Abstract. Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver dysfunction, and its prevalence has markedly increased. We previously evaluated the expression of fatty acid metabolism-related genes in NAFLD and reported changes in expression that could contribute to increased fatty acid synthesis. In the present study, we evaluated the expression of additional fatty acid metabolism-related genes in larger groups of NAFLD (n=26) and normal liver (n=10) samples. The target genes for real-time PCR analysis were as follows: acetyl-CoA carboxylase (ACC) 1, ACC2, fatty acid synthase (FAS), sterol regulatory element-binding protein 1c (SREBP-1c), and adipose differentiation-related protein (ADRP) for evaluation of de novo synthesis and uptake of fatty acids; carnitine palmitoyltransferase 1a (CPT1a), long-chain acyl-CoA dehydrogenase (LCAD), long-chain L-3-hydroxyacylcoenzyme A dehydrogenase α (HADHα), uncoupling protein 2 (UCP2), straight-chain acyl-CoA oxidase (ACOX), branched-chain acyl-CoA oxidase (BOX), cytochrome P450 2E1 (CYP2E1), CYP4A11, and peroxisome proliferatoractivated receptor (PPAR)α for oxidation in the mitochondria, peroxisomes and microsomes; superoxide dismutase (SOD), catalase, and glutathione synthetase (GSS) for antioxidant pathways; and diacylglycerol O-acyltransferase 1 (DGAT1), PPARγ, and hormone-sensitive lipase (HSL) for triglyceride synthesis and catalysis. In NAFLD, although fatty acids accumulated in hepatocytes, their de novo synthesis and uptake were up-regulated in association with increased expression of ACC1, FAS, SREBP-1c, and ADRP. Fatty acid oxidation-related genes, LCAD, HADHα, UCP2, ACOX, BOX, CYP2E1, and CYP4A11, were all overexpressed, indicating that oxidation was enhanced in NAFLD, whereas the expression of CTP1a and PPARα was decreased. Furthermore, SOD and catalase were also overexpressed, indicating that antioxidant pathways are activated to neutralize reactive oxygen species (ROS), which are overproduced during oxidative processes. The expression of DGAT1 was up-regulated without increased PPARγ expression, whereas the expression of HSL was decreased. Our data indicated the following regarding NAFLD: i) increased de novo synthesis and uptake of fatty acids lead to further fatty acid accumulation in hepatocytes; ii) mitochondrial fatty acid oxidation is decreased or fully activated; iii) in order to complement the function of mitochondria (ß-oxidation), peroxisomal (ß-oxidation) and microsomal (ω-oxidation) oxidation is up-regulated to decrease fatty acid accumulation; iv) antioxidant pathways including SOD and catalase are enhanced to neutralize ROS overproduced during mitochondrial, peroxisomal, and microsomal oxidation; and v) lipid droplet formation is enhanced due to increased DGAT expression and decreased HSL expression. Further studies will be needed to clarify how fatty acid synthesis is increased by SREBP-1c, which is under the control of insulin and AMP-activated protein kinase. IntroductionNonalcoholic fatty liver disease...

