High-fat Diet-induced Non-alcoholic Steatohepatitis Research Articles

Role of 11β-hydroxysteroid dehydrogenase type 1 inhibition in the antiobesity effect of J2H-1702 on adipocytes and a high-fat diet-induced NASH model.

Obesity due to excessive body fat accumulation remains a global problem. Patients with obesity have high cortisol levels, and its dysregulation is caused by increased 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) levels. The effects and mechanism of J2H-1702, an 11β-HSD1 inhibitor, on nonalcoholic steatohepatitis (NASH) were explored. This study compared the antiadipogenic effects of J2H-1702, elafibranor (PPARα/δ agonist), and BVT14225 (selective 11β-HSD1 inhibitor) using mouse 3T3-L1 pre-adipocytes. J2H-1702, elafibranor, and BVT14225 inhibited adipocyte differentiation and intracellular lipid accumulation in 3T3-L1 cells by downregulating phospho-extracellular signal-regulated kinase, extracellular signal-regulated kinase, phospho-c-Jun-N-terminal Kinase, c-Jun-N-terminal Kinase, phospho-P38 (P-P38), P38, CCAAT/enhancer-binding proteins alpha and β, peroxisome proliferator-activated receptor γ, and glucocorticoid receptor. Additionally, J2H-1702, elafibranor, and BVT14225 treatments effectively inhibited 11β-HSD1 activity, as revealed by cortisol concentrations, and inhibited cortisone-induced adipocyte differentiation and intracellular lipid accumulation in 3T3-L1 cells. These effects were associated with 11β-HSD1 protein inhibition. Furthermore, J2H-1702 and BVT14225 increased the expression of Akt and phosphoinositide 3-kinase involved in insulin resistance in 3T3L-1 adipocytes. In the LX-2 human hepatic stellate cell line, the relative expression of N-cadherin, 11β-HSD1, collagen1α (COLA1), α-actin of smooth muscle (α-SMA) genes in LX-2 activated with TGF-β increased significantly, and after treatment with J2H-1702, it was significantly reduced. The expression of E-cadherin is decreased in TGF-β-treated LX-2 cells and increased after treatment with J2H-1702. We tested the potential of J2H-1702 as a therapeutic agent for NASH using a high-fat diet-induced NASH model, with obeticholic acid, an FXR agonist, and elafibranor as reference drugs. All drugs significantly decreased the elevated triglyceride levels in the livers of high-fat, high-carbohydrate (HFHC-fed mice. The results may add to the benefits of targeting 11β-HSD1 inhibitors with antiadipogenic activity in developing a therapeutic agent for obesity treatment.

OM-85 attenuates high-fat diet-induced obesity, insulin resistance, gut dysbiosis and nonalcoholic steatohepatitis in a murine model

BackgroundObesity is a global epidemic that is tied to a wide range of human disorders. Chronic consumption of a high-fat diet is linked to disruption of the intestinal microbiome, which drives obesity-related pathophysiology. Broncho-Vaxom® (OM-85), a bacterial lysate used for prophylaxis of recurrent respiratory tract infections, has both immunostimulatory and immunomodulatory functions. MethodsMale C57Bl/6 mice were maintained on normal control vs. high-fat diets for 8 weeks and treated or untreated with OM-85 or with the probiotic Lactobacillus plantarum, as a positive control. Mice were evaluated for weight gain, glucose tolerance, insulin tolerance, gut microbiome composition and non-alcoholic steatohepatitis (NASH). ResultsHigh-fat diet mice developed obesity, insulin resistance, NASH and gut dysbiosis with a shift from the Bacteroidetes phylum, such as Bacteroidales order and Muribaculaceae family organisms to Firmicutes groups, such as the Clostridium and Blautia genuses. Treatment with OM-85 led to 1) prevention of obesity, 2) prevention of insulin resistance, 3) attenuation of NASH and 4) attenuation of gut dysbiosis, with decreased levels of the organisms mentioned above and increases in Verrucomicrobiae phylum organisms such as Akkermansia family microbes as well as Muribaculaceae organisms. These shifts in the gut microbiome predict favorable effects on the short chain fatty acid profile in the gut and increased integrity of the intestinal barrier. Pathway analysis showed that OM-85 decreases rates of carbohydrate metabolism, providing an additional mechanism whereby OM-85 prevents obesity. ConclusionImmune modulators such as OM-85 should be investigated for their potential therapeutic effects on metabolism.

