Nonalcoholic Steatohepatitis Subjects Research Articles

Circulating Citrate Is Associated with Liver Fibrosis in Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is associated with mitochondrial damage. Circulating mitochondrial metabolites may be elevated in NAFLD but their associations with liver damage is not known. This study aimed to assess the association of key mitochondrial metabolites with the degree of liver fibrosis in the context of NAFLD and nonalcoholic steatohepatitis (NASH). Cross-sectional analyses were performed on two cohorts of biopsy-proven NAFLD and/or NASH subjects. The association of circulating mitochondrial metabolite concentrations with liver fibrosis was assessed using linear regression analysis. In the single-center cohort of NAFLD subjects (n = 187), the mean age was 54.9 ±13.0 years, 40.1% were female and 86.1% were White. Type 2 diabetes (51.3%), hypertension (43.9%) and obesity (72.2%) were prevalent. Those with high citrate had a higher proportion of moderate/significant liver fibrosis (stage F ≥ 2) (68.4 vs. 39.6%, p = 0.001) and advanced fibrosis (stage F ≥ 3) (31.6 vs. 13.6%, p = 0.01). Citrate was associated with liver fibrosis independent of age, sex, NAFLD activity score and metabolic syndrome (per 1 SD increase: β = 0.19, 95% CI: 0.03-0.35, p = 0.02). This association was also observed in a cohort of NASH subjects (n = 176) (β = 0.21, 95% CI: 0.07-0.36, p = 0.005). The association of citrate with liver fibrosis was observed in males (p = 0.005) but not females (p = 0.41). In conclusion, circulating citrate is elevated and associated with liver fibrosis, particularly in male subjects with NAFLD and NASH. Mitochondrial function may be a target to consider for reducing the progression of liver fibrosis and NASH.

Acetyl-CoA carboxylase inhibitor increases LDL-apoB production rate in NASH with cirrhosis: prevention by fenofibrate

Treatment with acetyl-CoA carboxylase inhibitors (ACCi) in nonalcoholic steatohepatitis (NASH) may increase plasma triglycerides (TGs), with variable changes in apoB concentrations. ACC is rate limiting in de novo lipogenesis and regulates fatty acid oxidation, making it an attractive therapeutic target in NASH. Our objectives were to determine the effects of the ACCi, firsocostat, on production rates of plasma LDL-apoB in NASH and the effects of combined therapy with fenofibrate. Metabolic labeling with heavy water and tandem mass spectrometric analysis of LDL-apoB enrichments was performed in 16 NASH patients treated with firsocostat for 12 weeks and in 29 NASH subjects treated with firsocostat and fenofibrate for 12 weeks. In NASH on firsocostat, plasma TG increased significantly by 17% from baseline to week 12 (P = 0.0056). Significant increases were also observed in LDL-apoB fractional replacement rate (baseline to week 12: 31 ± 20.2 to 46 ± 22.6%/day, P = 0.03) and absolute synthesis rate (ASR) (30.4–45.2 mg/dl/day, P = 0.016) but not plasma apoB concentrations. The effect of firsocostat on LDL-apoB ASR was restricted to patients with cirrhosis (21.0 ± 9.6 at baseline and 44.2 ± 17 mg/dl/day at week 12, P = 0.002, N = 8); noncirrhotic patients did not change (39.8 ± 20.8 and 46.3 ± 14.8 mg/dl/day, respectively, P = 0.51, N = 8). Combination treatment with fenofibrate and firsocostat prevented increases in plasma TG, LDL-apoB fractional replacement rate, and ASR. In summary, in NASH with cirrhosis, ACCi treatment increases LDL-apoB100 production rate and this effect can be prevented by concurrent fenofibrate therapy.

