Nonalcoholic Steatohepatitis Patients Research Articles

SAT-429 - Morphometric analysis of sonic hedgehog in non-alcoholic fatty liver steatohepatitis patients by MorphoQuant: a potential biomarker of disease severity

SAT-429 - Morphometric analysis of sonic hedgehog in non-alcoholic fatty liver steatohepatitis patients by MorphoQuant: a potential biomarker of disease severity

SAT-407 - A stepwise screening approach using non-invasive tests to identify phenotypic non-alcoholic steatohepatitis (NASH) patients with fibrosis for clinical trials

SAT-407 - A stepwise screening approach using non-invasive tests to identify phenotypic non-alcoholic steatohepatitis (NASH) patients with fibrosis for clinical trials

LBO-05 - Pegozafermin for the treatment of non-alcoholic steatohepatitis patients with F2/F3 fibrosis: a multi-center, randomized, double-blind, placebo-controlled Phase 2b trial (ENLIVEN)

LBO-05 - Pegozafermin for the treatment of non-alcoholic steatohepatitis patients with F2/F3 fibrosis: a multi-center, randomized, double-blind, placebo-controlled Phase 2b trial (ENLIVEN)

OS-027 - Performance of vibration controlled transient elastography and related scores to identify at-risk non-alcoholic steatohepatitis patients: combined data from multiple trials including more than 5,000 patients (in collaboration with NAIL-NIT consortium)

OS-027 - Performance of vibration controlled transient elastography and related scores to identify at-risk non-alcoholic steatohepatitis patients: combined data from multiple trials including more than 5,000 patients (in collaboration with NAIL-NIT consortium)

SAT-453 - Utilization of fibroscan-AST scoring system to risk-stratify non-alcoholic steatohepatitis patients for clinical trials and novel therapies

SAT-453 - Utilization of fibroscan-AST scoring system to risk-stratify non-alcoholic steatohepatitis patients for clinical trials and novel therapies

Insulin Resistance Is the Main Characteristic of Metabolically Unhealthy Obesity (MUO) Associated with NASH in Patients Undergoing Bariatric Surgery.

(1) Background: Metabolically healthy obesity (MHO) is a concept that applies to obese patients without any elements of metabolic syndrome (metS). In turn, metabolically unhealthy obesity (MUO) defines the presence of elements of metS in obese patients. The components of MUO can be divided into subgroups regarding the elements of inflammation, lipid and glucose metabolism and cardiovascular disease. MUO patients appear to be at greater risk of developing non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) compared to MHO patients. The aim of this study was to evaluate the influence of different MUO components on NAFLD and NASH in patients with morbid obesity undergoing bariatric surgery. (2) Methods: 141 patients undergoing bariatric surgery from September 2015 and October 2021 at RWTH Aachen university hospital (Germany) were included. Patients were evaluated pre-operatively for characteristics of metS and MUO (HbA1c, HOMA, CRP, BMI, fasting glucose, LDL, TG, HDL and the presence of arterial hypertension). Intraoperatively, a liver biopsy was taken from the left liver lobe and evaluated for the presence of NAFLD or NASH. In ordinal regression analyses, different factors were evaluated for their influence on NAFLD and NASH. (3) Results: Mean BMI of the patients was 52.3 kg/m2 (36-74.8, SD 8.4). Together, the parameters HbA1c, HOMA, CRP, BMI, fasting glucose, LDL, TG, HDL and the presence of arterial hypertension accounted for a significant amount of variance in the outcome, with a likelihood ratio of χ2 (9) = 41.547, p < 0.001, for predicting the presence of NASH. Only HOMA was an independent predictor of NASH (B = 0.102, SE = 0.0373, p = 0.007). Evaluation of steatosis showed a similar trend (likelihood ratio χ2 (9) = 40.272, p < 0.001). Independent predictors of steatosis were HbA1c (B = 0.833, SE = 0.343, p = 0.015) and HOMA (B = 0.136, SE = 0.039, p < 0.001). (4) Conclusions: The above-mentioned model, including components of MUO, was significant for diagnosing NASH in patients with morbid obesity undergoing bariatric surgery. Out of the different subitems, HOMA independently predicted the presence of NASH and steatosis, while HbA1c independently predicted steatosis and fibrosis. Taken together, the parameter of glucose metabolism appears to be more accurate for the prediction of NASH than the parameters of lipid metabolism, inflammation or the presence of cardiovascular disease.

