Progression Of Steatohepatitis Research Articles

Suppression of insulin-induced gene 1 (INSIG1) function promotes hepatic lipid remodelling and restrains NASH progression

ObjectiveNon-alcoholic fatty liver disease (NAFLD) is a silent pandemic associated with obesity and the metabolic syndrome, and also increases cardiovascular- and cirrhosis-related morbidity and mortality. A complete understanding of adaptive compensatory metabolic programmes that modulate non-alcoholic steatohepatitis (NASH) progression is lacking. Methods and resultsTranscriptomic analysis of liver biopsies in patients with NASH revealed that NASH progression is associated with rewiring of metabolic pathways, including upregulation of de novo lipid/cholesterol synthesis and fatty acid remodelling. The modulation of these metabolic programmes was achieved by activating sterol regulatory element-binding protein (SREBP) transcriptional networks; however, it is still debated whether, in the context of NASH, activation of SREBPs acts as a pathogenic driver of lipotoxicity, or rather promotes the biosynthesis of protective lipids that buffer excessive lipid accumulation, preventing inflammation and fibrosis. To elucidate the pathophysiological role of SCAP/SREBP in NASH and wound-healing response, we used an Insig1 deficient (with hyper-efficient SREBPs) murine model challenged with a NASH-inducing diet. Despite enhanced lipid and cholesterol biosynthesis, Insig1 KO mice had similar systemic metabolism and insulin sensitivity to Het/WT littermates. Moreover, activating SREBPs resulted in remodelling the lipidome, decreased hepatocellular damage, and improved wound-healing responses. ConclusionsOur study provides actionable knowledge about the pathways and mechanisms involved in NAFLD pathogenesis, which may prove useful for developing new therapeutic strategies. Our results also suggest that the SCAP/SREBP/INSIG1 trio governs transcriptional programmes aimed at protecting the liver from lipotoxic insults in NASH.

Liver X Receptor Alpha Activation Inhibits Autophagy and Lipophagy in Hepatocytes by Dysregulating Autophagy-Related 4B Cysteine Peptidase and Rab-8B, Reducing Mitochondrial Fuel Oxidation.

Fat accumulation results from increased fat absorption and/or defective fat metabolism. Currently, the lipid-sensing nuclear receptor that controls fat utilization in hepatocytes is elusive. Liver X receptor alpha (LXRα) promotes accumulation of lipids through the induction of several lipogenic genes. However, its effect on lipid degradation is open for study. Here, we investigated the inhibitory role of LXRα in autophagy/lipophagy in hepatocytes and the underlying basis. In LXRα knockout mice fed a high-fat diet, or cell models, LXRα activation suppressed the function of mitochondria by inhibiting autophagy/lipophagy and induced hepatic steatosis. Gene sets associated with "autophagy" were enriched in hepatic transcriptome data. Autophagy flux was markedly augmented in the LXRα knockout mouse liver and primary hepatocytes. Mechanistically, LXRα suppressed autophagy-related 4B cysteine peptidase (ATG4B) and Rab-8B, responsible for autophagosome and -lysosome formation, by inducing let-7a and microRNA (miR)-34a. Chromatin immunoprecipitation assay enabled us to find LXRα as a transcription factor of let-7a and miR-34a. Moreover, 3' untranslated region luciferase assay substantiated the direct inhibitory effects of let-7a and miR-34a on ATG4B and Rab-8B. Consistently, either LXRα activation or the let-7a/miR-34a transfection lowered mitochondrial oxygen consumption rate and mitochondrial transmembrane potential and increased fat levels. In obese animals or nonalcoholic fatty liver disease (NAFLD) patients, let-7a and miR-34a levels were elevated with simultaneous decreases in ATG4B and Rab-8B levels. LXRα inhibits autophagy in hepatocytes through down-regulating ATG4B and Rab-8B by transcriptionally activating microRNA let-7a-2 and microRNA 34a genes and suppresses mitochondrial biogenesis and fuel consumption. This highlights a function of LXRα that culminates in the progression of liver steatosis and steatohepatitis, and the identified targets may be applied for a therapeutic strategy in the treatment of NAFLD.