The Growing Economic and Clinical Burden of Nonalcoholic Steatohepatitis (NASH) in the United States

Nonalcoholic steatohepatitis (NASH) is a cause of chronic liver disease. Model the burden of NASH in the United States according to obesity. The discrete-time Markov model comprised adult NASH subjects moving through 9 health states and 3 absorbing death states (liver, cardiac, and other deaths) with 1-year cycles and a 20-year horizon. Given that reliable natural history data for NASH are not available, transition probabilities were estimated from the literature and population-based data. These rates were disaggregated to determine age-obesity group rates by applying estimated age-obesity patterns. The model considers 2019 prevalent NASH cases and new incident NASH cases (2020-2039), assuming that recent trends will continue. Annual per-patient costs by health state were based on published data. Costs were standardized to 2019 US dollars and inflated by 3% annually. NASH cases in the United States are forecasted to increase by+82.6%, from 11.61 million (2020) to 19.53 million (2039). During the same period, cases of advanced liver disease increased+77.9%, from 1.51 million to 2.67 million, while its proportion remained stable (13.46%-13.05%). Similar patterns were observed in both obese and non-obese NASH. Among NASH, 18.71 million overall deaths, 6.72 million cardiac-specific deaths, and 1.71 million liver-specific deaths were observed by 2039. During this period, the projected cumulative direct healthcare costs were $1208.47 billion (obese NASH) and $453.88 billion (non-obese NASH). By 2039, the projected NASH attributable healthcare cost per patient increased from $3636 to $6968. There is a substantial and growing clinical and economic burden of NASH in the United States.

Antioxidative Self-Assembling Nanoparticles Attenuate the Development of Steatohepatitis and Inhibit Hepatocarcinogenesis in Mice.

Oxidative stress (OS) contributes to nonalcoholic steatohepatitis (NASH) and hepatocarcinogenesis. We investigated whether antioxidative self-assembling nanoparticles (SMAPoTN) could reduce the development of NASH and hepatocellular carcinoma (HCC) in p62/Sqstm1 and Nrf2 double knockout (DKO) mice and studied protective mechanisms. We measured disease development in male DKO mice fed a normal chow (NASH model) or a 60% high-fat diet (HFD; HCC model) with or without SMAPoTN administration for 26 weeks. SMAPoTN inhibited liver fibrosis in both groups and prevented HCC development (0% vs. 33%, p < 0.05) in the HFD group. SMAPoTN reduced OS, inflammatory cytokine signaling, and liver fibrosis. RNA-sequencing revealed that SMAPoTN decreased endoplasmic reticulum stress signaling genes in both groups, HCC driver genes, and cancer pathway genes, especially PI3K-AKT in the HFD groups. In the SMAPoTN treatment HFD group, serum lipopolysaccharide levels and liver lipopolysaccharide-binding protein expression were significantly lower compared with those in the nontreatment group. SMAPoTN improved the α-diversity of gut microbiota, and changed the microbiota composition. Oral SMAPoTN administration attenuated NASH development and suppressed hepatocarcinogenesis in DKO mice by improving endoplasmic reticulum stress in the liver and intestinal microbiota. SMAPoTN may be a new therapeutic option for NASH subjects and those with a high HCC risk.

Multimodal NASH prognosis using 3D imaging flow cytometry and artificial intelligence to characterize liver cells

To improve the understanding of the complex biological process underlying the development of non-alcoholic steatohepatitis (NASH), 3D imaging flow cytometry (3D-IFC) with transmission and side-scattered images were used to characterize hepatic stellate cell (HSC) and liver endothelial cell (LEC) morphology at single-cell resolution. In this study, HSC and LEC were obtained from biopsy-proven NASH subjects with early-stage NASH (F2-F3) and healthy controls. Here, we applied single-cell imaging and 3D digital reconstructions of healthy and diseased cells to analyze a spatially resolved set of morphometric cellular and texture parameters that showed regression with disease progression. By developing a customized autoencoder convolutional neural network (CNN) based on label-free cell transmission and side scattering images obtained from a 3D imaging flow cytometer, we demonstrated key regulated cell types involved in the development of NASH and cell classification performance superior to conventional machine learning methods.

Dysregulation of S-adenosylmethionine Metabolism in Nonalcoholic Steatohepatitis Leads to Polyamine Flux and Oxidative Stress.

Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease worldwide, with 25% of these patients developing nonalcoholic steatohepatitis (NASH). NASH significantly increases the risk of cirrhosis and decompensated liver failure. Past studies in rodent models have shown that glycine-N-methyltransferase (GNMT) knockout results in rapid steatosis, fibrosis, and hepatocellular carcinoma progression. However, the attenuation of GNMT in subjects with NASH and the molecular basis for its impact on the disease process is still unclear. To address this knowledge gap, we show the reduction of GNMT protein levels in the liver of NASH subjects compared to healthy controls. To gain insight into the impact of decreased GNMT in the disease process, we performed global label-free proteome studies on the livers from a murine modified amylin diet-based model of NASH. Histological and molecular characterization of the animal model demonstrate a high resemblance to human disease. We found that a reduction of GNMT leads to a significant increase in S-adenosylmethionine (AdoMet), an essential metabolite for transmethylation reactions and a substrate for polyamine synthesis. Further targeted proteomic and metabolomic studies demonstrated a decrease in GNMT transmethylation, increased flux through the polyamine pathway, and increased oxidative stress production contributing to NASH pathogenesis.

Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH.

Safety, Tolerability, and Biologic Activity of AXA1125 and AXA1957 in Subjects With Nonalcoholic Fatty Liver Disease.

INTRODUCTION:AXA1125 and AXA1957 are novel, orally administered endogenous metabolic modulator compositions, specifically designed to simultaneously support multiple metabolic and fibroinflammatory pathways associated with nonalcoholic fatty liver disease (NAFLD). This study assessed safety, tolerability, and biologic activity of AXA1125 and AXA1957 in NAFLD.METHODS:In this multicenter, 16-week, placebo-controlled, single-blind, randomized clinical study in subjects with NAFLD stratified by type 2 diabetes, AXA1125 24 g, AXA1957 13.5 g or 20.3 g, or placebo was administered twice daily. Key metabolism (MRI-proton density fat fraction [MRI-PDFF] and homeostasis model assessment of insulin resistance [HOMA-IR]) and fibroinflammation markers (alanine aminotransferase [ALT], corrected T1 [cT1], keratin-18 [K-18] M65, and N-terminal type III collagen propeptide [Pro-C3]) were evaluated. Safety outcomes included adverse events and standard laboratory assessments.RESULTS:Baseline characteristics of the 102 enrolled subjects, including 40 with type 2 diabetes, were consistent with presumed nonalcoholic steatohepatitis. AXA1125 showed consistently greater biologic activity than AXA1957 or placebo. Week 16 changes from baseline with AXA1125 vs placebo: MRI-PDFF −22.9% vs −5.7%, HOMA-IR −4.4 vs +0.7, ALT −21.9% vs −7.2%, K-18 M65 −13.6% vs +20.1%, cT1 −69.6 vs +18.3 ms (P < 0.05), and Pro-C3 −13.6% vs −3.6%. Week 16 changes from baseline with AXA1957 20.3 g: MRI-PDFF −8.1%, HOMA-IR +8.4, ALT −20.7%, K-18 M65 6.6%, cT1 −34.7 ms, and Pro-C3 −15.6%. A greater proportion of subjects treated with AXA1125 achieved clinically relevant thresholds: ≥30% MRI-PDFF, ≥17-IU/L ALT, and ≥80-ms cT1 reductions at week 16. Study products were safe and well tolerated with stable lipid and weight profiles.DISCUSSION:Both compositions showed multitargeted activity on relevant NAFLD pathways. AXA1125 demonstrated the greatest activity over 16 weeks, warranting continued clinical investigation in nonalcoholic steatohepatitis subjects.

Plasminogen activator inhibitor is significantly elevated in liver transplant recipients with decompensated NASH cirrhosis

BackgroundNon-alcoholic fatty liver disease is a prohaemostatic state with abnormal primary, secondary and tertiary haemostasis. Plasminogen activator inhibitor (PAI)-1 is the best-established marker for prohaemostasis in non-alcoholic fatty liver disease....

Gut Microbiome and Intestinal Permeability Are Promising Targets for Treating Nonalcoholic Fatty Liver Disease

