Environmental toxicants modulate disease severity in pediatric metabolic dysfunction-associated steatohepatitis.

OBJECTIVES: Metabolic dysfunction-associated steatotic liver disease (MASLD) is common in children. We hypothesized environmental toxins could drive progression to metabolic dysfunction-associated steatohepatitis (MASH), and assayed serum toxins and metabolites in children with histologically characterized MASLD/MASH. METHODS: Environmental chemicals, common in household items, perfluoroalkyl substances (PFAS), brominated flame retardants (PBDEs), and metabolic profiles were assayed in children enrolled in the multicenter NASH Clinical Research Network Pediatric Database 2. Mixture models, using repeated holdout weighted quantile sum regression (WQSrh) were run in addition to single chemical/metabolite logistic regression. For metabolomic analyses, random subset version of WQSrh was used for the large number of predictors versus participants. Nominal and false discovery rate (FDR) p-values (two-sided) were computed. RESULTS: Four hundred and thirty-five children distributed across MASH (n = 293) and MASLD (n = 142), with 304 (69.9%) males. Mean (standard deviation) for Nonalcoholic Steatohepatitis Score (NAS) and alanine aminotransferase (ALT) for MASLD were 3.1 (1.0), 67.9 (43.4), and for MASH 4.2 (1.4), 144 (121). There was an inverse association between PFAS/PBDE mixture and MASH versus MASLD, lobular inflammation (p = 0.026), NAS (p = 0.009, FDR p = 0.04), and log-transformed ALT (p = 0.005, FDR p = 0.025) driven by perfluorohexane sulfonate (PFHxS). Metabolites from positive hydrophilic interaction liquid chromatography mode, biliverdin (p = 0.002) and 1-methylhistidine (associated with meat ingestion, p = 0.02) and reverse phase negative mode, hippuric acid (solvent exposure, p = 0.022) significantly associated with MASH. CONCLUSIONS: Significant negative PFAS/PBDE mixture effect and odds of MASH were dominated by PHFXS. Several metabolites are significantly associated with MASH which inform mechanistic pathways and could drive key therapeutic and diagnostic strategies in children.

Fibrosis, biomarkers and liver biopsy in AAT deficiency and relation to liver Z protein polymer accumulation.

BACKGROUND AND AIMS: The course of adults with ZZ alpha-1-antitrypsin deficiency (AATD) liver disease is unpredictable. The utility of markers, including liver biopsy, is undefined. METHODS: A prospective cohort, including protocol liver biopsies, was enrolled to address these questions. RESULTS: We enrolled 96 homozygous ZZ AATD adults prospectively at three US sites with standardized clinical evaluations, and protocol liver biopsies. Fibrosis was scored using Ishak (stages 0-6). Also, 51% of the 96 subjects had Ishak score >1 fibrosis (49% Ishak 0-1, 36% Ishak 2-3 and 15% ≥4). Elevated aspartate aminotransferase (AST) more than alanine aminotransferase (ALT), high body mass index (BMI), obesity, AST platelet ratio index and elevated serum Z alpha 1 antitrypsin (AAT) polymer levels were associated with increased fibrosis. Steatosis did not correlate to fibrosis. Increased fibrosis was associated with increased mutant Z polymer globular inclusions (p = .002) and increased diffuse cytoplasmic Z polymer on biopsy (p = .0029) in a direct relationship. Increased globule Z polymer was associated with increased serum AST (p = .007) and increased periportal inflammation on histopathology (p = .004), but there was no relationship of Z polymer hepatocellular accumulation with ALT, gamma glutamine transferase, inflammation in other parts of the lobule, necrosis or steatosis. Serum Z polymer levels were directly correlated to hepatic Z protein polymer content. Lung function, smoking and alcohol consumption patterns were not associated with fibrosis. CONCLUSION: In AATD high BMI, obesity and elevated AST are associated with increased fibrosis. Liver biopsy features are correlated to some serum tests. Serum Z AAT polymer levels could be a future biomarker to detect fibrosis early and is directly correlated to liver Z content.

Liver stiffness progression in biopsy-proven metabolic dysfunction-associated steatotic disease among people with diabetes versus people without diabetes: A prospective multicenter study.

