Masao Honda

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.)

Increased oxidative stress precedes the onset of high-fat diet-induced insulin resistance and obesity.

Insulin resistance is a key pathophysiological feature of metabolic syndrome. However, the initial events triggering the development of insulin resistance and its causal relations with dysregulation of glucose and fatty acids metabolism remain unclear. We investigated biological pathways that have the potential to induce insulin resistance in mice fed a high-fat diet (HFD). We demonstrate that the pathways for reactive oxygen species (ROS) production and oxidative stress are coordinately up-regulated in both the liver and adipose tissue of mice fed an HFD before the onset of insulin resistance through discrete mechanism. In the liver, an HFD up-regulated genes involved in sterol regulatory element binding protein 1c-related fatty acid synthesis and peroxisome proliferator-activated receptor alpha-related fatty acid oxidation. In the adipose tissue, however, the HFD down-regulated genes involved in fatty acid synthesis and up-regulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Furthermore, increased ROS production preceded the elevation of tumor necrosis factor-alpha and free fatty acids in the plasma and liver. The ROS may be an initial key event triggering HFD-induced insulin resistance.

Activation of lipogenic pathway correlates with cell proliferation and poor prognosis in hepatocellular carcinoma

BACKGROUND/AIMSnMetabolic dysregulation is one of the risk factors for the development of hepatocellular carcinoma (HCC). We investigated the activated metabolic pathway in HCC to identify its role in HCC growth and mortality.nnnMETHODSnGene expression profiles of HCC tissues and non-cancerous liver tissues were obtained by serial analysis of gene expression. Pathway analysis was performed to characterize the metabolic pathway activated in HCC. Suppression of the activated pathway by RNA interference was used to evaluate its role in HCC in vitro. Relation of the pathway activation and prognosis was statistically examined.nnnRESULTSnA total of 289 transcripts were up- or down-regulated in HCC compared with non-cancerous liver (P<0.005). Pathway analysis revealed that the lipogenic pathway regulated by sterol regulatory element binding factor 1 (SREBF1) was activated in HCC, which was validated by real-time RT-PCR. Suppression of SREBF1 induced growth arrest and apoptosis whereas overexpression of SREBF1 enhanced cell proliferation in human HCC cell lines. SREBF1 protein expression was evaluated in 54 HCC samples by immunohistochemistry, and Kaplan-Meier survival analysis indicated that SREBF1-high HCC correlated with high mortality.nnnCONCLUSIONSnThe lipogenic pathway is activated in a subset of HCC and contributes to cell proliferation and prognosis.

Different signaling pathways in the livers of patients with chronic hepatitis B or chronic hepatitis C.

The clinical manifestations of chronic hepatitis B (CH‐B) and chronic hepatitis C (CH‐C) are different. We previously reported differences in the gene expression profiles of liver tissue infected with CH‐B or CH‐C; however, the signaling pathways underlying each condition have yet to be clarified. Using a newly constructed cDNA microarray consisting of 9614 clones selected from 256,550 tags of hepatic serial analysis of gene expression (SAGE) libraries, we compared the gene expression profiles of liver tissue from 24 CH‐B patients with those of 23 CH‐C patients. Laser capture microdissection was used to isolate hepatocytes from liver lobules and infiltrating lymphoid cells from the portal area, from 16 patients, for gene expression analysis. Furthermore, the comprehensive gene network was analyzed using SAGE libraries of CH‐B and CH‐C. Supervised and nonsupervised learning methods revealed that gene expression was correlated more with the infecting virus than any other clinical parameters such as histological stage and disease activity. Pro‐apoptotic and DNA repair responses were predominant in CH‐B with p53 and 14‐3‐3 interacting genes having an important role. In contrast, inflammatory and anti‐apoptotic phenotypes were predominant in CH‐C. These differences would evoke different oncogenic factors in CH‐B and CH‐C. In conclusion, we describe the different signaling pathways induced in the livers of patients with CH‐B or CH‐C. The results might be useful in guiding therapeutic strategies to prevent the development of hepatocellular carcinoma in cases of CH‐B and CH‐C. (HEPATOLOGY 2006;44:1122–1138.)

Obesity Upregulates Genes Involved in Oxidative Phosphorylation in Livers of Diabetic Patients

Obesity is a major cause of insulin resistance and contributes to the development of type 2 diabetes. The altered expression of genes involved in mitochondrial oxidative phosphorylation (OXPHOS) has been regarded as a key change in insulin‐sensitive organs of patients with type 2 diabetes. This study explores possible molecular signatures of obesity and examines the clinical significance of OXPHOS gene expression in the livers of patients with type 2 diabetes. We analyzed gene expression in the livers of 21 patients with type 2 diabetes (10 obese and 11 nonobese patients; age, 53.0 ± 2.1 years; BMI, 24.4 ± 0.9 kg/m2; fasting plasma glucose, 143.0 ± 10.6 mg/dl) using a DNA chip. We screened 535 human pathways and extracted those metabolic pathways significantly altered by obesity. Genes involved in the OXPHOS pathway, together with glucose and lipid metabolism pathways, were coordinately upregulated in the liver in association with obesity. The mean centroid of OXPHOS gene expression was significantly correlated with insulin resistance indices and the hepatic expression of genes involved in gluconeogenesis, reactive oxygen species (ROS) generation, and transcriptional factors and nuclear co‐activators associated with energy homeostasis. In conclusion, obesity may affect the pathophysiology of type 2 diabetes by upregulating genes involved in OXPHOS in association with insulin resistance markers and the expression of genes involved in hepatic gluconeogenesis and ROS generation.

