Spatial molecular and cellular determinants of STAT3 activation in liver fibrosis progression in non-alcoholic fatty liver disease.

Background & Aims: The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH), is increasing. Individuals with NASH often develop liver fibrosis and advanced liver fibrosis is the main determinant of mortality in individuals with NASH. We and others have reported that STAT3 contributes to liver fibrosis and hepatocellular carcinoma in mice. Methods: Here, we explored whether STAT3 activation in hepatocyte and non-hepatocyte areas, measured by phospho-STAT3 (pSTAT3), is associated with liver fibrosis progression in 133 patients with NAFLD. We further characterized the molecular and cellular determinants of STAT3 activation by integrating spatial distribution and transcriptomic changes in fibrotic NAFLD livers.Results: pSTAT3 scores in non-hepatocyte areas progressively increased with fibrosis severity (r = 0.53, p <0.001). Correlation analyses between pSTAT3 scores and expression of 1,540 immune- and cancer-associated genes revealed a large effect of STAT3 activation on gene expression changes in non-hepatocyte areas and confirmed a major role for STAT3 activation in fibrogenesis. Digital spatial transcriptomic profiling was also performed on 13 regions selected in hepatocyte and non-hepatocyte areas from four NAFLD liver biopsies with advanced fibrosis, using a customized panel of markers including pSTAT3, PanCK+CK8/18, and CD45. The regions were further segmented based on positive or negative pSTAT3 staining. Cell deconvolution analysis revealed that activated STAT3 was enriched in hepatic progenitor cells (HPCs) and sinusoidal endothelial cells. Regression of liver fibrosis upon STAT3 inhibition in mice with NASH resulted in a reduction of HPCs, demonstrating a direct role for STAT3 in HPC expansion. Conclusion: Increased understanding of the spatial dependence of STAT3 signaling in NASH and liver fibrosis progression could lead to novel targeted treatment approaches. Impact and implications: Advanced liver fibrosis is the main determinant of mortality in patients with NASH. This study showed using liver biopsies from 133 patients with NAFLD, that STAT3 activation in non-hepatocyte areas is strongly associated with fibrosis severity, inflammation, and progression to NASH. STAT3 activation was enriched in hepatic progenitor cells (HPCs) and sinusoidal endothelial cells (SECs), as determined by innovative technologies interrogating the spatial distribution of pSTAT3. Finally, STAT3 inhibition in mice resulted in reduced liver fibrosis and depletion of HPCs, suggesting that STAT3 activation in HPCs contributes to their expansion and fibrogenesis in NAFLD.

Selective disruption of NRF2-KEAP1 interaction leads to NASH resolution and reduction of liver fibrosis in mice.

Background & Aims: Oxidative stress is recognized as a major driver of non-alcoholic steatohepatitis (NASH) progression. The transcription factor NRF2 and its negative regulator KEAP1 are master regulators of redox, metabolic and protein homeostasis, as well as detoxification, and thus appear to be attractive targets for the treatment of NASH. Methods: Molecular modeling and X-ray crystallography were used to design S217879 - a small molecule that could disrupt the KEAP1-NRF2 interaction. S217879 was highly characterized using various molecular and cellular assays. It was then evaluated in two different NASH-relevant preclinical models, namely the methionine and choline-deficient diet (MCDD) and diet-induced obesity NASH (DIO NASH) models. Results: Molecular and cell-based assays confirmed that S217879 is a highly potent and selective NRF2 activator with marked anti-inflammatory properties, as shown in primary human peripheral blood mononuclear cells. In MCDD mice, S217879 treatment for 2 weeks led to a dose-dependent reduction in NAFLD activity score while significantly increasing liver Nqo1 mRNA levels, a specific NRF2 target engagement biomarker. In DIO NASH mice, S217879 treatment resulted in a significant improvement of established liver injury, with a clear reduction in both NAS and liver fibrosis. αSMA and Col1A1 staining, as well as quantification of liver hydroxyproline levels, confirmed the reduction in liver fibrosis in response to S217879. RNA-sequencing analyses revealed major alterations in the liver transcriptome in response to S217879, with activation of NRF2-dependent gene transcription and marked inhibition of key signaling pathways that drive disease progression. Conclusions: These results highlight the potential of selective disruption of the NRF2-KEAP1 interaction for the treatment of NASH and liver fibrosis. Impact and implications: We report the discovery of S217879 - a potent and selective NRF2 activator with good pharmacokinetic properties. By disrupting the KEAP1-NRF2 interaction, S217879 triggers the upregulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved in NASH disease progression, leading ultimately to the reduction of both NASH and liver fibrosis progression in mice.

Stellate cell expression of SPARC-related modular calcium-binding protein 2 is associated with human non-alcoholic fatty liver disease severity.

