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Background and aims Thrombin, the active enzyme of the coagulation system, plays a critical role in the pathogenesis of atherosclerosis. Vascular repair promoted by stromal cell-derived factor-1 is a protective process in atherosclerosis. Consumption of low amount of alcohol is believed to reduce the risk of atherosclerotic cardiovascular disease but the mechanism is unclear. This study evaluated whether alcohol can modulate the expression of stromal cell-derived factor-1 and the pro-atherosclerotic activity of thrombin. Methods Hepatocytes, monocytes, vascular endothelial and vascular smooth muscle cells were pre-treated with increasing concentrations of ethanol before stimulation with thrombin. The expression of cytokines, chemokines, cell adhesion molecules and epigenetic factors, including histone deacetylases and sirtuins, was evaluated. Results Thrombin stimulation significantly enhanced the expression of pro-inflammatory cytokines, chemokines and cell adhesion molecules, but significantly decreased the expression of stromal cell-derived factor-1. Pre-treatment of cells with a low dose of ethanol significantly decreased thrombin-induced production of pro-inflammatory cytokines and chemokines, and significantly increased the production of stromal cell-derived factor-1 compared to cells treated with thrombin alone. Ethanol significantly counteracted the decreased expression of histone deacetylases and sirtuins induced by thrombin. Inhibition of histone deacetylase-2 with trichostatin A or with specific siRNA abolished the stimulatory activity of low-dose ethanol on stromal cell-derived factor-1. Conclusions Low-dose of ethanol attenuates the inflammatory response and counteracts the reduced expression of stromal cell-derived factor-1 induced by thrombin via an epigenetic mechanism, providing a potential explanation for the protective activity of low dose of alcohol in atherosclerosis.

Objective Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity, dyslipidemia and type-2 diabetes mellitus. Bile acids (BAs) bind to the farnesoid X receptor (FXR) and G protein-coupled receptor 5 (TGR5), which are involved in lipid and glucose metabolism and energy expenditure. The present study aimed to determine associations between the circulating BAs and the skeletal muscle volume (SMV), and lipid and glucose metabolism in patients with NAFLD. Methods Serum BAs and metabolic parameters were measured in 55 patients with NAFLD (median age, 55 years). The changes (Δ) in serum BA (ΔBA) and metabolic parameters were determined in 17 patients (male, n=10; female, n=7) who received nutritional counseling for 12 months. Results Spearman's test revealed that the levels of 12α-hydroxysterol (12α-OH) BAs, including deoxycholic acid (DCA), were inversely correlated with the SMV of the upper and lower limbs and the total SMV. A multivariate analysis revealed that the level of DCA was correlated with a reduced total SMV, whereas non-12α-OH BAs, including chenodeoxycholic acid (CDCA), were correlated with an increased SMV of the lower limbs. Changes in CDCA were positively correlated with the ΔSMV of the lower limbs, and inversely correlated with the Δwaist-hip ratio and Δtotal cholesterol. Changes in the total non-12α-OH BA level were positively correlated with the ΔSMV of the lower limbs. Conclusion Circulating BAs were associated with SMV. The 12α-OH BAs, including DCA were associated with reduced SMV levels, whereas non-12α-OH BAs including CDCA were associated with increased SMV levels. The molecular mechanisms underlying the association between the BA levels and the SMV remain to be explored.

The aim of this study was to clarify the relationships among psychometric testing results, blood ammonia (NH3) levels, electrolyte abnormalities, and degree of inflammation, and their associations with the development of overt hepatic encephalopathy (HE) in liver cirrhosis (LC) patients. The relationships between covert HE and blood NH3, sodium (Na), and C-reactive protein (CRP) were examined in 40 LC patients. The effects of elevated NH3, hyponatremia, and elevated CRP on the development of overt HE were also investigated. The covert HE group had significantly lower serum Na levels and significantly higher serum CRP levels. During the median observation period of 11 months, 10 patients developed overt HE, and the results of multivariate analysis showed that covert HE and elevated blood NH3 were factors contributing to the development of overt HE. Electrolyte abnormalities and mild inflammation are involved in the pathogenesis of HE. Abnormal psychometric testing results and hyperammonemia are linked to subsequent development of overt HE.