Exploring Key Genes with Diagnostic Value for Nonalcoholic Steatohepatitis Based on Bioinformatics Analysis.

We aimed to screen specific genes in liver tissue samples of patients with nonalcoholic steatohepatitis (NASH) with clinical diagnostic value based on bioinformatics analysis. The datasets of liver tissue samples from healthy individuals and NASH patients were retrieved for consistency cluster analysis to obtain the NASH sample typing, followed by verification of the diagnostic value of sample genotyping-specific genes. All samples were subjected to logistic regression analysis, followed by the establishment of the risk model, and then, the diagnostic value was determined by receiver operating characteristic curve analysis. NASH samples could be divided into cluster 1, cluster 2, and cluster 3, which could predict the nonalcoholic fatty liver disease activity score of patients. A total of 162 sample genotyping-specific genes were extracted from patient clinical parameters, and the top 20 core genes in the protein interaction network were obtained for logistic regression analysis. Five sample genotyping-specific genes (WD repeat and HMG-box DNA-binding protein 1 [WDHD1], GINS complex subunit 2 [GINS2], replication factor C subunit 3 (RFC3), secreted phosphoprotein 1 [SPP1], and spleen tyrosine kinase [SYK]) were extracted to construct the risk models with high diagnostic value in NASH. Compared with the low-risk group, the high-risk group of the model showed increased lipoproduction and decreased lipolysis and lipid β oxidation. The risk models based on WDHD1, GINS2, RFC3, SPP1, and SYK have high diagnostic value in NASH, and this risk model is closely related to lipid metabolism pathways.

GRP78/BiP alleviates oxLDL-induced hepatotoxicity in familial hypercholesterolemia caused by missense variants of LDLR in a HepG2 cellular model

Background and aimsThe accumulation of misfolded proteins, encoded by genetic variants of functional genes leads to Endoplasmic Reticulum (ER) stress, which is a critical consequence in human disorders such as familial hypercholesterolemia, cardiovascular and hepatic diseases. In addition to the identification of ER stress as a contributing factor to pathogenicity, extensive studies on the role of oxidized Low-Density Lipoprotein (oxLDL) and its ill effects in expediting cardiovascular diseases and other metabolic comorbidities are well documented. However, the current understanding of its role in hepatic insults needs to be revised. This study elucidates the molecular mechanisms underlying the progression of oxLDL and ER stress-induced cytotoxicity in HepG2.MethodsHepG2 cells stably expressing wild-type Low-Density lipoprotein receptor (WT-LDLR) and missense variants of LDLR that are pathogenically associated with familial hypercholesterolemia were used as the in vitro models. The relative mRNA expression and protein profiles of ER stress sensors, inflammatory and apoptotic markers, together with cytotoxic assays and measurement of mitochondrial membrane potential, were carried out in HepG2 cells treated with 100 µg per ml oxLDL for 24 to 48 h. 1-way or 2-way ANOVA was used for statistical analyses of datasets.ResultsER stress responses are elicited along all three arms of the unfolded protein response (UPR), with adverse cytotoxic and inflammatory responses in oxLDL-treated conditions.Interestingly, oxLDL-treated ER-stressed HepG2 cells manifested intriguingly low expression of BiP- the master regulator of ER stress, as observed earlier by various researchers in liver biopsies of Non-Alcoholic Steatohepatitis (NASH) patients. This study shows that overexpression of BiP rescues hepatic cells from cytotoxic and inflammatory mechanisms instigated by ER stress in combination with oxLDL, along the ER and mitochondrial membrane and restores cellular homeostasis.ConclusionThe data provide interesting leads that identify patients with familial hypercholesterolemia conditions and potentially other Endoplasmic Reticulum Associated Degradation (ERAD) diseases as highly susceptible to developing hepatic insults with molecular signatures like those manifested in Non-Alcoholic Fatty Liver Disease (NAFLD) and NASH.Limitations and future perspectivesAlthough the use of HepG2 cells as the model is a major caveat of the study, the findings of this research may be used as the pilot study to expand further investigations in primary hepatocytes or iPSC- derived cellular models.