Background & Aims: Histological assessment of liver biopsies is the gold standard for diagnosis of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD), despite its well-established limitations. Therefore, non-invasive biomarkers that can offer an integrated view of the liver are needed to improve diagnosis and reduce sampling bias. Hepatic stellate cells (HSCs) are central in the development of hepatic fibrosis, a hallmark of NASH. Secreted HSC-specific proteins may, therefore, reflect disease state in the NASH liver and serve as non-invasive diagnostic biomarkers. Methods: We performed RNA-sequencing on liver biopsies from a histologically characterised cohort of obese patients (n = 30, BMI >35 kg/m2) to identify and evaluate HSC-specific genes encoding secreted proteins. Bioinformatics was used to identify potential biomarkers and their expression at single-cell resolution. We validated our findings using single-molecule fluorescence in situ hybridisation (smFISH) and ELISA to detect mRNA in liver tissue and protein levels in plasma, respectively. Results: Hepatic expression of SPARC-related modular calcium-binding protein 2 (SMOC2) was increased in NASH compared to no-NAFLD (p.adj <0.001). Single-cell RNA-sequencing data indicated that SMOC2 was primarily expressed by HSCs, which was validated using smFISH. Finally, plasma SMOC2 was elevated in NASH compared to no-NAFLD (p <0.001), with a predictive accuracy of AUROC 0.88. Conclusions: Increased SMOC2 in plasma appears to reflect HSC activation, a key cellular event associated with NASH progression, and may serve as a non-invasive biomarker of NASH. Impact and implications: Non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic steatohepatitis (NASH), are the most common forms of chronic liver diseases. Currently, liver biopsies are the gold standard for diagnosing NAFLD. Blood-based biomarkers to complement liver biopsies for diagnosis of NAFLD are required. We found that activated hepatic stellate cells, a cell type central to NAFLD pathogenesis, upregulate expression of the secreted protein SPARC-related modular calcium-binding protein 2 (SMOC2). SMOC2 was elevated in blood samples from patients with NASH and may hold promise as a blood-based biomarker for the diagnosis of NAFLD.

Effect of pegbelfermin on NASH and fibrosis-related biomarkers and correlation with histological response in the FALCON 1 trial.

Background & Aims: FALCON 1 was a phase IIb study of pegbelfermin in patients with non-alcoholic steatohepatitis (NASH) and stage 3 fibrosis. This FALCON 1 post hoc analysis aimed to further assess the effect of pegbelfermin on NASH-related biomarkers, correlations between histological assessments and non-invasive biomarkers, and concordance between the week 24 histologically assessed primary endpoint response and biomarkers. Methods: Blood-based composite fibrosis scores, blood-based biomarkers, and imaging biomarkers were evaluated for patients with available data from FALCON 1 at baseline through week 24. SomaSignal tests assessed protein signatures of NASH steatosis, inflammation, ballooning, and fibrosis in blood. Linear mixed-effect models were fit for each biomarker. Correlations and concordance were assessed between blood-based biomarkers, imaging, and histological metrics. Results: At week 24, pegbelfermin significantly improved blood-based composite fibrosis scores (ELF, FIB-4, APRI), fibrogenesis biomarkers (PRO-C3 and PC3X), adiponectin, CK-18, hepatic fat fraction measured by MRI-proton density fat fraction, and all four SomaSignal NASH component tests. Correlation analyses between histological and non-invasive measures identified four main categories: steatosis/metabolism, tissue injury, fibrosis, and biopsy-based metrics. Concordant and discordant effects of pegbelfermin on the primary endpoint vs. biomarker responses were observed; the most clear and concordant effects were on measures of liver steatosis and metabolism. A significant association between hepatic fat measured histologically and by imaging was observed in pegbelfermin arms. Conclusions: Pegbelfermin improved NASH-related biomarkers most consistently through improvement of liver steatosis, though biomarkers of tissue injury/inflammation and fibrosis were also improved. Concordance analysis shows that non-invasive assessments of NASH support and exceed the improvements detected by liver biopsy, suggesting that greater consideration should be given to the totality of available data when evaluating the efficacy of NASH therapeutics. Clinical trial number: Post hoc analysis of NCT03486899. Impact and implications: FALCON 1 was a study of pegbelfermin vs. placebo in patients with non-alcoholic steatohepatitis (NASH) without cirrhosis; in this study, patients who responded to pegbelfermin treatment were identified through examination of liver fibrosis in tissue samples collected through biopsy. In the current analysis, non-invasive blood- and imaging-based measures of fibrosis, liver fat, and liver injury were used to determine pegbelfermin treatment response to see how they compared with the biopsy-based results. We found that many of the non-invasive tests, particularly those that measured liver fat, identified patients who responded to pegbelfermin treatment, consistent with the liver biopsy findings. These results suggest that there may be additional value in using data from non-invasive tests, along with liver biopsy, to evaluate how well patients with NASH respond to treatment.

A randomized, double-blind, placebo-controlled phase IIa trial of efruxifermin for patients with compensated NASH cirrhosis.