Protein S is an anticoagulant factor that also regulates inflammation and cell apoptosis. The effect of protein S on diabetes and its complications is unknown. This study compared the development of diabetes between wild-type and transgenic mice overexpressing human protein S and the development of diabetic glomerulosclerosis between mice treated with and without human protein S and between wild-type and protein S transgenic mice. Mice overexpressing protein S showed significant improvements in blood glucose level, glucose tolerance, insulin sensitivity, and insulin secretion compared with wild-type counterparts. Exogenous protein S improved insulin sensitivity in adipocytes, skeletal muscle, and liver cell lines in db/db mice compared with controls. Significant inhibition of apoptosis with increased expression of BIRC3 and Bcl-2 and enhanced activation of Akt/PKB was induced by protein S in islet β-cells compared with controls. Diabetic wild-type mice treated with protein S and diabetic protein S transgenic mice developed significantly less severe diabetic glomerulosclerosis than controls. Patients with type 2 diabetes had significantly lower circulating free protein S than healthy control subjects. This study shows that protein S attenuates diabetes by inhibiting apoptosis of β-cells and the development of diabetic nephropathy.

Diabetes mellitus (DM), non-alcoholic fatty liver (NAFL), and obesity are associated with elevated branched-chain amino acid (BCAA) levels, but the mechanism and significance of this has not been elucidated. Eighty-four subjects were enrolled including 43 with DM. Serum BCAA levels were positively correlated with waist-hip ratio and ALT. Serum BCAA levels in subjects with DM were higher than non-DM and those in subjects with NAFL were also higher than non-NAFL. Treatment with pioglitazone and alogliptin (19 of 43 DM subjects) improved serum haemoglobin A1c and decreased BCAA levels. The decrease in BCAAs with improved glucose metabolism suggests that abnormal glucose metabolism is also a factor in elevated BCAA levels.

Transarterial chemoembolization (TACE) and radiofrequency ablation (RFA) are effective treatments for hepatocellular carcinoma (HCC). However, the extent of treatment depends on hepatic functional reserve. L-Carnitine is a vitamin-like substance and several reports have described the usefulness of L-carnitine supplementation in cases of cirrhosis, with confirmed effectiveness against refractory hepatic encephalopathy. On the other hand, we have previously reported that in patients who underwent TACE or RFA, administration of branched-chain amino acids (BCAAs) pre-intervention significantly reduced inflammatory reactions. We first determined serum levels of total, free, and acyl-carnitine before and at 7 d after performing TACE in 10 HCC patients. We administered levocarnitine (L-carnitine chloride, a biologically active form of carnitine) at 900 mg/d to 69 consecutive HCC patients hospitalized to undergo TACE and/or RFA, and compared changes in blood test values with those in 119 consecutive patients not administered this drug. Sixty-seven patients had a history of using BCAAs at the time of admission. We found that after 7 d of TACE, serum levels of total and acyl-carnitine are significantly decreased. On comparing the four groups, the carnitine+BCAA, carnitine-alone, and BCAA-alone groups showed significantly higher values for changes in NH3 when compared with the non-dosed group. The decrease in albumin (Alb) was significantly suppressed in the carnitine+BCAA and BCAA-alone groups. We also conducted the same examinations in a subset of patients classified as Child-Pugh class A, and noted the same trends. Administration of levocarnitine and/or BCAAs during invasive treatments reduced blood NH3 concentrations and suppressed decreases in Alb.