NIS2+™, an optimisation of the blood-based biomarker NIS4® technology for the detection of at-risk NASH: A prospective derivation and validation study

NIS4® is a blood-based non-invasive test designed to effectively rule in/rule out at-risk non-alcoholic steatohepatitis (NASH), defined as non-alcoholic fatty liver disease activity score ≥4 and significant fibrosis (stage ≥2), among patients with metabolic risk factors. Robustness of non-invasive test scores across characteristics of interest including age, type 2 diabetes mellitus, and sex, and optimised analytical aspects are critical for large-scale implementation in clinical practice. We developed and validated NIS2+™, an optimisation of NIS4®, specifically designed to improve score robustness. A well-balanced training cohort (n= 198) included patients from the GOLDEN-505 trial. The validation (n= 684) and test (n= 2,035) cohorts included patients from the RESOLVE-IT trial. Well-matched subgroups were created to avoid potential confounding effects during modelling and analysis of score robustness. Models were trained using logistic regressions for at-risk NASH detection and compared using Bayesian information criteria. Performance of NIS2+™ was compared with that of NIS4®, Fibrosis-4, and alanine aminotransferase using area under the receiver operating characteristic curve, and robustness was analysed through score distribution. Using the training cohort to compare all combinations of NIS4® biomarkers, NIS2 (miR-34a-5p, YKL-40) was identified as the best combination of parameters. To correct for the sex effect on miR-34a-5p (validation cohort), sex and sex ∗ miR-34a-5p parameters were added, creating NIS2+™. In the test cohort, NIS2+™ exhibited a statistically higher area under the receiver operating characteristic curve (0.813) vs. NIS4® (0.792; p= 0.0002), Fibrosis-4 (0.653; p <0.0001), and alanine aminotransferase (0.699; p <0.0001). NIS2+™ scores were not affected by age, sex, BMI, or type 2 diabetes mellitus status, providing robust clinical performances irrespective of patient characteristics. NIS2+™ constitutes a robust optimisation of NIS4® technology for the detection of at-risk NASH. The development of non-invasive tests for accurate, large-scale detection of patients with at-risk non-alcoholic steatohepatitis (NASH; defined as NASH with non-alcoholic fatty liver disease activity score ≥4 and fibrosis stage ≥2) - who are at higher risk for disease progression and for developing liver-related life-threatening outcomes - is critical for identifying this patient population in the clinical setting and improving the screening process of NASH clinical trials. We report the development and validation of NIS2+™, a diagnostic test designed as an optimisation of NIS4® technology, a blood-based panel currently used to detect at-risk NASH in patients with metabolic risk factors. NIS2+™ showed improved performance for the detection of at-risk NASH compared with NIS4® and other non-invasive liver tests that was not impacted by patients' characteristics of interest, such as age, sex, type 2 diabetes mellitus, BMI, dyslipidaemia, and hypertension. This makes NIS2+™ a robust and reliable tool for the diagnosis of at-risk NASH among patients with metabolic risk factors, and an effective candidate for large-scale implementation in clinical practice and clinical trials.

