Adiponectin prevents progression of steatohepatitis in mice by regulating oxidative stress and Kupffer cell phenotype polarization

Aim:  We reported previously that hypoadiponectinemia enhances hepatic oxidative stress and accelerates progression of nonalcoholic steatohepatitis (NASH) in mice. However, the precise mechanism and preventive effects of adiponectin on NASH remain unclear. The aim of this study was to examine the effects of adiponectin on steatohepatitis using adiponectin-knockout (KO) mice and adenovirus-mediated adiponectin expression system.
Methods:  We used male KO mice and C57BL6/J (WT) mice fed methionine choline-deficient (MCD)-diet as a steatohepatitis model. Liver histology, hepatic oxidative stress markers, and hepatic gene expression levels were investigated. In addition, Hepa 1–6 cells, a mouse liver cell line, were cultured with or without recombinant adiponectin, and gene expressions were investigated by real-time RT-PCR.
Results:  After 2-week feeding of MCD diet, hepatic steatosis was enhanced and plasma alanine aminotransferase elevated in KO mice than in WT mice. In KO mice liver, thiobarbituric acid reactive substances increased, glutathione levels decreased, and mRNA expression levels of antioxidant enzymes (catalase, superoxide dismutase-1) downregulated. Adenovirus-mediated adiponectin expression prevented these changes in KO mice. Moreover, Kupffer cell infiltration was enhanced and mRNA levels of anti-inflammatory M2 macrophage markers (interleukin-10, arginase-1) were decreased in KO mice liver. In the in vitro study, adiponectin significantly increased catalase gene expression in Hepa 1–6 cells.
Conclusions:  Lack of adiponectin enhanced, and adiponectin administration prevented steatohepatitis progression in mice. These changes were due to the anti-oxidative effects of adiponectin, and its effects on Kupffer cells recruitment and phenotype polarization. Augmentation of adiponectin effects could be a useful preventive approach for NASH progression.     

Mechanisms of ductular reaction in non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is a disease spectrum caused in part by insulin resistance and genetic predisposition. This disease is primarily characterized by excessive lipid accumulation in hepatocytes in the absence of alcohol abuse and other causes of liver damage. Histologically, NAFLD is divided into several periods: simple steatosis, non-alcoholic steatohepatitis (NASH), hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. With the increasing prevalence of obesity and hyperlipidemia, NAFLD has become the main cause of chronic liver disease worldwide. As a result, the pathogenesis of this disease is drawing increasing attention. Ductular reaction (DR) is a reactive bile duct hyperplasia caused by liver injury that involves hepatocytes, cholangiocytes, and hepatic progenitor cells. Recently, DR is shown to play a pivotal role in simple steatosis progression to NASH or liver fibrosis, providing new research and treatment options. This study reviews several DR signaling pathways, including Notch, Hippo/YAP-TAZ, Wnt/β-catenin, Hedgehog, HGF/c-Met, and TWEAK/Fn14, and their role in the occurrence and development of NASH.     

Regulation of synthesis and oxidation of fatty acids by adiponectin receptors (AdipoR1/R2) and insulin receptor substrate isoforms (IRS-1/-2) of the liver in a nonalcoholic steatohepatitis animal model

Nonalcoholic steatohepatitis (NASH) is one of the most frequent causes of abnormal liver dysfunction associated with synthesis and oxidation of fatty acids. Adiponectin receptors (AdipoR1/R2) and insulin receptor substrates (IRS-1/-2) are known as modulators of these fatty acid metabolisms in the liver; however, the regulatory roles of these receptors in the synthesis and oxidation of fatty acids are unclear in the liver of NASH. In this study, we examined the roles of hepatic AdipoR1/R2 and IRS-1/-2 in NASH using an animal model. After feeding a high-fat and high-cholesterol diet to obese fa/fa Zucker rats for 8 weeks, rats showed fatty liver spontaneously with inflammation and fibrosis that are characteristic of NASH. The expression levels of AdipoR1/R2 and IRS-2 were significantly decreased, whereas IRS-1 was significantly increased, in NASH. As a result of the decrease of AdipoR1/R2 expression, the messenger RNA expression levels of genes located downstream of AdipoR1/R2, adenosine monophosphate-activated protein kinase α1/α2, which inhibits fatty acid synthesis, and peroxisome proliferator-activated receptor α, which activates fatty acid oxidation, also decreased. Expression level of sterol regulatory element binding protein-1c was found to be elevated, suggesting the up-regulation of IRS-1, and resulted in increased fatty acid synthesis. Furthermore, increase of forkhead box protein A2 expression was observed, which might be associated with the down-regulation of IRS-2, facilitating fatty acid oxidation. Taken together, increased synthesis and oxidation of fatty acids by up- or down-regulation of AdipoR or IRS may contribute to the progression of NASH. Thus, AdipoR and IRS might be crucially important regulators for the synthesis and oxidation of fatty acids in the liver of NASH.     