Background & Aims: Efruxifermin has shown clinical efficacy in patients with non-alcoholic steatohepatitis (NASH) and F1-F3 fibrosis. The primary objective of the BALANCED Cohort C was to assess the safety and tolerability of efruxifermin in patients with compensated NASH cirrhosis. Methods: Patients with NASH and stage 4 fibrosis (n = 30) were randomized 2:1 to receive efruxifermin 50 mg (n = 20) or placebo (n = 10) once-weekly for 16 weeks. The primary endpoint was safety and tolerability of efruxifermin. Secondary and exploratory endpoints included evaluation of non-invasive markers of liver injury and fibrosis, glucose and lipid metabolism, and changes in histology in a subset of patients who consented to end-of-study liver biopsy. Results: Efruxifermin was safe and well-tolerated; most adverse events (AEs) were grade 1 (n = 7, 23.3%) or grade 2 (n = 19, 63.3%). The most frequent AEs were gastrointestinal, including transient, mild to moderate diarrhea, and/or nausea. Significant improvements were noted in key markers of liver injury (alanine aminotransferase) and glucose and lipid metabolism. Sixteen-week treatment with efruxifermin was associated with significant reductions in non-invasive markers of fibrosis including Pro-C3 (least squares mean change from baseline [LSMCFB] -9 µg/L efruxifermin vs. -3.4 µg/L placebo; p = 0.0130) and ELF score (-0.4 efruxifermin vs. +0.4 placebo; p = 0.0036), with a trend towards reduced liver stiffness (LSMCFB -5.7 kPa efruxifermin vs. -1.1 kPa placebo; n.s.). Of 12 efruxifermin-treated patients with liver biopsy after 16 weeks, 4 (33%) achieved fibrosis improvement of at least one stage without worsening of NASH, while an additional 3 (25%) achieved resolution of NASH, compared to 0 of 5 placebo-treated patients. Conclusions: Efruxifermin appeared safe and well-tolerated with encouraging improvements in markers of liver injury, fibrosis, and glucose and lipid metabolism following 16 weeks of treatment, warranting confirmation in larger and longer term studies. Lay summary: Cirrhosis resulting from non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease, represents a major unmet medical need. Currently there are no approved drugs for the treatment of NASH. This proof-of-concept randomized, double-blind clinical trial demonstrated the potential therapeutic benefit of efruxifermin treatment compared to placebo in patients with cirrhosis due to NASH. Clinical Trial Number: NCT03976401.

Indian National Association for Study of the Liver (INASL) Guidance Paper on Nomenclature, Diagnosis and Treatment of Nonalcoholic Fatty Liver Disease (NAFLD).

Nonalcoholic fatty liver disease (NAFLD) is a major cause of chronic liver disease globally and in India. The already high burden of NAFLD in India is expected to further increase in the future in parallel with the ongoing epidemics of obesity and type 2 diabetes mellitus. Given the high prevalence of NAFLD in the community, it is crucial to identify those at risk of progressive liver disease to streamline referral and guide proper management. Existing guidelines on NAFLD by various international societies fail to capture the entire landscape of NAFLD in India and are often difficult to incorporate in clinical practice due to fundamental differences in sociocultural aspects and health infrastructure available in India. A lot of progress has been made in the field of NAFLD in the 7 years since the initial position paper by the Indian National Association for the Study of Liver on NAFLD in 2015. Further, the ongoing debate on the nomenclature of NAFLD is creating undue confusion among clinical practitioners. The ensuing comprehensive review provides consensus-based, guidance statements on the nomenclature, diagnosis, and treatment of NAFLD that are practically implementable in the Indian setting.

Characterization and Pharmacological Validation of a Preclinical Model of NASH in Göttingen Minipigs.

Background: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which is associated with features of metabolic syndrome. NAFLD may progress in a subset of patients into nonalcoholic steatohepatitis (NASH) with liver injury resulting ultimately in cirrhosis and potentially hepatocellular carcinoma. Today, there is no approved treatment for NASH due to, at least in part, the lack of preclinical models recapitulating features of human disease. Here, we report the development of a dietary model of NASH in the Göttingen minipig. Methods: First, we performed a longitudinal characterization of diet-induced NASH and fibrosis using biochemical, histological, and transcriptional analyses. We then evaluated the pharmacological response to Obeticholic acid (OCA) treatment for 8 weeks at 2.5mg/kg/d, a dose matching its active clinical exposure. Results: Serial histological examinations revealed a rapid installation of NASH driven by massive steatosis and inflammation, including evidence of ballooning. Furthermore, we found the progressive development of both perisinusoidal and portal fibrosis reaching fibrotic septa after 6 months of diet. Histological changes were mechanistically supported by well-defined gene signatures identified by RNA Seq analysis. While treatment with OCA was well tolerated throughout the study, it did not improve liver dysfunction nor NASH progression. By contrast, OCA treatment resulted in a significant reduction in diet-induced fibrosis in this model. Conclusions: These results, taken together, indicate that the diet-induced NASH in the Göttingen minipig recapitulates most of the features of human NASH and may be a model with improved translational value to prioritize drug candidates toward clinical development.

Hepatoprotective Effects of Aureobasidium pullulans Derived ß 1,3-1,6 Glucans in a Murine Model of Non-alcoholic Steatohepatitis.