Probiotic Supplementation, Hepatic Fibrosis, and the Microbiota Profile in Patients with Nonalcoholic Steatohepatitis: A Randomized Controlled Trial

BackgroundPromising results in improvement of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH) have been identified following probiotic (PRO) treatment. ObjectivesTo evaluate PRO supplementation on hepatic fibrosis, inflammatory and metabolic markers, and gut microbiota in NASH patients. MethodsIn a double-blind, placebo-controlled clinical trial, 48 patients with NASH with a median age of 58 y and median BMI of 32.7 kg/m2 were randomly assigned to receive PROs (Lactobacillus acidophilus 1 × 109 colony forming units and Bifidobacterium lactis 1 × 109 colony forming units) or a placebo daily for 6 mo. Serum aminotransferases, total cholesterol and fractions, C-reactive protein, ferritin, interleukin-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, and leptin were assessed. To evaluate liver fibrosis, Fibromax was used. In addition, 16S rRNA gene-based analysis was performed to evaluate gut microbiota composition. All assessments were performed at baseline and after 6 mo. For the assessment of outcomes after treatment, mixed generalized linear models were used to evaluate the main effects of the group-moment interaction. For multiple comparisons, Bonferroni correction was applied (α = 0.05/4 = 0.0125). Results for the outcomes are presented as mean and SE. ResultsThe AST to Platelet Ratio Index (APRI) score was the primary outcome that decreased over time in the PRO group. Aspartate aminotransferase presented a statistically significant result in the group-moment interaction analyses, but no statistical significance was found after the Bonferroni correction. Liver fibrosis, steatosis, and inflammatory activity presented no statistically significant differences between the groups. No major shifts in gut microbiota composition were identified between groups after PRO treatment. ConclusionsPatients with NASH who received PRO supplementation for 6 mo presented improvement in the APRI score after treatment. These results draw attention to clinical practice and suggest that supplementation with PROs alone is not sufficient to improve enzymatic liver markers, inflammatory parameters, and gut microbiota in patients with NASH.This trial was registered at clinicaltrials.gov as NCT02764047.

Limosilactobacillus fermentum, Lactococcus lactis and Thomasclavelia ramosa are enriched and Methanobrevibacter smithii is depleted in patients with non-alcoholic steatohepatitis

Non-alcoholic fatty liver (NAFLD), and its complicated form, non-alcoholic steatohepatitis (NASH), have been associated with gut dysbiosis with specific signatures. Endogenous ethanol production by Klebsiella pneumoniae or yeasts has been identified as a potential physio-pathological mechanism. A species-specific association between Lactobacillus and obesity and metabolic diseases has been reported. In this study, the microbial composition of ten cases of NASH and ten controls was determined using v3v4 16S amplicon sequencing as well as quantitative PCR (qPCR). Using different statistical approaches, we found an association of Lactobacillus and Lactoccocus with NASH, and an association of Methanobrevibacter, Faecalibacterium and Romboutsia with controls. At the species level, Limosilactobacillus fermentum and Lactococcus lactis, two species producing ethanol, and Thomasclavelia ramosa, a species already associated with dysbiosis, were associated with NASH. Using qPCR, we observed a decreased frequency of Methanobrevibacter smithii and confirmed the high prevalence of L. fermentum in NASH samples (5/10), while all control samples were negative (p = 0.02). In contrast, Ligilactobacillus ruminis was associated with controls. This supports the critical importance of taxonomic resolution at the species level, notably with the recent taxonomic reclassification of the Lactobacillus genus. Our results point towards the potential instrumental role of ethanol-producing gut microbes in NASH patients, notably lactic acid bacteria, opening new avenues for prevention and treatment.

Hepatic Pin1 Expression, Particularly in Nuclei, Is Increased in NASH Patients in Accordance with Evidence of the Role of Pin1 in Lipid Accumulation Shown in Hepatoma Cell Lines.