Background: Nonalcoholic fatty liver disease (NAFLD) is a leading liver disease worldwide with a prevalence of approximately 24% among adult population. It is a spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Obesity, insulin resistance, inflammation, disrupted gut microbiome (dysbiosis), and increased intestinal permeability (“leaky gut”) are important risk factors associated with and/or contributing to NAFLD. In the absence of specific drugs to treat NAFLD, management relies mainly on lifestyle changes (diet and exercise). Gut microbiome and intestinal permeability are becoming promising targets for the treatment of several diseases including NAFLD. Methods: A systematic search of literature was conducted using the search terms nonalcoholic fatty liver disease, obesity, insulin resistance, inflammation, gut microbiome, intestinal permeability, and targeted therapy. Results: Several clinical studies have shown the association of qualitative and quantitative changes in gut microbiome (e.g., increased Lactobacillus and Gram-negative bacteria) with NAFLD and its severity. The increased gut microbiome taxa may produce more short-chain fatty acids (SCFAs), alcohol, and lipopolysaccharides (LPS). Increased SCFAs, alcohol, and LPS (endotoxins) associated with increased intestinal permeability are implicated in the pathogenesis of NAFLD and its evolution to NASH (promotion of obesity and inflammation). Gut microbiome and intestinal permeability can be modified with diet, prebiotics, probiotics, synbiotics, and fecal microbiota transplantation. A high-fiber diet for 6 months in NAFLD subjects caused a decrease in intestinal permeability (as assessed by serum zonulin) and a reduction of liver enzymes and fatty liver. Treatment with a prebiotic (oligofructose) for 8 weeks in NASH subjects showed a significant decrease in markers of liver inflammation. Administration of probiotics (e.g., Lactobacillus, Bifidobacterium, and VSL#3) for 8 to 16 weeks in NAFLD subjects had several beneficial effects including reduction of liver enzymes and fatty liver. Clinical studies using fecal microbiota transplantation in NAFLD subjects are currently ongoing. To optimize the efficacy of therapies using prebiotics, probiotics, synbiotics, and fecal microbiota transplantation in NAFLD subjects, focus should be on the altered gut microbiome taxa responsible for high SCFAs, alcohol, and LPS production. Conclusion: NAFLD is the most common chronic liver disease. NAFLD subjects have disrupted gut microbiome associated with increased intestinal permeability. In addition to weight loss (when excess body weight is present), targeting gut microbiome and intestinal permeability with diet, prebiotics, probiotics, synbiotics, and fecal microbiota transplantation represents a promising novel approach in the treatment of NAFLD.

Thrombospondin1 as a potential therapeutic target for human nonalcoholic fatty liver disease.

Thrombospondin1 as a potential therapeutic target for human nonalcoholic fatty liver disease.

Targeting of Secretory Proteins as a Therapeutic Strategy for Treatment of Nonalcoholic Steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) is defined as a progressive form of nonalcoholic fatty liver disease (NAFLD) and is a common chronic liver disease that causes significant worldwide morbidity and mortality, and has no approved pharmacotherapy. Nevertheless, growing understanding of the molecular mechanisms underlying the development and progression of NASH has suggested multiple potential therapeutic targets and strategies to treat this disease. Here, we review this progress, with emphasis on the functional role of secretory proteins in the development and progression of NASH, in addition to the change of expression of various secretory proteins in mouse NASH models and human NASH subjects. We also highlight secretory protein-based therapeutic approaches that influence obesity-associated insulin resistance, liver steatosis, inflammation, and fibrosis, as well as the gut–liver and adipose–liver axes in the treatment of NASH.

Quantitative Proteomics and Mechanistic Modeling of Transporter-Mediated Disposition in Nonalcoholic Fatty Liver Disease.

Understanding transporter-mediated drug disposition and pharmacokinetics (PK) in patients with nonalcoholic fatty liver disease (NAFLD) is critical in developing treatment options. Here, we quantified the expression levels of major drug transporters in healthy, steatosis, and nonalcoholic steatohepatitis (NASH) liver samples, via liquid-chromatography tandem mass spectrometry-based proteomics, and used the data to predict the PK of substrate drugs in the disease state. Expression of organic anion transporting polypeptides (OATPs) and multidrug resistance-associated protein (MRP)2 is significantly lower in NASH livers; whereas MRP3 is induced while no change was observed for organic cation transporter (OCT)1. Physiologically-based pharmacokinetic models verified with PK data from healthy subjects well recovered the PK in NASH subjects for morphine (involving OCT1) and its glucuronide metabolites (MRP2/MRP3/OATP1B), 99m TC-mebrofenen (OATP1B/MRP2/MRP3), and rosuvastatin (OATP1B/breast cancer resistance protein). Overall, considerations to altered protein expression can enable quantitative prediction of PK changes in subjects with NAFLD.