BACKGROUND AND AIMS: There are limited data on the progression of liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE) in people with type 2 diabetes mellitus (T2DM) versus those without T2DM in biopsy-proven metabolic dysfunction-associated steatotic liver disease. We examined LSM progression in participants with T2DM versus those without T2DM in a large, prospective, multicenter cohort study. APPROACH AND RESULTS: This study included 1231 adult participants (62% female) with biopsy-proven metabolic dysfunction-associated steatotic liver disease who had VCTEs at least 1 year apart. LSM progression and regression were defined by a ≥20% increase and an upward or downward change, respectively, in the LSM category in the Baveno VII categories for compensated advanced chronic liver disease, compared between participants with T2DM (n = 680) versus no T2DM (n = 551) at baseline. The mean (±SD) age and body mass index were 51.8 (±12.0) years and 34.0 (±6.5) kg/m 2 , respectively. The median (IQR) time between the first and last VCTE measurements was 4.1 (2.5-6.5) years. Participants with T2DM had higher LSM progression at 4 years (12% vs. 10%), 6 years (23% vs. 16%), and 8 years (50% vs. 39%), p = 0.04. Using a multivariable Cox proportional hazards model adjusted for multiple confounders, the presence of T2DM remained an independent predictor of LSM progression (adjusted HR: 1.35, 95% CI: 1.01-1.81, p = 0.04). T2DM was not associated with LSM regression ( p = 0.71). Mean HbA1c was significantly associated with LSM progression ( p = 0.003) and regression ( p = 0.02). CONCLUSIONS: Using serial VCTE data from a multicenter study of participants with biopsy-proven metabolic dysfunction-associated steatotic liver disease, we demonstrate that T2DM and HbA1c are associated with LSM progression.

Increases and decreases in liver stiffness measurement are independently associated with the risk of liver-related events in NAFLD.

BACKGROUND & AIMS: The clinical significance of change in liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE) in patients with non-alcoholic fatty liver disease (NAFLD) is not well-understood. We prospectively defined rates of progression to and regression from LSM-defined compensated advanced chronic liver disease (cACLD) and their associations with liver-related events (LREs). METHODS: Participants in the NASH Clinical Research Network-led NAFLD Database 2 and 3 studies were included. Progression to cACLD was defined as reaching LSM ≥10 kPa in participants with LSM <10 kPa on initial VCTE; regression from cACLD was defined as reaching LSM <10 kPa in participants with baseline LSM ≥10 kPa. LREs were defined as liver-related death, liver transplant, hepatocellular carcinoma, MELD >15, development of varices, or hepatic decompensation. Univariate and multivariable interval-censored Cox regression analyses were used to compare the cumulative LRE probability by LSM progression and regression status. RESULTS: In 1,403 participants, 89 LREs developed over a mean follow-up of 4.4 years, with an annual incidence rate for LREs of 1.5 (95% CI 1.2-1.8). In participants at risk, progression to LSM ≥10 or ≥15 kPa occurred in 29% and 17%, respectively, whereas regression to LSM <10 or <15 kPa occurred in 44% and 49%, respectively. Progressors to cACLD (≥10 kPa) experienced a higher cumulative LRE rate vs. non-progressors (16% vs. 4%, adjusted hazard ratio 4.0; 95% (1.8-8.9); p <0.01). Regressors from cACLD (to LSM <10 kPa) experienced a lower LRE rate than non-regressors (7% vs. 32%, adjusted hazard ratio 0.25; 95% CI 0.10-0.61; p <0.01). CONCLUSIONS: Change in LSM over time is independently and bi-directionally associated with risk of LRE and is a non-invasive surrogate for clinical outcomes in patients with NAFLD. IMPACT AND IMPLICATIONS: The prognostic value of change in LSM in patients with NAFLD is not well understood. In this large prospective study of patients with NAFLD and serial vibration-controlled transient elastography exams, baseline and dynamic changes in LSM were associated with the risk of developing liver-related events. LSM is a useful non-invasive surrogate of clinical outcomes in patients with NAFLD.

PNPLA3 rs738409, age, diabetes, sex, and advanced fibrosis jointly contribute to the risk of major adverse liver outcomes in metabolic dysfunction-associated steatotic liver disease.

