Takotsubo syndrome in a heart transplant recipient with poor cardiac sympathetic reinnervation.

Takotsubo syndrome (TTS), also referred to as stress cardiomyopathy, is characterized by transient left ventricular apical ballooning in the absence of obstructive coronary artery disease. Catecholamine-induced cardiac injury or vasospasm has been implicated in this pathophysiology. We present a case of a 67-year-old man 10 years after heart transplantation diagnosed with TTS. Sympathetic reinnervation could not be detected by iodine-123 meta iodobenzylguanidine uptake, suggesting that TTS can occur in the absence of functional sympathetic nerve systems reconstruction.

Toll-like receptor-4 is upregulated in plaque debris of patients with acute coronary syndrome more than Toll-like receptor-2.

Atherosclerosis is a disease 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.

Pathophysiological analysis of primary biliary cirrhosis focusing on choline/phospholipid metabolism.

BACKGROUND & AIMS: Injury to biliary epithelial cells caused by disorders in bile composition may be the initial step in the pathogenesis of primary biliary cirrhosis (PBC). We therefore examined choline/phospholipid metabolism in livers of patients with PBC. METHODS: Hepatic levels of mRNA encoded by choline metabolism-related genes in early stage PBC patients were quantified by real-time RT-PCR. Serum cholesterol and triglyceride concentrations in each lipoprotein compartment and serum/tissue choline levels were also measured. OCT1 expression was quantified by genotype (rs683369 and rs622342). RESULTS: Serum choline concentrations were significantly higher in PBC patients than in normal individuals, with the concentrations in the former lowered by treatment with fibrates. Hepatic choline levels were markedly lower in PBC patients than in controls. The levels of expression of genes associated with choline uptake (OCT1 and CTL1), phosphatidylcholine synthesis (PEMT and BHMT), and phosphatidylcholine transport (MDR3) were significantly upregulated in PBC compared with control livers. Serum cholesterol concentrations and the cholesterol/triglyceride ratio in serum very low density lipoprotein were markedly higher in PBC patients than in controls. In PBC liver, OCT1 protein levels were lower in patients with minor (CG/GG at rs683369 and/or CC at rs622342) than major (CC at rs683369 and AA at rs622342) genotypes of the OCT1 gene. CONCLUSION: During early stage PBC, hepatocellular choline uptake and PC synthesis become dysregulated. OCT1 genotypes may influence the pathogenesis of PBC.

Association of ITPA polymorphism with outcomes of peginterferon-α plus ribavirin combination therapy.

AIM: To analyzed the association between inosine triphosphatase (ITPA) (rs1127354) genotypes and sustained virological response (SVR) rates in peginterferon (Peg-IFN)α + ribavirin (RBV) treatment. METHODS: Patients who underwent Peg-IFNα + RBV combination therapy were enrolled (n = 120) and they had no history of other IFN-based treatments. Variation in hemoglobin levels during therapy, cumulative reduction of RBV dose, frequency of treatment withdrawal, and SVR rates were investigated in each ITPA genotype. RESULTS: In patients with ITPA CC genotype, hemoglobin decline was significantly greater and the percentage of patients in whom total RBV dose was < 60% of standard and/or treatment was withdrawn was significantly higher compared with CA/AA genotype. However, SVR rates were equivalent between CC and CA/AA genotypes, and within a subset of patients with Interleukin 28B (IL28B) (rs8099917) TT genotype, SVR rates tended to be higher in patients with ITPA CC genotype, although the difference was not significant. CONCLUSION: ITPA CC genotype was a disadvantageous factor for Peg-IFNα + RBV treatment in relation to completion rates and RBV dose. However, CC genotype was not inferior to CA/AA genotype for SVR rates. When full-length treatment is accomplished, it is plausible that more SVR is achieved in patients with ITPA CC variant, especially in a background of IL28B TT genotype.

