Non-alcoholic fatty liver disease is a multifaceted disease that progresses through multiple phases; it involves metabolic as well as structural changes. These alterations can be measured directly or indirectly through blood, non-invasive imaging, and/or tissue analyses. While some studies have evaluated the correlations between two sets of measurements (e.g., histopathology with cross-sectional imaging or blood biomarkers), the interrelationships, if any, among histopathology, clinical blood profiles, cross-sectional imaging, and metabolomics in a pediatric cohort remain unknown. We created a multiparametric clinical MRI-histopathologic NMR network map of pediatric NAFLD through multimodal correlation networks, in order to gain insight into how these different sets of measurements are related. We found that leptin and other blood markers were correlated with many other measurements; however, upon filtering out the blood biomarkers, the network was decomposed into three independent hubs centered around histopathological features, each with associated MRI and plasma metabolites. These multi-modality maps could serve as a framework for characterizing disease status and progression and could potentially guide medical interventions.

With rising prevalence of obesity and diabetes, nonalcoholic fatty liver disease (NAFLD) is now a leading cause of chronic liver disease. One-third of obese or diabetic men have subnormal free and bioavailable testosterone concentrations.¹ Several studies have further shown low testosterone to be associated with imaging-confirmed NAFLD in men,² although it is unknown whether low testosterone confers increased risk of more clinically relevant manifestations of NAFLD, including nonalcoholic steatohepatitis (NASH) and NASH fibrosis. We therefore aimed to evaluate the association of testosterone with histologic features of NAFLD among a representative cohort of men from the multicenter NASH Clinical Research Network (NASH CRN).

Aims

Fish and ω-3 fatty acids are reported to be beneficial in pediatric nonalcoholic fatty liver disease (NAFLD), but no studies have assessed their relation to histological severity. The objectives of this study were to evaluate the dietary intake of fish and ω-3 fatty acids in children with biopsy-proven NAFLD, and examine their association with serological and histological indicators of disease.

Methods

This was a cross-sectional analysis of 223 children (6-18 years) who participated in the Treatment of Nonalcoholic Fatty Liver Disease in Children trial or the NAFLD Database study conducted by the Nonalcoholic Steatohepatitis Clinical Research Network. The distribution of fish and ω-3 fatty acid intake was determined from responses to the Block Brief 2000 Food Frequency Questionnaire, and analyzed for associations with serum alanine aminotransferase, histological features of fatty liver disease, and diagnosis of steatohepatitis after adjusting for demographic, anthropometric, and dietary variables.

Results

The minority of subjects consumed the recommended 8 ounces of fish per week (22/223 [10%]) and 200 mg of long-chain ω-3 fatty acids per day (12/223 [5%]). Lack of fish and long-chain ω-3 fatty acid intake was associated with greater portal (P = 0.03 and P = 0.10, respectively) and lobular inflammation (P = 0.09 and P = 0.004, respectively) after controlling for potential confounders.

Conclusions

Fish and ω-3 fatty acid intake was insufficient in children with NAFLD, which may increase susceptibility to hepatic inflammation. Patients with pediatric NAFLD should be encouraged to consume the recommended amount of fish per week.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in children and adults. This study examined the relationship between hepatic nuclear receptor (NR) expression and histologic features of NAFLD. Drugs targeting a variety of NRs for nonalcoholic steatohepatitis (NASH) are in clinical trials. Liver messenger RNA was isolated from 40 children (10-19 years) undergoing end-of-treatment biopsy in the Treatment of NAFLD in Children (TONIC) trial. High-throughput quantitative polymerase chain reaction assayed NR messenger RNA. Cluster analysis was used to group 36 NRs, and NR levels were related to histologic measures of specific NAFLD features. Cluster analysis determined five groupings of NRs. Significant (P < 0.05) differential expressions of specific NRs associated with histologic measures include farnesoid X receptor alpha and retinoic acid receptor (RARβ and RARβ) for steatosis; estrogen receptor alpha (ERα) and peroxisome proliferator-activated receptor gamma 3 (PPARγ3) for hepatocellular ballooning; ER and PPARγ2 for lobular inflammation; PPARα/δ/γ1/γ2, ERα, constitutive androstane receptor, chicken ovalbumin upstream promoter transcription factor 1, RARα, RARβ1, retinoid X receptor, pregnane X receptor, thyroid hormone receptors α and β, and nuclear receptor related-1 for fibrosis; and ERα and RARβ/β1/α for diagnosis of NASH. Conclusion: Differential expression of specific NRs correlates with histologic severity of specific NAFLD features. These NRs are pleiotropic transactivators regulating basal metabolic functions and inflammatory responses. Derangement of activity of these receptors in NAFLD provides a rationale for exploiting their ability with receptor-specific ligands to ameliorate NASH and its consequences.