Lessons from animal models of NASH.

Studies of animals with obesity-related liver disease have taught us much about the mechanisms that mediate this pathology. Our work with genetically obese, insulin-resistant ob/ob mice demonstrates that hepatocytes become steatotic and die at increased rates. Thus, ob/ob mice develop non-alcoholic steatohepatitis (NASH) spontaneously. NASH is intimately related to the insulin resistance (i.e., metabolic) syndrome, a constellation of disorders that result from abnormal production of hormones and cytokines that regulate inflammatory responses. Like humans with the metabolic syndrome, ob/ob mice exhibit increased tumor necrosis factor (TNF) but relatively low levels of adiponectin. Because TNF and adiponectin typically antagonize each other, the combination of increased TNF and decreased adiponectin promotes a state of high TNF activity. Consequently, hepatocytes generate excessive reactive oxygen species (ROS), have altered viability, accumulate lipid and are resistant to insulin. Treatments that inhibit TNF activity or that increase adiponectin improve NASH in ob/ob mice, other mice and humans with NASH. Hence, there is no doubt that cytokine and hormonal imbalances play a key role in the pathogenesis of NASH. However, the fundamental cellular events involved are still poorly understood. Even within very small areas of livers with NASH, most hepatocytes are merely steatotic, while others are ballooned (pre-necrotic), and still others have succumbed to apoptosis. This observation suggests cell-to-cell variability in the response to chronic inflammatory stress. In NASH, most steatotic hepatocytes survive by inducing adaptive, cytoprotective factors. However, such cells respond to super-imposed toxic and mitogenic stimuli differently than (3)na?ve(2) (un-adapted) hepatocytes. Fatty hepatocytes tend to be more vulnerable to ATP depletion and less proliferative, perpetuating chronic liver injury while encouraging the expansion of liver progenitor populations that may become neoplastic. Finally, like other causes of chronic injury, NASH increases the risk for cirrhosis. Studies of ob/ob mice demonstrate that progression to cirrhosis is potentiated by leptin. Leptin probably acts at multiple levels to promote hepatic fibrosis, including direct activation of stellate cells via leptin receptors, regulation of pro- and anti-fibrogenic cytokine production by innate immune cells, and modulation of other neuronal factors that regulate stellate cell activation. The latter two mechanisms seem to dominate because stellate cell activation, fibrogenic cytokine production, collagen gene expression and fibrosis can all be induced by manipulating cytokines and neuronal factors in ob/ob mice (that are genetically deficient in leptin). Thus, studies in mice have uncovered several basic mechanisms that explain the dysfunction that occurs in different types of liver cells during the metabolic syndrome. This has important therapeutic implications for human NASH.     

Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis

In the early stages of nonalcoholic fatty liver disease (NAFLD), triglycerides accumulate in hepatocytes. Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in hepatocyte triglyceride biosynthesis. DGAT2 antisense oligonucleotide (ASO) treatment improved hepatic steatosis dramatically in a previous study of obese mice. According to the 2-hit hypothesis for progression of NAFLD, hepatic steatosis is a risk factor for nonalcoholic steatohepatitis (NASH) and fibrosis. To evaluate this hypothesis, we inhibited DGAT2 in a mouse model of NASH induced by a diet deficient in methionine and choline (MCD). Six-week-old genetically obese and diabetic male db/db mice were fed either the control or the MCD diet for 4 or 8 weeks. The MCD diet group was treated with either 25 mg/kg DGAT2 ASO or saline intraperitoneally twice weekly. Hepatic steatosis, injury, fibrosis, markers of lipid peroxidation/oxidant stress, and systemic insulin sensitivity were evaluated. Hepatic steatosis, necroinflammation, and fibrosis were increased in saline-treated MCD diet-fed mice compared to controls. Treating MCD diet-fed mice with DGAT2 ASO for 4 and 8 weeks decreased hepatic steatosis, but increased hepatic free fatty acids, cytochrome P4502E1, markers of lipid peroxidation/oxidant stress, lobular necroinflammation, and fibrosis. Progression of liver damage occurred despite reduced hepatic expression of tumor necrosis factor alpha, increased serum adiponectin, and striking improvement in systemic insulin sensitivity. CONCLUSION: Results from this mouse model would suggest accumulation of triglycerides may be a protective mechanism to prevent progressive liver damage in NAFLD.     

Is adiponectin involved in the pathogenesis of nonalcoholic steatohepatitis? A preliminary human study

BACKGROUND: Animal studies have suggested that adiponectin may play a role in the pathogenesis of alcoholic and nonalcoholic fatty liver disease. Studies are limited that evaluated the role of adiponectin in the pathogenesis of nonalcoholic steatohepatitis (NASH). METHODS: To further our understanding of the role of adiponectin in the pathogenesis of NASH, the following studies were conducted. Serum adiponectin was measured and correlated with anthropometric and nutritional variables in 21 patients with biopsy-proven NASH and 19 age-, gender-, body mass index-, and body fat-matched controls. The effect of a mixed meal on serum adiponectin levels in a subgroup of patients (n = 24) with NASH and controls was assessed. In a separate cohort, liver samples belonging to healthy (n = 11), steatotic (n = 12), and NASH (n = 12) patients were used to further explore the role of adiponectin by measuring the expression of adiponectin and adiponectin receptor (AdipoR2) mRNA. RESULTS: Patients with NASH had significantly lower levels of serum adiponectin than controls (4.9 +/- 2.7 vs. 7.3 +/- 3.5 microg/mL, P = 0.02). While no significant correlation existed between serum adiponectin and anthropometric or nutritional variables, it was independently associated with age, high density lipoprotein, and triglycerides. Mixed meal had no effect on serum adiponectin either in patients with NASH or in controls. There was no expression of adiponectin mRNA in any of liver samples studied. However, AdipoR2 mRNA expression was higher in NASH than in steatotic and normal liver tissue. CONCLUSION: These data show that adiponectin may have a role in the pathogenesis of human NASH and should be investigated further.     

Adiponectin and its receptors in patients with chronic hepatitis C

BACKGROUND/AIMS: There is increasing interest in the influence of excess body weight and associated metabolic factors on the liver. In patients with non-alcoholic steatohepatitis, lower levels of adiponectin were associated with higher grades of hepatic steatosis and necroinflammatory activity, suggesting a pathophysiological role for this adipokine in liver disease. METHODS: We studied 194 consecutive patients with untreated chronic HCV, to assess the relationship between adiponectin and its receptors and hepatic steatosis, fibrosis and inflammation. RESULTS: Significant negative correlations between serum adiponectin and male gender, body mass index and serum insulin were observed. However, there was no association between serum adiponectin and stage of fibrosis and lower levels of serum adiponectin were associated with the presence of steatosis in males only. In contrast, there was a significant increase in serum adiponectin and hepatic adiponectin immunoreactivity with increasing inflammation. The hepatic mRNA expression of the adiponectin receptors, AdipoR1 and AdipoR2, displayed significant but opposite associations with phosphoenolpyruvate carboxykinase (PEPCK) gene expression, a substitute marker of hepatic insulin sensitivity. CONCLUSIONS: In patients with chronic HCV, adiponectin was associated with steatosis only in males and was paradoxically increased with inflammation. Our results suggest that the role of adiponectin in chronic liver diseases may be linked to gender and etiology.     

