Shigeyuki Takeshita

A snack enriched with oral branched-chain amino acids prevents a fall in albumin in patients with liver cirrhosis undergoing chemoembolization for hepatocellular carcinoma

Nutritional support may play an important role in management of liver cirrhosis (LC) associated with unresectable hepatocellular carcinoma (HCC). Total protein and albumin deteriorate in patients with LC undergoing trans-arterial chemoembolization (TACE). Therefore, in this study, we examined the hypothesis that short-term administration of branched-chain amino acids (BCAA) will prevent a fall in total protein and albumin in the perioperative period. The subjects were 56 patients who underwent TACE for HCC between 2004 and 2005 at Nagasaki University Hospital. The patients were randomly placed in the BCAA group (n = 28) or a control group (n = 28). The patients in the BCAA group consumed a snack containing 50 g of BCAA once a day at 10:00 pm starting 1 day before TACE and continuing until 2 weeks after TACE. A comparison of baseline and end point data showed greater decreases in the concentrations of total protein, albumin, cholinesterase, and total cholesterol and in the red blood cell count in the control group compared to the BCAA group. Ammonia levels decreased in the BCAA group and increased in the control group. Our findings indicate that a BCAA supplement taken orally as a late evening snack prevents suppression of liver function by TACE in patients with LC complicated with HCC during the 2-week period after TACE.

Predictive value of suppressor of cytokine signal 3 (SOCS3) in the outcome of interferon therapy in chronic hepatitis C

Aims:  Suppressor of cytokine signaling 3 (SOCS3) can suppress Janus kinase (JAK)‐signal transducers and activators of transcription (STAT) signaling by blocking an IFN‐induced protein. In this study, the relationship between SOCS3 and phosphorylation of STAT1 in the liver and outcome of interferon therapy were examined.

Interferon-α-induced mTOR activation is an anti-hepatitis C virus signal via the phosphatidylinositol 3-kinase-Akt-independent pathway

ObjectThe interferon-induced Jak-STAT signal alone is not sufficient to explain all the biological effects of IFN. The PI3-K pathways have emerged as a critical additional component of IFN-induced signaling. This study attempted to clarify that relationship between IFN-induced PI3-K-Akt-mTOR activity and anti-viral action.ResultWhen the human normal hepatocyte derived cell line was treated with rapamycin (rapa) before accretion of IFN-α, tyrosine phosphorylation of STAT-1 was diminished. Pretreatment of rapa had an inhibitory effect on the IFN-α-induced expression of PKR and p48 in a dose dependent manner. Rapa inhibited the IFN-α inducible IFN-stimulated regulatory element luciferase activity in a dose-dependent manner. However, wortmannin, LY294002 and Akt inhibitor did not influence IFN-α inducible luciferase activity. To examine the effect of PI3-K-Akt-mTOR on the anti-HCV action of IFN-α, the full-length HCV replication system, OR6 cells were used. The pretreatment of rapa attenuated its anti-HCV replication effect in comparison to IFN-α alone, whereas the pretreatment with PI3-K inhibitors, wortmannin and LY294002 and Akt inhibitor did not influence IFN-induced anti-HCV replication.ConclusionIFN-induced mTOR activity, independent of PI3K and Akt, is the critical factor for its anti-HCV activity. Jak independent mTOR activity involved STAT-1 phosphorylation and nuclear location, and then PKR is expressed in hepatocytes.

Study of liver-targeted regulatory T cells in hepatitis B and C virus in chronically infected patients.

Introduction: Regulatory T cells (Tregs) play a critical role in chronic viral infections. The role of Tregs in chronic hepatitis B (CHB) and chronic hepatitis C (CHC) is unknown. This study examined the distribution and frequency of forkhead box p3+ (Foxp3+) Tregs in the liver tissue and compared the clinicopathological characteristics of CHB and CHC patients.

