Background & Aims There is evidence from clinical studies that compromised intestinal epithelial permeability contributes to the development of non-alcoholic steatohepatitis (NASH), but the exact mechanisms are not clear. Mice with disruption of the gene (F11r) encoding junctional adhesion molecule A (JAM-A) have defects in intestinal epithelial permeability. We used these mice to study how disruption of the intestinal epithelial barrier contributes to NASH. Methods Male C57BL/6 (control) or F11r−/− mice were fed a normal diet or a diet high in saturated fat, fructose and cholesterol (HFCD) for 8 weeks. Liver and intestinal tissues were collected and analyzed by histology, quantitative reverse transcription PCR and flow cytometry. Intestinal epithelial permeability was assessed in mice by measuring permeability to fluorescently labeled dextran. The intestinal microbiota were analyzed using 16S rRNA sequencing. We also analyzed biopsies from proximal colon of 30 patients with non-alcoholic fatty liver disease (NAFLD) and 19 subjects without NAFLD (controls) undergoing surveillance colonoscopy. Results F11r−/− mice fed a HFCD, but not a normal diet, developed histologic and pathologic features of severe NASH including steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis, whereas control mice fed a HFCD developed only modest steatosis. Interestingly, there were no differences in body weight, ratio of liver weight:body weight, or glucose homeostasis between control and F11r−/− mice fed a HFCD. In these mice, liver injury was associated with significant increases in mucosal inflammation, tight junction disruption and intestinal epithelial permeability to bacterial endotoxins, compared with control mice or F11r−/− mice fed a normal diet. The HFCD led to a significant increase in inflammatory microbial taxa in F11r−/− mice, compared with control mice. Administration of oral antibiotics or sequestration of bacterial endotoxins with sevelamer hydrochloride reduced mucosal inflammation and restored normal liver histology in F11r−/− mice fed a HFCD. Protein and transcript levels of JAM-A were significantly lower in the intestinal mucosa of patients with NAFLD than without NAFLD; decreased expression of JAM-A correlated with increased mucosal inflammation. Conclusions Mice with defects in intestinal epithelial permeability develop more severe steatohepatitis following a HFCD than control mice; colon tissues from patients with NAFLD have lower levels of JAM-A and higher levels of inflammation than subjects without NAFLD. These findings indicate that intestinal epithelial barrier function and microbial dysbiosis contribute to development of NASH. Restoration of intestinal barrier integrity and manipulation of gut microbiota might be developed as therapeutic strategies for patients with NASH.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome (MetS). Up to a third of NAFLD subjects are at risk for developing nonalcoholic steatohepatitis (NASH). Many rodent models fail to replicate both MetS and NASH. The purpose of this study was to develop a reliable mouse model of NASH and MetS using a diet containing cholesterol, saturated fat and carbohydrate that is reflective of Western diets of North Americans. Experimental design: We used adult male C57BL/6 J 4- to 5-week-old mice and administered a solid diet containing 0.2% cholesterol, 45% of its calories from fat, with 30% of the fat in the form of partially hydrogenated vegetable oil. We also provided carbohydrate largely as high-fructose corn syrup equivalent in water. In a separate cohort, we gave the identical diet in the absence of cholesterol. Glucose and insulin tolerance testing was conducted throughout the feeding period. The feeding was conducted for 16 weeks, and the mice were sacrificed for histological analysis, markers of MetS, liver inflammation, circulating lipids, as well as liver staining for fibrosis and alpha smooth muscle actin (α-SMA). Results: We found that cholesterol significantly increased serum leptin, interleukin-6, liver weight and liver weight/body weight ratio, fibrosis and liver α-SMA. Conclusions: Mice administered a diet accurately reflecting patterns associated with humans afflicted with MetS can reliably replicate features of MetS, NASH and significant liver fibrosis. The model we describe significantly reduces the time by several months for development of stage 3 hepatic fibrosis.

SYNOPSIS Adiponectin is protective against hepatic fibrosis, while leptin promotes fibrosis. In hepatic stellate cells (HSCs), leptin signals via a Janus Kinase 2/Signal Transducers and Activators of Transcription 3 (Jak2/Stat3) pathway, producing effects that enhance extracellular matrix deposition. Suppressors of Cytokine Signaling-3 (SOCS-3) and Protein Tyrosine Phosphatase-1B (PTP1B) are both negative regulators of Jak/Stat signaling, and recent studies demonstrated a role for adiponectin in regulating SOCS-3 expression. In this study we investigated mechanisms whereby adiponectin dampens leptin signaling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad−/−) and wild-type mice with leptin and/or carbon tetrachloride (CCl4), or saline. We analyzed Jak2 and Ob-Rb phosphorylation, and PTP1B expression and activity. We also explored potential mechanisms through which adiponectin regulates SOCS-3/Ob-Rb association. Adiponectin inhibited leptin-stimulated Jak2 activation and Ob-Rb phosphorylation in HSCs, while both were increased in Ad−/− mice. Adiponectin stimulated PTP1B expression and activity, in vitro, while PTP1B expression was lower in Ad−/−mice than in wild-type mice. Adiponectin also promoted SOCS-3/Ob-R association, and blocked leptin-stimulated formation of extracellular TIMP-1/MMP-1 complexes, in vitro. These data suggest two novel mechanisms whereby adiponectin inhibits hepatic fibrosis: by promoting binding of SOCS-3 to Ob-Rb, and stimulating PTP1B expression and activity, thus inhibiting Jak2-Stat3 signaling at multiple points.