Adiponectin and its receptors in non-alcoholic steatohepatitis

BACKGROUND: Adiponectin, an adipocyte derived polypeptide, has been shown to alleviate steatosis and inflammation in mice with non-alcoholic fatty liver disease. AIM: In the present study, we wished to define liver expression of adiponectin and its receptors in morbidly obese patients undergoing bariatric surgery. Patients with non-alcoholic steatohepatitis (NASH) or simple steatosis were investigated to test whether dysregulation of this system might be involved in these disorders. PATIENTS AND METHODS: Liver mRNA expression of adiponectin and its recently cloned receptors RI and RII (adipoRI and adipoRII) were analysed by fluorescence based real time polymerase chain reaction in 13 patients with NASH and nine with simple steatosis. Adiponectin and adipoRII protein expression were assessed by immunohistochemistry in a subgroup of patients. RESULTS: Adiponectin and adipoRII mRNA expression were significantly reduced in liver biopsies of patients with NASH compared with simple steatosis while no difference was found in adipoRI mRNA expression. In NASH, adipoRII mRNA expression was negatively correlated with serum aspartate aminotransferase levels, serum alanine aminotransferase levels, and grade of fibrosis. Liver adiponectin protein expression was mainly found in endothelial cells of portal vessels and liver sinusoids whereas adipoRII expression was seen in hepatocytes only. Adiponectin and adipoRII staining were lower in biopsies of subjects with NASH compared with simple steatosis. CONCLUSION: Reduced hepatic expression of adiponectin and adipoRII might be of pathophysiological relevance in non-alcoholic fatty liver diseases.     

Adiponectin and its receptors in rodent models of fatty liver disease and liver cirrhosis

INTRODUCTION Obesity and especially visceral fat accumulation cause insulin resistance, a common risk factor for hepatic steatosis. Fatty liver is thought to represent the first step towards the subsequent development of liver fibrosis. Impaired mitochond…     

Hypoadiponectinemia accelerates hepatic tumor formation in a nonalcoholic steatohepatitis mouse model

BACKGROUND/AIMS: Adipose tissue produces a number of adipocytokines, including adiponectin, leptin, and tumor necrosis factor-alpha. Obesity, which is associated with low plasma adiponectin levels, is an independent risk factor for various liver diseases including nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the effects of adiponectin on the progression of NASH to cirrhosis and tumor formation using adiponectin-knockout (KO) mice. METHODS: Using a choline-deficient L-amino acid-defined (CDAA) diet-induced mouse NASH model, liver histology and oxidative stress markers were investigated in KO and wild-type (WT) mice. RESULTS: Hepatic steatosis was enhanced to a greater extent in KO mice, compared to WT mice after a 1-week CDAA diet. After 24 weeks, 6 out of 14 KO mice developed liver cirrhosis and hepatic tumors, whereas the 15 WT mice showed only simple steatosis. In KO mice, hepatic cytochrome P450 2E1 levels were upregulated, and markers of oxidative stress (thiobarbituric acid reactive substances, 8-hydroxydeoxyguanosine-positive cells) were significantly increased compared with WT mice. CONCLUSIONS: Our results indicate that lack of adiponectin enhances the progression of hepatic steatosis, fibrosis, and hepatic tumor formation in an animal model of NASH. Hypoadiponectinemia in obesity could be a risk factor for NASH-related hepatic tumor formation.     

Eicosapentaenoic Acid Attenuates Progression of Hepatic Fibrosis with Inhibition of Reactive Oxygen Species Production in Rats Fed Methionine- and Choline-Deficient Diet

Background and Aims  

Nonalcoholic steatohepatitis (NASH) is associated with fat accumulation in the liver, and develops to cirrhosis with the progression of hepatic fibrosis. Eicosapentaenoic acid (EPA) is used to treat hyperlipidemia, and suppresses hepatic fat accumulation. As the effect of EPA on NASH remains unclear, we assessed the therapeutic effect of EPA and its mechanisms in an animal model of NASH.     