Impact of diabetes on recurrence of hepatocellular carcinoma after surgical treatment in patients with viral hepatitis.

OBJECTIVES: Consensus has been reached that diabetes is a risk factor for development of HCC, but the impact on postoperative recurrence is still controversial. To clarify this point, we analyzed the relationship of postoperative recurrence rate of HCC and coexistence of diabetes in the patients with viral hepatitis.METHODS: A total of 90 patients who had undergone curative resection for HCC were analyzed. They were divided into two groups with and without diabetes, and the recurrence-free survival rates after surgical treatment and the factors contributing to recurrence were examined.RESULTS: Kaplan-Meier survival analysis showed the recurrence-free survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P = 0.005) and overall survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P = 0.005). These results were emphasized in the analysis of patients infected with hepatitis C virus. Univariate and multivariate analyses showed diabetes was a significant factor contributing to HCC recurrence after treatment. Furthermore, multivariate analysis in HCC patients with diabetes showed Child-Pugh classification B (P = 0.001) and insulin therapy (P = 0.049) were significant factors contributing to HCC recurrence after treatment.CONCLUSIONS: The results of the present study suggest that diabetes is a risk factor for the recurrence of HCV-related HCC and decreases the overall survival rates after surgical treatment. HCV-related HCC patients with diabetes should be closely followed for postoperative recurrence.

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 (pu2009=u20090.04). 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.

Identification of novel candidate tumour marker genes for intrahepatic cholangiocarcinoma

BACKGROUND/AIMSnSpecific markers are required for early detection and diagnosis of intrahepatic cholangiocarcinoma (ICC); however, the tumour markers currently in use are not specific for ICC.nnnMETHODSnWe compared an ICC cDNA library with that of hepatocellular carcinoma (HCC) by serial analysis of gene expression (SAGE). The expression patterns in each were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunohistochemical analysis of 74 samples including 16 ICC samples.nnnRESULTSnA comparison of the two libraries revealed distinct gene expression patterns for each type of liver cancer. In addition to the known tumour markers, we detected nine novel genes associated with ICC. By comparing the mean transcript abundance in the ICC library with those in other libraries, including gastric, colon, prostate and breast cancer, together with our RT-PCR results, we identified three genes as specific markers of ICC: biglycan, insulin-like growth factor-binding protein 5 and claudin-4. Immunoblotting and immunohistochemical analyses showed that claudin-4 was highly expressed in ICC. Moreover, discrimination analysis revealed that a combination of these genes could be used to distinguish ICC from HCC or metastatic adenocarcinoma.nnnCONCLUSIONSnWe identified novel marker genes of ICC that are potentially useful for the diagnosis of liver cancer.

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis C virus and its susceptibility to interferon

We developed a reverse genetics system of hepatitis C virus (HCV) genotypes 1a and 2a using infectious clones and human hepatocyte chimeric mice. We inoculated cell culture‐produced genotype 2a (JFH‐1) HCV intravenously. We also injected genotype 1a CV‐H77C clone RNA intrahepatically. Mice inoculated with HCV by both procedures developed measurable and transmissible viremia. Interferon (IFN) alpha treatment resulted in greater reduction of genotype 2a HCV levels than genotype 1a, as seen in clinical practice. Genetically engineered HCV infection system should be useful for analysis of the mechanisms of resistance of HCV to IFN and other drugs.

Protein expression profile characteristic to hepatocellular carcinoma revealed by 2D-DIGE with supervised learning

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Although several major risks related to HCC, e.g., hepatitis B and/or hepatitis C virus infection, aflatoxin B1 exposure, alcohol drinking and genetic defects have been revealed, the molecular mechanisms leading to the initiation and progression of HCC have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to detect the proteins which are associated with tumor progression and may be useful as potential therapeutic or diagnosis targets. However, previous studies have not yet revealed the associations among HCC cells, histological grade and AFP. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) combined with MS for 18 HCC patients. To focus not on individual proteins but on multiple proteins associated with pathogenesis, we introduce the supervised feature selection based on stochastic gradient boosting (SGB) for identifying protein spots that discriminate HCC/non HCC, histological grade of moderate/well and high alpha-fetoprotein (AFP)/low AFP level without arbitrariness. We detected 18, 25 and 27 protein spots associated with HCC, histological grade and AFP level, respectively. We confirmed that SGB is able to identify the known HCC-related proteins, e.g., heat shock proteins, carbonic anhydrase 2. Moreover, we identified the differentially expressed proteins associated with histological grade of HCC and AFP level and found that aldo-keto reductase 1B10 (AKR1B10) is related to well differentiated HCC, keratin 8 (KRT8) is related to both histological grade and AFP level and protein disulfide isomerase-associated 3 (PDIA3) is associated with both HCC and AFP level. Our pilot study provides new insights on understanding the pathogenesis of HCC, histological grade and AFP level.