Exercise suppresses NLRP3 inflammasome activation in mice with diet-induced NASH: a plausible role of adropin

AbstractNLRP3 inflammasome activation, which can be triggered by reactive oxygen species (ROS), contributes to nonalcoholic steatohepatitis (NASH) progression. Exercise is an effective therapeutic strategy for NASH. However, whether exercise prevents NLRP3 activation in NASH has not been investigated. Here, we investigated the effect of exercise on NLRP3 inflammasome in mice with high-fat diet (HFD)-induced or methionine and choine-deficient (MCD) diet-induced NASH and explored whether adropin, a metabolic peptide hormone shown to inhibit inflammation, mediates an exercise-induced benefit against NLRP3 inflammasome activation. Exercise alleviated diet-induced hepatic steatosis, inflammation, and fibrosis. Importantly, exercise significantly reduced the expression of NLRP3 inflammasome components, decreased Caspase-1 enzymatic activity, normalized IL-1β production, and suppressed ROS overproduction in HFD-fed and MCD diet-fed mice. The exercise-elicited NLRP3 inflammasome inhibition was accompanied by increased adropin levels. Moreover, serum adropin levels were negatively correlated with serum IL-1β levels. We further explored the effect of adropin on the NLRP3 inflammasome in palmitic acid (PA)-treated hepatocytes and Kupffer cells. Although adropin treatment did not significantly decrease the levels of all inflammasome components, it reduced the active Caspase-1 level, decreased Caspase-1 activity and downregulated IL-1β expression in hepatocytes and Kupffer cells (KCs) treated with PA. Moreover, ROS levels in PA-stimulated hepatocytes and Kupffer cells were reduced upon adropin treatment. In summary, we demonstrated that the inhibitory effect of exercise on NLRP3 inflammasome activation was associated with adropin induction, resulting in NASH improvement.This study shows that exercise increases adropin levels and inhibits NLRP3 inflammasome activation in mice with diet-induced nonalcoholic steatohepatitis (NASH). Furthermore, adropin suppresses palmitic acid-induced NLRP3 inflammasome activation in hepatocytes and Kupffer cells. These results indicate that exercise may inhibit NLRP3 inflammasome activation via adropin induction, resulting in NASH improvement.

The roles of gap junctional intercellular communication in non-alcoholic steatohepatitis (NASH) and liver fibrosis

Non-alcoholic steatohepatitis (NASH) is a common risk factor for fibrosis, cirrhosis, and a predisposing factor for the development of hepatocellular carcinoma. Recently, incidence of NASH has increased due to an increase in metabolic syndrome. Connexin (Cx)32, a hepatocyte gap-junction protein, plays an important role in liver tissue homeostasis; Cx32 dominant-negative transgenic rat (Cx32ΔTg) has much decreased gap-junctional intercellular communication, and high susceptibility to carcinogens. We found for the first time that Cx32 has play suppressive roles in inflammation and fibrosis of NASH using Cx32ΔTg received methionine-choline deficient diet (MCDD). Elevation of reactive oxygen species (ROS) play important roles in progression of NASH and elimination of ROS by antioxidant luteolin inhibited NASH in the Cx32ΔTg-MCDD model. This model had histological changes similar to those of human NASH, but was not accompanied by the metabolic syndrome such as obesity and insulin resistance. Therefore, we further established an improved NASH model. Cx32ΔTg rats and wild-type rats were fed a high-fat diet (HFD) and dimethylnitrosamine to induce NASH with metabolic syndrome. The HFD and DMN increased body, liver, and visceral fat weights in both genotypes. Serum insulin level and HOMA-IR score in Cx32ΔTg rats were higher than those in wild-type rats. Elevation of serum hepatic enzymes (AST, ALT), inflammatory cytokine expressions, α-smooth muscle actin expression, progression of steatohepatitis and fibrosis were induced by HFD and dimethylnitrosamine especially in Cx32ΔTg rats. These results indicate Cx32 dysfunction promoted the development of NASH and fibrosis accompanied by metabolic syndrome through accumulation of oxidative stress.

