Hirofumi Misu

Lipid-induced oxidative stress causes steatohepatitis in mice fed an atherogenic diet.

Recently, nonalcoholic steatohepatitis (NASH) was found to be correlated with cardiovascular disease events independently of the metabolic syndrome. The aim of this study was to investigate whether an atherogenic (Ath) diet induces the pathology of steatohepatitis necessary for the diagnosis of human NASH and how cholesterol and triglyceride alter the hepatic gene expression profiles responsible for oxidative stress. We investigated the liver pathology and plasma and hepatic lipids of mice fed the Ath diet. The hepatic gene expression profile was examined with microarrays and real‐time polymerase chain reactions. The Ath diet induced dyslipidemia, lipid peroxidation, and stellate cell activation in the liver and finally caused precirrhotic steatohepatitis after 24 weeks. Cellular ballooning, a necessary histological feature defining human NASH, was observed in contrast to existing animal models. The addition of a high‐fat component to the Ath diet caused hepatic insulin resistance and further accelerated the pathology of steatohepatitis. A global gene expression analysis revealed that the Ath diet up‐regulated the hepatic expression levels of genes for fatty acid synthesis, oxidative stress, inflammation, and fibrogenesis, which were further accelerated by the addition of a high‐fat component. Conversely, the high‐fat component down‐regulated the hepatic gene expression of antioxidant enzymes and might have increased oxidative stress. Conclusion: The Ath diet induces oxidative stress and steatohepatitis with cellular ballooning. The high‐fat component induces insulin resistance, down‐regulates genes for antioxidant enzymes, and further aggravates the steatohepatitis. This model suggests the critical role of lipids in causing oxidative stress and insulin resistance leading to steatohepatitis. (HEPATOLOGY 2007.)

A liver-derived secretory protein, selenoprotein P, causes insulin resistance.

The liver may regulate glucose homeostasis by modulating the sensitivity/resistance of peripheral tissues to insulin, by way of the production of secretory proteins, termed hepatokines. Here, we demonstrate that selenoprotein P (SeP), a liver-derived secretory protein, causes insulin resistance. Using serial analysis of gene expression (SAGE) and DNA chip methods, we found that hepatic SeP mRNA levels correlated with insulin resistance in humans. Administration of purified SeP impaired insulin signaling and dysregulated glucose metabolism in both hepatocytes and myocytes. Conversely, both genetic deletion and RNA interference-mediated knockdown of SeP improved systemic insulin sensitivity and glucose tolerance in mice. The metabolic actions of SeP were mediated, at least partly, by inactivation of adenosine monophosphate-activated protein kinase (AMPK). In summary, these results demonstrate a role of SeP in the regulation of glucose metabolism and insulin sensitivity and suggest that SeP may be a therapeutic target for type 2 diabetes.

Genes involved in oxidative phosphorylation are coordinately upregulated with fasting hyperglycaemia in livers of patients with type 2 diabetes.

Aims/hypothesisMitochondrial oxidative phosphorylation (OXPHOS) plays an important role in the pathophysiology of type 2 diabetes. Genes involved in OXPHOS have been reported to be down-regulated in skeletal muscle from patients with type 2 diabetes; however, hepatic regulation is unknown.Materials and methodsWe analysed expression of genes involved in OXPHOS from the livers of 14 patients with type 2 diabetes and 14 subjects with NGT using serial analysis of gene expression (SAGE) and DNA chip analysis. We evaluated the correlation between expression levels of genes involved in OXPHOS and the clinical parameters of individuals with type 2 diabetes and NGT.ResultsBoth gene analyses showed that genes involved in OXPHOS were significantly upregulated in the type 2 diabetic liver. In the SAGE analysis, tag count comparisons of mitochondrial transcripts showed that ribosomal RNAs (rRNA) were 3.5-fold over-expressed, and mRNAs were 1.2-fold over-expressed in the type 2 diabetes library. DNA chip analysis revealed that expression of genes involved in OXPHOS, which correlated with several nuclear factors, including estrogen-related receptor-α or peroxisome proliferator-activated receptor-γ, was a predictor of fasting plasma glucose levels, independently of age, BMI, insulin resistance and fasting insulin levels (p = 0.04). Surprisingly, genes involved in OXPHOS did not correlate with peroxisome proliferator-activated receptor-γ coactivator-1α or nuclear respiratory factor 1.Conclusions/interpretationOur results indicate that upregulation of genes involved in OXPHOS in the liver, which are regulated by different mechanisms from genes in the skeletal muscle, is associated with fasting hyperglycaemia in patients with type 2 diabetes.