Background: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are highly prevalent conditions characterized by inflammation and fibrosis of the liver, which can progress to cirrhosis and hepatocellular carcinoma if left untreated. Conventional modalities are mainly symptomatic, with no definite solution. Beta-glucan-based biological response modifiers are a potential strategy in lieu of their beneficial metabolic effects. Aureobasidium pullulans strains AFO-202 and N-163 beta-glucans were evaluated for anti-fibrotic and anti-inflammatory hepatoprotective potentials in a NASH animal model in this study. Methods: In the STAM™ murine model of NASH, five groups were studied for 8 weeks: (1) vehicle (RO water), (2) AFO-202 beta-glucan; (3) N-163 beta-glucan, (4) AFO-202+N-163 beta-glucan, and (5) telmisartan (standard pharmacological intervention). Evaluation of biochemical parameters in plasma and hepatic histology including Sirius red staining and F4/80 immunostaining were performed. Results: AFO-202 beta-glucan significantly decreased inflammation-associated hepatic cell ballooning and steatosis. N-163 beta-glucan decreased fibrosis and inflammation significantly (P value < 0.05). The combination of AFO-202 with N-163 significantly decreased the NAFLD Activity Score (NAS) compared with other groups. Conclusion: This preclinical study supports the potential of N-163 and AFO-202 beta-glucans alone or in combination as potential preventive and therapeutic agent(s), for NASH.

Pharmacotherapeutic Impact on Nonalcoholic Steatohepatitis Histology: A Systematic Review and Network Meta-analysis.

Background: Due to lack of targeted treatment options and inconsistent utilization of histologic endpoints among clinical trials, identifying efficacious pharmacotherapies for nonalcoholic steatohepatitis [NASH] has proven challenging. Methods: A thorough systematic review and frequentist random-effects network meta-analysis was performed across all randomized clinical trials reporting a pharmacotherapeutic intervention on biopsy-proven NASH. Primary outcomes were based on the most current, up-to-date recommended histologic endpoints. Results: A total of 40 RCTs were identified including 6593 total patients. The most effective and statistically significant treatment interventions for minimum two-point improvement in NAFLD Activity Score were aldafermin 1 mg [RR 7.69, 95% CI 2.00; 29.57], vitamin E 800 IU in combination with pioglitazone 45 mg [RR 3.38, 95% CI 1.88; 6.07], pioglitazone 45 mg [RR 3.29, 95% CI 1.74; 6.22], vitamin E 800 IU [RR 2.06, 95% CI 1.33; 3.18], resmetirom 80 mg [RR 1.74, 95% CI 1.03; 2.94], obeticholic acid 25 mg [RR 1.63, 95% CI 1.32; 2.01], and obeticholic acid 10 mg [RR 1.31, 95% CI 1.02; 1.67]). The most robust pharmacotherapies for NASH resolution without worsening fibrosis were found to be aldafermin 1 mg [RR 5.77, 95% CI 1.48; 22.51], pioglitazone 45 mg [RR 2.65, 95% CI 1.43; 4.91], vitamin E 800 IU in combination with pioglitazone 45 mg [RR 2.64, 95% CI 1.36; 5.12], pioglitazone 30 mg [RR 2.46, 95% CI 1.56; 3.88], vitamin E 800 IU [RR 1.90, 95% CI 1.20; 3.00], and obeticholic acid 25 mg [RR 1.52, 95% CI 1.03; 2.23]). Obeticholic acid had a significant improvement on fibrosis. Multiple interventions were found to improve individual histologic scores across secondary outcome analyses and are detailed below. Conclusion: This novel systematic review and network meta-analysis represents the most comprehensive investigation to date regarding the pharmacotherapeutic options for biopsy-proven NASH using current recommended histologic endpoints.

FibroScan-AST (FAST) Score for Nonalcoholic Steatohepatitis - Validation in an Indian Cohort.