BACKGROUND AND AIMS: The patatin-like phospholipase domain-containing protein 3 ( PNPLA3 ) rs738409 variant is associated with steatotic liver disease and its progression. We examined the association between PNPLA3 and the development of major adverse liver outcomes (MALOs) and how nonmodifiable and modifiable conditions modify this relationship. APPROACH AND RESULTS: A total of 2075 adults with biopsy-confirmed metabolic dysfunction-associated steatotic liver disease (MASLD) were enrolled in the metabolic dysfunction-associated steatohepatitis Clinical Research Network (MASH CRN) studies and followed prospectively until death, transplant, or withdrawal of consent. One hundred four MALOs were recorded during an average of 4.3 years. PNPLA3 G-allele (Adj. sub-hazard ratio (sHR): 1.4, 95% CI: 1.07-1.8), advanced fibrosis (AF) (Adj. sHR: 7.8, 95% CI: 4.4-13.8), age >60 years (Adj. sHR: 2.9, 95% CI: 1.3-6.8), and type 2 diabetes mellitus (Adj. sHR: 2.8, 95% CI: 1.8-4.2) were associated with MALO. Among participants with AF, those carrying the G-allele displayed the highest cumulative incidence of MALO (85%) versus noncarriers (53%), p =0.03, and p -value for interaction <0.01. The strength of the association between PNPLA3 and MALO was statistically significantly greater among older than 60 years (sHR: 2.1, 95% CI: 1.5-2.8), women (sHR: 1.4, 95% CI: 1.1-1.9), and those with AF (sHR: 1.9, 95% CI: 1.5-2.4) or type 2 diabetes mellitus (sHR: 2.1, 95% CI: 1.5-2.8) as compared with their counterparts, p -value for interaction between PNPLA3 and each factor<0.01. CONCLUSIONS: The deleterious effects of PNPLA3 rs738409 on the risk of MALO are significantly worsened by AF, age, type 2 diabetes mellitus, and sex.

High Concordance Between Nonalcoholic Fatty Liver Disease and Metabolic Dysfunction-Associated Steatotic Liver Disease in the TARGET-NASH Real-World Cohort.

INTRODUCTION: This study investigates the applicability of the new metabolic dysfunction-associated steatotic liver disease (MASLD) nomenclature to the real-world TARGET-NASH US adult cohort. METHODS: The new MASLD/metabolic steatohepatitis nomenclature was applied to patients enrolled with pragmatic diagnoses of nonalcoholic fatty liver and nonalcoholic steatohepatitis (NASH), and NASH cirrhosis and concordance were determined between the definitions. RESULTS: Approximately 99% of TARGET-NASH participants met the new MASLD diagnostic criteria. Approximately 1,484/1,541 (96.3%, kappa 0.974) nonalcoholic fatty liver patients (metabolic dysfunction-associated steatotic liver), 2,195/2,201 (99.7%, kappa 0.998) NASH patients (metabolic steatohepatitis), and 1,999/2,003 (99.8%, kappa 0.999) NASH cirrhosis patients met the new criteria. DISCUSSION: The new MASLD nomenclature is highly concordant with the previous TARGET-NASH pragmatic definitions.

Metabolic dysfunction-associated steatotic liver disease: Update and impact of new nomenclature on the American Association for the Study of Liver Diseases practice guidance on nonalcoholic fatty liver disease.

This commentary discusses how clinicians and various stakeholders can utilize the recently published American Association for the Study of Liver Diseases nonalcoholic fatty liver disease (AASLD NAFLD) Practice Guidance in light of the change in the nomenclature to steatotic liver disease and its subcategories. The new terminologies explained in this commentary make it easier for the readers to interchangeably use metabolic dysfunction-associated steatotic liver disease (MASLD) in place of NAFLD and metabolic-dysfunction associated steatohepatitis (MASH) instead of nonalcoholic steatohepatitis (NASH), respectively, as they read the NAFLD Practice Guidance. The guidance document is relevant and can be utilized for the diagnosis, risk stratification, and management of patients with MASLD. This commentary serves as an accompanying article to the NAFLD Practice Guidance and helps it clinical application in the light of the new nomenclature.