Our previous studies using rodent models have suggested an essential role for Pin1 in the pathogenesis of non-alcoholic steatohepatitis (NASH). In addition, interestingly, serum Pin1 elevation has been reported in NASH patients. However, no studies have as yet examined the Pin1 expression level in human NASH livers. To clarify this issue, we investigated the expression level and subcellular distribution of Pin1 in liver specimens obtained using needle-biopsy samples from patients with NASH and healthy liver donors. Immunostaining using anti-Pin1 antibody revealed the Pin1 expression level to be significantly higher, particularly in nuclei, in the livers of NASH patients than those of healthy donors. In the samples from patients with NASH, the amount of nuclear Pin1 was revealed to be negatively related to serum alanine aminotransferase (ALT), while tendencies to be associated with other serum parameters such as aspartate aminotransferase (AST) and platelet number were noted but did not reach statistical significance. Such unclear results and the lack of a significant relationship might well be attributable to our small number of NASH liver samples (n = 8). Moreover, in vitro, it was shown that addition of free fatty acids to medium induced lipid accumulation in human hepatoma HepG2 and Huh7 cells, accompanied with marked increases in nuclear Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), in accordance with the aforementioned observations in human NASH livers. In contrast, suppression of Pin1 gene expression using siRNAs attenuated the free fatty acid-induced lipid accumulation in Huh7 cells. Taken together, these observations strongly suggest that increased expression of Pin1, particularly in hepatic nuclei, contributes to the pathogenesis of NASH with lipid accumulation.

The causes of death in patients with nonalcoholic steatohepatitis following liver transplantation stratified using pre-liver transplant BMI.

Determining the effects of pre-liver transplant (LT) BMI independent of underlying ascites on the post-LT outcomes of patients with nonalcoholic steatohepatitis (NASH) is needed to clarify the paradoxical and protective effects of obesity on post-LT endpoints. In order to accomplish this, we used graded severities of ascites to stratify the NASH-LT population and to perform an ascites-specific strata analysis with differing pre-LT BMI levels. 2005-2019 United Network for Organ Sharing (UNOS) Standard Transplant Analysis and Research (STAR) database was queried to select patients with NASH, who were categorized into specific sets of ascites severity: no ascites (n = 1188), mild ascites (n = 4463), and moderate ascites (n = 3525). Then, BMI classification (underweight: < 18.5, normal: 18.5-25, overweight: 25-30, obese: ≥ 30kg/m2) was used to stratify each ascites-specific group and to compare to the post-LT mortality endpoints. Those under 18years old and those who received living/multi-organ transplants were excluded. Among each ascites category, there were the following numbers of normal, underweight, overweight, and obese BMI patients respectively; no ascites: 161, 4, 359, 664; mild ascites: 643, 28, 1311, 2481; and moderate ascites: 529, 25, 1030, 1941. The obese BMI cohort was at a lower risk of all-cause mortality compared to recipients with normal BMI with mild ascites (aHR: 0.79, 95% Confidence Interval (CI) 0.65-0.94, p-value = 0.010; case-incidence 47.10 vs 56.81 deaths per 1000 person-years) and moderate ascites (aHR: 0.77, 95% CI 0.63-0.94, p-value = 0.009; case-incidence 53.71 vs 66.17 deaths per 1000 person-years). In addition, the overweight BMI cohort with mild ascites demonstrated a lower hazard of all-cause mortality (aHR: 0.80, 95% CI 0.66-0.97, p-value = 0.03; case-incidence 49.09 vs 56.81 deaths per 1000 person-years). There was no difference in graft failure for the three BMI groups (underweight, overweight, and obese) in comparison to normal BMI. Furthermore, the overweight BMI group with mild ascites cohort demonstrated a lower hazard of death due to general infectious causes (aHR: 0.51, 95% CI 0.32-0.83, p = 0.006; case-incidence 6.12 vs 11.91 deaths per 1000 person-years) and sepsis (aHR: 0.49, 95% CI 0.27-0.86, p = 0.01; case-incidence 4.31 vs 8.50 deaths per 1000 person-years). The paradoxical effects of obesity in reducing the risks of all-cause death appears to be in part modulated by ascites. The current study emphasizes the need to evaluate BMI with concomitant ascites severity pre-LT to accurately prognosticate post-LT outcomes when evaluating NASH patients with advanced liver disease.

Semaglutide Has Beneficial Effects on Non-Alcoholic Steatohepatitis in Ldlr-/-.Leiden Mice.