Background Chronic progression of diabetes is associated with decreased pancreatic islet mass due to apoptosis of β-cells. Patients with diabetes have increased circulating matrix metalloproteinase-2 (MMP2); however, the physiological significance has remained elusive. This study tested the hypothesis that MMP2 inhibits cell apoptosis, including islet β-cells. Methods Samples from diabetic patients and newly developed transgenic mice overexpressing human MMP2 (hMMP2) were harnessed, and diabetes was induced with streptozotocin. Results Circulating hMMP2 was significantly increased in diabetic patients compared to controls and significantly correlated with the serum C-peptide levels. The diabetic hMMP2 transgenic mice showed significant improvements in glycemia, glucose tolerance and insulin secretion compared to diabetic wild type mice. Importantly, the increased hMMP2 levels in mice correlated with significant reduction in islet β-cell apoptosis compared to wild-type counterparts, and an inhibitor of hMMP2 reversed this mitigating activity against diabetes. The increased activation of Akt and BAD induced by hMMP2 in β-cells compared to controls, links this signaling pathway to the anti-apoptotic activity of hMMP2, a property that was reversible by both an hMMP2 inhibitor and antibody against integrin-β3. Conclusion Overall, this study demonstrates that increased expression of hMMP2 may attenuate the severity of diabetes by protecting islet β-cells from apoptosis through an integrin-mediated activation of the Akt/BAD pathway.

Aims: Chronic hepatitis C virus (HCV) infection is often associated with glucose metabolic disorders and iron overload. It has recently been shown that reactive oxygen species (ROS) increase gluconeogenesis in hepatocytes through the forkhead box-containing protein O subfamily-1 (FOXO1)-dependent pathway. The aim of this study is proving a cause-and-effect relationship between iron-mediated ROS production and insulin resistance (IR) in chronic hepatitis C (CH-C) patients. Methods: The study included 42 patients with CH-C (22 males and 20 females, median age 53 years). Homeostasis model assessment of insulin resistance (HOMA-IR) value was assessed for each patient at entry. Gene expression levels in the biopsied liver tissues were determined by quantitative reverse transcription-polymerase chain reaction (RT- PCR). In addition, the effect of ROS on gluconeogenesis was assessed using HepG2 cells treated with a well-known ROS generator, diethylmaleate (DEM). Results: The serum ferritin levels were significantly correlated with the serum aspartate aminotransferase level, alanine aminotransferase level, HOMA-IR value, grade of fatty accumulation, total hepatic iron score, and 8-OH-deoxy-2’-guanosine (8-OHdG)-positive cell count. FOXO1 expression was correlated with 8-OHdG-positive cell count, phosphoenolpyruvate carboxykinase (PEPCK) expression, and HOMA-IR. In HepG2 cells, the gene transcription of FOXO1 and PEPCK was increased by DEM treatment, which was associated with an increase in non-phosphorylated FOXO1 protein in the nuclear fraction. Conclusions: Iron-mediated ROS production enhances gluconeogenesis through the FOXO1-mediated pathway and is an affecting factor to IR in patients with CH-C.

Objective Although the prognosis is known to be poor in cirrhosis patients associated with sarcopenia, the relationships among skeletal muscle, visceral fat, and the liver have not yet been thoroughly investigated. Therefore, the prognosis and its associations with body composition and the severity of liver disease were examined in patients with cirrhosis. Methods The skeletal muscle mass and visceral fat area were measured in 161 patients with cirrhosis, the effects of body composition on the prognosis were analyzed, and any factors that contribute to changes in body composition were assessed. Results During the mean observation period of 1,005 days, 73 patients died. Patients with sarcopenia or sarcopenic obesity had a poor prognosis, and this difference was pronounced in the subset of patients classified as Child-Pugh class A. A decreased skeletal muscle mass was strongly correlated with decreased serum albumin levels. Sarcopenia is a common feature of advanced cirrhosis, and transitions were observed from normal body composition to sarcopenia and from obese to sarcopenic obesity. Conclusion The body composition is a prognostic factor for cirrhosis, and a better body composition may be advantageous for obtaining a long-term survival in patients with cirrhosis.