Spirulina platensis and its ingredient biopterin glucoside improved insulin sensitivity in non-alcoholic steatohepatitis model.

Non-alcoholic steatohepatitis is the chronic liver disease leading to cirrhosis and cancer and its prevalence is increasing. Some agents are under clinical trials for non-alcoholic steatohepatitis treatment. We previously reported Spirulina (Arthrospira) platensis effectively prevented non-alcoholic steatohepatitis progression in our model rats. The contribution of phycocyanin, an ingredient of Spirulina (Arthrospira) platensis, was limited. We, therefore, have looked for more active components of Spirulina (Arthrospira) platensis. In this study, we pursued the effect of biopterin glucoside, another bioactive ingredient of Spirulina (Arthrospira) platensis. We found Spirulina (Arthrospira) platensis and biopterin glucoside oral administrations effectively alleviated oxidative stress, inflammation and insulin signal failure, and prevented fibroblast growth factor 21 gene overexpression in non-alcoholic steatohepatitis rat livers. We concluded biopterin glucoside is a major component of Spirulina (Arthrospira) platensis action.

Use of Ultrasound Microscopy for Ex Vivo Analysis of Acoustic Impedance in Mouse Liver with Steatohepatitis

Scanning acoustic microscopy reveals information on histology and acoustic impedance through tissues. The objective of the present study was to investigate whether acoustic impedance values in the liver over time reflect the progression of steatohepatitis through different grades and stages, and whether this approach can visualize histologic features of the disease. Mice were divided into two groups: a control group and a steatohepatitis group prepared by keeping the mice on a methionine and choline-deficient diet for 56 weeks. The hepatic lobe was excised for measurement of impedance and observation of microscopic structure using a commercially available scanning acoustic microscopy system with a central frequency of 320 MHz. Scanning acoustic microscopy revealed that acoustic impedance through liver tissue with steatohepatitis temporarily decreased with the degree of fat deposition and then increased in parallel with the progression of inflammation and fibrosis. However, the acoustic images obtained did not allow discrimination of detailed microstructures from those seen using light microscopy. In conclusion, estimation of acoustic impedance appears to have potential clinical applications, such as for monitoring or follow-up studies.

Hepatocyte-specific expression of human carboxylesterase 2 attenuates nonalcoholic steatohepatitis in mice.

Human carboxylesterase 2 (CES2) has triacylglycerol hydrolase (TGH) activities and plays an important role in lipolysis. In this study, we aim to determine the role of human CES2 in the progression or reversal of steatohepatitis in diet-induced or genetically obese mice. High-fat/high-cholesterol/high-fructose (HFCF) diet-fed C57BL/6 mice or db/db mice were intravenously injected with an adeno-associated virus expressing human CES2 under the control of an albumin promoter. Human CES2 protected against HFCF diet-induced nonalcoholic fatty liver disease (NAFLD) in C57BL/6J mice and reversed steatohepatitis in db/db mice. Human CES2 also improved glucose tolerance and insulin sensitivity. Mechanistically, human CES2 reduced hepatic triglyceride (T) and free fatty acid (FFA) levels by inducing lipolysis and fatty acid oxidation and inhibiting lipogenesis via suppression of sterol regulatory element-binding protein 1. Furthermore, human CES2 overexpression improved mitochondrial respiration and glycolytic function, and inhibited gluconeogenesis, lipid peroxidation, apoptosis, and inflammation. Our data suggest that hepatocyte-specific expression of human CES2 prevents and reverses steatohepatitis. Targeting hepatic CES2 may be an attractive strategy for treatment of NAFLD.NEW & NOTEWORTHY Human CES2 attenuates high-fat/cholesterol/fructose diet-induced steatohepatitis and reverses steatohepatitis in db/db mice. Mechanistically, human CES2 induces lipolysis, fatty acid and glucose oxidation, and inhibits hepatic glucose production, inflammation, lipid oxidation, and apoptosis. Our data suggest that human CES2 may be targeted for treatment of non-alcoholic steatohepatitis (NASH).