Relationship between Regulatory T Cells and the Combination of Pegylated Interferon and Ribavirin for the Treatment of Chronic Hepatitis Type C

Background/Aim: The frequency of regulatory T cells (Tregs) may be related to persistent hepatitis C virus (HCV) infection. We studied the alteration of the Treg ratio in peripheral blood mononuclear cells (PBMCs) from chronic hepatitis C patients during combination therapy compared with the Treg ratio in liver-infiltrating lymphocytes (LILs) before therapy. Method: The study group consisted of 20 patients who were treatment-naive and had high virus titers of HCV genotype 1. Blood samples were collected prior to treatment and at several time points during treatment. All patients received a liver biopsy prior to treatment. Forkhead box P3 (Foxp3)+, CD3+, CD4+ and CD8+ cells in PBMCs and LILs were stained by specific antibodies. Results: Ten patients had a sustained virological response (SVR), and 10 patients were non-responders. The SVR group had a significant increase in the Foxp3+/CD4+ ratio in PBMCs at 8 and 12 weeks as well as a significant decrease in the Foxp3+/CD4+ ratio and increase in the CD8+/Foxp3+ ratio in LILs. Conclusion: The evaluation of Tregs, a potentially significant factor for persistent HCV infection, in LILs prior to treatment and in PBMCs during treatment could predict the result of combination therapy.

Branched-chain amino acid deficiency stabilizes insulin-induced vascular endothelial growth factor mRNA in hepatocellular carcinoma cells.

Abnormal sugar metabolism is closely related to chronic liver diseases, including hepatocellular carcinoma (HCC). We previously reported that fasting hyperinsulinemia is a poor prognostic factor for HCC patients. A recent large‐scale study has shown that long‐term administration of branched chain amino acids (BCAA) reduces the risk of HCC development in obese cirrhotic patients who have been diagnosed with diabetes mellitus, although the mechanism by which it does so is unclear. In this study, we analyzed the expression of vascular endothelial growth factor (VEGF) in HepG2 cells under high‐insulin culture conditions, and examined the effect of BCAA on VEGF expression. VEGF secretion was significantly increased by 200 nM of insulin under BCAA deficient conditions, but it was repressed by the addition of BCAA. BCAA activated the mTOR pathway and increase HIF‐1α expression under high‐insulin culture conditions, however quantitative PCR analysis showed that insulin‐induced expression of VEGF mRNAs (VEGF121 and VEGF165) decreased 2 h after the addition of BCAA. The half‐lives of both VEGF121 and 165 mRNAs were shortened in the presence of BCAA compared to the absence of BCAA. Therefore it is thought that BCAA regulate VEGF expression mainly at the post‐transcriptional level. We also examined which of the Valine, Leucine, and Isoleucine components of BCAA were essential for VEGF mRNA degradation. All three BCAA components were required for acceleration of insulin‐induced VEGF mRNA degradation. These results suggest that administration of BCAA may downregulate VEGF expression in patients who have hyperinsulinemia and are in the process of developing HCC. J. Cell. Biochem. 113: 3113–3121, 2012.

Predictive Value of the Phosphorylation of Signal Transducers and Activators of Transcription in the Outcome of Interferon Therapy for Chronic Hepatitis C

Objective: Signal transducers and activators of transcription (STATs) play a critical role in antiviral defense. To better understand pegylated interferon (IFN)-α and ribavirin combination therapy resistance, we examined the STAT expression in the liver. Methods: We immunostained Phospho-STAT1 (P-STAT1) and Phospho-STAT3 (P-STAT3) in 59 hepatitis C virus (HCV)-infected liver tissues and compared the expression of these STATs proteins and the clinicopathological factors. Results: The number of P-STAT1 observed correlated significantly with the body mass index (BMI; p = 0.03) and homeostatic model assessment (p = 0.007). The number of P-STAT3 observed correlated significantly with the ALT level (p = 0.002) and platelet count (p = 0.002). The number of P-STAT1 nuclei in the sustained virological response (SVR) group was significantly larger than in the non-SVR group (p = 0.003). On multivariance analysis, the number of P-STAT1 nuclei (p = 0.004) and age (p = 0.016) were significant predictors of SVR. Conclusions: P-STAT1 in the liver tissue prior to IFN therapy correlated with an increased BMI and increased insulin resistance, and might be a useful predictor of HCV clearance by IFN therapy. On the other hand, P-STAT3 might play a critical role in the hepatocellular response against inflammatory damage.