Background: The FibroScan-AST (FAST) score was recently described to detect patients with nonalcoholic steatohepatitis (NASH) having elevated nonalcoholic fatty liver disease (NAFLD) activity score (NAS ≥ 4) and significant fibrosis (≥ F2) on liver biopsy (NASH+ NAS ≥ 4 + F ≥ 2). Aim: The aim of this study was to validate the FAST score in Indian patients with NAFLD and to derive optimal cut-offs. Methods: Sixty patients with biopsy-proven NAFLD [men: 38 (63.3%), age 40 (32-52) years] with all parameters for assessing the FAST score within 3 months of liver histology were retrospectively analysed. Results: Histological NASH was present in 17 patients (28.3%), while 11 (18.3%) patients had NASH + NAS ≥ 4 + F ≥ 2. The area under the curve (AUROC) of the FAST score for discriminating NASH + NAS ≥ 4 + F ≥ 2 was 0.81. Using cut-offs by Newsome et al, the rule-out cut-off (FAST: ≤ 0.35) had a negative predictive value (NPV) of 0.88 [sensitivity: 0.91, specificity: 0.14, negative likelihood ratio (LR): 0.64], while the rule-in cut-off (FAST: ≥ 0.67) had a positive predictive value (PPV) of 0.33 (sensitivity: 0.73, specificity: 0.67, positive LR: 2.22). Fifteen (25%) patients were correctly classified as per histology, while 28 (46.67%) patients fell in the grey zone. On recalculating the optimal cut-offs for our patients, the rule-out cut-off (FAST: ≤ 0.55) had an NPV of 0.95 (sensitivity: 0.90, specificity: 0.45, negative LR: 0.21), while the optimal rule-in cut-off (FAST: ≥ 0.78) had a PPV of 0.70 (sensitivity: 0.64, specificity 0.94, positive LR: 10.39). With these cut-offs, 27 (45%) patients fell in the grey zone and 29 (48.3%) were correctly classified as per histology, performing better than the cut-offs by Newsome et al (P < 0.001). Conclusion: The FAST score demonstrates good AUROC for detecting NASH with significant fibrosis and inflammation on histology. Cut-offs should be recalibrated based on prevalence of disease. Lay summary: India has a high burden of NAFLD with an estimated 25 million patients at potential risk for significant liver disease. Liver biopsy remains the gold standard for diagnosing NASH, although its application in routine clinical practice is limited. Noninvasive tests for the simultaneous detection of steatosis, inflammation and fibrosis are thus the need of the hour. The FAST score has been recently suggested for the noninvasive detection of NASH with significant fibrosis (≥ F2) and inflammation (NAS ≥ 4) on liver biopsy. We validated the utility of the FAST score for detecting NASH with significant fibrosis and inflammation on liver biopsy in Indian patients with NAFLD. This noninvasive, easy-to-use and nonproprietary FAST score can correctly classify disease severity in more than 50% patients. However, our results suggest that cut-offs should be recalibrated based on the anticipated prevalence of NASH + NAS ≥ 4 + F ≥ 2 in the given population.

Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: Diagnostic and mechanistic relevance.

BACKGROUND & AIMS: Serum microRNA (miRNA) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages. METHODS: We profiled 2,083 serum miRNAs in a discovery cohort (183 cases with NAFLD representing the complete NAFLD spectrum and 10 population controls). miRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional cases with NAFLD and 15 population controls by quantitative reverse transcriptase PCR. RESULTS: Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages, but miR-193a-5p consistently showed increased levels in all comparisons. Relative to NAFL/non-alcoholic steatohepatitis (NASH) with mild fibrosis (stage 0/1), 3 miRNAs (miR-193a-5p, miR-378d, and miR378d) were increased in cases with NASH and clinically significant fibrosis (stages 2-4), 7 (miR193a-5p, miR-378d, miR-378e, miR-320b, miR-320c, miR-320d, and miR-320e) increased in cases with NAFLD activity score (NAS) 5-8 compared with lower NAS, and 3 (miR-193a-5p, miR-378d, and miR-378e) increased but 1 (miR-19b-3p) decreased in steatosis, activity, and fibrosis (SAF) activity score 2-4 compared with lower SAF activity. The significant findings for miR-193a-5p were replicated in the additional cohort with NAFLD. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n = 80); liver GPX8 levels correlated positively with serum miR-193a-5p. CONCLUSIONS: Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD. LAY SUMMARY: MicroRNAs (miRNAs) are small pieces of nucleic acid that may turn expression of genes on or off. These molecules can be detected in the blood circulation, and their levels in blood may change in liver disease including non-alcoholic fatty liver disease (NAFLD). To see if we could detect specific miRNA associated with advanced stages of NAFLD, we carried out miRNA sequencing in a group of 183 patients with NAFLD of varying severity together with 10 population controls. We found that a number of miRNAs showed changes, mainly increases, in serum levels but that 1 particular miRNA miR-193a-5p consistently increased. We confirmed this increase in a second group of cases with NAFLD. Measuring this miRNA in a blood sample may be a useful way to determine whether a patient has advanced NAFLD without an invasive liver biopsy.

Bile acids contribute to the development of non-alcoholic steatohepatitis in mice.

BACKGROUND & AIMS: Through FXR and TGR5 signaling, bile acids (BAs) modulate lipid and glucose metabolism, inflammation and fibrosis. Hence, BAs returning to the liver after enteric secretion, modification and reabsorption may contribute to the pathogenesis of non-alcoholic steatohepatitis (NASH). Herein, we characterized the enterohepatic profile and signaling of BAs in preclinical models of NASH, and explored the consequences of experimental manipulation of BA composition. METHODS: We used high-fat diet (HFD)-fed foz/foz and high-fructose western diet-fed C57BL/6J mice, and compared them to their respective controls. Mice received a diet supplemented with deoxycholic acid (DCA) to modulate BA composition. RESULTS: Compared to controls, mice with NASH had lower concentrations of BAs in their portal blood and bile, while systemic BA concentrations were not significantly altered. Notably, the concentrations of secondary BAs, and especially of DCA, and the ratio of secondary to primary BAs were strikingly lower in bile and portal blood of mice with NASH. Hence, portal blood was poor in FXR and TGR5 ligands, and conferred poor anti-inflammatory protection in mice with NASH. Enhanced primary BAs synthesis and conversion of secondary to primary BAs in NASH livers contributed to the depletion in secondary BAs. Dietary DCA supplementation in HFD-fed foz/foz mice restored the BA concentrations in portal blood, increased TGR5 and FXR signaling, improved the dysmetabolic status, protected from steatosis and hepatocellular ballooning, and reduced macrophage infiltration. CONCLUSIONS: BA composition in the enterohepatic cycle, but not in systemic circulation, is profoundly altered in preclinical models of NASH, with specific depletion in secondary BAs. Dietary correction of the BA profile protected from NASH, supporting a role for enterohepatic BAs in the pathogenesis of NASH. LAY SUMMARY: This study clearly demonstrates that the alterations of enterohepatic bile acids significantly contribute to the development of non-alcoholic steatohepatitis in relevant preclinical models. Indeed, experimental modulation of bile acid composition restored perturbed FXR and TGR5 signaling and prevented non-alcoholic steatohepatitis and associated metabolic disorders.

