl.1):1

Exercise training-induced effects on abdominal subcutaneous adipose tissue gene

and protein expression and adipose tissue morphology in obese humans
Stinkens R, Brouwers B, Jocken JW, Blaak EE, Hesselink M, Schrauwen P,
Goossens GH.
Obesity Facts, 2016; 6(9) (Suppl 1):38

LCZ696 improves lipid mobilization from adipose tissue: A randomized, double-

blind, active-controlled, parallel-group study in obese hypertensive patients
Jordan J, Stinkens R, Jax T, Engeli S, Haufe S, Blaak EE, May M, Havekes B,
Schindler C, Albrecht D, Pal P, Schaper N, van der Kolk B, Tegtbur U, Heise T,
Goossens GH, Langenickel TH.
Obesity Facts, 2016; 6(9) (Suppl 1):152

LCZ696 improves lipid mobilization from adipose tissue: a randomized, double-

blind, active-controlled, parallel-group study in obese hypertensive patients
Jordan J, Stinkens R, Jax T, Engeli S, Haufe S, Blaak EE, May M, Havekes B,
Schindler C, Albrecht D, Pal P, Schaper N, van der Kolk B, Tegtbur U, Heise T,
Goossens GH, Langenickel TH.
Circulation, 2015; 132 (Suppl 3):A15144

Metabolic benefits of LCZ696: a randomized, double-blind, active-controlled,

parallel-group study in obese hypertensive patients
Jordan J, Stinkens R, Jax T, Engeli S, Blaak EE, May M, Havekes B, Schindler C,
Albrecht D, Pal P, Heise T, Goossens GH, Langenickel TH.
Diabetes, 2015; 64 (Suppl 1):A496-A574

272