Chronic hepatic encephalopathy is a characteristically reversible neuropsychiatric disorder that occurs mainly in patients with liver cirrhosis. The brain regions critically involved in the pathophysiology of cirrhosis are not clear. Magnetic resonance imaging (MRI) with voxel-based morphometry (VBM) is a valuable tool for evaluating structural brain changes in many neurodegenerative diseases. We performed an MRI scan on 18 patients with liver cirrhosis and 16 age-matched healthy controls. We evaluated brain regional structural changes, regional differences and the relationship of these changes with the blood levels of ammonia and the results of neuropsychological tests in patients with cirrhosis. The VBM showed reduction in the volume of gray matter in the cerebellum and occipital lobe and in the volume of white matter in the cingulate, parietal, temporal, occipital lobe and precentral area in cirrhotic patients compared with controls. There were significant correlations between the volume of these regions with the plasma levels of ammonia and the results of neuropsychological tests. Voxel-based analysis of MRI revealed evidence for structural abnormalities of brain in patients with cirrhosis. Abnormal function in the above regions may account for the ammonia-mediated changes and neuropsychological deficits in hepatic encephalopathy.

Ninety seven patients with chronic hepatitis C (CHC) and 72 with non-alcoholic fatty liver disease (NAFLD) were enrolled. Increased visceral fat area (VFA) was associated with high values of HbA1c. The variables associated with a high risk of new-onset diabetes had a VFA>101 cm(2) in CHC, but not in NAFLD.

Cachexia, or disease-related loss of muscle mass, is a complication of chronic liver disease that modifies its clinical course. The aim of this study was to determine whether improvement in liver function and cachexia through control of the hepatitis B virus (HBV) increases skeletal muscle mass.The blood tests and cross-sectional area (mm(2)) of the psoas major muscle on computed tomography were measured before and after long-term entecavir therapy (median, 39 mo; range, 14-76 mo) in patients with hepatitis B (17 men, 13 women; mean age, 63 ± 13 y).The anti-HBV effect was good in 30 patients given entecavir, and most patients had undetectable serum HBV-DNA levels (93%) and alanine aminotransferase normalization (83%) within a median of 32 mo. Overall, no significant change in the area of the psoas major muscle was seen in any of the patients, although a significant increase was seen when limited to cases of protein malnutrition defined as serum albumin (Alb)4 g/dL. A positive correlation was seen for the amount of change (Δ) in the psoas major muscle and the amount of change (Δ) in Alb.The present findings suggest that skeletal muscle mass may fluctuate in parallel with Alb levels. An improvement in low muscle mass may thus be expected from antiviral therapy for viral liver disease, especially in patients with cachexia.

Summary Background Alcohol consumption is a major cause of liver injury but the mechanisms are not completely understood. Protein S (PS) is an anticoagulant glycoprotein with multiple functions. The role of PS in liver injury is unknown. Objectives This study investigated the role of PS in acute alcoholic hepatitis. Methods A mouse overexpressing human PS (hPS-TG) was generated in which acute hepatitis was induced by intraperitoneal injection of ethanol. Results The levels of serum liver enzymes and liver tissue inflammatory cytokines and the degree of hepatic steatosis were significantly increased in hPS-TG mice treated with ethanol compared with ethanol-treated wild type (WT) mice. Cell expansion, activation and inhibition of apoptosis were significantly augmented in natural killer T (NKT) cells from hPS-TG mice compared with WT mice. Liver mononuclear cells from hPS-TG mice express higher levels of inflammatory cytokines than those from WT mice after stimulation with a specific stimulant of NKT cells in vitro. In a co-culture system of hepatocytes and NKT cells, the effects of PS on ethanol-mediated cell injury were suppressed by a CD1d neutralizing antibody. Alcoholic liver injury was significantly improved in mice pre-treated with PS siRNA and anti-protein S antibody compared with control mice. Patients with alcoholic hepatitis showed significantly increased plasma PS levels and enhanced liver expression of PS and CD1d compared with controls. Conclusions The results of this study suggest that PS exacerbates acute alcoholic hepatitis by inhibiting apoptosis of activated NKT cells.