Nonalcoholic steatohepatitis severity is defined by a failure in compensatory antioxidant capacity in the setting of mitochondrial dysfunction

AIM To comprehensively evaluate mitochondrial(dys) function in preclinical models of nonalcoholic steatohepatitis(NASH).METHODS We utilized two readily available mouse models of nonalcoholic fatty liver disease(NAFLD) with or without progressive fibrosis: Lep~(ob)/Lep~(ob)(ob/ob) and FATZO mice on high trans-fat, high fructose and high cholesterol(AMLN) diet. Presence of NASH was assessed using immunohistochemical and pathological techniques, and gene expression profiling. Morphological features of mitochondria were assessed via transmission electron microscopy and immunofluorescence, and function was assessed by measuring oxidative capacity in primary hepatocytes, and respiratory control and proton leak in isolated mitochondria. Oxidative stress was measured by assessing activity and/or expression levels of Nrf1, Sod1, Sod2, catalase and 8-OHdG. RESULTS When challenged with AMLN diet for 12 wk, ob/ob and FATZO mice developed steatohepatitis in the presence of obesity and hyperinsulinemia. NASH development was associated with hepatic mitochondrial abnormalities, similar to those previously observed in humans, including mitochondrial accumulation and increased proton leak. AMLN diet also resulted in increased numbers of fragmented mitochondria in both strains of mice. Despite similar mitochondrial phenotypes, we found that ob/ob mice developed more advanced hepatic fibrosis. Activity of superoxide dismutase(SOD) was increased in ob/ob AMLN mice, whereas FATZO mice displayed increased catalase activity, irrespective of diet. Furthermore, 8-OHd G, a marker of oxidative DNA damage, was significantly increased in ob/ob AMLN mice compared to FATZO AMLN mice. Therefore, antioxidant capacity reflected as the ratio of catalase:SOD activity was similar between FATZO and C57 BL6 J control mice, but significantly perturbed in ob/ob mice. CONCLUSION Oxidative stress, and/or the capacity to compensate for increased oxidative stress, in the setting of mitochondrial dysfunction, is a key factor for development of hepatic injury and fibrosis in these mouse models.     

Palmitic acid induces production of proinflammatory cytokine interleukin-8 from hepatocytes

Obesity and the metabolic syndrome are closely correlated with hepatic steatosis. Simple hepatic steatosis in nonalcoholic fatty liver disease can progress to nonalcoholic steatohepatitis (NASH), which can be a precursor to more serious liver diseases, such as cirrhosis and hepatocellular carcinoma. The pathogenic mechanisms underlying progression of steatosis to NASH remain unclear; however, inflammation, proinflammatory cytokines, and oxidative stress have been postulated to play key roles. We previously reported that patients with NASH have elevated serum levels of proinflammatory cytokines, such as interleukin-8 (IL-8), which are likely to contribute to hepatic injury. This study specifically examines the effect of hepatic steatosis on IL-8 production. We induced lipid accumulation in hepatocytes (HepG2, rat primary hepatocytes, and human primary hepatocytes) by exposing them to pathophysiologically relevant concentrations of palmitic acid to simulate the excessive influx of fatty acids into hepatocytes. Significant fat accumulation was documented morphologically by Oil Red O staining in cells exposed to palmitic acid, and it was accompanied by an increase in intracellular triglyceride levels. Importantly, palmitic acid was found to induce significantly elevated levels of biologically active neutrophil chemoattractant, IL-8, from steatotic hepatocytes. Incubation of the cells with palmitate led to increased IL-8 gene expression and secretion (both mRNA and protein) through mechanisms involving activation of nuclear factor kappaB (NF-kappaB) and c-Jun N-terminal kinase/activator protein-1. CONCLUSION: These data demonstrate for the first time that lipid accumulation in hepatocytes can stimulate IL-8 production, thereby potentially contributing to hepatic inflammation and consequent liver injury.     