Ileal microbial microbiome and its secondary bile acids modulate susceptibility to nonalcoholic steatohepatitis in dairy goats

BackgroundLiver damage from nonalcoholic steatohepatitis (NASH) presents a significant challenge to the health and productivity of ruminants. However, the regulatory mechanisms behind variations in NASH susceptibility remain unclear. The gut‒liver axis, particularly the enterohepatic circulation of bile acids (BAs), plays a crucial role in regulating the liver diseases. Since the ileum is the primary site for BAs reabsorption and return to the liver, we analysed the ileal metagenome and metabolome, liver and serum metabolome, and liver single-nuclei transcriptome of NASH-resistant and susceptible goats together with a mice validation model to explore how ileal microbial BAs metabolism affects liver metabolism and immunity, uncovering the key mechanisms behind varied NASH pathogenesis in dairy goats.ResultsIn NASH goats, increased total cholesterol (TC), triglyceride (TG), and primary BAs and decreased secondary BAs in the liver and serum promoted hepatic fat accumulation. Increased ileal Escherichia coli, Erysipelotrichaceae bacterium and Streptococcus pneumoniae as well as proinflammatory compounds damaged ileal histological morphology, and increased ileal permeability contributes to liver inflammation. In NASH-tolerance (NASH-T) goats, increased ursodeoxycholic acid (UDCA), isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) in the liver, serum and ileal contents were attributed to ileal secondary BAs-producing bacteria (Clostridium, Bifidobacterium and Lactobacillus) and key microbial genes encoding enzymes. Meanwhile, decreased T-helper 17 (TH17) cells and increased regulatory T (Treg) cells proportion were identified in both liver and ileum of NASH-T goats. To further validate whether these key BAs affected the progression of NASH by regulating the proliferation of TH17 and Treg cells, the oral administration of bacterial UDCA, isoDCA and isoLCA to a high-fat diet-induced NASH mouse model confirmed the amelioration of NASH through the TH17 cell differentiation/IL-17 signalling/PPAR signalling pathway by these bacterial secondary BAs.ConclusionThis study revealed the roles of ileal microbiome and its secondary BAs in resilience and susceptibility to NASH by affecting the hepatic Treg and TH17 cells proportion in dairy goats. Bacterial UDCA, isoDCA and isoLCA were demonstrated to alleviate NASH and could be novel postbiotics to modulate and improve the liver health in ruminants.A8SM95maBgMZc1-WqV_tnDVideo

Homotherapy for heteropathy: therapeutic effect of Butein in NLRP3-driven diseases

BackgroundAberrant inflammatory responses drive the initiation and progression of various diseases, and hyperactivation of NLRP3 inflammasome is a key pathogenetic mechanism. Pharmacological inhibitors of NLRP3 represent a potential therapy for treating these diseases but are not yet clinically available. The natural product butein has excellent anti-inflammatory activity, but its potential mechanisms remain to be investigated. In this study, we aimed to evaluate the ability of butein to block NLRP3 inflammasome activation and the ameliorative effects of butein on NLRP3-driven diseases.MethodsLipopolysaccharide (LPS)-primed bone-marrow-derived macrophages were pretreated with butein and various inflammasome stimuli. Intracellular potassium levels, ASC oligomerization and reactive oxygen species production were also detected to evaluate the regulatory mechanisms of butein. Moreover, mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis were used to test whether butein has protective effects on these NLRP3-driven diseases.ResultsButein blocks NLRP3 inflammasome activation in mouse macrophages by inhibiting ASC oligomerization, suppressing reactive oxygen species production, and upregulating the expression of the antioxidant pathway nuclear factor erythroid 2-related factor 2 (Nrf2). Importantly, in vivo experiments demonstrated that butein administration has a significant protective effect on the mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis.ConclusionOur study illustrates the connotation of homotherapy for heteropathy, i.e., the application of butein to broaden therapeutic approaches and treat multiple inflammatory diseases driven by NLRP3.

Pseudotargeted lipidomics analysis of scoparone on glycerophospholipid metabolism in non-alcoholic steatohepatitis mice by LC-MRM-MS.

