biomolecules

Review
Factors Influencing Gallstone Formation: A Review of
the Literature
Hao Sun 1 , Jonathan Warren 2 , James Yip 3 , Yu Ji 1,4 , Shaolong Hao 4 , Wei Han 1,4, * and Yuchuan Ding 2,5, *

1 Central Laboratories, Beijing Luhe Clinical Institute, Capital Medical University, Beijing 101199, China;
m18810253032@163.com (H.S.); doc.jiyu@foxmail.com (Y.J.)
2 Department of Neurosurgery, Wayne State University School of Medicine, Detroit, MI 48201, USA;
jonathan.warren2@med.wayne.edu
3 Department of Pathology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96831, USA;
jyip@med.wayne.edu
4 Department of General Surgery, Beijing Luhe Clinical Institute, Capital Medical University,
Beijing 101199, China; haoshaolong2014@163.com
5 John D. Dingell VA Medical Center, 4646 John R Street, Detroit, MI 48201, USA
* Correspondence: dr-han1972@mail.ccmu.edu.cn (W.H.); yding@med.wayne.edu (Y.D.)

Abstract: Gallstone disease is a common pathology of the digestive system with nearly a 10–20%
incidence rate among adults. The mainstay of treatment is cholecystectomy, which is commonly
associated with physical pain and may also seriously affect a patient’s quality of life. Clinical research
suggests that cholelithiasis is closely related to the age, gender, body mass index, and other basic
physical characteristics of patients. Clinical research further suggests that the occurrence of cholelithi-
asis is related to obesity, diabetes, non-alcoholic fatty liver, and other diseases. For this reason, we
reviewed the following: genetic factors; excessive liver cholesterol secretion (causing cholesterol
supersaturation in gallbladder bile); accelerated growth of cholesterol crystals and solid cholesterol



crystals; gallbladder motility impairment; and cardiovascular factors. Herein, we summarize and


analyze the causes and mechanisms of cholelithiasis, discuss its correlation with the pathogenesis of
Citation: Sun, H.; Warren, J.; Yip, J.;
related diseases, and discuss possible mechanisms.
Ji, Y.; Hao, S.; Han, W.; Ding, Y.
Factors Influencing Gallstone
Keywords: gallstone disease; bile acids; obesity; diabetes; non-alcoholic fatty liver; cardiovascular disease
Formation: A Review of the
Literature. Biomolecules 2022, 12, 550.
https://doi.org/10.3390/
biom12040550
1. Introduction
Academic Editor: Jorge Joven
Gallstone disease is a common pathology of the digestive system. Nearly 75% of
Received: 29 January 2022 patients with gallstones have no obvious symptoms in the initial stages. As the gallstones
Accepted: 31 March 2022 progress in development, they may trigger symptoms such as nausea, epigastric colic,
Published: 6 April 2022 diarrhea, anorexia, etc. Eventually, gallstone obstruction can lead to life-threatening con-
Publisher’s Note: MDPI stays neutral
ditions such as acute cholangitis, acute cholecystitis, and biliary pancreatitis [1]. At this
with regard to jurisdictional claims in
point, cholecystectomy is necessary. However, this treatment has complications that can
published maps and institutional affil-
seriously threaten a patient’s health and their overall quality of life [2,3]. In 2010, five
iations. factors were proposed to promote the formation of gallstones. These include genetic factors;
excessive liver cholesterol secretion (resulting in cholesterol supersaturation in gallbladder
bile); factors that accelerate the growth of cholesterol crystals and solid cholesterol crystals;
impairment of gallbladder motility; and intestinal factors [4]. Additionally, the formation of
Copyright: © 2022 by the authors. gallstones is related to factors such as age, gender, weight, and bacterial infection [5–8]. This
Licensee MDPI, Basel, Switzerland. review describes and summarizes the causes, formation process and influencing factors of
This article is an open access article gallstones, and discusses potential mechanisms for their formation.
distributed under the terms and
conditions of the Creative Commons 2. Classification and Formation of Gallstones
Attribution (CC BY) license (https://
Gallstones are categorized into several types, including cholesterol, pigment, and
creativecommons.org/licenses/by/
mixed stones [9]. Evidence suggests that 90% of cholelithiasis patients have cholesterol
4.0/).