A Current Understanding of Bile Acids in Chronic Liver Disease.

Chronic liver disease (CLD) is one of the leading causes of disability-adjusted life years in many countries. A recent understanding of nuclear bile acid receptor pathways has increased focus on the impact of crosstalk between the gut, bile acids, and liver on liver pathology. While conventionally used in cholestatic disorders and to dissolve gallstones, the discovery of bile acids' influence on the gut microbiome and human metabolism offers a unique potential for their utility in early and advanced liver diseases because of diverse etiologies. Based on these findings, preclinical studies using bile acid-based molecules have shown encouraging results at addressing liver inflammation and fibrosis. Emerging data also suggest that bile acid profiles change distinctively across various causes of liver disease. We summarize the current knowledge and evidence related to bile acids in health and disease and discuss culminated and ongoing therapeutic trials of bile acid derivatives in CLD. In the near future, further evidence in this area might help clinicians better detect and manage liver diseases.

The burden of non-alcoholic steatohepatitis: A systematic review of health-related quality of life and patient-reported outcomes.

Background & Aims: Non-alcoholic steatohepatitis (NASH) is associated with increased mortality and a high clinical burden. NASH adversely impacts patients' health-related quality of life (HRQoL), but published data on the humanistic burden of disease are limited. This review aimed to summarise and critically evaluate studies reporting HRQoL or patient-reported outcomes (PROs) in populations with NASH and identify key gaps for further research. Methods: Medline, EMBASE, the Cochrane Library and PsycINFO were searched for English-language publications published from 2010 to 2021 that reported HRQoL/PRO outcomes of a population or subpopulation with NASH. Results: Twenty-five publications covering 23 unique studies were identified. Overall, the data showed a substantial impact of NASH on HRQoL, particularly in terms of physical functioning and fatigue, with deterioration of physical and mental health as NASH progresses. Prevalent symptoms, including fatigue, abdominal pain, anxiety/depression, cognition problems, and poor sleep quality, adversely impact patients' ability to work and perform activities of daily living and the quality of relationships. However, some patients fail to attribute symptoms to their disease because of a lack of patient awareness and education. NASH is associated with high rates of comorbidities such as obesity and type 2 diabetes, which contribute to reduced HRQoL. Studies were heterogeneous in terms of diagnostic methods, population, outcomes, follow-up time, and measures of HRQoL/utility. Most studies were rated 'moderate' at quality assessment, and all evaluable studies had inadequate control of confounders. Conclusions: NASH is associated with a significant HRQoL burden that begins early in the disease course and increases with disease progression. More robust studies are needed to better understand the humanistic burden of NASH, with adequate adjustment for confounders that could influence outcomes. Lay summary: Non-alcoholic steatohepatitis (NASH) has a significant impact on quality of life, with individuals experiencing worse physical and mental health compared with the general population. NASH and its symptoms, which include tiredness, stomach pain, anxiety, depression, poor focus and memory, and impaired sleep, affect individuals' relationships and ability to work and perform day-to-day tasks. However, not all patients are aware that their symptoms may be related to NASH. Patients would benefit from more education on their disease, and the importance of good social networks for patient health and well-being should be reinforced. More studies are needed to better understand the patient burden of NASH.

Historical narrative from fatty liver in the nineteenth century to contemporary NAFLD - Reconciling the present with the past.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder worldwide. This historical narrative traces the evolution from basic descriptions of fatty liver in the nineteenth century to our contemporary understanding of NAFLD in the twentieth and twenty-first centuries. A detailed historiographic review of fatty liver from 1800s onwards was performed alongside a brief review of contemporary associations. Archived published literature dating back to the 1800s describe clinicopathological features of fatty liver. In the nineteenth century, doyens of medicine associated fatty liver with alcohol, malnutrition or wasting conditions, and subsequently adiposity, unhealthy diets and sedentary lifestyle. Microscopically, fatty liver was described when 5% or more hepatocytes were distended with fat. Recommendations to reverse fatty liver included reducing consumption of fat, sugar, starchy carbohydrates and alcohol, plus increasing physical exercise. Fatty liver was associated with liver fibrosis and cirrhosis in the late 1800s, and with diabetes in the early 1900s. The diagnostic labels NAFLD and non-alcoholic steatohepatitis (NASH) were introduced in the late 1900s. Metabolic dysfunction-associated fatty liver disease (MAFLD) was recently proposed to update the nosology of fatty liver, recognising the similar metabolic pathogenesis evident in individuals with typical NAFLD and those with heterogenous "secondary" co-factors including alcohol and other aetiologies. Fatty liver has emerged from being considered a disorder of nutritional extremes or alcohol excess to contemporary recognition as a complex metabolic disorder that risks progression to cirrhosis and hepatocellular carcinoma. The increasing prevalence of NAFLD and our growing understanding of its lifestyle and metabolic determinants justify the current exercise of re-examining the evolution of this common metabolic disorder.