Add-on therapy of pitavastatin and eicosapentaenoic acid improves outcome of peginterferon plus ribavirin treatment for chronic hepatitis C.

Despite the use of pegylated-interferon (peg-IFN) plus ribavirin combination therapy, many patients infected with hepatitis C virus (HCV)-1b remain HCV-positive. To determine whether addition of pitavastatin and eicosapentaenoic acid (EPA) is beneficial, the "add-on" therapy option (add-on group) was compared retrospectively with unmodified peg-IFN/ribavirin therapy (standard group). Association of host- or virus-related factors with sustained virological response was assessed. In HCV replicon cells, the effects of pitavastatin and/or EPA on HCV replication and expression of innate-immunity- and lipid-metabolism-associated genes were investigated. In patients infected with HCV-1b, sustained virological response rates were significantly higher in the add-on than standard group. In both groups, sustained virological response rates were significantly higher in patients with genotype TT of IL-28B (rs8099917) than in those with non-TT genotype. Among the patients with non-TT genotype, sustained virological response rates were markedly higher in the add-on than standard group. By multivariate analysis, genome variation of IL28B but not add-on therapy remained as a predictive factor of sustained virological response. In replicon cells, pitavastatin and EPA suppressed HCV replication. Activation of innate immunity was obvious in pitavastatin-treated cells and EPA suppressed the expression of sterol regulatory element binding protein-1c and low-density lipoprotein receptor. Addition of pitavastatin and EPA to peg-IFN/ribavirin treatment improved sustained virological response in patients infected with HCV-1b. Genotype variation of IL-28B is a strong predictive factor in add-on therapy.

Association between lipid accumulation and the cannabinoid system in Huh7 cells expressing HCV genes.

Evidence from clinical and laboratory studies has accumulated indicating that the activation of the cannabinoid system is crucial for steatosis, especially in non-alcoholic fatty liver disease. However, the association between hepatitis C virus (HCV) infection and the cannabinoid system has not been well investigated and it is unclear whether steatosis in chronic hepatitis C develops via activation of the endocannabinoid/cannabinoid receptor signaling pathway. In this study, we examined the expression of a cannabinoid receptor (CB1) and the lipid accumulation in the hepatic Huh7 cell line, expressing HCV genes. We utilized Huh7/Rep-Feo-1b cells stably expressing HCV non-structural proteins (NS) 3, NS4, NS5A, and NS5B, as well as Tet-On Core-2 cells, in which the HCV core protein expression is inducible. Significantly higher levels of stored triglycerides were found in Huh7/Rep-Feo-1b cells compared to Huh7 cells. Also, triglyceride accumulation and CB1 receptor expression were down-regulated in Huh7/Rep-Feo-1b cells after HCV reduction by IFNα. Moreover, lipid accumulation appeared to increase after CB1 agonist treatment, while it decreased after CB1 antagonist treatment, although significant differences were not found compared to untreated cells. In Tet-On Core-2 cells, induction of HCV core protein expression did not affect CB1 expression or triglyceride accumulation. The results of this study in cultured cells suggest that HCV infection may activate the cannabinoid system and precede steatosis, but the core protein by itself may not have any effect on the cannabinoid system.

Expression profile of lipid metabolism-associated genes in hepatitis C virus-infected human liver.