Vesicular ATP release from hepatocytes plays a role in the progression of nonalcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is becoming a growing public health problem along with the increase of metabolic syndrome worldwide. Extracellular nucleotides are known to serve as a danger signal by initiating purinergic signaling in many inflammatory disorders, although the role of purinergic signaling in the progression of NASH remains to be clarified. Vesicular nucleotide transporter (VNUT) is a key molecule responsible for vesicular ATP release to initiate purinergic signaling. Here, we studied the role of VNUT in the progression of nonalcoholic steatohepatitis. VNUT was expressed in mouse hepatocytes and associated, at least in part, with apolipoprotein B (apoB)-containing vesicles. High glucose stimulation evoked release of appreciable amount of ATP from hepatocytes, which disappeared in hepatocytes of Vnut knockout (Vnut−/−) mice. Glucose treatment also stimulated triglyceride secretion from hepatocytes, which was inhibited by PPADS and MRS211, antagonists of P2Y receptors, and clodronate, a VNUT inhibitor, and was significantly reduced in Vnut−/− mice. In vivo, postprandial secretion of triglyceride from hepatocytes was observed, while the serum triglyceride level was significantly reduced in Vnut−/− mice. On a high-fat diet, the liver of wild type mice exhibited severe inflammation, fibrosis, and macrophage infiltration, which is similar to NASH in humans, while this NASH pathology was not observed in Vnut−/− mice. These results suggest that VNUT-mediated vesicular ATP release regulates triglyceride secretion and involves in chronic inflammation in hepatocytes. Since blockade of vesicular ATP release protects against progression of steatohepatitis, VNUT may be a pharmacological target for NASH.

Transporter Activity Changes in Nonalcoholic Steatohepatitis: Assessment with Plasma Coproporphyrin I and III.

Expression and functional changes in the organic anion transporting polypeptide (OATP)-multidrug resistance-associated protein (MRP) axis of transporters are well reported in patients with nonalcoholic steatohepatitis (NASH). These changes can impact plasma and tissue disposition of endo- and exogenous compounds. The transporter alterations are often assessed by administration of a xenobiotic or by transporter proteomic analysis from liver biopsies. Using gene expression, proteomics, and endogenous biomarkers, we show that the gene expression and activity of OATP and MRP transporters are associated with disease progression and recovery in humans and in preclinical animal models of NASH. Decreased OATP and increased MRP3/4 gene expression in two cohorts of patients with steatosis and NASH, as well as gene and protein expression in multiple NASH rodent models, have been established. Coproporphyrin I and III (CP I and III) were established as substrates of MRP4. CP I plasma concentration increased significantly in four animal models of NASH, indicating the transporter changes. Up to a 60-fold increase in CP I plasma concentration was observed in the mouse bile duct-ligated model compared with sham controls. In the choline-deficient amino acid-defined high-fat diet (CDAHFD) model, CP I plasma concentrations increased by >3-fold compared with chow diet-fed mice. In contrast, CP III plasma concentrations remain unaltered in the CDAHFD model, although they increased in the other three NASH models. These results suggest that tracking CP I plasma concentrations can provide transporter modulation information at a functional level in NASH animal models and in patients. SIGNIFICANCE STATEMENT: Our analysis demonstrates that multidrug resistance-associated protein 4 (MRP4) transporter gene expression tracks with nonalcoholic steatohepatitis (NASH) progression and intervention in patients. Additionally, we show that coproporphyrin I and III (CP I and III) are substrates of MRP4. CP I plasma and liver concentrations increase in different diet- and surgery-induced rodent NASH models, likely explained by both gene- and protein-level changes in transporters. CP I and III are therefore potential plasma-based biomarkers that can track NASH progression in preclinical models and in humans.