Pegbelfermin in Patients With Nonalcoholic Steatohepatitis and Stage 3 Fibrosis (FALCON 1): A Randomized Phase 2b Study.

BACKGROUND & AIMS: Pegbelfermin is a polyethlene glycol-conjugated analog of human fibroblast growth factor 21, a nonmitogenic hormone that regulates energy metabolism. This phase 2b study evaluated 48-week pegbelfermin treatment in patients with nonalcoholic steatohepatitis (NASH) and stage 3 (bridging) fibrosis. METHODS: The FALCON 1 study (NCT03486899) was a multicenter, randomized (1:1:1:1), double-blind, placebo-controlled study. Patients with biopsy-confirmed NASH and stage 3 fibrosis (N = 197) received weekly subcutaneous pegbelfermin (10, 20, or 40 mg) or placebo injections for 48 weeks. The week 24 primary endpoint was a ≥1-point decrease in fibrosis score without NASH worsening or NASH improvement without fibrosis worsening; pegbelfermin dose response was assessed using a Cochran-Armitage trend test across proportions (1-sided α = 0.05). Secondary/exploratory endpoints included histological and noninvasive measures of steatosis, fibrosis, and liver injury/inflammation. RESULTS: At week 24, the primary endpoint was met by 14% (placebo) vs 24%-31% (pegbelfermin arms); statistical significance was not reached due to lack of pegbelfermin dose response (P = .134). At weeks 24 and 48, more patients who received pegbelfermin had ≥30% relative reductions in hepatic fat fraction (magnetic resonance imaging-proton density fat fraction) vs placebo, although no differences reached statistical significance. In the pegbelfermin arms, improvements in liver fibrosis (magnetic resonance elastography and N-terminal type III collagen propeptide) and liver injury/inflammation (alanine aminotransferase, aspartate aminotransferase) were observed vs placebo. Adverse events occurred at similar frequencies across arms. No treatment-related serious adverse events were observed. CONCLUSIONS: The FALCON 1 study did not meet its primary endpoint; a ≥1-point decrease in fibrosis score without NASH worsening or NASH improvement without fibrosis worsening assessed via biopsy. Pegbelfermin was generally well tolerated during 48 weeks of treatment.

Pegbelfermin in Patients With Nonalcoholic Steatohepatitis and Compensated Cirrhosis (FALCON 2): A Randomized Phase 2b Study.

BACKGROUND & AIMS: Pegbelfermin is a polyethylene glycol-conjugated analog of human fibroblast growth factor 21, a nonmitogenic hormone that regulates energy metabolism. This phase 2b study evaluated 48-week pegbelfermin treatment in patients with nonalcoholic steatohepatitis (NASH) with compensated cirrhosis. METHODS: FALCON 2 (NCT03486912) was a randomized (1:1:1:1), double-blind, placebo-controlled study. Eligible adults had biopsy-confirmed NASH and stage 4 fibrosis. Pegbelfermin (10, 20, or 40 mg) or placebo was injected subcutaneously once weekly. The primary endpoint was 1 or more stages of improvement in the NASH Clinical Research Network fibrosis score without NASH worsening at week 48; pegbelfermin dose response was assessed using a Cochran-Armitage trend test across proportions (1-sided α = .05). Additional endpoints included histologic and noninvasive measures of steatosis, fibrosis, and liver injury/inflammation. RESULTS: Overall, 155 patients were randomized, and 154 patients received treatment. At week 48, 24% to 28% of the pegbelfermin arms had primary endpoint responses vs 31% of the placebo arm (P = .361). Nonalcoholic fatty liver disease activity score improvements were more frequent with pegbelfermin vs placebo and were driven primarily by reduced lobular inflammation. Numerically higher proportions of the pegbelfermin arms had liver stiffness (magnetic resonance elastography) and steatosis (magnetic resonance imaging-proton density fat fraction) improvements vs placebo; these differences were not statistically significant. Mean N-terminal type III collagen propeptide, alanine aminotransferase, and aspartate aminotransferase values were numerically lower in the 20- and/or 40-mg pegbelfermin arms compared with placebo. Serious adverse events were more frequent with pegbelfermin vs placebo, although none were treatment related. One patient (40-mg pegbelfermin) discontinued treatment because of a treatment-emergent adverse event (worsening ascites). CONCLUSIONS: FALCON 2 did not meet its primary endpoint of 1 or more stages of improvement in the NASH Clinical Research Network fibrosis without NASH worsening assessed via biopsy. Pegbelfermin generally was well tolerated in this advanced NASH population.