Hepatitis C virus kinetics during the first phase of pegylated interferon-α-2b with ribavirin therapy in patients with living donor liver transplantation

Aim:  To identify the problems of pegylated interferon (PEG IFN) with ribavirin therapy against hepatitis C virus (HCV) reinfection in living donor liver transplantation (LDLT) patients. HCV kinetics during the PEG IFN with ribavirin therapy were analyzed in LDLT patients, as well as in chronic hepatitis C (CHC) patients.

Insulin-induced mTOR activity exhibits anti-hepatitis C virus activity

The mammalian target of rapamycin (mTOR) is one of the influential molecules for the anti-hepatitis C virus (HCV) action of interferon (IFN). IFN-induced mTOR activity, independent of phosphatidylinositol-3-kinase (PI3K) and Akt, is a critical factor for anti-HCV activity. mTOR activity is involved in signal transducers and activators of transcription (STAT)-1 phosphorylation and nuclear localization, and then double-stranded RNA-dependent protein kinase (PKR) is expressed in hepatocytes. Insulin (INS) is a major cytokine for metabolism and regulates the PI3K-Akt-mTOR signaling pathway in hepatocytes. Changes in mTOR activity have been reported in chronic HCV-infected patients with excess nutrition and INS resistance. Therefore, this experiment investigated whether INS increases anti-HCV activity via mTOR activity. This study used a genome-length HCV RNA (strain O of genotype 1b) replicon reporter system (OR6), derived from HuH7 cells. OR6 cells were pre-treated with rapamycin or LY294002 or siRNA, and the cells were treated with INS (0-300 nmol/l) or IFN (0-50 IU/ml) for 30 min to 48 h. The cells were lysed and analyses were carried out using the Renilla luciferase assay, western blotting or ELISA. INS induced the anti-HCV effects via mTOR activity, independently of STAT-1 tyrosine phosphorylation, in a dose- and time-dependent manner. INS-induced mTOR activation was found to be PI3K-Akt-dependent in OR6 cells. The combination of IFN and INS had an additive anti-HCV effect. The INS-induced mTOR activity was identified to be an anti-HCV signal independent of the STAT pathway in this study. mTOR activity may be associated with the HCV life cycle. Future studies should, therefore, attempt to identify new agents that activate mTOR to promote anti-HCV activity.

Geranylgeranylacetone has anti-hepatitis C virus activity via activation of mTOR in human hepatoma cells

BackgroundGeranylgeranylacetone (GGA), an isoprenoid compound which includes retinoids, has been used orally as an anti-ulcer drug in Japan. GGA acts as a potent inducer of anti-viral gene expression by stimulating ISGF3 formation in human hepatoma cells. This drug has few side effects and reinforces the effect of IFN when administered in combination with peg-IFN and ribavirin. This study verified the anti-HCV activity of GGA in a replicon system. In addition, mechanisms of anti-HCV activity were examined in the replicon cells.MethodsOR6 cells stably harboring the full-length genotype 1 replicon containing the Renilla luciferase gene, ORN/C-5B/KE, were used to examine the influence of the anti-HCV effect of GGA. After treatment, the cells were harvested with Renilla lysis reagent and then subjected to a luciferase assay according to the manufacturer’s protocol.ResultThe results showed that GGA had anti-HCV activity. GGA induced anti-HCV replicon activity in a time- and dose-dependent manner. GGA did not activate the tyrosine 701 and serine 727 on STAT-1, and did not induce HSP-70 in OR6 cells. The anti-HCV effect depended on the GGA induced mTOR activity, not STAT-1 activity and PKR. An additive effect was observed with a combination of IFN and GGA.ConclusionsGGA has mTOR dependent anti-HCV activity. There is a possibility that the GGA anti-HCV activity can be complimented by IFN. It will be necessary to examine the clinical effectiveness of the combination of GGA and IFN for HCV patients in the future.