Background and Aims The proinflammatory cytokine IL‐1β has been implicated in the pathophysiology of nonalcoholic and alcoholic steatohepatitis. How IL‐1β promotes liver injury in these diseases is unclear, as no IL‐1β receptor‐linked death pathway has been identified. Autophagy functions in hepatocyte resistance to injury and death, and findings of decreased hepatic autophagy in many liver diseases suggest a role for impaired autophagy in disease pathogenesis. Recent findings that autophagy blocks mouse liver injury from lipopolysaccharide led to an examination of autophagy’s function in hepatotoxicity from proinflammatory cytokines. Approach and Results AML12 cells with decreased autophagy from a lentiviral autophagy‐related 5 (Atg5) knockdown were resistant to toxicity from TNF, but sensitized to death from IL‐1β, which was markedly amplified by TNF co‐treatment. IL‐1β/TNF death was necrosis by trypan blue and propidium iodide positivity, absence of mitochondrial death pathway and caspase activation, and failure of a caspase inhibitor or necrostatin‐1s to prevent death. IL‐1β/TNF depleted autophagy‐deficient cells of ATP, and ATP depletion and cell death were prevented by supplementation with the energy substrate pyruvate or oleate. Pharmacological inhibitors and genetic knockdown studies demonstrated that IL‐1β/TNF‐induced necrosis resulted from lysosomal permeabilization and release of cathepsins B and L in autophagy‐deficient cells. Mice with a tamoxifen‐inducible, hepatocyte‐specific Atg5 knockout were similarly sensitized to cathepsin‐dependent hepatocellular injury and death from IL‐1β/TNF in combination, but neither IL‐1β nor TNF alone. Knockout mice had increased hepatic inflammation, and IL‐1β/TNF‐treated, autophagy‐deficient AML12 cells secreted exosomes with proinflammatory damage–associated molecular patterns. Conclusions The findings delineate mechanisms by which decreased hepatocyte autophagy promotes IL‐1β/TNF‐induced necrosis from impaired energy homeostasis and lysosomal permeabilization and inflammation through the secretion of exosomal damage–associated molecular patterns.

Liver fibrosis is a growing global health problem characterized by excess deposition of fibrillar collagen, and activation of hepatic stellate cells (HSCs). Adiponectin is known to possess anti-fibrotic properties; however a high physiological concentration and multiple forms circulating in blood prohibit clinical use. Recently, an adiponectin-like small synthetic peptide agonist (ADP355: H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH2) was synthesized for the treatment of murine breast cancer. The present study was designed to evaluate the efficacy of ADP355 as an anti-fibrotic agent in the in vivo carbon tetrachloride (CCl4)-induced liver fibrosis model. Liver fibrosis was induced in eight-week old male C57BL/6J mice by CCl4-gavage every other day for four weeks before injection of a nanoparticle-conjugated with ADP355 (nano-ADP355). Control gold nanoparticles and nano-ADP355 were administered by intraperitoneal injection for two weeks along with CCl4-gavage. All mice were sacrificed after 6 weeks, and serum and liver tissue were collected for biochemical, histopathologic and molecular analyses. Biochemical studies suggested ADP355 treatment attenuates liver fibrosis, determined by reduction of serum aspartate aminotransferase (AST), alanine aminotransferase ALT) and hydroxyproline. Histopathology revealed chronic CCl4-treatment results in significant fibrosis, while ADP355 treatment induced significantly reversed fibrosis. Key markers for fibrogenesis–α-smooth muscle actin (α-SMA), transforming growth factor-beta1 (TGF-β1), connective tissue growth factor (CTGF), and the tissue inhibitor of metalloproteinase I (TIMP1) were also markedly attenuated. Conversely, liver lysates from ADP355 treated mice increased phosphorylation of both endothelial nitric oxide synthase (eNOS) and AMPK while AKT phosphorylation was diminished. These findings suggest ADP355 is a potent anti-fibrotic agent that can be an effective intervention against liver fibrosis.

The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), a major transcriptional regulator of endoplasmic reticulum (ER) stress-mediated apoptosis, is implicated in lipotoxicity-induced ER stress and hepatocyte apoptosis in non-alcoholic fatty liver disease (NAFLD). We have previously demonstrated that the glucagon like peptide 1 (GLP-1) agonist, liraglutide, protects steatotic hepatocytes from lipotoxicity-induced apoptosis by improved handling of free fatty acid (FFA)-induced ER stress. In the present study, we investigated whether CHOP is critical for GLP-1 mediated restoration of ER homeostasis and mitigation of hepatocyte apoptosis in a murine model of NASH (non-alcoholic steatohepatitis). Our data show that despite similar caloric intake, CHOP KO (CHOP−/−) mice fed a diet high in fat, fructose, and cholesterol (HFCD) for sixteen weeks developed more severe histological features of NASH compared with wild type (WT) controls. Severity of NASH in HFCD-fed CHOP−/− mice correlated with significant decrease in peroxisomal β-oxidation, and increased de novo lipogenesis and ER stress-mediated hepatocyte apoptosis. Four weeks of liraglutide treatment markedly attenuated steatohepatitis in HFCD-fed WT mice by improving insulin sensitivity, and suppressing de novo lipogenesis and ER stress-mediated hepatocyte apoptosis. However, in the absence of CHOP, liraglutide did not improve insulin sensitivity, nor suppress peroxisomal β-oxidation or ER stress- mediated hepatocyte apoptosis. Taken together, these data indicate that CHOP protects hepatocytes from HFCD-induced ER stress, and plays a significant role in the mechanism of liraglutide-mediated protection against NASH pathogenesis.

No abstract

No abstract