AIM: Recent studies have shown that lipid metabolic pathways are required for the entry, replication and secretion of hepatitis C virus (HCV). Although little is known about the life cycle of HCV in humans, the activation of cholesterol and fatty acid biosynthesis may be critical for HCV proliferation. METHODS: We assessed the transcription levels of genes essential for cholesterol and fatty acid biosynthesis in liver samples obtained from patients with chronic hepatitis C and determined their correlations. The serum levels of low-density lipoprotein (LDL) cholesterol and HCV core antigen were also measured. RESULTS: The gene expression of the LDL receptor (LDLR) was suppressed, whereas that of SREBP1c, liver X receptor-α (LXRα), fatty acid synthase (FASN), and HMG-CoA reductase and synthase (HMGR and HMGS) was significantly increased, and SREBP2 transcription was comparable in HCV-infected liver compared with normal liver. Positive correlations were found for LDLR versus HMGR, HMGR versus SREBP1c, and LDLR versus SREBP2 in the HCV-infected and control liver. Although the LXRα-SREBP1c-FASN pathway was upregulated, proteasome activator 28γ (PA28γ) was downregulated at the transcriptional level in HCV-infected liver, and was not significantly correlated with the other genes examined. The serum LDL cholesterol level was negatively correlated with LDLR and HMGR expression. CONCLUSION: 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.

Changes in the expression of cholesterol metabolism-associated genes in HCV-infected liver: a novel target for therapy?

Recent investigations indicate that hepatitis C virus (HCV) infection is closely associated with hepatocytic lipid metabolism and induces hepatic steatosis. However, the actual lipid metabolism in HCV-infected liver has not been extensively investigated in humans. In this study, we evaluated the expression of lipid metabolism-associated genes in patients with HCV infection by real-time PCR. Sterol regulatory element-binding protein (SREBP)-2 expression was unchanged and low density lipoprotein receptor expression was markedly reduced by 90% in HCV-infected liver. The expression of apolipoprotein B100, microsomal triglyceride transfer protein and ATP-binding cassette G5 was significantly increased. Up-regulation of cholesterol synthesis-associated genes, including HMG-CoA reductase, HMG-CoA synthase, farnesyl-diphosphate synthase and squalene synthase, confirmed enhanced de novo cholesterol synthesis. The expression of cholesterol 7alpha-hydroxylase and farnesoid X receptor was enhanced, while bile salt export pump expression was unchanged. Fatty acid synthase expression was increased which was accompanied by increased expression of liver X receptor alpha and SREBP-1c. In summary, the regulation of lipid metabolism was impaired and cholesterol and fatty acid synthesis continued to increase without negative feedback in HCV-infected liver. These changes may be beneficial for HCV replication.

The state of cholesterol metabolism in the liver of patients with primary biliary cirrhosis: the role of MDR3 expression.

AIM: Because dyslipidemia, such as hypercholesterolemia, is a characteristic of primary biliary cirrhosis (PBC), hepatic lipid metabolism may be disturbed in PBC patients. We examined the expression of lipid metabolism-associated genes in PBC liver. METHODS: All of the patients examined were in stage I or II PBC and without medication. RNA was isolated from liver specimens by needle biopsies of PBC patients and controls. The expression levels of various genes were measured by real-time RT-PCR. Multidrug resistance 3 (MDR3) expression was examined immunohistochemically. Statistical correlations between the gene expression levels and indices of blood testing were calculated. RESULTS: The expression levels of sterol regulatory element-binding protein (SREBP) 2 and LDL receptor were significantly lower, and those of apolipoprotein B, microsomal triglyceride transfer protein, ATP-binding cassette G5, and liver X receptor α (LXRα) were significantly higher in the PBC liver than in the normal control liver. The expression levels of bile acid synthesis- and excretion-associated genes did not change, and those of farnesoid X receptor, peroxisome proliferator-activated receptor α, and SREBP-1c were similar between the PBC and normal liver. MDR3 gene expression levels in the PBC liver were more than 4-fold higher than those in the control liver. Immunohistochemically, strong canalicular staining for MDR3 was observed in the PBC liver. LXRα expression was positively correlated with MDR3 levels. Serum levels of γ-glutamyl transpeptidase (GGT) and IgM were negatively correlated with MDR3 levels. CONCLUSIONS: Hepatocellular cholesterol metabolism was at least partially disturbed, even in the early stage of PBC. The most characteristic finding was a distinct elevation of MDR3 expression, and the MDR3 levels were negatively correlated with GGT and IgM levels.