Concentration-dependent Dual Effects of Hydrogen Peroxide on Insulin Signal Transduction in H4IIEC Hepatocytes

Background Oxidative stress induced by the accumulation of reactive oxygen species (ROS) has a causal role in the development of insulin resistance, whereas ROS themselves function as intracellular second messengers that promote insulin signal transduction. ROS can act both positively and negatively on insulin signaling, but the molecular mechanisms controlling these dual actions of ROS are not fully understood. Methodology/Principal Findings Here, we directly treated H4IIEC hepatocytes with hydrogen peroxide (H2O2), a representative membrane-permeable oxidant and the most abundant ROS in cells, to identify the key factors determining whether ROS impair or enhance intracellular insulin signaling. Treatment with high concentrations of H2O2 (25–50 µM) for 3 h reduced insulin-stimulated Akt phosphorylation, and increased the phosphorylation of both JNK and its substrate c-Jun. In contrast, lower concentrations of H2O2 (5–10 µM) enhanced insulin-stimulated phosphorylation of Akt. Moreover, lower concentrations suppressed PTP1B activity, suggesting that JNK and phosphatases such as PTP1B may play roles in determining the thresholds for the diametrical effects of H2O2 on cellular insulin signaling. Pretreatment with antioxidant N-acetyl-L-cysteine (10 mM) canceled the signal-promoting action of low H2O2 (5 µM), and it canceled out further impairment of insulin of insulin signaling induced by high H2O2 (25 µM). Conclusions/Significance Our results demonstrate that depending on its concentration, H2O2 can have the positive or negative effect on insulin signal transduction in H4IIEC hepatocytes, suggesting that the concentration of intracellular ROS may be a major factor in determining whether ROS impair or enhance insulin signaling.

Metformin Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model. Methodology/Principal Findings Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH. Conclusions/Significance These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance.

Proteasome Dysfunction Mediates Obesity-Induced Endoplasmic Reticulum Stress and Insulin Resistance in the Liver

Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. However, the molecular link between obesity and ER stress remains to be identified. Proteasomes are important multicatalytic enzyme complexes that degrade misfolded and oxidized proteins. Here, we report that both mouse models of obesity and diabetes and proteasome activator (PA)28-null mice showed 30–40% reduction in proteasome activity and accumulation of polyubiquitinated proteins in the liver. PA28-null mice also showed hepatic steatosis, decreased hepatic insulin signaling, and increased hepatic glucose production. The link between proteasome dysfunction and hepatic insulin resistance involves ER stress leading to hyperactivation of c-Jun NH2-terminal kinase in the liver. Administration of a chemical chaperone, phenylbutyric acid (PBA), partially rescued the phenotypes of PA28-null mice. To confirm part of the results obtained from in vivo experiments, we pretreated rat hepatoma-derived H4IIEC3 cells with bortezomib, a selective inhibitor of the 26S proteasome. Bortezomib causes ER stress and insulin resistance in vitro—responses that are partly blocked by PBA. Taken together, our data suggest that proteasome dysfunction mediates obesity-induced ER stress, leading to insulin resistance in the liver.

Inverse Correlation between Serum Levels of Selenoprotein P and Adiponectin in Patients with Type 2 Diabetes

Background We recently identified selenoprotein P (SeP) as a liver-derived secretory protein that causes insulin resistance in the liver and skeletal muscle; however, it is unknown whether and, if so, how SeP acts on adipose tissue. The present study tested the hypothesis that SeP is related to hypoadiponectinemia in patients with type 2 diabetes. Methodology/Principal Findings We compared serum levels of SeP with those of adiponectin and other clinical parameters in 36 patients with type 2 diabetes. We also measured levels of blood adiponectin in SeP knockout mice. Circulating SeP levels were positively correlated with fasting plasma glucose (r = 0.35, P = 0.037) and negatively associated with both total and high-molecular adiponectin in patients with type 2 diabetes (r = −0.355, P = 0.034; r = −0.367, P = 0.028). SeP was a predictor of both total and high-molecular adiponectin, independently of age, body weight, and quantitative insulin sensitivity index (β = −0.343, P = 0.022; β = −0.357, P = 0.017). SeP knockout mice exhibited an increase in blood adiponectin levels when fed regular chow or a high sucrose, high fat diet. Conclusions/Significance These results suggest that overproduction of liver-derived secretory protein SeP is connected with hypoadiponectinemia in patients with type 2 diabetes.