As the inflammatory subtype of nonalcoholic fatty liver disease (NAFLD), the progression of nonalcoholic steatohepatitis (NASH) is associated with disorders of glycerophospholipid metabolism. Scoparone is the major bioactive component in Artemisia capillaris which has been widely used to treat NASH in traditional Chinese medicine. However, the underlying mechanisms of scoparone against NASH are not yet fully understood, which hinders the development of effective therapeutic agents for NASH. Given the crucial role of glycerophospholipid metabolism in NASH progression, this study aimed to characterize the differential expression of glycerophospholipids that is responsible for scoparone's pharmacological effects and assess its efficacy against NASH. Liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) was performed to get the concentrations of glycerophospholipids, clarify mechanisms of disease, and highlight insights into drug discovery. Additionally, pathologic findings also presented consistent changes in high-fat diet-induced NASH model, and after scoparone treatment, both the levels of glycerophospholipids and histopathology were similar to normal levels, indicating a beneficial effect during the observation time. Altogether, these results refined the insights on the mechanisms of scoparone against NASH and suggested a route to relieve NASH with glycerophospholipid metabolism. In addition, the current work demonstrated that a pseudotargeted lipidomic platform provided a novel insight into the potential mechanism of scoparone action.

A study of the anti-fibrotic effect of ghrelin on mice with nonalcoholic steatohepatitis

Objective: To study the anti-fibrotic effect of ghrelin on high-fat diet-induced non-alcoholic steatohepatitis (NASH) in mice. Methods: 24 male C57BL/6 mice were randomly divided into a normal diet group, a normal diet + ghrelin group, a high-fat diet group, and the high-fat diet + ghrelin group. The HFD and HFD+ghrelin groups were fed high-fat diet for 16 weeks to induce non-alcoholic steatohepatitis. Among them, the NCD+ghrelin group and HFD+ghrelin group were continuously given ghrelin intervention (11nmol·kg(-1)·d(-1)) for 2 weeks after feeding for 14 weeks. 16 mice were euthanized on weekends. The plasma levels of alanine aminotransferase (ALT) and hyaluronic acid (HA) were measured in mice. The content of hydroxyproline (Hyp) was determined in liver tissue. RT-qPCR was used to detect the mRNA expression levels of transforming growth factor β1 (TGF-β1) and collagen types I, III, and IV in liver tissue. A Western blot was used to detect the expression level of the α-smooth muscle actin (α-SMA) protein in liver tissue. HE staining was used to observe the morphological changes in liver tissue. VG staining was used to observe the fibrotic condition in liver tissue. Results: Compared with the NCD group, plasma ALT (266.80±146.80)U/L, HA (219.00±39.47) ng/ml levels, Hyp content (0.35±0.05)μg/mg prot (P < 0.05), mRNA expression levels of transforming growth factor β1 (TGF-β1) and collagen types I, III, IV (P < 0.05), and the expression level of α-SMA protein in the HFD group were significantly increased (P < 0.05), with congestion in the hepatic central lobular veins, hepatocytes swelling, and deposition of a large amount of collagen fibers in liver tissue. Compared with the HFD group, plasma ALT (57.17±20.88)U/L, HA (75.68±8.40)μg/mg levels, Hyp content (0.19±0.07)μg/mg prot, mRNA expression levels of transforming growth factor β1 (TGF-β1) and collagen types I, III, IV (P < 0.05), and the expression level of α-SMA protein in the HFD+ghrelin mice group was significantly reduced (P < 0.05), with only mild sinusoidal congestion in the liver tissue but significant improvement and reduction in liver injury and collagen fiber deposition. Conclusion: Ghrelin has a significant improvement effect on liver fibrosis in NASH mice.

Rotundic acid improves nonalcoholic steatohepatitis in mice by regulating glycolysis and the TLR4/AP1 signaling pathway

BackgroundSteatosis and inflammation are the hallmarks of nonalcoholic steatohepatitis (NASH). Rotundic acid (RA) is among the key triterpenes of Ilicis Rotundae Cortex and has exhibited multipronged effects in terms of lowering the lipid content and alleviating inflammation. The study objective is to systematically evaluate the potential mechanisms through which RA affects the development and progression of NASH.MethodsTranscriptomic and proteomic analyses of primary hepatocytes isolated from the control, high-fat diet-induced NASH, and RA treatment groups were performed through Gene Ontology analysis and pathway enrichment. Hub genes were identified through network analysis. Integrative analysis revealed key RA-regulated pathways, which were verified by gene and protein expression studies and cell assays.ResultsHub genes were identified and enriched in the Toll-like receptor 4 (TLR4)/activator protein-1 (AP1) signaling pathway and glycolysis pathway. RA reversed glycolysis and attenuated the TLR4/AP1 pathway, thereby reducing lipid accumulation and inflammation. Additionally, lactate release in L-02 cells increased with NaAsO2-treated and significantly decreased with RA treatment, thus revealing that RA had a major impact on glycolysis.ConclusionsRA is effective in lowering the lipid content and reducing inflammation in mice with NASH by ameliorating glycolysis and TLR4/AP1 pathways, which contributes to the existing knowledge and potentially sheds light on the development of therapeutic interventions for patients with NASH.