Semaglutide, a glucagon-like peptide-1 receptor agonist, is an antidiabetic medication that has recently been approved for the treatment of obesity as well. Semaglutide is postulated to be a promising candidate for the treatment of non-alcoholic steatohepatitis (NASH). Here, Ldlr-/-.Leiden mice received a fast-food diet (FFD) for 25 weeks, followed by another 12 weeks on FFD with daily subcutaneous injections of semaglutide or vehicle (control). Plasma parameters were evaluated, livers and hearts were examined, and hepatic transcriptome analysis was performed. In the liver, semaglutide significantly reduced macrovesicular steatosis (-74%, p < 0.001) and inflammation (-73%, p < 0.001) and completely abolished microvesicular steatosis (-100%, p < 0.001). Histological and biochemical assessment of hepatic fibrosis showed no significant effects of semaglutide. However, digital pathology revealed significant improvements in the degree of collagen fiber reticulation (-12%, p < 0.001). Semaglutide did not affect atherosclerosis relative to controls. Additionally, we compared the transcriptome profile of FFD-fed Ldlr-/-.Leiden mice with a human gene set that differentiates human NASH patients with severe fibrosis from those with mild fibrosis. In FFD-fed Ldlr-/-.Leiden control mice, this gene set was upregulated as well, while semaglutide predominantly reversed this gene expression. Using a translational model with advanced NASH, we demonstrated that semaglutide is a promising candidate with particular potential for the treatment of hepatic steatosis and inflammation, while for the reversal of advanced fibrosis, combinations with other NASH agents may be necessary.

Representative Rodent Models for Renal Transporter Alterations in Human Nonalcoholic Steatohepatitis.

Alterations in renal elimination processes of glomerular filtration and active tubular secretion by renal transporters can result in adverse drug reactions. Nonalcoholic steatohepatitis (NASH) alters hepatic transporter expression and xenobiotic elimination, but until recently, renal transporter alterations in NASH were unknown. This study investigates renal transporter changes in rodent models of NASH to identify a model that recapitulates human alterations. Quantitative protein expression by surrogate peptide liquid chromatography-coupled mass spectrometry (LC-MS/MS) on renal biopsies from NASH patients was used for concordance analysis with rodent models, including methionine/choline deficient (MCD), atherogenic (Athero), or control rats and Leprdb/db MCD (db/db), C57BL/6J fast-food thioacetamide (FFDTH), American lifestyle-induced obesity syndrome (ALIOS), or control mice. Demonstrating clinical similarity to NASH patients, db/db, FFDTH, and ALIOS showed decreases in glomerular filtration rate (GFR) by 76%, 28%, and 24%. Organic anion transporter 3 (OAT3) showed an upward trend in all models except the FFDTH (from 3.20 to 2.39 pmol/mg protein), making the latter the only model to represent human OAT3 changes. OAT5, a functional ortholog of human OAT4, significantly decreased in db/db, FFDTH, and ALIOS (from 4.59 to 0.45, 1.59, and 2.83 pmol/mg protein, respectively) but significantly increased for MCD (1.67 to 4.17 pmol/mg protein), suggesting that the mouse models are comparable to human for these specific transport processes. These data suggest that variations in rodent renal transporter expression are elicited by NASH, and the concordance analysis enables appropriate model selection for future pharmacokinetic studies based on transporter specificity. These models provide a valuable resource to extrapolate the consequences of human variability in renal drug elimination. SIGNIFICANCE STATEMENT: Rodent models of nonalcoholic steatohepatitis that recapitulate human renal transporter alterations are identified for future transporter-specific pharmacokinetic studies to facilitate the prevention of adverse drug reactions due to human variability.

Abstract 321: Altered Epha2 Signaling In Non-alcoholic Fatty Liver Disease Progression