Cenicriviroc Lacked Efficacy to Treat Liver Fibrosis in Nonalcoholic Steatohepatitis: AURORA Phase III Randomized Study.

BACKGROUND AND AIMS: Cenicriviroc (CVC) is a novel, orally administered, chemokine receptor type 2 and 5 antagonist that showed antifibrotic potential in preclinical and phase IIb studies of nonalcoholic steatohepatitis (NASH). Herein, we report efficacy and safety results from the phase III study. METHODS: The AURORA (A Study for the Efficacy and Safety of CVC for the Treatment of Liver Fibrosis in Adults With NASH) study was a phase III, randomized, double-blind, placebo-controlled, 2-part study of patients with NASH and stage 2/3 liver fibrosis. Adults, 18-75 years of age, were randomized to CVC 150 mg or placebo once daily for 12 months (part 1) or 60 months (part 2). Liver biopsies were performed at screening, month 12, and early study discontinuation or termination. The primary efficacy endpoint was the proportion of patients with fibrosis improvement ≥1 stage without worsening of steatohepatitis at month 12 relative to screening. Adverse events were assessed throughout the study. RESULTS: A total of 1778 patients were randomized and discontinued (part 1: n = 1293; part 2: n = 485). In part 1, at month 12, a similar proportion of patients receiving CVC or placebo achieved the primary endpoint (22.3% vs 25.5%; odds ratio, 0.84; 95% confidence interval, 0.63-1.10; P = .21) and complete resolution of steatohepatitis without worsening of fibrosis (23.0% vs 27.2%; P = .21). The safety profile was generally comparable across treatment groups. CONCLUSIONS: This study did not demonstrate the efficacy of CVC for treating liver fibrosis assessed by histology in adults with NASH; however, CVC was safe and well tolerated in patients with NASH and liver fibrosis. (ClinicalTrials.gov, Number: NCT03028740).

Age, BMI, and Type 2 Diabetes Modify the Relationship Between PNPLA3 and Advanced Fibrosis in Children and Adults With NAFLD.

BACKGROUND & AIMS: PNPLA3 G-allele is an important determinant of disease severity in nonalcoholic fatty liver disease (NAFLD). Here, we investigated the effect of age, body mass index (BMI), and type 2 diabetes mellitus (T2DM) on the relationship between PNPLA3 G-allele and advanced fibrosis in adults and children with histologically characterized NAFLD. METHODS: A total of 1047 children and 2057 adults were included. DNA was genotyped for rs738409 in duplicate. Primary outcome of interest was advanced fibrosis (fibrosis stage ≥3). Regression analyses were performed after controlling for relevant covariates. An additive model was used to assess the effect of PNPLA3 G-allele (CC vs CG vs GG). RESULTS: PNPLA3 G-allele was significantly associated with advanced fibrosis in children (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.16-2.09) and adults (OR, 1.55; 95% CI, 1.16-1.54). Across the cohort, older age significantly increased the risk for advanced fibrosis for PNPLA3 CC (OR, 1.019; 95% CI, 1.013-1.026), CG (OR, 1.024; 95% CI, 1.018-1.030), and GG (OR, 1.03; 95% CI, 1.023-1.037) genotypes. BMI significantly increased the relationship between PNPLA3 genotypes and advanced fibrosis in children and adults. A BMI of 30 kg/m2 was the cutoff beyond which PNPLA3 G-allele had exponential effect on the risk for advanced fibrosis in children and adults. T2DM significantly worsened the relationship between PNPLA3 G-allele and advanced fibrosis in children and adults (interaction P < .01 for both). CONCLUSIONS: Age, BMI, and T2DM modify the risk of advanced fibrosis associated with PNPLA3 G-allele. Preventing or reversing T2DM and obesity in persons carrying PNPLA3 G-allele may lower the risk for advanced fibrosis in NAFLD.