GPAT1 Deficiency in Mice Modulates NASH Progression in a Model-Dependent Manner

Nonalcoholic fatty liver disease (NAFLD), and its more severe form, nonalcoholic steatohepatitis (NASH), is the leading cause for liver failure and liver cancer. Although the etiology is likely multifactorial, genes involved in regulating lipid metabolism are enriched in human NAFLD genome-wide association studies (GWAS), pointing to dysregulated lipid metabolism as a major pathogenic factor. Glycerol-3-phosphate acyltransferase 1 (GPAT1), encoded by GPAM, converts acyl-CoAs and glycerol-3-phosphate into lysophosphatidic acid and has been shown to regulate lipid accumulation in the liver. However, its role in mediating the progression from NAFLD to NASH has not been explored. GPAT1-deficient mice were generated and challenged with diets inducing hepatic steatosis and NASH. Effects of GPAT1 deficiency on lipid and systemic metabolic end points were evaluated. Ablating GPAT1 globally or specifically in mouse hepatocytes reduced hepatic steatosis in the context of diet-induced or genetic obesity. Interestingly, blunting of progression from NAFLD to NASH in global GPAT1 knockout (KO) mice was model dependent. GPAT1 KO mice were protected from choline deficient, amino acid defined high-fat diet-induced NASH development, but not from the high fat, high carbohydrate, and high cholesterol diet-induced NASH. Our preclinical data support the notion that lipid metabolism pathways regulated by GPAT1 in hepatocytes play an essential role in NASH progression, albeit in a model-dependent manner.

Curcumin Alleviates High-fat Diet-induced Nonalcoholic Steatohepatitis via Improving Hepatic Endothelial Function with Microbial Biotransformation in Rats.

Hepatic endothelial function is central to the development of nonalcoholic steatohepatitis (NASH). Curcumin (Cur) is reportedly hepatoprotective, however, it remains unknown whether Cur improves hepatic endothelial function in NASH. Additionally, the poor bioavailability of Cur renders it difficult to elucidate its hepatoprotective effect, hence, its biotransformation should be considered. Herein, we investigated the effects and mechanisms of Cur and its bioconversion on hepatic endothelial function against high-fat diet-induced NASH in rats. The results revealed that Cur improved hepatic lipid accumulation, inflammation, and endothelial dysfunction by inhibiting NF-κB and PI3K/Akt/HIF-1α pathways, however, these effects were weakened via antibiotic addition, which was closely related to reduced tetrahydrocurcumin (THC) produce in the liver and intestinal content. Moreover, THC exerted a better effect than Cur on restoring liver sinusoidal endothelial cells function to attenuate steatosis and injury in L02 cells. Thus, these findings indicate that the effect of Cur on NASH is closely related to hepatic endothelial function improvement with intestinal microbial biotransformation.

Akkermansia muciniphila inhibits nonalcoholic steatohepatitis by orchestrating TLR2-activated γδT17 cell and macrophage polarization

ABSTRACT Current evidence indicates that the next-generation probiotic Akkermansia muciniphila (A. muciniphila) has therapeutic potential for nonalcoholic fatty liver disease (NAFLD), especially its inflammatory stage known as nonalcoholic steatohepatitis (NASH). However, the mechanisms of A. muciniphila in NASH prevention remain unknown. Here, A. muciniphila supplementation prevented hepatic inflammation in high-fat diet-induced NASH mice, characterized by reduced hepatic proinflammatory macrophages (M1) and γδT and γδT17 cells. Consistently, hepatic M1 and γδT cells were enriched in biopsy-proven NASH patients and high-fat/high-carbohydrate diet-induced NASH mice. Antibiotics reduced hepatic M1, γδT and γδT17 cells in NASH mice. Furthermore, A. muciniphila inhibited intestinal barrier disruption and accordingly downregulated hepatic Toll-like receptor 2 (TLR2) expression in NASH mice. The activation of TLR2 by lipoteichoic acid enriched hepatic γδT17 cells (not M1) in normal diet-fed mice and neutralized the γδT cell-lowering and liver inflammation-protecting effects of A. muciniphila in NASH mice. Additionally, activated γδT cells could promote macrophage polarization via IL−17. Our study first supported that A. muciniphila prevented NASH by modulating TLR2-activated γδT17 cells and further macrophage polarization, facilitating clinical therapeutic applications.