Biomolecules 2022, 12, 550. https://doi.org/10.3390/biom12040550 https://www.mdpi.com/journal/biomolecules

Biomolecules 2022, 12, 550 2 of 13

stones [10]. Cholesterol sources in the human body mainly consist of ab initio synthesis of
acetyl coenzyme, enterohepatic circulation, and food intake. However, because human and
animal tissues do not possess enzymes that can degrade the ring structure of this sterol,
cholesterol cannot be metabolized to CO2 and water in the body. Therefore, to prevent a
potentially hazardous accumulation of cholesterol in the body, excess cholesterol must be
metabolized into other compounds and/or excreted in the feces.
Cholesterol can be secreted into bile by the liver, and is then carried by bile acid
micelles and lecithin, which increase the cholesterol content in bile. Excess cholesterol
is carried by cholesterol-rich lecithin cholesterol vesicles which have characteristics of
affinity aggregation. They fuse with each other to form large vesicles, resulting in high local
cholesterol concentration, and finally form cholesterol hydrate crystals. These cholesterol
hydrate crystals then form the initial aggregation nucleus of cholesterol stones, which is an
initial and necessary step in the formation of cholesterol stones. In addition, there exists a
transitional form for carrying excess cholesterol, phospholipid lamella (disk-like particles),
which also play a role in the nucleation of cholesterol crystals [11]. When the cholesterol
crystals appear, concentrated granulocytes are recruited, and neutrophils extrude their
DNA. This extruded DNA adheres to different cholesterol crystals and, over time, the
crystals with neutrophil DNA wrapped around them will coalesce with nearby DNA-
wrapped crystals. This pulls the individual DNA-wrapped crystals together, eventually
forming larger stones [12]. Clinical studies have found that cholesterol-supersaturated bile
is an essential prerequisite for the precipitation of solid cholesterol monohydrate crystals
and the formation of cholesterol gallstones.

3. Cholesterol and Bile Acid Circulation

3.1. Cholesterol Circulation
Cholesterol in the liver can travel through the biliary tract to the small intestine,
where some cholesterol is reabsorbed and enters the bloodstream through the lymphatic
system, eventually returning to the liver. There are two main sources of cholesterol in
the body: biosynthesis and absorption. Cholesterol biosynthesis mainly occurs in the
liver. This process converts acetyl-CoA into cholesterol molecules through enzymatic
reactions. Endoplasmic reticulum transmembrane protein 3-Hydroxy-3-Methylglutaryl-
CoA Reductase (HMGCR) and squalene monooxygenase are rate-limiting enzymes in this
process [13]. Part of the cholesterol in hepatocytes is converted into bile salts, and the other
part of free cholesterol is pumped out by hepatocytes through ABCG5/8 into the biliary
tract. Here, phospholipids form microclumps that are excreted into the intestine through
bile secretion [14].
Biosynthetic and dietary cholesterol are absorbed by Niemann–Pick C1-Like Protein1
(NPC1L1) on the membranes of intestinal epithelium and further esterified by ACAT (the
alternative name is acyl-coenzymeA: cholesterol acyltransferase), which then enters the
bloodstream through the lymphatic system and is eventually absorbed by the liver as
chylomicrons [15,16]. The proportion of total cholesterol from diet also depends mainly
on the efficiency of absorption of cholesterol from the intestinal tract and the amount
of cholesterol consumed daily. Cholesterol absorbed in the small intestine may regulate
cholesterol synthesis in the liver through a negative regulatory mechanism, depending on
daily food intake.

3.2. Bile Acid Cycle

There is an equilibrium between cholesterol and bile acid in bile. The liver is the only
organ that synthesizes bile acids (Figure 1). Bile acids are synthesized by cholesterol in liver
parenchymal cells, including the classical pathway mediated by cholesterol 7α -hydroxylase
(CYP7A1) and the non-classical pathway mediated by sterol 27-hydroxylase (CYP27A1) [17].
The non-classical pathway mainly produces chenodeoxycholic acid (CDCA), while the
classical pathway produces CDCA and cholic acid (CA). The newly synthesized binding
Biomolecules 2022, 12, 550 3 of 13

bile acids are secreted into the capillary bile duct of hepatocytes by the bile salt export
pump (BESP (ABCB11)), eventually making their way into the intestine [18].