Aims

Fish and ω-3 fatty acids are reported to be beneficial in pediatric nonalcoholic fatty liver disease (NAFLD), but no studies have assessed their relation to histological severity. The objectives of this study were to evaluate the dietary intake of fish and ω-3 fatty acids in children with biopsy-proven NAFLD, and examine their association with serological and histological indicators of disease.

Methods

This was a cross-sectional analysis of 223 children (6-18 years) who participated in the Treatment of Nonalcoholic Fatty Liver Disease in Children trial or the NAFLD Database study conducted by the Nonalcoholic Steatohepatitis Clinical Research Network. The distribution of fish and ω-3 fatty acid intake was determined from responses to the Block Brief 2000 Food Frequency Questionnaire, and analyzed for associations with serum alanine aminotransferase, histological features of fatty liver disease, and diagnosis of steatohepatitis after adjusting for demographic, anthropometric, and dietary variables.

Results

The minority of subjects consumed the recommended 8 ounces of fish per week (22/223 [10%]) and 200 mg of long-chain ω-3 fatty acids per day (12/223 [5%]). Lack of fish and long-chain ω-3 fatty acid intake was associated with greater portal (P = 0.03 and P = 0.10, respectively) and lobular inflammation (P = 0.09 and P = 0.004, respectively) after controlling for potential confounders.

Conclusions

Fish and ω-3 fatty acid intake was insufficient in children with NAFLD, which may increase susceptibility to hepatic inflammation. Patients with pediatric NAFLD should be encouraged to consume the recommended amount of fish per week.

Importance

Nonalcoholic fatty liver disease (NAFLD) is a major chronic liver disease in children in the United States and is associated with insulin resistance. In adults, NAFLD is also associated with type 2 diabetes. To our knowledge, the prevalence of type 2 diabetes in children with NAFLD is unknown.

Objective

To determine the prevalence of type 2 diabetes and prediabetes in children with NAFLD and assess type 2 diabetes and prediabetes as risk factors for nonalcoholic steatohepatitis (NASH).

Design, setting, and participants

This was a multicenter, cross-sectional study at 12 pediatric clinical centers across the United States participating in the National Institute of Diabetes and Digestive and Kidney Diseases NASH Clinical Research Network. Children younger than 18 years with biopsy-confirmed NAFLD enrolled in the NASH Clinical Research Network.

Main outcomes and measures

The presence of type 2 diabetes and prediabetes as determined by American Diabetes Association screening criteria using clinical history and fasting laboratory values.

Results

There were 675 children with NAFLD included in the study with a mean age of 12.6 years and mean body mass index (calculated as weight in kilograms divided by height in meters squared) of 32.5. Most of the children were boys (480 of 675) and Hispanic (445 of 675).The estimated prevalence of prediabetes was 23.4% (95% CI, 20.2%-26.6%), and the estimated prevalence of type 2 diabetes was 6.5% (95% CI, 4.6%-8.4%). Girls with NAFLD had 1.6 (95% CI, 1.04-2.40) times greater odds of having prediabetes and 5.0 (95% CI, 2.49-9.98) times greater odds of having type 2 diabetes than boys with NAFLD. The prevalence of NASH was higher in those with type 2 diabetes (43.2%) compared with prediabetes (34.2%) or normal glucose (22%) (P < .001). The odds of having NASH were significantly higher in those with prediabetes (OR, 1.9; 95% CI, 1.21-2.9) or type 2 diabetes (OR, 3.1; 95% CI, 1.5-6.2) compared with those with normal glucose.

Conclusions and relevance

In this study, nearly 30% of children with NAFLD also had type 2 diabetes or prediabetes. These children had greater odds of having NASH and thus were at greater long-term risk for adverse hepatic outcomes.