LECT2 functions as a hepatokine that links obesity to skeletal muscle insulin resistance

Recent articles have reported an association between fatty liver disease and systemic insulin resistance in humans, but the causal relationship remains unclear. The liver may contribute to muscle insulin resistance by releasing secretory proteins called hepatokines. Here we demonstrate that leukocyte cell–derived chemotaxin 2 (LECT2), an energy-sensing hepatokine, is a link between obesity and skeletal muscle insulin resistance. Circulating LECT2 positively correlated with the severity of both obesity and insulin resistance in humans. LECT2 expression was negatively regulated by starvation-sensing kinase adenosine monophosphate-activated protein kinase in H4IIEC hepatocytes. Genetic deletion of LECT2 in mice increased insulin sensitivity in the skeletal muscle. Treatment with recombinant LECT2 protein impaired insulin signaling via phosphorylation of Jun NH2-terminal kinase in C2C12 myocytes. These results demonstrate the involvement of LECT2 in glucose metabolism and suggest that LECT2 may be a therapeutic target for obesity-associated insulin resistance.

Characteristics of hepatic fatty acid compositions in patients with nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is closely related to insulin resistance and lipid metabolism. Recent studies have suggested that the quality of fat accumulated in the liver is associated with the development of nonalcoholic steatohepatitis (NASH). In this study, we investigated the fatty acid composition in liver tissue and its association with the pathology in NAFLD patients.

Metformin Suppresses Expression of the Selenoprotein P Gene via an AMP-activated Kinase (AMPK)/FoxO3a Pathway in H4IIEC3 Hepatocytes

Background: The suppression of selenoprotein P production may be a novel therapeutic target for reducing insulin resistance. Results: Selenoprotein P expression was suppressed by metformin treatment, but co-administration of AMPK inhibitor or FoxO3a siRNA cancelled this suppression. Conclusion: Metformin suppresses selenoprotein P expression via the AMPK/FoxO3a pathway. Significance: The AMPK/FoxO3a pathway in the liver may be a therapeutic target for type 2 diabetes. Selenoprotein P (SeP; encoded by SEPP1 in humans) is a liver-derived secretory protein that induces insulin resistance in type 2 diabetes. Suppression of SeP might provide a novel therapeutic approach to treating type 2 diabetes, but few drugs that inhibit SEPP1 expression in hepatocytes have been identified to date. The present findings demonstrate that metformin suppresses SEPP1 expression by activating AMP-activated kinase (AMPK) and subsequently inactivating FoxO3a in H4IIEC3 hepatocytes. Treatment with metformin reduced SEPP1 promoter activity in a concentration- and time-dependent manner; this effect was cancelled by co-administration of an AMPK inhibitor. Metformin also suppressed Sepp1 gene expression in the liver of mice. Computational analysis of transcription factor binding sites conserved among the species resulted in identification of the FoxO-binding site in the metformin-response element of the SEPP1 promoter. A luciferase reporter assay showed that metformin suppresses Forkhead-response element activity, and a ChIP assay revealed that metformin decreases binding of FoxO3a, a direct target of AMPK, to the SEPP1 promoter. Transfection with siRNAs for Foxo3a, but not for Foxo1, cancelled metformin-induced luciferase activity suppression of the metformin-response element of the SEPP1 promoter. The overexpression of FoxO3a stimulated SEPP1 promoter activity and rescued the suppressive effect of metformin. Metformin did not affect FoxO3a expression, but it increased its phosphorylation and decreased its nuclear localization. These data provide a novel mechanism of action for metformin involving improvement of systemic insulin sensitivity through the regulation of SeP production and suggest an additional approach to the development of anti-diabetic drugs.