Dysregulation of murine long noncoding single-cell transcriptome in nonalcoholic steatohepatitis and liver fibrosis.

LncRNAs comprise a heterogeneous class of RNA-encoding genes typified by low expression, nuclear enrichment, high tissue-specificity, and functional diversity, but the vast majority remain uncharacterized. Here, we assembled the mouse liver noncoding transcriptome from >2000 bulk RNA-seq samples and discovered 48,261 liver-expressed lncRNAs, a majority novel. Using these lncRNAs as a single-cell transcriptomic reference set, we elucidated lncRNA dysregulation in mouse models of high fat diet-induced nonalcoholic steatohepatitis and carbon tetrachloride-induced liver fibrosis. Trajectory inference analysis revealed lncRNA zonation patterns across the liver lobule in each major liver cell population. Perturbations in lncRNA expression and zonation were common in several disease-associated liver cell types, including nonalcoholic steatohepatitis-associated macrophages, a hallmark of fatty liver disease progression, and collagen-producing myofibroblasts, a central feature of liver fibrosis. Single-cell-based gene regulatory network analysis using bigSCale2 linked individual lncRNAs to specific biological pathways, and network-essential regulatory lncRNAs with disease-associated functions were identified by their high network centrality metrics. For a subset of these lncRNAs, promoter sequences of the network-defined lncRNA target genes were significantly enriched for lncRNA triplex formation, providing independent mechanistic support for the lncRNA-target gene linkages predicted by the gene regulatory networks. These findings elucidate liver lncRNA cell-type specificities, spatial zonation patterns, associated regulatory networks, and temporal patterns of dysregulation during hepatic disease progression. A subset of the liver disease-associated regulatory lncRNAs identified have human orthologs and are promising candidates for biomarkers and therapeutic targets.

Effect of High-intensity Intermittent Swimming Training on peroxisome proliferator-activated receptors-αand Liver Enzymes in Non-alcoholic Steatohepatitis Male Rats

Introduction: Accumulation of fat in the liver tissue is known as the most important cause of non-alcoholic steatohepatitis, which is associated with a decrease in the protein of hepatic peroxisome proliferator-activated receptors (PPAR-α). This study aimed to investigate the effect of eight weeks of high-intensity interval training (HIST) on PPAR-α and liver enzymes in high-fat diet-induced non-alcoholic steatohepatitis rats.&#x0D; Methods: The current research was semi-experimental. 40 Spraguedauli male rats (age: 6 to 8 weeks and weight: 230 ± 20 grams) were selected and divided into two equal groups, healthy and high-fat diet (HFD). The HFD group was subjected to gavage for eight weeks with the aim of inducing the disease. Rats were divided into four groups: control-healthy (n=9), healthy-swimming (n=9), control- disease (n=9) and disease -swimming (n=9). The training groups performed HIST for eight weeks (three sessions per week, each session lasting 30 minutes). In the end, liver PPAR-α was measured by western blot analysis; and ALT, AST and GGT enzymes in serum level were measured by photometric method. One-way analysis of variance and Bonferroni's post hoc test were used to analyze the data. Significant level (P≥0.05) and all statistical methods were performed using SPSS16 software.&#x0D; Results: HIST controlled the weight gain caused by HFD and there was a significant decrease in serum ALT and AST in the healthy-swimming and sick-swimming groups compared to the control-patient group and a significant decrease in serum GGT in the healthy-swimming group compared to the control-patient (P≤0.001) and patient-swimming (P≤0.044) were observed. A significant increase in hepatic PPAR-α was observed in healthy-swimming patients compared to the control-patients (P≤0.002).&#x0D; Conclusion: HIST can control body weight and reduce liver enzymes. However, more research is needed to obtain definitive results.