Objective

Approximately 18 million people in the U.S. have coexisting type 2 diabetes and nonalcoholic fatty liver disease (NAFLD). It is not known who among these patients has nonalcoholic steatohepatitis (NASH) with advanced fibrosis. Therefore, we aimed to determine factors that are associated with both NASH and advanced fibrosis in patients with diabetes and NAFLD in order to identify who should be prioritized for referral to a hepatologist for further diagnostic evaluation and treatment.

Research design and methods

This study was derived from the NASH Clinical Research Network studies and included 1,249 patients with biopsy-proven NAFLD (including a model development cohort of 346 patients and an independent validation cohort of 100 patients with type 2 diabetes as defined by the American Diabetes Association criteria). Outcome measures were presence of NASH or advanced fibrosis (stage 3 or 4) using cross-validated, by jackknife method, multivariable-adjusted area under the receiver operating characteristic curve (AUROC) and 95% CI.

Results

The mean ± SD age and BMI of patients with diabetes and NAFLD was 52.5 ± 10.3 years and 35.8 ± 6.8 kg/m(2), respectively. The prevalence of NASH and advanced fibrosis was 69.2% and 41.0%, respectively. The model for NASH included white race, BMI, waist, alanine aminotransferase (ALT), Aspartate aminotransferase (AST), albumin, HbA1c, HOMA of insulin resistance, and ferritin with an AUROC of 0.80 (95% CI 0.75-0.84, P = 0.007). The specificity, sensitivity, negative predictive values (NPVs), and positive predictive values (PPVs) were 90.0%, 56.8%, 47.7%, and 93.2%, respectively, and the model correctly classified 67% of patients as having NASH. The model for predicting advanced fibrosis included age, Hispanic ethnicity, BMI, waist-to-hip ratio, hypertension, ALT-to-AST ratio, alkaline phosphatase, isolated abnormal alkaline phosphatase, bilirubin (total and direct), globulin, albumin, serum insulin, hematocrit, international normalized ratio, and platelet count with an AUROC of 0.80 (95% CI 0.76-0.85, P < 0.001). The specificity, sensitivity, NPV, and PPV were 90.0%, 57%, 75.1%, and 80.2%, respectively, and the model correctly classified 76.6% of patients as having advanced fibrosis. Results remained consistent for both models in the validation cohort. The proposed model performed better than the NAFLD fibrosis score in detecting advanced fibrosis.

Conclusions

Routinely available clinical variables can be used to quantify the likelihood of NASH or advanced fibrosis in adult diabetic patients with NAFLD. The clinical models presented can be used to guide clinical decision making about referrals of patients with diabetes and NAFLD to hepatologists.

Background and aims

Emerging data from a single-center study suggests that a 30% relative reduction in liver fat content as assessed by magnetic resonance imaging-proton density fat fraction (MRI-PDFF) from baseline may be associated with histologic improvement in nonalcoholic steatohepatitis (NASH). There are limited multicenter data comparing an active drug versus placebo on the association between the quantity of liver fat reduction assessed by MRI-PDFF and histologic response in NASH. This study aims to examine the association between 30% relative reduction in MRI-PDFF and histologic response in obeticholic acid (OCA) versus placebo-treated patients in the FLINT (farnesoid X receptor ligand obeticholic acid in NASH trial).

Approach and results

This is a secondary analysis of the FLINT trial including 78 patients with MRI-PDFF measured before and after treatment along with paired liver histology assessment. Histologic response was defined as a 2-point improvement in nonalcoholic fatty liver disease activity score without worsening of fibrosis. OCA (25 mg orally once daily) was better than placebo in improving MRI-PDFF by an absolute difference of -3.4% (95% confidence interval [CI], -6.5 to -0.2%, P value = 0.04) and relative difference of -17% (95% CI, -34 to 0%, P value = 0.05). The optimal cutoff point for relative decline in MRI-PDFF for histologic response was 30% (using Youden's index). The rate of histologic response in those who achieved less than 30% decline in MRI-PDFF versus those who achieved a 30% or greater decline in MRI-PDFF (MRI-PDFF responders) relative to baseline was 19% versus 50%, respectively. Compared with MRI-PDFF nonresponders, MRI-PDFF responders demonstrated both a statistically and clinically significant higher odds 4.86 (95% CI, 1.4-12.8, P value < 0.009) of histologic response, including significant improvements in both steatosis and ballooning.