Non-invasive diagnosis of non-alcoholic steatohepatitis and fibrosis with the use of omics and supervised learning: A proof of concept study

BackgroundNon-alcoholic fatty liver disease (NAFLD) affects 25–30% of the general population and is characterized by the presence of non-alcoholic fatty liver (NAFL) that can progress to non-alcoholic steatohepatitis (NASH), liver fibrosis and cirrhosis leading to hepatocellular carcinoma. To date, liver biopsy is the gold standard for the diagnosis of NASH and for staging liver fibrosis. This study aimed to train models for the non-invasive diagnosis of NASH and liver fibrosis based on measurements of lipids, glycans and biochemical parameters in peripheral blood and with the use of different machine learning methods. MethodsWe performed a lipidomic, glycomic and free fatty acid analysis in serum samples of 49 healthy subjects and 31 patients with biopsy-proven NAFLD (15 with NAFL and 16 with NASH). The data from the above measurements combined with measurements of 4 hormonal parameters were analyzed with two different platforms and five different machine learning tools. Results365 lipids, 61 glycans and 23 fatty acids were identified with mass-spectrometry and liquid chromatography. Robust differences in the concentrations of specific lipid species were observed between healthy, NAFL and NASH subjects. One-vs-Rest (OvR) support vector machine (SVM) models with recursive feature elimination (RFE) including 29 lipids or combining lipids with glycans and/or hormones (20 or 10 variables total) could differentiate with very high accuracy (up to 90%) between the three conditions. In an exploratory analysis, a model consisting of 10 lipid species could robustly discriminate between the presence of liver fibrosis or not (98% accuracy). ConclusionWe propose novel models utilizing lipids, hormones and glycans that can diagnose with high accuracy the presence of NASH, NAFL or healthy status. Additionally, we report a combination of lipids that can diagnose the presence of liver fibrosis. Both models should be further trained prospectively and validated in large independent cohorts.

1097 Are There Demographic Variations Between Obese and Non-Obese (Lean) Patients With Non-alcoholic Steatohepatitis?

INTRODUCTION: Non-alcoholic steatohepatitis (NASH) in the absence of overweight and/or obesity is designated as non-obese (lean) NASH. Several studies suggested that lean NASH is a variant phenotype of NASH, where its features metabolically resemble obese NASH individuals with insulin resistance, and metabolic syndrome plays the pathogenetic role in both phenotypes. Limited data suggested that Asians have propensity to develop NASH at a comparatively lower BMI, mostly due to body composition and fat distribution. We aimed at assessing baseline demographic variations between obese and non-obese (lean) patients with NASH. METHODS: Based on fibroscan reports with CAP and fibrosis scores, 124 patients with NAFLD had been identified between 09/2018 and 05/2019 at the Liver Associates of Texas Hepatology Clinics in Houston. Standardized fibroscan report (steatosis, and fibrosis stage) was determined by expert clinicians. Demographic information, chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), baseline body mass index (BMI), presence of major comorbidities and metabolic syndrome were recorded for all patients. For continuous variables, we used the Student’s t test to compare differences in the mean values. The chi-squared test was used to compare the proportions of risk factors between groups. RESULTS: According to the baseline body mass index, patients were classified into obese (BMI ≥30) and non-obese (BMI &lt; 30). With the consideration of presence and absence of NASH and obesity, participants were further classified into four groups as seen in Table 1. Our data indicated that Asians (47.1%) especially men (57.1%), P = .06 are more likely to be classified as non-obese/NASH. No significant variation between all groups, however, with respect to age at diagnosis or gender distribution. CONCLUSION: No significant difference between obese and lean patients with NASH regarding age, gender or ethnicity. NASH should be considered in the differential diagnosis of unexplained liver disease even in presence of normal BMI. Management strategy, and surveillance protocol for NASH should be evaluated and validated in non-obese (lean) NASH subjects.

Hepatic sonic hedgehog protein expression measured by computer assisted morphometry significantly correlates with features of non-alcoholic steatohepatitis