Flavonoids from Scutellaria amoena C. H. Wright alleviate mitochondrial dysfunction and regulate oxidative stress via Keap1/Nrf2/HO-1 axis in rats with high-fat diet-induced nonalcoholic steatohepatitis

BackgroundNonalcoholic steatohepatitis (NASH) is among the most common liver diseases in the world. Flavonoids from Scutellaria amoena (SAF) are used in the treatment of hepatopathy in China. However, the effect and mechanism against NASH remain unclear. We investigated the alleviating effect of SAF on NASH via regulating mitochondrial dysfunction and oxidative stress. MethodsThe effects of SAF on NASH were evaluated using in vitro and in vivo methods. L02 cells were induced by fat emulsion to establish an adipocytes model, followed by treatment with SAF for 24 h. NASH rat models were established by the administration of a high-fat diet for 12 weeks and were administered SAF for six weeks. Changes in body weight, organ indexes, lipid levels, inflammatory cytokines, mitochondrial indicators, and fatty acid metabolism were investigated. ResultsSAF significantly improved body weight, organ indexes, lipid levels, liver injury, and inflammatory infiltration in NASH rats. SAF notably regulated interleukin-6, tumor necrotic factor-alpha, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), kelch-like ECH-associated protein 1 (Keap1), nuclear factor-erythroid factor 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1). Additionally, SAF improved mitochondrial dysfunction, increased the levels of GSH, SOD, ATP synthase, complex I and II, and decreased the level of MDA in liver mitochondria. SAF regulated the expression of β-oxidation genes, including peroxisome proliferator-activated receptor -gamma coactivator-1alpha (PGC-1α), carnitine palmitoyltransferase-1 (CPT1) A, CPT1B, medium-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, very long-chain acyl-CoA dehydrogenase, and PPARα. ConclusionSAF can alleviate NASH by regulating mitochondrial function and oxidative stress via the Keap1/Nrf2/HO-1 axis.

Loss of GPR40 in LDL receptor-deficient mice exacerbates high-fat diet-induced hyperlipidemia and nonalcoholic steatohepatitis.

GPR40, a G protein-coupled receptor for free fatty acids (FFAs), is considered as a therapeutic target for type 2 diabetes mellitus (T2DM) since GPR40 activation in pancreatic beta cells enhances glucose-stimulated insulin secretion. Nonalcoholic fatty liver disease (NAFLD) is a common complication of T2DM or metabolic syndrome (MetS). However, the role of GPR40 in NAFLD associated with T2DM or MetS has not been well established. Given that it is known that cholesterol and FFAs are critically involved in the pathogenesis of nonalcoholic steatohepatitis (NASH) and LDL receptor (LDLR)-deficient mice are a good animal model for human hyperlipidemia including high cholesterol and FFAs, we generated GPR40 and LDLR double knockout (KO) mice in this study to determine the effect of GPR40 KO on hyperlipidemia-promoted NASH. We showed that GPR40 KO increased plasma levels of cholesterol and FFAs in high-fat diet (HFD)-fed LDLR-deficient mice. We also showed that GPR40 KO exacerbated HFD-induced hepatic steatosis, inflammation and fibrosis. Further study demonstrated that GPR40 KO led to upregulation of hepatic CD36 and genes involved in lipogenesis, fatty acid oxidation, fibrosis and inflammation. Finally, our in vitro mechanistic studies showed that while CD36 was involved in upregulation of proinflammatory molecules in macrophages by palmitic acid (PA) and lipopolysaccharide (LPS), GPR40 activation in macrophages exerts anti-inflammatory effects. Taken together, this study demonstrated for the first time that loss of GPR40 in LDLR-deficient mice exacerbated HFD-induced hyperlipidemia, hepatic steatosis, inflammation and fibrosis potentially through a CD36-dependent mechanism, suggesting that GPR40 may play a beneficial role in hyperlipidemia-associated NASH in LDLR-deficient mice.

Phosphorylation of 17β-hydroxysteroid dehydrogenase 13 at serine 33 attenuates nonalcoholic fatty liver disease in mice

17β-hydroxysteroid dehydrogenase-13 is a hepatocyte-specific, lipid droplet-associated protein. A common loss-of-function variant of HSD17B13 (rs72613567: TA) protects patients against non-alcoholic fatty liver disease with underlying mechanism incompletely understood. In the present study, we identify the serine 33 of 17β-HSD13 as an evolutionally conserved PKA target site and its phosphorylation facilitates lipolysis by promoting its interaction with ATGL on lipid droplets. Targeted mutation of Ser33 to Ala (S33A) decreases ATGL-dependent lipolysis in cultured hepatocytes by reducing CGI-58-mediated ATGL activation. Importantly, a transgenic knock-in mouse strain carrying the HSD17B13 S33A mutation (HSD17B1333A/A) spontaneously develops hepatic steatosis with reduced lipolysis and increased inflammation. Moreover, Hsd17B1333A/A mice are more susceptible to high-fat diet-induced nonalcoholic steatohepatitis. Finally, we find reproterol, a potential 17β-HSD13 modulator and FDA-approved drug, confers a protection against nonalcoholic steatohepatitis via PKA-mediated Ser33 phosphorylation of 17β-HSD13. Therefore, targeting the Ser33 phosphorylation site could represent a potential approach to treat NASH.