Conclusion

OCA was better than placebo in reducing liver fat. This multicenter trial provides data regarding the association between 30% decline in MRI-PDFF relative to baseline and histologic response in NASH.

Background and aims

Predictive, noninvasive tools are needed to monitor key features of nonalcoholic fatty liver disease (NAFLD) in children that relate to improvement in liver histology. The purpose of this study was to evaluate the relationship between liver chemistries and liver histology using data from the CyNCh (Cysteamine Bitartrate Delayed-Release for the Treatment of NAFLD in Children) clinical trial.

Approach and results

This study included 146 children. Improvement in liver histology, defined as decrease in nonalcoholic fatty liver disease (NAFLD) Activity Score ≥2 points without worsening of fibrosis, occurred in 43 participants (30%). There were 46 participants with borderline zone 1 nonalcoholic steatohepatitis (NASH) at baseline, with resolution in 28% (12 of 46). Multivariate models were constructed using baseline and change in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) at 52 weeks, for improvement in (1) liver histology primary outcome, (2) borderline zone 1 NASH, and (3) fibrosis. For improvement in histology, the model (P < 0.0001) retained baseline and change in GGT (area under the receiver operating characteristic [AUROC], 0.79; 95% confidence interval [CI], 0.71-0.87). For borderline zone 1 NASH, the model (P = 0.0004) retained baseline and change in ALT (AUROC, 0.80; 95% CI, 0.67-0.93). For fibrosis, the model (P < 0.001) retained baseline and change in ALT (AUROC, 0.80; 95% CI, 0.67-0.93). Additional clinical parameters were added to the models using Akaike's information criterion selection, and significantly boosted performance: improvement in histology with AUROC of 0.89 (95% CI, 0.82-0.95), borderline zone 1 NASH with AUROC of 0.91 (95% CI, 0.83-0.99), and fibrosis with AUROC of 0.89 (95% CI, 0.82-0.94). Models were validated using data from the TONIC (Treatment of Nonalcoholic Fatty Liver Disease in Children) trial.

Conclusions

In children with NAFLD, dynamic changes in serum ALT and GGT are associated with change in liver histology and appear to be powerful indicators of histological response.

Background & aims

Cross-sectional studies have associated serum levels of the keratin 18 (K18) fragment with histologic features of liver in individuals with nonalcoholic fatty liver disease (NAFLD). We investigated the relationship between changes in serum levels of K18 and changes in liver histology in adults and children with NAFLD.

Methods

We measured levels of K18 in stored serum samples collected at baseline and various time points from 231 adults with nonalcoholic steatohepatitis and 152 children with NAFLD who participated in 2 separate prospective randomized clinical trials. Liver biopsy specimens collected at baseline and week 96 were reviewed centrally.

Results

There were greater decreases in serum levels of K18 in adults with histologic improvement at week 96 than in those without histologic improvement at week 16 (decrease, 193 ± 293 vs 139 ± 467 U/L; P < .001), week 48 (decrease, 232 ± 360 vs 113 ± 425 U/L; P < .001), or week 96 (decrease, 269 ± 368 vs 97 ± 400 U/L; P < .001). There were greater decreases in serum levels of K18 in children with histologic improvements than in those without histologic improvements at week 48 (decrease, 197 ± 467 vs 47 ± 350 U/L; P = .005) and week 96 (decrease, 206 ± 432 vs 2 ± 474 U/L; P < .001). However, reductions in serum levels of K18 were not better than reductions in levels of alanine aminotransferase in identifying adults with histologic improvement (area under the receiver operator characteristic [AUROC], 0.71; 95% confidence interval [CI], 0.63-0.80; vs AUROC, 0.68; 95% CI, 0.61-0.79; P = .34) or children (AUROC, 0.72; 95% CI, 0.63-0.81; vs AUROC, 0.79; 95% CI, 0.70-0.87; P = .42).

Conclusions

Decreases in serum levels of K18 are associated strongly with improved liver histologies in adults or children with NAFLD. However, reductions in K18 do not perform better than those in alanine aminotransferase level in identifying histologic changes in NAFLD.