Nutrition assessment and MASH severity in children using the Healthy Eating Index.

BACKGROUND: Pediatric metabolic-associated fatty liver disease (MAFLD) is a global health problem, with lifestyle modification as its major therapeutic strategy. Rigorous characterization of dietary content on MAFLD in children is lacking. We hypothesized an objectively measured healthier diet would positively modulate MAFLD. METHODS: Diet was assessed using the Nutrition Data System for Research in children enrolled from 10 tertiary clinical centers to determine the Healthy Eating Index (HEI, 0-100) and individual food components. RESULTS: In all, 119 children were included (13.3 ± 2.7 y), 80 (67%) male, 67 (18%) White, and 90 (76%) Hispanic, with an average body mass index Z-score of 2.2 ± 0.5. Diet was classified as low HEI < 47.94 (n = 39), mid HEI ≥ 47.94 and < 58.89 (n = 41), or high HEI ≥ 58.89 (n=39). Children with high HEI (healthier diet) had lower body weight (p = 0.005) and more favorable lipids. Mean serum triglycerides for low, mid, and high HEI were 163, 148, and 120 mg/dL, respectively; p = 0.04 mid versus high, p = 0.01 low versus high. Mean HDL was 38, 41 and 43 mg/dL; p = 0.02 low vs high. Less severe steatosis was noted with added sugar ≤ 10% of calories (p = 0.03). Higher lobular inflammation is associated with a higher percentage of calories from fat (OR (95% CI) = 0.95 (0.91-1.00), p = 0.04). CONCLUSIONS: In children with MAFLD, high HEI is associated with lower body weight and more favorable lipids, while added sugar and fat intake has individual histologic features. Differential consumption of major dietary components may modify both metabolic risk factors and histologic liver injury, highlighting the importance of objective diet assessments in children with MAFLD.

Inhibition of integrin binding to ligand arg-gly-asp motif induces AKT-mediated cellular senescence in hepatic stellate cells.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play an essential role in liver fibrogenesis. The induction of cellular senescence has been reported to inhibit HSC activation. Previously, we demonstrated that CWHM12, a small molecule arginine-glycine-aspartic acid (RGD) peptidomimetic compound, inhibits HSC activation. This study investigated whether the inhibitory effects of CWHM12 on HSCs affected cellular senescence. METHODS: The immortalized human HSC lines, LX-2 and TWNT-1, were used to evaluate the effects of CWHM12 on cellular senescence via the disruption of RGD-mediated binding to integrins. RESULTS: CWHM12 induces cell cycle arrest, senescence-associated beta-galactosidase activity, acquisition of senescence-associated secretory phenotype (SASP), and expression of senescence-associated proteins in HSCs. Further experiments revealed that the phosphorylation of AKT and murine double minute 2 (MDM2) was involved in the effects of CWHM12, and the inhibition of AKT phosphorylation reversed these effects of CWHM12 on HSCs. CONCLUSIONS: Pharmacological inhibition of RGD-mediated integrin binding induces senescence in activated HSCs.

Diagnostic performance of circulating biomarkers for non-alcoholic steatohepatitis.

There are no approved diagnostic biomarkers for at-risk non-alcoholic steatohepatitis (NASH), defined by the presence of NASH, high histological activity and fibrosis stage ≥2, which is associated with higher incidence of liver-related events and mortality. FNIH-NIMBLE is a multi-stakeholder project to support regulatory approval of NASH-related biomarkers. The diagnostic performance of five blood-based panels was evaluated in an observational (NASH CRN DB2) cohort (n = 1,073) with full spectrum of non-alcoholic fatty liver disease (NAFLD). The panels were intended to diagnose at-risk NASH (NIS4), presence of NASH (OWLiver) or fibrosis stages >2, >3 or 4 (enhanced liver fibrosis (ELF) test, PROC3 and FibroMeter VCTE). The prespecified performance metric was an area under the receiver operating characteristic curve (AUROC) ≥0.7 and superiority over alanine aminotransferase for disease activity and the FIB-4 test for fibrosis severity. Multiple biomarkers met these metrics. NIS4 had an AUROC of 0.81 (95% confidence interval: 0.78-0.84) for at-risk NASH. The AUROCs of the ELF test, PROC3 and FibroMeterVCTE for clinically significant fibrosis (≥stage 2), advanced fibrosis (≥stage 3) or cirrhosis (stage 4), respectively, were all ≥0.8. ELF and FibroMeter VCTE outperformed FIB-4 for all fibrosis endpoints. These data represent a milestone toward qualification of several biomarker panels for at-risk NASH and also fibrosis severity in individuals with NAFLD.