Figure 1. Cholesterol synthesis metabolism and bile acid hepatoenteric circulation. Cholesterol can
be de novo synthesized. Part of the cholesterol in hepatocytes is converted into bile salts, and the
other part of free cholesterol is pumped out by hepatocytes through ABCG5/8 into the biliary tract.
Cholesterol is absorbed by NPC1L1 on the membranes of intestinal epithelium, which then enters the
bloodstream through the lymphatic system. The newly synthesized binding bile acids are secreted
into the capillary bile duct of hepatocytes by ABCB11, eventually making their way into the intestine.
Binding bile acids can be effectively reabsorbed into intestinal epithelial cells at the terminal ileum
by ASBT, which binds to ileal bile acid binding protein for transport to the basement membrane.
In the presence of heterodimer OSTα/β at the terminal cavity of the basement membrane, bile
acids are reabsorbed and transported to the liver via the portal vein. Orange represents cholesterol.
Dark green represents the bile acid cycle. NPC1L1:Niemann–Pick C1-Like Protein1; ABCG5/8:
ATP Binding Cassette Subfamily G Member 5/8; CYP7A1: cholesterol 7α–hydroxylase; CYP27A1:
sterol 27-hydroxylase; ABCB11: ATP-Binding Cassette Sub-Family B Member 11; ASBT: the apical
sodium-dependent bile acid transporter; OSTα/β: organic solute transporter α/β.

Binding bile acids can be effectively reabsorbed into intestinal epithelial cells at the
terminal ileum by the apical sodium-dependent bile acid transporter (ASBT), which binds
to the ileal bile acid binding protein for transport to the basement membrane. In the
presence of heterodimer organic solute transporter α/β (OSTα/β) at the terminal cavity
of the basement membrane, bile acids are reabsorbed and transported to the liver via
the portal vein. At the liver, bile acids are again absorbed from the sinusoidal space of
hepatocytes by Na+/taurocholic acid cotransport polypeptide (NTCP (SLC10A1)) [19,20].
The reabsorbed bile acids are then secreted into the biliary system by BESP (ABCB11) in
the capillary bile duct of hepatocytes, together with the newly synthesized binding bile
acids. Clinically, NTCP is primarily responsible for the uptake of conjugated bile acids
(>80%), while members of the Organic Anion Transporting Polypeptide (OATP) family
are primarily responsible for the transport of unconjugated or sulphated bile acids to the
liver [21].
In addition, free bile acids are passively reabsorbed into intestinal epithelial cells in the
small intestine and colon. From there, they are absorbed through the sinusoidal membrane
of hepatocytes by OATP and finally processed and secreted into the capillary bile ducts [22].
Bile acids enter the intestinal lumen through the biliary tract, completing the bile acid
enterohepatic circulation. Bile acids that are not reabsorbed by liver cells spread throughout
the body and may eventually be excreted through the kidneys.
Biomolecules 2022, 12, 550 4 of 13

4. Factors Influencing Gallstone Formation

Dysfunction of the gallbladder or other parts of the bile-secretion pathway can result
in gallstone formation. Given that the bile-secretion pathway is a complex process, there are
many reasons for the formation of gallstones. Evidence suggests that gallstones are related
to age, gender, female physiological status, obesity, cardiovascular disease, microbiome,
sugar metabolism, and various environmental exposures (Figure 2). Based on a large num-
ber of mouse and human studies, the interaction of five main factors were proposed. The
pathogenesis of cholesterol gallstone disease is precipitated by: genetic factors; excessive
cholesterol secretion by the liver (leading to supersaturation of cholesterol in gallbladder
bile); rapid phase change by accelerating the growth of cholesterol crystals and solid choles-
terol crystals; impairment of gallbladder motility; and intestinal factors. Intestinal factors
can be further broken down into two categories: increased cholesterol absorption from the
small intestine to the liver, eventually resulting in increased bile secretion, and microbiota
that inhabit the intestinal tract. These factors will increase the production or growth of
cholesterol crystals, eventually leading to the formation of stones [4–7,23,24].

Figure 2. Influence factors of gallstones. CVD: cardiovascular disease.