Primary Versus Secondary NAFLD: Perspective on Advanced Fibrosis.

BACKGROUND: The presence of macrovesicular steatosis on liver biopsy is the commonest histopathological finding. Nonalcoholic fatty liver disease (NAFLD) is the presence of ≥5% macrovesicular steatosis without significant alcohol use. It is subdivided into primary and secondary NAFLD; information on their differences is limited. AIM: To determine the histopathological differences between primary and secondary NAFLD and establish whether the prevalence of advanced fibrosis varies between the two types. METHODOLOGY: Three years of retrospective study of 90 liver biopsies with ≥5% macrovesicular steatosis. Age, gender, clinical history, serum transaminase levels were noted. The biopsy was reviewed for steatosis, inflammation, and fibrosis. Differences between primary and secondary NAFLD for age, gender, AST/ALT ratio, histopathological features were determined. Descriptive statistical analysis, 2-tailed Student's t test, Chi-square test, Fisher's exact test were used, where p < 0.05 was considered significant. RESULT: Primary and secondary NAFLD were 42 (46.7%) and 48 (53.3%), respectively. Inflammation was noted in 50 (55.5%) and fibrosis in 31 (34.4%). The prevalence of advanced fibrosis was 24.4%. Primary and secondary NAFLD differed significantly on ballooning degeneration, Mallory Denk bodies (MDBs), glycogenated nuclei, and fibrosis stage (p < 0.05). There were no significant differences among AST/ALT ratio, steatosis, and inflammation grade. CONCLUSION: Primary NAFLD is a more severe type of liver disease. On histopathology, ballooning degeneration, MDBs, glycogenated nuclei, and advanced fibrosis was more prevalent in primary than secondary NAFLD.

Characterization of adipose tissue-derived stromal cells of mice with nonalcoholic fatty liver disease and their use for liver repair.

INTRODUCTION: Freshly isolated uncultured adipose tissue-derived stromal cells (u-ADSCs), containing miscellaneous cells like the relatively abundant mesenchymal stem cells, are attractive for repair and regenerative therapy. However, the detailed characteristics and therapeutic efficacy of u-ADSCs obtained from disease-affected hosts are unknown. We compared the properties of u-ADSCs obtained from wild-type mice and from a mouse model of non-alcoholic steatohepatitis (NASH). METHODS: The NASH model was established by feeding C57BL/6J mice an atherogenic high-fat diet for 4 (NASH (4w)) or 12 weeks (NASH (12w)), followed by the isolation and characterization of u-ADSCs. Wild-type u-ADSCs or NASH-derived u-ADSCs were administered to mice with NASH cirrhosis, followed by analyses of hepatic inflammatory cells, antigen profiles, fibrosis, and gene expression. RESULTS: Wild-type u-ADSCs and NASH-derived u-ADSCs did not show marked differences in surface antigen profiles. In NASH (4w) u-ADSCs, but not NASH (12w) u-ADSCs, the frequencies of the leukocyte markers CD11b, CD45, and CD44 were elevated; furthermore, we observed an increase in the M1/M2 macrophage ratio only in NASH (12w) u-ADSCs. Only in NASH-4w u-ADSCs, the expression levels cell cycle-related genes were higher than those in u-ADSCs. Wild-type u-ADSCs administered to mice with NASH-related cirrhosis decreased the infiltration of CD11b+, F4/80+, and Gr-1+ inflammatory cells, ameliorated fibrosis, and had a restorative effect on liver tissues, as determined by gene expression profiles and the NAFLD activity score. The therapeutic effects of NASH (4w) u-ADSCs and NASH (12w) u-ADSCs on NASH-related cirrhosis were highly similar to the effect of wild-type u-ADSCs, including reductions in inflammation and fibrosis. CONCLUSIONS: NASH-derived u-ADSCs, similar to wild-type u-ADSCs, are applicable for reparative and regenerative therapy in mice with NASH.

Prospective comparison of transient elastography, MRI and serum scores for grading steatosis and detecting non-alcoholic steatohepatitis in bariatric surgery candidates.