Background: Nonalcoholic fatty liver disease (NAFLD), the leading cause of chronic liver disease worldwide, results from diet-induced hepatic steatosis that can result in hepatocyte ballooning, inflammation and fibrosis driving nonalcoholic steatohepatitis (NASH). The Eph receptors, the largest family of receptor tyrosine kinases in the mammalian genome, affect inflammation and fibrosis in other model systems, and we have shown that EphA2 deletion reduces atherosclerosis despite increasing plasma cholesterol levels. While a GWAS study has linked EphA2 to NAFLD, the potential role of EphA2 in NAFLD has never been investigated. Methods: EphA2 expression and signaling was assessed in mice fed a high fat diet (HFD) for 8 and 24 weeks, in human liver samples from NASH patients, and in HuH7 cells treated with palmitic acid (FA), low density lipoprotein (LDL), and/or fructose. Global EphA2 KO mice were fed a HFD for 12 weeks followed by histological and molecular analyses. Results: Mice with early-stage non-alcoholic fatty liver disease (NAFLD) showed elevated EphA2 expression along with markers of EphA2 ligand-dependent signaling (EphA2 tyrosine phosphorylation). Consistently, HuH7 cells treated with FA, LDL, and/or fructose displayed a similar enhancement of EphA2 expression upon treatment. However, a mouse model of late-stage NASH exhibited reduced expression of the EphA2 ligand ephrinA1 and elevated markers of ligand-independent signaling (Ser897 phosphorylation). Similarly, human liver samples from NASH patients show reduced ephrinA1 expression and elevated markers of EphA2 ligand independent signaling. Consistent with a role for EphA2 in NAFLD/NASH, global EphA2 KO mice show significantly lower indices of NAFLD following high fat diet feeding, including reduced liver-to-body weight ratios, hepatic steatosis, and inflammation. Conclusion: Taken together, our data demonstrate that EphA2 expression and signaling are altered during NAFLD/NASH pathogenesis and suggest that EphA2 inhibition may reduce NAFLD/NASH disease progression.

Looking ahead to potential incretin combination therapies for nonalcoholic steatohepatitis in patients with diabetes

ABSTRACT Introduction There are no drugs approved by regulatory agencies for the treatment of nonalcoholic fatty liver disease (NAFLD); incretin combination therapies are being developed for treatment of type 2 diabetes and research has moved to test their usefulness in NAFLD. Areas covered We reviewed the literature on the effectiveness of dual and triple peptides combining receptor agonists of the glucagon-like peptide 1, the glucose-dependent insulinotropic peptide, and glucagon to treat NAFLD and its associated metabolic diseases, and/or the cardiovascular risk intimately connected with the cluster of the metabolic syndrome. Other combination peptides involved the glucagon-like peptide 2 receptor, the fibroblast growth factor 21, the cholecystokinin receptor 2, and the amylin receptor. Expert opinion Both dual and triple agonists are promising, based on animal, pharmacokinetic and proof-of concept studies, showing effectiveness both in the presence and the absence of diabetes on a few validated surrogate NAFLD biomarkers, but the majority of studies are still in progress. Considering the long natural history of NAFLD, final proof of their efficacy on primary clinical liver outcomes might be also derived from the analysis of large databases of National Healthcare Systems or Insurance companies, when used in diabetes for improving glycemic control, after careful propensity-score matching.

Circulating bone morphogenetic protein 8A is a novel biomarker to predict advanced liver fibrosis

Background & AimsAdvanced hepatic fibrosis is the main risk factor of liver-related morbidity and mortality in patients with chronic liver disease. In this study, we assessed the potential role of bone morphogenetic protein 8A (BMP8A) as a novel target involved in liver fibrosis progression.MethodsHistological assessment and BMP8A expression were determined in different murine models of hepatic fibrosis. Furthermore, serum BMP8A was measured in mice with bile duct ligation (BDL), in 36 subjects with histologically normal liver (NL) and in 85 patients with biopsy-proven non-alcoholic steatohepatitis (NASH): 52 with non- or mild fibrosis (F0-F2) and 33 with advanced fibrosis (F3-F4). BMP8A expression and secretion was also determined in cultured human hepatocyte-derived (Huh7) and human hepatic stellate (LX2) cells stimulated with transforming growth factor ꞵ (TGFꞵ).ResultsBmp8a mRNA levels were significantly upregulated in livers from fibrotic mice compared to control animals. Notably, serum BMP8A levels were also elevated in BDL mice. In addition, in vitro experiments showed increased expression and secretion to the culture supernatant of BMP8A in both Huh7 and LX2 cells treated with TGFꞵ. Noteworthy, we found that serum BMP8A levels were significantly higher in NASH patients with advanced fibrosis than in those with non- or mild fibrosis. In fact, the AUROC of circulating BMP8A concentrations to identify patients with advanced fibrosis (F3-F4) was 0.74 (p˂0.0001). Moreover, we developed an algorithm based on serum BMP8A levels that showed an AUROC of 0.818 (p˂0.0001) to predict advanced fibrosis in NASH patients.ConclusionThis study provides experimental and clinical evidence indicating that BMP8A is a novel molecular target linked to liver fibrosis and introduces an efficient algorithm based on serum BMP8A levels to screen patients at risk for advanced hepatic fibrosis.