4.1. Genetic Mechanism of Gallstone Formation

Gallstone-susceptible mice (C57L/J) and gallstone-resistant mice (AKR/J) have been
used to better elucidate the genetic components of gallstone formation. Using these mice,
it has been described that lithogenic genes 1 and 2 (Lith1 and Lith2) may play a role in
gallstone formation. Lith1, located on mouse chromosome 2, plays a major role in the
determination of liver cholesterol hypersecretion. Lith2, located on mouse chromosome 19,
regulates the bile salt-dependent flow of bile [25]. The functional counterparts of Mouse
Lith1 and Lith 2 are ABCG5 and ABCG8 in the human equivalents. ABCG5 and ABCG8 are
ATP-binding cassette (ABC) transporters with significant expression in hepatocytes and
intestinal cells [26]. These two proteins form heterodimers in the endoplasmic reticulum
and are subsequently transported to the apical membrane. In hepatocytes, they transport
neutral sterols to bile or they promote active efflux of cholesterol from the enterocyte
back into the intestinal lumen for fecal excretion [27]. Inactivation of ABCG5/G8 will
Biomolecules 2022, 12, 550 5 of 13

result in significantly reduced cholesterol secretion in bile, making the level of cholesterol
in liver and plasma very sensitive to changes in dietary cholesterol content. Because of
this, hypercholesterolemia, phytosterolemia, and premature coronary heart disease may
result [28,29]. However, the overexpression of ABCG5/G8 protein increases cholesterol
content in the gallbladder, thus increasing the likelihood of cholesterol crystal precipita-
tion [30]. Subsequently, ABCG5/G8 was found to be associated with cholesterol gallstone
disease in patients, and two gallstone associated variants in ABCG5/G8 (ABCG5-R50C and
ABCG8-D19H) were identified in Germans, Chileans, Chinese, and Indians. Taking this
information into account, these may be the primary promoter genes of gallstones.

4.2. Gallbladder Contraction

After ingesting a large amount of food containing fat and protein, the neuroenteropep-
tide hormone cholecystokinin (CCK), released by endocrine cells of the duodenum, reaches
the gallbladder and directly binds with the CCK1 receptor (CCK-1R) on the smooth muscle
cells of the gallbladder wall. This triggers contraction of the gallbladder and discharges
the concentrated bile into the intestine. CCK-1R is also located in the sphincter of Oddi,
pancreas, small intestine, gastric mucosa, and pyloric sphincter. It is responsible for CCK
regulation of pancreatic secretion, small intestine transport, gastric emptying, and other
digestive processes. Observing CCK or CCK-1R gene knockout mice shows that gall-
bladder emptying and bile cholesterol metabolism are inhibited, intestinal absorption of
cholesterol is increased, and cholesterol stone formation is significantly increased [31]. This
suggests that CCK can regulate gallbladder and small intestine motility through the CCK-
1R signal cascade, promote small intestine transport, and regulate intestinal cholesterol
absorption. This also explains why the abnormal gallbladder motility caused by exogenous
cholecystokinin is mainly found in patients with cholesterol stones [32,33].
Clinical studies have found that glucagon-like peptide 1 (GLP-1) receptor agonists
have achieved good results in the treatment of type II diabetes, obesity, and other diseases.
However, such drugs have a negative impact on the gallbladder and seem to increase the
risk of gallbladder-related diseases [34]. In one study after acute injection of the glp-1r
agonist exendin-4, there was no significant change in gallbladder volume in mice. When
combined with CCK injection, exendin-4 reduced the emptying ability of the gallbladder.
The effect of the glp-1r agonist for 12 weeks on patients with type 2 diabetes mellitus was
not significant. In addition, the mRNA transcription level of GLP-1R in the gallbladder of
mice was low, suggesting that GLP-1 has a more indirect effect on the gallbladder. This
further suggests that GLP-1 may be related to slowing down of upper gastrointestinal
motility. However, the molecular mechanism is still unclear [35].

4.3. Microbiome
The various flora in the body are in a dynamic balance and, when disturbed, many
tissues and organs are affected. This complex system of microorganisms also exists in bile,
and the occurrence of gallstones is closely related to abnormalities with flora. In almost all
stages of bile formation, the microbiota of the gastrointestinal and biliary tracts are involved,
including the regulation of lipid metabolism, cholesterol metabolism, biotransformation,
and enterohepatic circulation of bile acids [24].
Microbiome in the biliary tract. Studies have shown the presence of living bacteria
in gallstones. The flora in the biliary tract and duodenum are highly homologous and
closely related to the formation of gallstones. Microorganisms can enter the biliary system
from the duodenum by migrating through the sphincter of Oddi. They can also spread
hematogenously to the liver and from there into bile [36,37]. When in bile, microorganisms
play an important role as nucleating factors, leading to the formation of pigment and
cholesterol gallstones [38].
The properties of bacteria that reside in the gallbladder can control the formation of
gallstones. Bacteria that produce beta-glucuronidase and phospholipase in the bile yield a
higher percentage of pigment in the stones, while bacteria that cause mucus abnormalities
Biomolecules 2022, 12, 550 6 of 13