Differential TM4SF5-mediated SIRT1 modulation and metabolic signaling in nonalcoholic steatohepatitis progression.

Nonalcoholic fatty liver disease is a chronic condition involving steatosis, steatohepatitis and fibrosis, and its progression remains unclear. Although the tetraspanin transmembrane 4 L six family member 5 (TM4SF5) is involved in hepatic fibrosis and cancer, its role in nonalcoholic steatohepatitis (NASH) progression is unknown. We investigated the contribution of TM4SF5 to liver pathology using transgenic and KO mice, diet- or drug-treated mice, in vitro primary cells, and in human tissue. TM4SF5-overexpressing mice exhibited nonalcoholic steatosis and NASH in an age-dependent manner. Initially, TM4SF5-positive hepatocytes and liver tissue exhibited lipid accumulation, decreased Sirtuin 1 (SIRT1), increased sterol regulatory-element binding proteins (SREBPs) and inactive STAT3 via suppressor of cytokine signaling (SOCS)1/3 upregulation. In older mice, TM4SF5 promoted inflammatory factor induction, SIRT1 expression and STAT3 activity, but did not change SOCS or SREBP levels, leading to active STAT3-mediated ECM production for NASH progression. A TM4SF5-associated increase in chemokines promoted SIRT1 expression and progression to NASH with fibrosis. Suppression of the chemokine CCL20 reduced immune cell infiltration and ECM production. Liver tissue from high-fat diet- or CCl4 -treated mice and human patients exhibited TM4SF5-dependent steatotic or steatohepatitic livers with links between TM4SF5-mediated SIRT1 modulation and SREBP or SOCS/STAT3 signaling axes. TM4SF5-mediated STAT3 activation in fibrotic NASH livers increased collagen I and laminin γ2. Both collagen I α1 and laminin γ2 suppression resulted in reduced SIRT1 and active STAT3, but no change in SREBP1 or SOCS, and abolished CCl4 -mediated mouse liver damage. TM4SF5-mediated signaling pathways that involve SIRT1, SREBPs and SOCS/STAT3 promoted progression to NASH. Therefore, TM4SF5 and its downstream effectors may be promising therapeutic targets to treat nonalcoholic fatty liver disease. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Role of polar extracts from two quinoa varieties in prevention of steatohepatitis and cardiovascular diseases and improving glucose tolerance in rats.

Introduction: Steatohepatitis, which is the deposition of fat in the liver with inflammation and starting of necrosis, can induce cardiovascular diseases (CVDs). The aim of this research was to study the preventive effects of steatohepatitis and CVD by ethanol extract of two quinoa varieties (quinoa 1 and hualhuas) in rats. Methods: Phenolic and flavonoid compounds were determined in the extracts utilizing colorimetric and high-performance liquid chromatography techniques. The 2,2-diphenyl-1-picryl-hydrazil (DPPH) scavenging activity was assessed for the extracts. Rats were divided into four groups, the first group was fed on a balanced diet (negative control), and other groups consumed a high fructose-fat diet (HFFD) to induce steatohepatitis and CVD. The second group served as a positive control; however, the third and fourth groups were treated by ethanol extract of quinoa 1 and hualhuas, respectively. Different biochemical changes, as well as liver and heart histopathology, were followed. Results: Results showed significant elevation in liver lipids, plasma malondialdehyde, total cholesterol (T-C), triglycerides and low-density lipoprotein cholesterol with reduction of high-density lipoprotein cholesterol (HDL-C) and total antioxidant as well as a significant increase in T-C/HDL-C in control positive group (P < 0.05) compared to control negative group. Plasma parameters and liver lipids were improved by the extracts; hualhuas was superior concerning the effect on lipid while quinoa 1 was more efficient in reducing oxidative stress. The oral glucose tolerance curve and the histopathology of the liver and heart tissues were improved by both extracts. Total phenolic and DPPH scavenging activity were higher in quinoa 1 than hualhuas. Protocatechuic and rutin were the major identified phenolic acid and flavonoid compounds, respectively in the extracts. Conclusion: Quinoa extracts are able to prevent the progression of steatohepatitis and CVD, and might be beneficial in patients with such diseases.