Angiotensin II type 1 receptor blocker inhibits fibrosis in rat nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is now the most frequent cause of chronic liver impairment in developed countries and is a suggested causative factor in the development of cryptogenic cirrhosis and hepatocellular carcinoma. At present there is no effective and accepted therapy for NASH. The renin-angiotensin system is involved in hepatic fibrosis through activation of hepatic stellate cells, major fibrogenic cells in the liver. Hepatic stellate cells are activated by liver injury to express excessive matrix proteins and profibrogenic cytokines such as transforming growth factor-beta 1. Medicines that inhibit this pathway may be of therapeutic potential in NASH. Using a methionine-choline-deficient rat model of NASH, we studied the potential utility of an angiotensin II type 1 receptor blocker (ARB), olmesartan, on biochemical, histologic, and antioxidant measures of disease activity. ARB significantly attenuated increases in aspartate aminotransferase, activation of hepatic stellate cells, oxidative stress, expression of transforming growth factor-beta 1, expression of collagen genes, and liver fibrosis. CONCLUSION: Our observations strongly suggest a potential preventive role for ARB in the progression of nonalcoholic steatohepatitis.     

The pathogenesis of nonalcoholic steatohepatitis and other fatty liver diseases: a four-step model including the role of lipid release and hepatic venular obstruction in the progression to cirrhosis

Fatty liver disease involves the accumulation of triglycerides in hepatocytes, necrosis of hepatocytes, inflammation, and often fibrosis with progression to cirrhosis. The two-hit model summarizes the important early metabolic events leading to hepatocellular necrosis in nonalcoholic steatohepatitis (NASH). In this article, we provide evidence of lipid release from hepatocytes in posttransplant fat necrosis and in NASH and quantify vascular obliteration in a series of biopsies with NASH. Obliteration of small hepatic veins (<30 microm) in small numbers is compensated by collateral flow. Obliteration of larger hepatic veins (>30 microm) is associated with fibrotic collapse lesions that are not easily resorbed. Based on these observations, we propose a new four-step model that includes the later events that lead to cirrhosis after necrosis has occurred. This model is applicable to nonalcoholic fatty liver disease (NAFLD), alcoholic disease, postjejunoileal bypass disease, and posttransplant fat necrosis. The first step is steatosis facilitated by insulin, and the second is necrosis induced by intracellular lipid toxicity or lipid peroxidation, or both, modified by alcohol, drugs, and ischemia. The third step is release of bulk lipid from hepatocytes into the interstitium leading to direct and inflammatory injury to hepatic veins. The fourth step is venous obstruction with secondary collapse and ultimately fibrous septation and cirrhosis.     

Hypoadiponectinemia predicts the severity of hepatic fibrosis and pancreatic Beta-cell dysfunction in nondiabetic nonobese patients with nonalcoholic steatohepatitis

OBJECTIVES: The relationships between the adipokines tumor necrosis factor (TNF)-alpha and adiponectin and the parameters of glucose homeostasis and severity of liver disease were assessed in nonobese nondiabetic subjects with nonalcoholic steatohepatitis (NASH). METHODS: A frequently sampled intravenous glucose tolerance test, serum cytokine measurement, and 7-day alimentary record were performed in 20 biopsy-proven NASH patients and 45 age-, sex-, and BMI-matched controls (30 insulin sensitive and 15 insulin resistant). RESULTS: Patients with NASH had impaired pancreatic beta-cell function compared with both insulin-sensitive (adaptation index, AI: 97.7 +/- 17.7 vs 307.4 +/- 24.1 min(-2) mmol(-1) L; p= 0.00001) and insulin-resistant (adaptation index, AI: 97.7 +/- 17.7 vs 201.4 +/- 41.1 min(-2) mmol(-1) L; p= 0.001) controls. Serum adiponectin levels were also significantly lower in the NASH group than in the two control groups and correlated with adaptation index and with the severity of hepatic steatosis, necroinflammation, and fibrosis. When NASH patients were grouped according to the severity of histological liver damage, adiponectin was the only variable discriminating patients with higher necroinflammatory grade and fibrosis score from those with milder lesions. CONCLUSIONS: Beta-cell secretory impairment is present in nonobese patients with NASH before glucose intolerance appears and may contribute to their increased risk for developing diabetes. Hypoadiponectinemia is a feature of NASH and may have a pathogenetic role in beta-cell dysfunction and in hepatic necroinflammation and fibrosis, independently of insulin resistance, visceral fat accumulation, TNF-alpha axis activity, and dietary habits. Our findings provide further rationale for therapeutic approaches aimed at increasing adiponectin levels together with restoring beta-cell function and insulin sensitivity.