are more likely to lead to the formation of cholesterol stones [39,40]. Biofilm-forming
bacteria in the gallbladder, bile, and gallstones are closely associated with gallstone forma-
tion [41]. For example, biofilms are formed during the formation of pigment stones, and
the aggregating factor in this case is the glycocalyx (anionic glycoprotein) [42]. Differences
in the functional metagenomes of microbial communities have been found by comparing
pigment gallstones and cholesterol gallstones. Gram-positive bacteria were predominant in
most of the cholesterol gallstones examined, whereas they were not found in the pigment
stones. A high proportion of genes involved in carbohydrate metabolism were found in
the pigment stones, whereas genes dominating protein metabolism were more active in
the cholesterol stones. Helicobacter pylori is a Gram-negative, spiral-shaped, motile mi-
croorganism [43]. The presence of H. pylori in patients with symptomatic gallstone disease
(GSD) has been shown to promote the formation of gallstones. However, this finding is still
controversial and more data are required for adequate discussion of this topic [44].
Oral flora. Microflora of the oral cavity affects the secretion of cholecystokinin [45],
the main factor involved in the emptying and filling of the gallbladder [46]. A microbiome
changes the expression of mucin genes (MUC1, MUC3, and MUC4 genes) through im-
munomodulation, thereby changing the accumulation of mucin gel, which is the nucleation
matrix for the formation of cholesterol gallstones in the gallbladder [46]. Studies have
shown that H. pylori and enterohepatic strains of Helicobacter contribute to the formation
of cholesterol gallstones [47–49].
The composition of the gut and biliary tract microbiome varies significantly in pa-
tients with GSD and in healthy subjects [50]. In patients with GSD, microbial diversity is
reduced, beneficial bacteria such as Roseburia are reduced, and an overgrowth of bacteria of
the Proteobacteria type—including a wide range of pathogenic microorganisms such as
Escherichia, Salmonella, Vibrio and Helicobacter—more easily occurs [51]. Disorders of bile
acid metabolism are the leading factors in the pathogenesis of cholesterol GSD [25].
Gut microbes. Gut microbiota-mediated biotransformation of the bile acid pool regu-
lates bile acid signaling by influencing the activation of host bile acid receptors, such as the
nuclear receptor farnesoid X receptor (FXR). The role of FXR in liver cells and intestinal
cells is recognized as a regulator of bile acid, lipid, and glucose balance [52,53]. In the
intestine, bile acids directly bind to and activate the bile acid receptors FXR and fibroblast
growth factor 15/19 (FGF15/19) gene expression. FGF15/19 inhibits the synthesis of bile
acids by reducing the expression of CYP7A1, which plays a negative feedback role in bile
acid synthesis [54]. In fact, disturbances in the intestinal microbiota and changes in the
composition of bile can adversely affect the metabolism of bile acids and the balance of
glucose and cholesterol, leading to the development of gallstones [55,56].
Gut microbes lower cholesterol in bile. Bifidobacteria have been proven to lower
cholesterol in bile by assimilation or precipitation [39,57]. A meta-analysis showed that pro-
biotics (L. acidophilus, B. lactis, VSL#3, and the L. plantarum group) can significantly reduce
total serum cholesterol [58]. The consumption of a BSH-positive strain of Lactobacillus
significantly reduced cholesterol in patients with hypercholesterolemia [59].