S1148 Effect of Cannabis Use on Progression of Non-Alcoholic Fatty Liver Disease in Obese Patients: A Propensity-Matched Retrospective Cohort Study

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) describes a condition of pathologic fat accumulation in hepatocytes when a secondary cause cannot be identified (e.g. excessive alcohol use). The spectrum of disease seen in NAFLD includes Non-Alcoholic Fatty Liver (NAFL), and the more pathologic variant, Non-Alcoholic Steatohepatitis (NASH). While NAFL is more common, the hepatocellular injury and inflammation that occurs in NASH contributes significantly to the increased risk of liver failure, cirrhosis, and hepatocellular carcinoma in NAFLD patients. Anti-inflammatory effects of cannabis are well described in experimental literature. Tetrahydrocannabinol (THC) specifically, has been shown to have anti-inflammatory and hepatoprotective activity in myofibroblast and stellate cells. How the hepatoprotective properties of cannabis affect disease manifestation and incidence of NAFLD is the subject of this investigation. The aim of this study is to determine how cannabis use affects the prevalence and progression of NAFLD in obese human subjects. METHODS: We studied the 2016 Healthcare Cost and Utilization Project’s (HCUP) National Inpatient Sample (NIS) discharge records, and gathered data on patients who were obese and at least 18 years of age (N = 879952). The main study outcome was prevalence of the four presentations of NAFLD: Steatosis, Steatohepatitis, Cirrhosis and Hepatocellular Carcinoma. We then compared the prevalence of disease stage between cannabis and non-cannabis users. Lastly, we further matched the non-cannabis group with demographic factors and other patient related confounders. All analysis was done using STATA 14 software. RESULTS: A total of 879,952 obese patients were admitted within the study period. Cannabis users 14,236 (1.6%) had less steatohepatitis (0.4% vs 0.7%, P < 0.001) and cirrhosis (1.1% vs 1.5%, P < 0.001) than non-users. After propensity matched analysis, cannabis use remained significantly associated with less steatohepatitis (0.4% vs 0.5%, P = 0.035). Post-match, there was no statistically significant difference in the prevalence of NAFL, cirrhosis and hepatocellular carcinoma. CONCLUSION: Results from this study suggest that cannabis use is associated with reduced prevalence and progression of steatohepatitis in obese patients. These findings could possibly be explained by the anti-inflammatory and hepatoprotective effect of cannabis on hepatocytes through the endocannabinoid system. Additional studies are needed to further explore this relationship.Table 1a.: Descriptive Statistics of Obese Population: Cannabis and Non-Cannabis Users and Propensity Score MatchTable 1b.: Outcome Statistics of Obese Population: Cannabis and Non-Cannabis Users and Propensity Score Match

Nonalcoholic fatty liver disease and alcohol-related liver disease: From clinical aspects to pathophysiological insights

Two major causes of steatohepatitis are alcohol and metabolic syndrome. Although the underlying causes of alcohol-related liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) differ, there are certain similarities in terms of the mode of disease progression and underlying pathophysiological mechanisms. Further, excessive alcohol consumption is often seen in patients with metabolic syndrome, and alcoholic hepatitis exacerbation by comorbidity with metabolic syndrome is an emerging clinical problem. There are certain ethnic differences in the development of both NAFLD and ALD. Especially, Asian populations tend to be more susceptible to NAFLD, and genetic polymorphisms in patatin-like phospholipase domain-containing 3 (PNPLA3) play a key role in both NAFLD and ALD. From the viewpoint of pathophysiology, cellular stress responses, including autophagy and endoplasmic reticulum (ER) stress, are involved in the development of cellular injury in steatohepatitis. Further, gut-derived bacterial products and innate immune responses in the liver most likely play a profound role in the pathogenesis of both ALD and NASH. Though the recent progress in the treatment of viral hepatitis has reduced the prevalence of viral-related development of hepatocellular carcinoma (HCC), non-viral HCC is increasing. Alcohol and metabolic syndrome synergistically exacerbate progression of steatohepatitis, resulting in carcinogenesis. The gut-liver axis is a potential therapeutic and prophylactic target for steatohepatitis and subsequent carcinogenesis.