Abstract. Nonalcoholic fatty liver disease (NAFLD) is a common liver disease whose prevalence has increased markedly. We reported previously that fatty acid synthesis was enhanced in NAFLD with the accumulation of fatty acids. To clarify the disorder, we evaluated the expression of genes regulating fatty acid synthesis by real-time PCR using samples from NAFLD (n=22) and normal liver (control; n=10). A major regulator of fatty acids synthesis is sterol regulatory element-binding protein-1c (SREBP-1c). Its expression was significantly higher in NAFLD, nearly 5-fold greater than the controls. SREBP-1c is positively regulated by insulin signaling pathways, including insulin receptor substrate (IRS)-1 and -2. In NAFLD, IRS-1 expression was enhanced and correlated positively with SREBP-1c expression. In contrast, IRS-2 expression decreased by 50% and was not correlated with SREBP-1c. Forkhead box protein A2 (Foxa2) is a positive regulator of fatty acid oxidation and is itself negatively regulated by IRSs. Foxa2 expression increased in NAFLD and showed a negative correlation with IRS-2, but not with IRS-1, expression. It is known that SREBP-1c is negatively regulated by AMPactivated protein kinase (AMPK) but expression levels of AMPK in NAFLD were almost equal to those of the controls. These data indicate that, in NAFLD, insulin signaling via IRS-1 causes the up-regulation of SREBP1-c, leading to the increased synthesis of fatty acids by the hepatocytes; negative feedback regulation via AMPK does not occur and the activation of Foxa2, following a decrease of IRS-2, upregulates fatty acid oxidation. IntroductionNonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of liver dysfunction (1-3) and its prevalence has been increasing markedly (4-6). Furthermore, nonalcoholic steatohepatitis (NASH), a severe form of NAFLD accompanied by hepatitis and fibrosis, may progress to cirrhosis and hepatic failure (7,8). NAFLD is often found in patients with obesity and/or insulin resistance, however, its precise cause remains unclear. Therefore, it is important to understand the features of lipid metabolism, particularly fatty acid metabolism, in NAFLD. Previously, we evaluated the expression levels of genes involved in fatty acid metabolism in the liver with NAFLD and found that the expression of those genes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), was up-regulated, indicating that fatty acid synthesis was enhanced in hepatocytes, leading to the accumulation of fatty acids (9,10).Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that regulate the expression of genes involved in lipid synthesis, and SREBP-1c positively regulates the expression of genes encoding lipogenic enzymes including ACC and FAS (11,12). Insulin is a well-known stimulator of lipogenesis and activates the hepatic expression of 14). Insulin receptor substrate (IRS) proteins, a family of docking molecules, connect insulin receptor activation to essential downstream cascad...

These results suggest that, in HCV-infected liver, the cholesterol load increases and cholesterol uptake is controlled, while de novo cholesterol synthesis is upregulated compared with the normal physiological state. The positive correlations in the expression levels of some cholesterol metabolism-associated genes indicate that not all of the metabolic pathways are dysregulated in HCV-infected liver.

Atherosclerosis is a dise ase characterized by inflammation in the arterial wall. Atherogenesis is dependent on the innate immune response involving activation of Toll-like receptors (TLRs) and the expression of inflammatory proteins, those may lead to acute coronary syndrome (ACS). We investigated the expression level of TLR-4 in ACS, as compared with TLR-2 and patients with stable angina. Fifty-eight consecutive patients who underwent primary percutaneous coronary intervention (PCI, n = 29) because of ACS and elective PCI (n = 29) because of stable angina using a filter-device distal protection device system were prospectively analyzed. mRNA levels of TLR-2 and TLR-4 in debris containing various inflammatory tissues entrapped in the filter device were altogether analyzed using real-time PCR. There were no significant differences in age, sex distribution, between stable angina and ACS groups. TLR-4 expression levels were higher in patients with ACS than in patients with stable angina. TLR-4 might play a more important role than TLR-2 in atherogenesis, especially in ACS.

Recent studies have demonstrated that several cellular factors are involved in entry of hepatitis C virus (HCV) into host cells. Detailed gene expression profiles of these factors in HCV-infected livers have not been reported for humans. Transcriptional levels of LDL receptor (LDLR), CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin genes in liver samples from patients with chronic hepatitis C were investigated. Serum levels of LDL-cholesterol (LDL-C) and HCV core antigen were also evaluated, and expression of claudin-1 and occludin were immunohistochemically analyzed. Compared with normal liver, transcription of LDLR and claudin-1 genes was significantly suppressed (P < 0.0001) and occludin transcription was significantly up-regulated in HCV-infected livers (P < 0.0001). Significant positive correlations were found for LDLR versus occludin, LDLR versus claudin-1, occludin versus claudin-1, and CD81 versus SR-BI in HCV-infected (P = 0.0012, P < 0.0001, P = 0.0004, and P < 0.0001, respectively) and normal livers (P < 0.0001, P = 0.0051, P < 0.0001, and P < 0.0001, respectively). Positive correlation was observed between serum levels of HCV core antigen and LDL-C (P = 0.0147), with their levels negatively correlated to LDLR (P = 0.0270 and P = 0.0021, respectively). Immunohistochemically, hepatocellular expression of claudin-1 and occludin was increased in HCV-infected livers. Different levels of expression were demonstrated at the mRNA and protein levels for occludin and claudin-1 in HCV-infected and normal livers. Correlation of elements associated with viral entry was comparable in HCV-infected and normal livers.