4.4. Effect of Estrogen in Gallstone Formation

According to grouping analysis of cholelithiasis, the number of female patients of
all ages with gallstones is significantly higher than that of men. The incidence rate of
gallbladder diseases in women is further increased during pregnancy, which has become the
second most common indication of non-obstetric intervention during pregnancy [60–62].
Furthermore, the incidence rate of gallbladder disease in women who have had multiple
pregnancies is higher than that of those who have been pregnant once [63]. The importance
of estrogen in terms of cholelithiasis is well documented. Estrogen, such as 17β-estradiol
(E2), is a major female steroid hormone which plays an important role in health and
disease [30]. As a steroid, estrogen has liposoluble properties which allows it to passively
diffuse into cells and play the role of a transcription factor. After entering cells, it directly
binds to ESR1 and ESR2 receptors and initiates changes in receptor tertiary and quaternary
Biomolecules 2022, 12, 550 7 of 13

structures. As a result, active complexes that regulate transcription are formed [64]. When
E2 reaches the liver, it also passively diffuses into cells and increases liver secretion of
cholesterol into bile, thus increasing the cholesterol saturation in bile and the risk of
cholelithiasis. It has been confirmed that ESR1, rather than ESR2, plays a more major role
in the formation of cholesterol gallstones in mice induced by high dose E2. E2 has also
been shown to play important roles in health and disease [65,66]. It regulates a wide range
of biological processes, including reproduction, cardiovascular function, hepatobiliary
secretion, metabolic processes, nerve function, and inflammation. There is a large amount of
clinical evidence suggesting that oral contraceptive steroids and conjugated estrogen play a
significant part in promoting cholesterol stone formation in premenopausal women [67–70].
The classical estrogen regulatory pathway involves E2 promotion of cholesterol biosynthesis
and liver secretion of bile cholesterol through the “e2-esr1-srebp-2” pathway. During
estrogen treatment or in times of increased blood estrogen concentration, synthesis of
cholesterol increases mainly by estrogen-induced stimulation of sterol regulatory element
binding protein-2 (SREBP-2) [71]. These changes lead to excessive secretion of newly
synthesized cholesterol, supersaturation of bile, and easily lead to cholesterol precipitation
and gallstone formation. Estrogen-activated ESR1 also stimulates the activity of ABCG5
and ABCG8, which are only expressed in hepatocytes and intestinal cells. These two
proteins form heterodimers in the endoplasmic reticulum and are then transported to the
apical membrane. There, they transport neutral sterols to bile or the intestinal lumen which
promotes the secretion of bile cholesterol, eventually leading to the supersaturation of
cholesterol in bile [29].
G protein-coupled receptor 30 (GPR30), a newly discovered estrogen receptor in
humans, is produced by the gallstone gene lith18 [4,72,73]. It was found that E2 can
effectively bind and activate GPR30 and ER-α [74]. In order to distinguish the role of
Er-α and GPR30 in stone formation, a mouse model of ovariectomized female wild type,
GPR30 gene knockout, ER-α gene knockout, and GPR30/ER-α double gene knockout
was constructed. It was found that E2 activated GPR30 and ER-α produced liquid crystal
and amorphous metastable intermediates. These evolved into cholesterol monohydrate
crystals from supersaturated bile. In addition, the cholesterol crystal of GPR30/ER-α
double knockout mice decreased significantly. This suggests that GPR30 and ER-α have
a synergistic effect on the formation of gallstones induced by E2 [73,75]. Because GPR30
is mainly located in the endoplasmic reticulum rather than the nucleus of hepatocytes,
E2 may activate GPR30 through the signal cascade of epidermal growth factor receptor,
thus inhibiting the classical pathway of hepatic cholesterol 7α-hydroxylase and bile acid
synthesis. This results in excessive cholesterol production, leading to increased cholesterol
secretion by the liver and increased likelihood of bile stone formation [75].

4.5. Obesity and Gallstone

Nonalcoholic fatty liver disease (NAFLD) is an important risk factor for gallstone
formation. The abnormally low expression of aquaporin 8 (AQP8) mediated by hypoxia
inducible factor-1α (HIF-1α) in NAFLD seems to explain this situation [76]. HIF-1α is an
important transcription factor regulating gene expression of oxygen transfer, cell growth,
and redox homeostasis that promotes an adaptive response to hypoxic conditions resulting
in greater cell survival [76,77]. Given this, it makes sense that in the liver HIF-1α mainly
exists in the area around the hepatic vein. During the development of hepatic steatosis,
lipid accumulation significantly increases the size of hepatocytes, thereby reducing hepatic
sinusoidal perfusion and microcirculation, and ultimately leading to liver hypoxia [78].
In one study examining the upregulated expression of AQP8, a water channel protein
responsible for the secretion of liver water into the bile duct [79–81], researchers found
a significant 35% increase in bile flow, diluted bile lipid concentration in gallbladder
and hepatobiliary juice by 36%, and alleviation of gallbladder inflammation. As a result,
cholesterol crystal formation was inhibited in the liver-specific HIF-1α knockout mice. On
the contrary, activation of the HIF-1α pathway in diet-induced fatty liver has been shown
Biomolecules 2022, 12, 550 8 of 13

to accelerate the formation of gallstones in wild-type mice. In addition, the increased

expression of HIF-1α and its downstream targets in the liver suggests that HIF-1α may play
an important role in the formation of cholesterol gallstones in patients with NAFLD [2].