High-Risk Groups for Non-alcoholic Fatty Liver and Non-alcoholic Steatohepatitis Development and Progression

This paper reviews the pathophysiology of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) development and the clinical and genetic risk factors associated with its progression. Lipotoxicity underlies the development and progression of NAFLD/NASH. Obesity, insulin resistance, metabolic syndrome, ethnicity, and genetics are associated with the severity and progression of NAFLD/NASH. Personalized approaches to NAFLD/NASH management should take these “high-risk” categories of NAFLD/NASH progression into account in order to reduce NAFLD/NASH-associated morbidity and mortality.

A Lipid-Based Nanocarrier Containing Active Vitamin D3 Ameliorates NASH in Mice via Direct and Intestine-Mediated Effects on Liver Inflammation.

The gut-liver axis may be involved in non-alcoholic steatohepatitis (NASH) progression. Pathogen-associated molecular patterns leak through the intestinal barrier to the liver via the portal vein to contribute to NASH development. Active vitamin D3 (1,25(OH)2D3) is a potential therapeutic agent to enhance the intestinal barrier. Active vitamin D3 also suppresses inflammation and fibrosis in the liver. However, the adverse effects of active vitamin D3 such as hypercalcemia limit its clinical use. We created a nano-structured lipid carrier (NLC) containing active vitamin D3 to deliver active vitamin D3 to the intestine and liver to elicit NASH treatment. We found a suppressive effect of the NLC on the lipopolysaccharide-induced increase in permeability of an epithelial layer in vitro. Using mice in which NASH was induced by a methionine and choline-deficient diet, we discovered that oral application of the NLC ameliorated the permeability increase in the intestinal barrier and attenuated steatosis, inflammation and fibrosis in liver at a safe dose of active vitamin D3 at which the free form of active vitamin D3 did not show a therapeutic effect. These data suggest that the NLC is a novel therapeutic agent for NASH.

AS013 - MBOAT7, HSD17B13 and metabolic risk factors in the progression of PNPLA3-associated steatohepatitis

AS013 - MBOAT7, HSD17B13 and metabolic risk factors in the progression of PNPLA3-associated steatohepatitis

Emerging insight into the role of cellular cholesterol in the pathogenesis of nonalcoholic steatohepatitis progression.

Emerging insight into the role of cellular cholesterol in the pathogenesis of nonalcoholic steatohepatitis progression.

Simultaneous Inhibition of Peripheral CB1R and iNOS Mitigates Obesity-Related Dyslipidemia Through Distinct Mechanisms.

Diabetic dyslipidemia, characterized by increased plasma triglycerides and decreased HDL cholesterol levels, is a major factor contributing to nonalcoholic steatohepatitis and cardiovascular risk in type 2 diabetes. Activation of the cannabinoid-1 receptor (CB1R) and activation of inducible nitric oxide synthase (iNOS) are associated with nonalcoholic steatohepatitis progression. Here, we tested whether dual-targeting inhibition of hepatic CB1R and iNOS improves diabetic dyslipidemia in mice with diet-induced obesity (DIO mice). DIO mice were treated for 14 days with (S)-MRI-1867, a peripherally restricted hybrid inhibitor of CB1R and iNOS. (R)-MRI-1867, the CB1R-inactive stereoisomer that retains iNOS inhibitory activity, and JD-5037, a peripherally restricted CB1R antagonist, were used to assess the relative contribution of the two targets to the effects of (S)-MRI-1867. (S)-MRI-1867 reduced hepatic steatosis and the rate of hepatic VLDL secretion, upregulated hepatic LDLR expression, and reduced the circulating levels of proprotein convertase subtilisin/kexin type 9 (PCSK9). The decrease in VLDL secretion could be attributed to CB1R blockade, while the reduction of PCSK9 levels and the related increase in LDLR resulted from iNOS inhibition via an mTOR complex 1-dependent mechanism. In conclusion, this approach based on the concomitant inhibition of CB1R and iNOS represents a promising therapeutic strategy for the treatment of dyslipidemia.