Ube4A maintains metabolic homeostasis and facilitates insulin signaling in vivo.

OBJECTIVE: Defining the regulators of cell metabolism and signaling is essential to design new therapeutic strategies in obesity and NAFLD/NASH. E3 ubiquitin ligases control diverse cellular functions by ubiquitination-mediated regulation of protein targets, and thus their functional aberration is associated with many diseases. The E3 ligase Ube4A has been implicated in human obesity, inflammation, and cancer. However, its in vivo function is unknown, and no animal models are available to study this novel protein. METHODS: A whole-body Ube4A knockout (UKO) mouse model was generated, and various metabolic parameters were compared in chow- and high fat diet (HFD)-fed WT and UKO mice, and in their liver, adipose tissue, and serum. Lipidomics and RNA-Seq studies were performed in the liver samples of HFD-fed WT and UKO mice. Proteomic studies were conducted to identify Ube4A's targets in metabolism. Furthermore, a mechanism by which Ube4A regulates metabolism was identified. RESULTS: Although the body weight and composition of young, chow-fed WT and UKO mice are similar, the knockouts exhibit mild hyperinsulinemia and insulin resistance. HFD feeding substantially augments obesity, hyperinsulinemia, and insulin resistance in both sexes of UKO mice. HFD-fed white and brown adipose tissue depots of UKO mice have increased insulin resistance and inflammation and reduced energy metabolism. Moreover, Ube4A deletion exacerbates hepatic steatosis, inflammation, and liver injury in HFD-fed mice with increased lipid uptake and lipogenesis in hepatocytes. Acute insulin treatment resulted in impaired activation of the insulin effector protein kinase Akt in liver and adipose tissue of chow-fed UKO mice. We identified the Akt activator protein APPL1 as a Ube4A interactor. The K63-linked ubiquitination (K63-Ub) of Akt and APPL1, known to facilitate insulin-induced Akt activation, is impaired in UKO mice. Furthermore, Ube4A K63-ubiquitinates Akt in vitro. CONCLUSION: Ube4A is a novel regulator of obesity, insulin resistance, adipose tissue dysfunction and NAFLD, and preventing its downregulation may ameliorate these diseases.

Enhancing Hepatic MBOAT7 Expression in Mice With Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Polymorphisms near the membrane bound O-acyltransferase domain containing 7 (MBOAT7) genes are associated with worsened nonalcoholic fatty liver (NASH), and nonalcoholic fatty liver disease (NAFLD)/NASH may decrease MBOAT7 expression independent of these polymorphisms. We hypothesized that enhancing MBOAT7 function would improve NASH. METHODS: Genomic and lipidomic databases were mined for MBOAT7 expression and hepatic phosphatidylinositol (PI) abundance in human NAFLD/NASH. Male C57BL6/J mice were fed either choline-deficient high-fat diet or Gubra Amylin NASH diet and subsequently infected with adeno-associated virus expressing MBOAT7 or control virus. NASH histological scoring and lipidomic analyses were performed to assess MBOAT7 activity, hepatic PI, and lysophosphatidylinositol (LPI) abundance. RESULTS: Human NAFLD/NASH decreases MBOAT7 expression and hepatic abundance of arachidonate-containing PI. Murine NASH models display subtle changes in MBOAT7 expression, but significantly decreased activity. After MBOAT7 overexpression, liver weights, triglycerides, and plasma alanine and aspartate transaminase were modestly improved by MBOAT7 overexpression, but NASH histology was not improved. Despite confirmation of increased activity with MBOAT7 overexpression, content of the main arachidonoylated PI species was not rescued by MBOAT7 although the abundance of many PI species was increased. Free arachidonic acid was elevated but the MBOAT7 substrate arachidonoyl-CoA was decreased in NASH livers compared to low-fat controls, likely due to the decreased expression of long-chain acyl-CoA synthetases. CONCLUSION: Results suggest decreased MBOAT7 activity plays a role in NASH, but MBOAT7 overexpression fails to measurably improve NASH pathology potentially due to the insufficient abundance of its arachidonoyl-CoA substrate.