4.6. Carbohydrate Metabolism

The classic role of bile in the digestion and absorption of fat is well documented.
In addition, the gallbladder also plays a physiological role in glucose, fat, and energy
homeostasis. Both GSD and cholecystectomy can reduce insulin sensitivity [82], which
suggests that obesity is not a correlate between gallstone and insulin resistance (IR) but is
a common risk factor for both. GSD and cholecystectomy increase triglyceride content in
the liver, and possibly increase IR in the liver as well. On the other hand, the gallbladder
not only regulates the secretion and transport of bile acids, but also affects the homeostasis
of lipids and glucose, which may affect whole body energy consumption [83,84]. In
addition, experimental evidence suggests that liver IR may promote GSD by increasing the
diagenesis of bile.

5. Gallstones, Cardiovascular and Cerebrovascular Diseases

Recent studies have found that GSD is also closely related to the occurrence of cardio-
vascular disease (CVD), and the presence of GSD increases the incidence of CVD [85,86].
According to a meta-analysis of 10 published study cohorts, Zhao et al., found that patients
with GSD were at higher risk of hypertension, diabetes, coronary heart disease, atrial
fibrillation, and hyperlipidemia. Additionally, they found that GSD was associated with a
1.23-fold increase in the incidence of cardiovascular and cerebrovascular diseases [87]. In a
cohort of 5,928 subjects established by Daniel et al., gallstone disease was associated with
all cardiovascular disease (hazard ratio (HR) 1.36, 95% confidence interval (CI) (1.17; 1.59))
and to the subgroups’ coronary artery (HR1.34, 95% CI (0.10; 1.64)), cerebrovascular (HR
1.22, 95% CI (0.97; 1.52)), and peripheral artery disease (HR 1.57,95% CI (1.15; 2.13)) [88].
Gallstones and cardiovascular disease share common risk factors such as age, sex, obe-
sity, and disorders of lipid and glucose metabolism, all of which are major risk factors
for metabolic syndrome. Metabolic syndrome is strongly associated with coronary artery
disease and gallstones may be considered a biliary feature of this syndrome [89,90].
It is well-known that cholesterol is mainly carried by lipoproteins in plasma and by
micelles and vesicles in bile. If excess cholesterol is accumulated in the arterial wall, it
leads to atherosclerosis and causes cardiovascular disease. If excess cholesterol cannot be
dissolved in bile by the bile salts and/or phospholipids, it precipitates as plate-like solid
cholesterol monohydrate crystals, thus leading to the formation of cholesterol gallstones in
the gallbladder and/or the bile duct [27].
Cholesterol is a major component of most gallstones and is a major component of
atherosclerotic plaques. Cholesterol accumulation is a major cause of atherosclerotic CVD
and GSD [91]. In the cardiovascular system, such metabolic abnormalities usually lead
to the accumulation of excessive cholesterol esters in the arterial wall, leading to clinical
atherosclerosis, which mainly occurs in the heart and brain, leading to cardiovascular and
cerebrovascular diseases [88]. Cholesterol is present in the blood in the form of lipoproteins
and is involved in the transport of lipids. High-density lipoprotein (HDL) is a protective
factor of CVD, which can transport cholesterol in surrounding tissues and convert it into
bile acid or be discharged directly from the intestinal tract through bile into the biliary
system. It has a significant negative correlation with the degree of arterial lumen stenosis.
Low density lipoprotein (LDL) carries cholesterol into peripheral tissue cells. When LDL is
excessive, the carried cholesterol accumulates in the arterial wall, increasing susceptibility
to arteriosclerosis, cardiovascular, and cerebrovascular diseases [92].
As mentioned above, ABG5/8 can transport cholesterol to the intestine and bile. Ab-
normally elevated expression of ABG5/8 can reduce cholesterol concentration in blood and
inhibit the occurrence of atherosclerosis, but it can increase cholesterol concentration in bile
and increase the risk of cholesterol-type stones. NPC1L1 mediates cholesterol uptake from
Biomolecules 2022, 12, 550 9 of 13