The role of AMPKα2 in the HFD-induced nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic steatosis, inflammation and liver fibrosis and has become one of the leading causes of hepatocellular carcinoma and liver failure. However, the underlying molecular mechanism of hepatic steatosis and the progression to nonalcoholic steatohepatitis (NASH) are not fully understood. Herein, we discovered that AMPKα2 catalytic subunit showed reduced expression in the liver following high fat diet (HFD) feeding to mice. Importantly, knockout of AMPKα2 in mice aggravated NAFLD, hepatic steatosis, inflammation and fibrosis. On the other hand, hepatocyte-targeted overexpression of AMPKα2 prevented or reversed NAFLD indications. In vivo mechanistic studies revealed that increased phosphorylation of IKKα/β and NF-κB in HFD-fed AMPKα2−/− mice compared to WT mice, and treatment of these mouse cohorts with an inhibitor of NF-κB signaling for 4 weeks, effectively attenuated the progression of steatohepatitis and metabolic disorder features. In summary, AMPKα2 provides a protective role in the process of hepatic steatosis to NASH progression through suppression of liver NF-κB signaling.

1833-P: A Novel Genetic Modified and Pegylated FGF21 Analog for the Treatment of Nonalcoholic Steatohepatitis in Nonhuman Primates

Nonalcoholic steatohepatitis (NASH) is becoming a major cause of liver diseases with no effective drugs in the clinic. Fibroblast growth factor 21 (FGF-21) has emerged as a potent metabolic regulator with therapeutic prospect to lipids and NASH in animal models and preclinical trials. FGF-21 and the agents mimicking its activity may be developed into the pharmacotherapeutic targets for NASH treatment. Here, we show that B1344, a site-specific PEGylated human FGF-21 analog, shifts physiology of NASH mice or cynomolgus monkeys towards that of the control animals fed with standard diet, and significantly improves their NASH phenotypes. To determine the role of B1344 in the progression of murine NAFLD independent of obesity, methionine and choline deficiency (MCD) diet-fed mouse model of NASH were generated. Both short- and long-term administration of B1344 with the doses of 0.125 mg/kg and 0.2 mg/kg in mice protect against lipotoxicity damage and liver fibrosis. Expression levels of proinflammatory and fibrotic genes are decreased in livers of mice treated with B1344. Moreover, pharmacologic efficacies of B1344 on NASH were evaluated in NAFLD cynomolgus monkeys fed with high-fat diet. Strikingly, administration of B1344 causes a potent reduction of hepatic steatosis and fibrosis as measured by magnetic resonance imaging proton density fat fraction (MRI-PDFF) and Sirius Red staining, and protects the monkeys from the progression of steatohepatitis. In addition, administration of B1344 results in a reduction of body weight and adiposity, and improvement of lipid profile and glycemic control. The biological properties of the novel FGF-21 analog B1344 is identical to human FGF-21. Together, these demonstrate impressive effects of B1344 on NASH and metabolic parameters, and may represent novel therapeutics for treating these diseases in humans. Disclosure A. Cui: None. J. Li: None. S. Ji: None. F. Ma: None. G. Wang: None. Y. Xue: None. Z. Liu: None. X. Ma: None. Y. Li: None. Funding Tasly Pharmaceutical Co. Ltd.; National Key Research and Development Program of China (2019YFA0802502); National Natural Science Foundation of China (81925008); Chinese Academy of Sciences (XDA12030210); Shanghai Science and Technology Commission (19140903300); National Science and Technology Major Projects (2019ZX09201001-001-001)