Iron overload and hyperglycemia are common findings in patients with chronic hepatitis C (CHC). The aim of this study was to determine the relation of serum ferritin and hepatic hepcidin expression with glucose metabolic parameters and to evaluate whether long term phlebotomy lowers the risk of new-onset diabetes in CHC patients.Hepatic hepcidin mRNA expression was measured in 28 CHC patients and their relation with clinical parameters and histological findings was evaluated. Ninety-two patients without type 2 diabetes were divided into two groups: a phlebotomy group underwent an initial period of phlebotomy and maintenance phlebotomy was performed; data obtained in CHC patients that declined to receive phlebotomy were used as control.Hepatic hepcidin expression was positively correlated with body mass index and glucose metabolic parameters. A total number of five patients had onset of type 2 diabetes over 38.9 months of follow-up. Long-term phlebotomy tended to lower the risk of new-onset diabetes compared with control CHC patients. In addition, high ferritin levels predicted further episodes of diabetes in control patients.Iron overload is a risk for the development of diabetes in patients with CHC and that reduction of body iron stores lowers this risk.

Objectives:Very few reports thus far have clinically elucidated the advantages of a nutrition support team (NST) in the!eld of liver diseases. The present study retrospectively analyzed whether nutrition therapy for liver cirrhosis (LC), performed by a multidisciplinary team that includes registered dieticians, improves survival rates. Methods:In study 1, we compared survival rates between two groups of patients with LC to elucidate the effects of nutrition management by registered dieticians. The!rst group was comprised of 101 patients that received no dietary counseling from a dietician, and the second group was comprised of 133 patients that received nutritional counseling following nutrition assessment. In study 2, we split the patients who received nutritional counseling in study 1 into two groups and compared their survival rates with the objective of investigating the effects of a multidisciplinary team approach on survival rate. The!rst group was comprised of 51 patients that, in addition to regular nutritional counseling given by a dietician, regularly attended courses on liver disease given every 3 to 6 mo. The second group was comprised of 82 patients that did not attend the liver-disease courses. Results:During study 1, 34 patients in the!rst group and 20 patients in the second group died, representing a signi!cant difference (P

Background: A moderate intake of alcohol is associated with lower cardiovascular mortality, and the role of circulating progenitor cells in the beneficial effect of alcohol on atherosclerosis is unclear. The hypothesis of this study was that alcohol ameliorates atherosclerosis by modulating the circulating levels of stromal cell-derived growth factor (SDF)-1 and vascular progenitor cells. Methods and Results: Atherosclerosis was induced by infusion of angiotensin II in apolipoprotein-E deficient mice, which were treated with high and low doses of ethanol for 28 days by intraperitoneal injection. Mice treated with low-dose ethanol had significantly less dilatation and fewer atheromatous lesions than mice receiving the high-dose ethanol. The number of circulating fibrocytes was significantly lower in mice treated with high-dose ethanol compared with mice with atherosclerosis untreated with ethanol. The plasma CXCL12/SDF-1 level was significantly increased in mice treated with low-dose ethanol compared with mice treated with a high dose, and the plasma concentration of transforming growth factor-β1 was significantly increased in mice treated with high-dose ethanol compared with control mice. Ethanol regulated the secretion of SDF-1 and vascular endothelial growth factor from fibroblasts in a dose-dependent and bimodal fashion. Conclusions: The circulating level of CXCL12/SDF-1 may be involved, at least in part, in the differential effects of alcohol consumption on atherosclerosis. (Circ J 2011; 75: 2269-2279)

Sir,The authors read with great interest the article by Dr Kukla et al. about the association of the serum levels of fibroblast growth factor-21 (FGF21) and retinol-binding protein 4 (RBP4) with hi...

Long-term supplementation with branched-chain amino acids (BCAA) is associated with prolonged survival and decreased frequency of development of hepatocellular carcinoma (HCC) in patients with liver cirrhosis. However, the pharmaceutical mechanism underlying this association is still unclear. We investigated whether continuous BCAA supplementation increases survival rate of rats exposed to a fibrogenic agent and influences the iron accumulation, oxidative stress, fibrosis, and gluconeogenesis in the liver. Further, the effects of BCAA on gluconeogenesis in cultured cells were also investigated. A significant improvement in cumulative survival was observed in BCAA-supplemented rats with advanced cirrhosis compared to untreated rats with cirrhosis (P