BACKGROUND & AIMS: Tools for the non-invasive diagnosis of non-alcoholic steatohepatitis (NASH) in morbidly obese patients with suspected non-alcoholic fatty liver disease (NAFLD) are an unmet clinical need. We prospectively compared the performance of transient elastography, MRI, and 3 serum scores for the diagnosis of NAFLD, grading of steatosis and detection of NASH in bariatric surgery candidates. METHODS: Of 186 patients screened, 152 underwent liver biopsy, which was used as a reference for NAFLD (steatosis [S]>5%), steatosis grading and NASH diagnosis. Biopsies were read by a single expert pathologist. MRI-based proton density fat fraction (MRI-PDFF) was measured in an open-bore, vertical field 1.0T scanner and controlled attenuation parameter (CAP) was measured by transient elastography, using the XL probe. Serum scores (SteatoTest, hepatic steatosis index and fatty liver index) were also calculated. RESULTS: The applicability of MRI was better than that of FibroScan (98% vs. 79%; p <0.0001). CAP had AUROCs of 0.83, 0.79, 0.73 and 0.69 for S>5%, S>33%, S>66% and NASH, respectively. Transient elastography had an AUROC of 0.80 for significant fibrosis (F0-F1 vs. F2-F3). MRI-PDFF had AUROCs of 0.97, 0.95, 0.92 and 0.84 for S>5%, S>33%, S>66% and NASH, respectively. When compared head-to-head in the 97 patients with all valid tests available, MRI-PDFF outperformed CAP for grading steatosis (S>33%, AUROC 0.97 vs. 0.78; p <0.0003 and S>66%, AUROC 0.93 vs. 0.75; p = 0.0015) and diagnosing NASH (AUROC 0.82 vs. 0.68; p = 0.0056). When compared in "intention to diagnose" analysis, MRI-PDFF outperformed CAP, hepatic steatosis index and fatty liver index for grading steatosis (S>5%, S>33% and S>66%). CONCLUSION: MRI-PDFF outperforms CAP for diagnosing NAFLD, grading steatosis and excluding NASH in morbidly obese patients undergoing bariatric surgery. LAY SUMMARY: Non-invasive tests for detecting fatty liver and steatohepatitis, the active form of the disease, have not been well studied in obese patients who are candidates for bariatric surgery. The most popular tests for this purpose are Fibroscan, which can be used to measure the controlled attenuation parameter (CAP), and magnetic resonance imaging, which can be used to measure the proton density fat fraction (MRI-PDFF). We found that, when taking liver biopsy as a reference, MRI-PDFF performed better than CAP for detecting and grading fatty liver as well as excluding steatohepatitis in morbidly obese patients undergoing bariatric surgery.

Metabolic dysfunction-associated fatty liver disease increases risk of adverse outcomes in patients with chronic hepatitis B.

BACKGROUND & AIMS: A recent consensus document has defined metabolic dysfunction-associated fatty liver disease (MAFLD) as hepatic steatosis together with overweight, diabetes, and/or a combination of other metabolic risk factors. The clinical relevance of this novel diagnosis is unknown among patients with chronic hepatitis B (CHB). We studied the association between MAFLD (with or without steatohepatitis) and adverse clinical outcomes in patients with CHB. METHODS: We performed a retrospective long-term follow-up cohort study at 2 tertiary hospitals in patients with CHB who underwent liver biopsy. Biopsies were reassessed for steatosis, degree of fibrosis, and presence of steatohepatitis. Associations with event-free hepatocellular carcinoma (HCC)-free and transplant-free survival were explored. RESULTS: In our cohort, 1076 patients were included, median follow-up was 9.8 years (25th-75th percentile: 6.6-14.0), and 107 events occurred in 78 patients, comprising death (n = 43), HCC (n = 36), liver decompensation (n = 21), and/or liver transplantation (n = 7). MAFLD was present in 296 (27.5%) patients and was associated with reduced event-free (adjusted hazard ratio [aHR] 2.00, 95% CI 1.26-3.19), HCC-free (aHR 1.93, 95% CI 1.17-3.21), and transplant-free survival (aHR 1.80, 95% CI 0.98-3.29) in multivariable analysis. Among patients with MAFLD, the presence of steatohepatitis (p = 0.95, log-rank test) was not associated with adverse outcomes. CONCLUSIONS: The presence of MAFLD in patients with CHB was associated with an increased risk for liver-related clinical events and death. Among patients with MAFLD, steatohepatitis did not increase the risk of adverse outcomes. Our findings highlight the importance of metabolic dysfunction in patients with CHB. LAY SUMMARY: Recently, metabolic dysfunction-associated fatty liver disease (MAFLD) has been defined as fatty liver disease with signs of metabolic dysfunction. Among patients with chronic hepatitis B, MAFLD was associated with liver-related events and death. Metabolic health assessment should be encouraged among patients with chronic hepatitis B, especially in those with fatty liver disease.

Case-finding strategies in non-alcoholic fatty liver disease.

Among the large population of patients with non-alcoholic fatty liver disease (NAFLD), identifying those with advanced disease remains challenging. Many patients are diagnosed late, following the development of liver-related complications, leading to poor clinical outcomes. Accumulating evidence suggests that using non-invasive tests for liver fibrosis in patients with metabolic risk factors improves the detection of patients in need of specialised management and is cost-effective. Because of the vast number of patients requiring evaluation, the active participation of general practitioners and physicians who manage patients with metabolic disorders, such as diabetologists, is crucial; this calls for the increased awareness of NAFLD beyond liver clinics. Non-invasive case-finding strategies will need to be further validated and generalised for upcoming drug therapies to have the required impact on the worldwide burden of NAFLD.