Design of Xenopus GLP-1-Based Long-Acting Dual GLP-1/Y2 Receptor Agonists.

GLP-1 receptor (GLP-1R) and neuropeptide Y2 receptor (Y2R) dual agonists have shown great potential to treat obesity and type 2 diabetes (T2DM). We developed a multitarget strategy to design monomeric agonists based on Xenopus GLP-1 (xGLP-1) and PYY3-36 analogues with dual activation activities on GLP-1R and Y2R. A novel peptide, 6q, was obtained via stepwise structure optimization and in vitro receptor screens. In db/db and diet-induced obesity (DIO) mice, 6q produced greater effects on long-term glycemic control and body weight reduction than GLP-1R and Y2R monoagonist counterparts. Notably, in high-fat diet-induced nonalcoholic steatohepatitis (NASH) mice, 6q treatment significantly reduced hepatic triglyceride and total cholesterol levels and reversed hepatic steatosis compared with GLP-1R monoagonist (liraglutide) treatment. Collectively, these data support the therapeutic potential of our GLP-1R/Y2R dual agonist 6q as a novel antidiabetic, antiobesity, and antisteatotic agent.

The NLRP3 inhibitor and Nrf2 Agonist, RRx-001, Ameliorates Non-alcoholic Fatty Liver Disease in Rats

Lead Author's Financial Disclosures EpicentRx. Study Funding EpicentRx Inc. Background/Synopsis The development and progression of non-alcoholic steatohepatitis (NASH), an increasingly prevalent disease for which no pharmacological therapy has been approved, is linked to oxidative stress, inflammation, and fibrosis of the liver. Here, we investigated the novel mechanism of RRx-001, an NLRP3 inhibitor and Nrf2 agonist, which is in Phase 3 clinical trials for the treatment of cancer, as a therapeutic option for ameliorating NASH in a diet-induced mice model. Objective/Purpose To investigate whether intraperitoneal administration of RRx-001 improved high-fat diet-induced NASH in rats. Methods Sprague-Dawley rats (6-weeks-old male; about 180g weight) were randomly divided into three groups, one subjected to normal diet (ND) and two groups in high-fat, atherogenic diet (HFD, 40 kcal % fat, 1.25% cholesterol, 0.5% sodium cholate) for 12 weeks. One of HFD groups was treated IP with (5 mg/kg of RRx-001 every other week) for the last 8 weeks before characterization. Several markers of inflammation and steatosis were evaluated. Results Every other week administrations of RRx-001 reduced hepatic steatosis scores, serum ALT and AST levels, and hepatic levels of cholesterol and triglycerides in rats fed a high-fat diet. These were correlated with the suppression of NLRP3 inflammasome activation by RRx-001 as evidenced by decreased levels of NLRP3, caspase-1, and IL-1β levels on ELISA. Conclusions The findings of this study demonstrate that RRx-001 reduced inflammation with an anti-lipidemic and anti-NASH effect mediated at least in part by inhibition of the NLRP3 pathway. Thus, targeting the NLRP3 inflammasome with RRx-001 may contribute to the prevention and treatment of NASH. EpicentRx.

Human Mesenchymal Stromal Cells Resolve Lipid Load in High Fat Diet-Induced Non-Alcoholic Steatohepatitis in Mice by Mitochondria Donation.

Mesenchymal stromal cells (MSC) increasingly emerge as an option to ameliorate non-alcoholic steatohepatitis (NASH), a serious disease, which untreated may progress to liver cirrhosis and cancer. Before clinical translation, the mode of action of MSC needs to be established. Here, we established NASH in an immune-deficient mouse model by feeding a high fat diet. Human bone-marrow-derived MSC were delivered to the liver via intrasplenic transplantation. As verified by biochemical and image analyses, human mesenchymal stromal cells improved high-fat-diet-induced NASH in the mouse liver by decreasing hepatic lipid content and inflammation, as well as by restoring tissue homeostasis. MSC-mediated changes in gene expression indicated the switch from lipid storage to lipid utilization. It was obvious that host mouse hepatocytes harbored human mitochondria. Thus, it is feasible that resolution of NASH in mouse livers involved the donation of human mitochondria to the mouse hepatocytes. Therefore, human MSC might provide oxidative capacity for lipid breakdown followed by restoration of metabolic and tissue homeostasis.