BackgroundHepatic expression of Sonic Hedgehog (SHH) is associated with Non-alcoholic fatty liver disease (NAFLD) and development of Non-alcoholic steatohepatitis (NASH). Hepatic SHH detection increases with the diagnosis of NASH. This pilot study was designed to confirm that staining for SHH is useful in NASH diagnosis and determine whether quantification of staining by computer assisted morphometry (CAM) can be used to assess severity of ballooning degeneration.MethodsSHH was detected by immunohistochemistry (IHC) on paraffin-embedded liver sections in subjects (N = 69) with biopsy proven NAFLD and no liver disease (control). Serum samples were also available for these subjects. Post-staining, a digitized image of the section was acquired and an area quantification algorithm was used to quantify the degree of SHH expression. Additionally, circulating M30, M65, and SHH were measured by ELISA.ResultsNotably, hepatic SHH expression correlated with histologic ballooning degeneration (rho = 0.62, p < 0.0001), steatosis grade (rho = 0.554, P < 0.001), Mallory-Denk bodies (rho = 0.54, P < 0.001), pericellular fibrosis (rho = 0.527, P < 0.001), and lymphocytic infiltration (rho = 0.435, P < 0.0002). Additionally, hepatic SHH expression correlated with circulating M65 (rho = 0.588, p < 0.0001), and circulating M30 (rho = 0.375, p = 0.001), as well as AST and ALT (rho = 0.43, p = 0.0004, and rho = 0.27, p = 0.03, respectively). Further, serum M30 was almost twice as high in NASH patients compared to non-NASH (539.1 ± 290.8 U/L vs. 287.6 ± 190.5 U/L; p = 0.0002), while M65 was almost three times higher in NASH patients compared to non-NASH (441.2 ± 464.2 U/L vs. 162.8 ± 353.1 U/L, P = 0.0006). Logistic modeling indicates hepatic SHH expression and presence of type 2 diabetes as independent predictors of advanced fibrosis (defined as portal and pericellular fibrosis > 2: OR = 1.986, p = 0.01, and OR = 3.280, p = 0.03, respectively).ConclusionThus, our findings show quantitation of SHH expression by CAM can provide a tool for quantifying changes in hepatocyte injury and assist in unambiguous staging/grading of NASH. Our study showed minimal interobserver variability using CAM based quantification. Once validated, CAM assessment of hepatic SHH could benefit clinical trials or long term outcomes studies of NASH subjects.

Systematic review with meta-analysis: risk of hepatocellular carcinoma in non-alcoholic steatohepatitis without cirrhosis compared to other liver diseases.

Given the lack of long-term prospective studies, it is challenging for clinicians to make informed decisions about screening and treatment decisions regarding the risk of hepatocellular carcinoma (HCC) in patients with non-alcoholic steatohepatitis (NASH) who do not have cirrhosis. To characterise the pooled risk of HCC in the non-cirrhosis population. Published studies were identified through April 2016 in MEDLINE, Scopus, Science Citation Index, AMED and the Cochrane Library. Two independent reviewers screened citations and extracted data. Random effect odds ratios (OR) were calculated to obtain aggregate estimates of effect size between NASH and non-NASH groups. Between-study variability and heterogeneity were assessed. Nineteen studies with 168571 participants were included. Eighty-six per cent of included subjects had cirrhosis. The prevalence of HCC in non-cirrhotic NASH was 38.0%; among other aetiologies in non-cirrhotics, it was 14.2% (P<0.001). Non-cirrhotic NASH subjects were at greater odds of developing HCC than non-cirrhotic subjects of other aetiologies (OR 2.61, 95% CI 1.27-5.35, P=0.009). When examining all NASH subjects either with or without cirrhosis, those with NASH as the underlying liver disease did not have a significantly increased risk of HCC (OR 1.43, 95% CI 0.77-2.65, P=0.250). In non-cirrhotic subjects, those with NASH have a higher risk of HCC compared to other aetiologies of liver disease. Further study investigating the risk factors of HCC among non-cirrhotic NASH patients is needed.

17. Elevated serum endotoxins increase inflammation via iNOS and COX-2 in non-alcoholic fatty liver disease patients

Background and Aims: Emerging data indicate that gut microbiota-derived endotoxin may contribute to low-grade systemic inflammation in non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH). This study aimed to examine the role of serum endotoxin and inflammatory markers in these patients. Methods: Serum and stool samples from NASH (n = 30), ASH (n = 20) patients with biopsy-proven NASH and (n = 20) control subjects were analysed for endotoxin, soluble TLR4, soluble tumour necrosis factor receptor II, IL-1, IL-6 and total Nitric oxide. The level of inflammatory markers, such as iNOS and COX-2, was determined by Western blotting in peripheral blood mononuclear cells (PBMC) and liver biopsies. Results: Endotoxin levels were significantly higher in patients with NASH compared with ASH and controls (NASH: 10.6 EU/mL; controls: 3.9 EU/mL and ASH: 6.2 EU/ml) were supported in some previous studies. Level of the serum Nitric Oxide (NO) was noticed to be higher in the NASH patients (2.1 mM/100 μl) as compared to the controls (1 mM/100 μl). The level of IL-1 (18.6 pg/ml) and IL-6 (17.5 pg/ml) were higher in NASH patients as compared to the healthy controls (4.6 fold and 4.2 fold respectively). A significant correlation between BMI of patients and serum endotoxin was observed (p = 0.008) in NASH patients. Both iNOS (4.2 fold) and COX-2 (4.3 fold) expression were higher in PBMCs and liver biopsies of NASH patients as compared to the healthy controls and ASH patients. A positive correlation was also observed between NO concentration and serum endotoxin in the NASH subjects (r2 = 0.23, p = 0.004). Conclusions: Our study suggests that iNOS and NO levels may play a role in the pathogenesis of the NASH in patients with higher BMI. The authors have none to declare.