Fibrosis Progression Rate in Biopsy-Proven Nonalcoholic Fatty Liver Disease Among People With Diabetes Versus People Without Diabetes: A Multicenter Study.

BACKGROUND & AIMS: There are limited data regarding fibrosis progression in biopsy-proven nonalcoholic fatty liver disease (NAFLD) in people with type 2 diabetes mellitus (T2DM) compared with people without T2DM. We assessed the time to fibrosis progression in people with T2DM compared with people without T2DM in a large, multicenter, study of people with NAFLD who had paired liver biopsies. METHODS: This study included 447 adult participants (64% were female) with NAFLD who had paired liver biopsies more than 1 year apart. Liver histology was systematically assessed by a central pathology committee blinded to clinical data. The primary outcome was the cumulative incidence of a ≥1-stage increase in fibrosis in participants with T2DM compared with participants without T2DM. RESULTS: The mean (SD) age and body mass index (calculated as weight in kilograms divided by the square of the height in meters) were 50.9 (11.5) years and 34.7 (6.3), respectively. The median time between biopsies was 3.3 years (interquartile range, 1.8-6.1 years). Participants with T2DM had a significantly higher cumulative incidence of fibrosis progression at 4 years (24% vs 20%), 8 years (60% vs 50%), and 12 years (93% vs 76%) (P = .005). Using a multivariable Cox proportional hazards model adjusted for multiple confounders, T2DM remained an independent predictor of fibrosis progression (adjusted hazard ratio, 1.69; 95% CI, 1.17-2.43; P = .005). The cumulative incidence of fibrosis regression by ≥1 stage was similar in participants with T2DM compared with participants without T2DM (P = .24). CONCLUSIONS: In this large, multicenter cohort study of well-characterized participants with NAFLD and paired liver biopsies, we found that fibrosis progressed faster in participants with T2DM compared with participants without T2DM. These data have important implications for clinical practice and trial design.

The nonalcoholic steatohepatitis extended hepatocyte ballooning score: histologic classification and clinical significance.

BACKGROUND AND AIMS: The NAFLD activity score was developed to measure histologic changes in NAFLD during therapeutic trials. Hepatocyte ballooning (HB) is the most specific feature in steatohepatitis diagnosis, yet the impact of variations in HB has not been incorporated. APPROACH AND RESULTS: Liver biopsies from patients enrolled in the NASH Clinical Research Network with an initial diagnosis of NASH or NAFL (n=1688) were evaluated to distinguish classic hepatocyte ballooning (cHB) from smaller, nonclassic hepatocyte ballooning (nHB), and also to designate severe ballooning and assign an extended hepatocyte ballooning (eB) score [0 points, no ballooning (NB); 1 point, few or many nHB; 2 points, few cHB; 3 points, many cHB; 4 points, severe cHB] to the biopsy assessment. The eB score was reproducible among NASH CRN liver pathologists (weighted kappa 0.76) and was significantly associated with older age (mean 52.1 y, cHB; 48.5 y, nHB, p<0.001), gender (72.3% female, cHB; 54.5% female, nHB, p<0.001), diabetes (49.8% diabetes, cHB; 28.2% diabetes, nHB, p<0.001), metabolic syndrome (68.5% metabolic syndrome, nHB; 50.2% metabolic syndrome, NB, p<0.001), and body mass index [33.2, 34.2, 35 mean body mass index (kg/m2); NB, nHB, and cHB, respectively, p<0.05]. Finally, fibrosis stage, as a marker of disease severity, was significantly correlated with the eB score (p<0.001). CONCLUSIONS: The eB score allows for a reproducible and more precise delineation of the range of ballooned hepatocyte morphology and corresponds with both clinical features of NASH and fibrosis stage.