Elevated Serum Alpha-fetoprotein Levels in Non-alcoholic Steatohepatitis: Possible Molecular Mechanisms and Potential Clinical Significance

With the improvement of living standards in recent years, up to 90% of obese patients have nonalcoholic fatty liver disease (NAFLD). The number of nonalcoholic steatohepatitis (NASH)-related deaths will gradually increase, and NASH is expected to be the most common cause of liver-related deaths in the future. Therefore, there is an urgent need to find effective and reliable serum biomarkers to distinguish simple hepatic steatosis (SS) from NASH. Liver cell regeneration, oxidative stress-induced DNA methylation, and biliary epithelial cell proliferation were reported to increase serum alpha-fetoprotein (AFP) levels. AFP has long been used as a tool to monitor liver cancer. However, the function of AFP in NAFLD, especially NASH, has not been clarified. Moreover, whether an elevated AFP level indicates the occurrence of NASH or serves as a serum biomarker remains to be elucidated. The miRNA-122 pathway, DNA methylation and DNA damage, and activation of resident stem cells and/or progenitor cells in the liver, as well as necrosis, regeneration, and repair of liver cells, may contribute to slight increases in AFP levels in the development of NASH in patients with NAFLD. Furthermore, mildly elevated AFP levels may indicate the development of NASH. This review explores the role of elevated AFP levels in the development of NASH, with a specific focus on the underlying molecular mechanisms and the clinical significance.

Noncompetitive immunoassay optimized for pharmacokinetic assessments of biologically active efruxifermin

Efruxifermin (EFX) is a homodimeric human IgG1 Fc-FGF21 fusion protein undergoing investigation for treatment of liver fibrosis due to nonalcoholic steatohepatitis (NASH), a prevalent and serious metabolic disease for which there is no approved treatment. Biological activity of FGF21 requires its intact C-terminus, which enables binding to its obligate co-receptor β-Klotho on the surface of target cells. This interaction is a prerequisite for FGF21 signal transduction through its canonical FGF receptors: FGFR1c, 2c, and 3c. Therefore, the C-terminus of each FGF21 polypeptide chain must be intact, with no proteolytic truncation, for EFX to exert its pharmacological activity in patients. A sensitive immunoassay for quantification of biologically active EFX in human serum was therefore needed to support pharmacokinetic assessments in patients with NASH. We present a validated noncompetitive electrochemiluminescent immunoassay (ECLIA) that employs a rat monoclonal antibody for specific capture of EFX via its intact C-terminus. Bound EFX is detected by a SULFO-TAG™-conjugated, affinity purified chicken anti-EFX antiserum. The ECLIA reported herein for quantification of EFX demonstrated suitable analytical performance, with a sensitivity (LLOQ) of 20.0 ng/mL, to support reliable pharmacokinetic assessments of EFX. The validated assay was used to quantify serum EFX concentrations in a phase 2a study of NASH patients (BALANCED) with either moderate-to-advanced fibrosis or compensated cirrhosis. The pharmacokinetic profile of EFX was dose-proportional and did not differ between patients with moderate-to-advanced fibrosis and those with compensated cirrhosis. This report presents the first example of a validated pharmacokinetic assay specific for a biologically active Fc-FGF21 fusion protein, as well as the first demonstration of use of a chicken antibody conjugate as a detection reagent specific for an FGF21 analog.