the gut to intestinal cells and liver reuptake of cholesterol from bile to hepatocytes, thus
counteracting ABG5/8 function. NPC1L1 is a transmembrane protein highly expressed
in the intestinal tract, especially in the lumen membrane of mammalian intestinal epithe-
lial cells, which mediates intestinal cholesterol absorption [93–95]. NPC1L1 is a target
molecule of ezetimibe, a cholesterol absorption inhibitor clinically used in the treatment
of dyslipidemia [96]. Inhibition of intestinal activity of NPC1L1 or genetic variants re-
duces cholesterol uptake, lowers plasma LDL cholesterol concentrations, and prevents
atherosclerosis and ischemic vascular diseases. Inhibition of hepatic NPC1L1 may lead to
an increase in bile cholesterol concentration, thereby promoting the formation of cholesterol
gallstones [97].
Abnormal inflammation is involved in the development of GSD and CVD. Reducing
the expression of certain inflammatory factors has been shown to improve CVD [98]. Some
inflammatory factors, including von Willebrand factor (vWF), lectin-like oxidized low-
density-lipoprotein receptor-1 (LOX-1), as well as soluble urokinase plasminogen activator
receptor (suPAR), have been proposed to be associated with CVD [99–102]. Inflammatory
processes in GSD may promote atherosclerosis, vascular lesions in the cerebrovascular
system, and increase the risk of cardiovascular disease. Related liver diseases, including
nonalcoholic fatty liver disease and suppurative liver abscesses, have been identified as
risk factors for subsequent cardiovascular disease [103].

6. Conclusions
New evidence continues to uncover the many roles the gallbladder plays in differ-
ent physiologic mechanisms and its importance in broader physiological function. The
relationship between cholelithiasis and disease processes such as diabetes mellitus, nonal-
coholic fatty liver disease, obesity, and insulin resistance suggests that the gallbladder is an
important link in the multi-directional communication between different tissues mediated
by bile acids, gut hormones, hepatocyte factors, and adipokines. The gallbladder serves as
a link in coordinating metabolic homeostasis, maintenance of normal body composition,
and insulin sensitivity. We summarized the important feedback mechanisms of FXR and
GLP-1 in cholesterol metabolism and insulin sensitivity, discussed the possible important
role of GLP-1 in gallbladder contraction disorders, and examined the relationship between
cholelithiasis and metabolism. With further understanding of the processes involved in
gallstone formation, one may be able to elucidate its role for better treatment of gallbladder
disease in the future.

Author Contributions: Conceptualization, H.S., J.W., W.H., Y.D. and J.Y.; methodology, H.S., J.W.,
J.Y., Y.J., S.H., W.H. and Y.D.; software, H.S.; validation, H.S., J.W., J.Y., Y.J., S.H., W.H. and Y.D.;
formal analysis, H.S., J.W. and J.Y.; investigation, H.S., J.W., J.Y., Y.J., S.H., W.H. and Y.D.; resources,
H.S. and Y.D.; data curation, H.S. and J.Y.; writing—original draft preparation, H.S.; writing—review
and editing, H.S., J.W., J.Y. and Y.D.; visualization, H.S., J.W., S.H., W.H. and Y.D.; supervision, H.S.,
W.H. and Y.D.; project administration, H.S., W.H. and Y.D.; funding acquisition, N/A. All authors
have read and agreed to the published version of the manuscript.
Funding: This work was supported in part by the Science and Technology Plan of Beijing Tongzhou
District (KJ2020CX006-11, KJ2021CX008-24, KJ2021CX008-22) and Youth Research and Development
Fund of Beijing Luhe Hospital, Capital Medical University (LHYY2020-JC10).
Institutional Review Board Statement: Not Applicable.
Informed Consent Statement: Not Applicable.
Data Availability Statement: No data was reported in this study.
Conflicts of Interest: The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with the subject matter or
materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock
ownership or options, expert testimony, grants or patents received or pending, or royalties.
Biomolecules 2022, 12, 550 10 of 13

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