Variation in the Plasma Levels of Polyunsaturated Fatty Acids in Control vis-à-vis Nonalcoholic Fatty Liver Disease Subjects and Its Possible Association with Gut Microbiome.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are becoming a major cause of chronic liver diseases globally. Polyunsaturated fatty acids (PUFA) have been postulated as a potential treatment for NAFLD. They can be obtained from diet and are also endogenously synthesized by enzymes delta-6-desaturase (D6D), delta-5-desaturase, and elongases. The current study is aimed at investigating the differences in the intake and levels of plasma PUFA between NAFLD patients and controls in Asian Indians. This correlation further propelled a pilot study to check for the differences in the gut microbiome of NASH subjects versus controls. One hundred forty-seven subjects were recruited and were grouped into healthy controls and cases. Subjects filled a food frequency questionnaire to assess PUFA intake. Plasma samples were subjected to gas chromatography analysis. For characterizing the gut microbiome, fecal samples of 20 NASH and healthy controls were analyzed by 16s rRNA gene sequencing. Alpha and beta diversity metrics and taxonomic analysis were carried out. Plasma levels of eicosapentaenoic acid, γ-linoleic acid and D6D enzyme activity were significantly lower in cases. Dietary intake of total n-6 and n-3 PUFA did not differ between the two groups. Relative abundance of Streptococcus and Clostridium ramosum was significantly higher in NASH patients compared with healthy controls. This study demonstrates, for the first time, decreased D6D enzyme activity and plasma PUFA levels in NAFLD patients. Furthermore, it demonstrates gut dysbiosis in histologically proven NASH patients vis-à-vis healthy controls.

Physical deconditioning is the common denominator in both obese and overweight subjects with nonalcoholic steatohepatitis.

Obesity and decreased physical activity mirror increasing prevalence of nonalcoholic fatty liver disease (NAFLD). We aimed to investigate associations between aerobic fitness, anthropometrics and disease parameters in patients with nonalcoholic steatohepatitis (NASH). We hypothesised that NASH subjects have lower aerobic power and capacity than untrained, sedentary, normal subjects. Forty subjects (60% obese, 40% overweight) with biopsy-confirmed NASH and NAFLD activity score (NAS) ≥4 were enrolled in a clinical trial where anthropometrics, laboratories, liver fat content by MRI, activity, and aerobic fitness by cycle ergometry data were obtained. NASH subjects were significantly deconditioned compared to 148 untrained, sedentary, healthy subjects from our laboratory in aerobic power (VO2peak) (NASH 16.8±6.6 vs control 28.4±10.6mL/kg/min, P<0.0001) and capacity (VO2 at lactate threshold [LT]) (NASH 8.3±2.5 vs control 14.1±5.9mL/kg/min, P<0.0001). NASH subjects' fitness was comparable to the "least fit" tertile of controls: VO2peak [NASH 16.8±6.6 vs "least fit" 17.3±3.3, P=0.64]) and VO2 at LT (NASH 8.3±2.5 vs "least fit" 9.3±2.1, P=0.31). Fitness was similar in obese compared to overweight subjects (adjusted for gender) and was not correlated with visceral adiposity or NAS. Engaging in dedicated cardiovascular activity correlated with higher VO2peak and VO2peak at LT. Aerobic deconditioning was universally present in NASH subjects. NASH subjects' fitness was similar to our laboratory's "least fit" untrained, sedentary control subjects. Further research investigating NASH patients' ability to improve low baseline aerobic fitness is warranted.