High-fat diet-induced nonalcoholic steatohepatitis is accelerated by low carnitine and impaired glucose tolerance in novel murine models

AbstractCarnitine deficiency and impaired glucose tolerance (IGT) exacerbate liver steatosis. Given the current lack of ideal murine nonalcoholic steatohepatitis (NASH) models, we investigated new NASH models using jvs/+ mice with low carnitine and wild-type mice with low-dose alloxan-induced IGT. The jvs/+ and wild-type mice were divided into jvs/+ mice fed a high-fat diet (HFD) from 3 weeks of age (HF hetero group), wild-type mice with low-dose alloxan treatment fed HFD (AL + HF wild group), wild-type mice fed HFD (HF wild group), and two types of mice fed a normal diet—jvs/+ and wild-type (intact group). All mice were sacrificed at 20 or 40 weeks of age. All male HFD-fed mice showed obesity, IGT, high blood insulin levels, homeostatic model assessment of insulin resistance (HOMA-IR), high liver enzyme levels, and high cholesterol levels. The degree of IGT was the worst in the AL + HF wild group, and blood insulin levels and HOMA-IR score were remarkably increased from 20 to 40 weeks of age. Almost all HFD-fed mice showed steatosis, fibrosis, and lobular inflammation in the centrilobular zone. These changes were accompanied by hepatocyte ballooning and were enhanced at 40 weeks of age. Furthermore, the incidence rate of nodular hyperplasia and adenoma in both the HF hetero and AL + HF wild groups was nearly 30%. We successfully established two novel murine models of NASH using male jvs/+ mice with low carnitine and male wild-type mice with IGT that eventually developed obesity, fatty liver, insulin resistance, liver fibrosis, and tumorigenesis. These results suggest that low carnitine levels and early-stage induction of IGT are important factors in the progression of NASH to tumorigenesis, similar to human NASH.

Melatonin Suppresses NLRP3 Inflammasome Activation via TLR4/NF-κB and P2X7R Signaling in High-Fat Diet-Induced Murine NASH Model.

BackgroundNLRP3 inflammasome activation plays a critical role in mediating inflammation and NASH (non-alcoholic steatohepatitis) progression that ultimately leads to cirrhosis and hepatocellular carcinoma. Melatonin (MLT) controls high-fat diet-induced NASH in the murine model by modulating NLRP3 mediated inflammation. P2X7R-mediated inflammasome activation is reported in several inflammatory models including NASH.ObjectiveThe role of MLT in P2X7R-mediated inflammation in the NASH model has not yet been explored. The present study investigated the role of MLT in amending high-fat diet-induced nonalcoholic steatohepatitis in the murine liver.MethodsTo evaluate the hepatological changes, mice were divided into four groups to investigate the improvement potential of this MLT (10 and 20 mg/kg) and to assess the experimental findings. Histology, biochemical assays, ELISA, FACS analysis, Western blotting, and IF were performed to assess the physical and molecular changes upon melatonin treatment.ResultsThe result demonstrated that MLT administration reduced HFD (high-fat diet)-induced non-alcoholic steatohepatitic indices, which successively restored the hepatic morphological architecture and other pathophysiological features too. Moreover, the application of MLT suppressed HFD-induced activation of the inflammasome and through TLR4/NF-κB signaling. Herein, we report that MLT significantly suppresses P2X7R expression and calcium influx along with inflammasome in both in vitro and in vivo. The docking study revealed a strong binding affinity of MLT with P2X7R. Moreover, the results also showed that the Nrf2 level was boosted which may normalize the expression of antioxidant proteins that safeguard against oxidative damage triggered by inflammation. Furthermore, some matrix metalloproteinases like MMP 2 and MMP 9 were repressed and TIMP-1 level was increased, which also signifies that MLT could improve liver fibrosis in this model.ConclusionBased on our findings, this study may conclude that MLT could be used as a therapeutic agent in the high-fat diet-induced NASH model as it has persuasive anti-inflammatory potential.