Impact of cholesterol metabolism and the LXRalpha-SREBP-1c pathway on nonalcoholic fatty liver disease.

We previously studied fatty acid metabolism in the liver of nonalcoholic fatty liver disease (NAFLD) and reported the activation of the LXRalpha-SREBP-1c pathway in hepatocytes. LXRalpha regulates cholesterol metabolism as well as fatty acid metabolism, and its agonistic ligands are oxysterols. Moreover, there is some evidence that excess cholesterol intake is involved in the onset of NAFLD. Therefore, in this study, we examined the expression of cholesterol metabolism-associated genes in the NAFLD liver by real-time PCR. Expression of LXRalpha and ACAT1 was up-regulated in NAFLD and this was more noticeable in non-obese rather than in obese patients. Although the expression of the LDL receptor, which acts on cholesterol uptake, and of SREBP-2, a positive key regulator of cholesterol, was suppressed, the expression of enzymes that promote cholesterol synthesis was uniformly increased in NAFLD. Gene expression of apoB100 and microsomal triglyceride transfer protein, which are associated with VLDL secretion, and ABCG5, which is involved in cholesterol excretion, was significantly elevated in NAFLD. Because cholesterol accumulates in hepatocytes in NAFLD liver, cholesterol uptake and synthesis should be physiologically down-regulated. However, cholesterol synthesis was activated in NAFLD liver, meaning that cholesterol metabolism is dysregulated in NAFLD. Overproduction of cholesterol may lead to an increased level of oxysterols, activation of LXRalpha and SREBP-1c, and enhanced fatty acid synthesis.

The significance of differences in fatty acid metabolism between obese and non-obese patients with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is considered to be associated with metabolic syndrome; however, a number of NAFLD patients are not obese. To explore any differences in lipid metabolism between obese and non-obese patients, we determined the expression of fatty acid metabolism-related genes. Expression levels of target genes were quantified by real-time PCR using liver biopsy samples from NAFLD patients and normal controls. Serum adipocytokine levels were also determined. The expression of genes related to fatty acid synthesis and uptake was generally up-regulated in NAFLD patients; however, no significant difference was seen between obese and non-obese groups. Most of the genes tested related to fatty acid and reactive oxygen species (ROS) elimination, were overexpressed in NAFLD and the levels were significantly higher in non-obese patients. As an exception, peroxisome proliferator-activated receptor alpha expression was suppressed in NAFLD and the levels were lower in the obese group. Triglyceride synthesis-related genes were up-regulated and lipolytic enzymes were decreased in NAFLD, but there was no significant difference between the obese and non-obese groups. In NAFLD, increased de novo synthesis and uptake of fatty acids led to further hepatocyte accumulation of fatty acids. The up-regulation of fatty acid oxidation and the antioxidant pathway and the suppression of lipolysis seemed to be involved in this process. Expression of genes related to fatty acid oxidation and ROS elimination were higher in the non-obese group than in the obese group, which contributes to the trend of more severe liver injury, insulin resistance and steatosis in obese patients.

SREBP-1c, regulated by the insulin and AMPK signaling pathways, plays a role in nonalcoholic fatty liver disease.

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 AMP-activated 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, up-regulates fatty acid oxidation.

Re-evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease.

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 alpha (HADHalpha), uncoupling protein 2 (UCP2), straight-chain acyl-CoA oxidase (ACOX), branched-chain acyl-CoA oxidase (BOX), cytochrome P450 2E1 (CYP2E1), CYP4A11, and peroxisome proliferator-activated receptor (PPAR)alpha 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), PPARgamma, 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, HADHalpha, UCP2, ACOX, BOX, CYP2E1, and CYP4A11, were all overexpressed, indicating that oxidation was enhanced in NAFLD, whereas the expression of CTP1a and PPARalpha 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 PPARgamma 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 (beta-oxidation), peroxisomal (beta-oxidation) and microsomal (omega-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.