Masao Shinohara

Mechanisms of Protection by the Betaine-Homocysteine Methyltransferase/Betaine System in HepG2 Cells and Primary Mouse Hepatocytes

Betaine‐homocysteine methyltransferase (BHMT) regulates homocysteine levels in the liver. We previously reported that the alteration of BHMT is associated with alcoholic liver steatosis and injury. In this study, we tested whether BHMT protects hepatocytes from homocysteine‐induced injury and lipid accumulation. Both BHMT transfectants of HepG2 cells and primary mouse hepatocytes with suppressed BHMT were generated. Comparisons were made between the cell models with respect to their response to homocysteine treatments. Homocysteine metabolism was impaired in HepG2 cells, and the expression of BHMT in HepG2 cells ameliorated the impairment and stabilized the levels of intracellular homocysteine after the addition of exogenous homocysteine. BHMT expression inhibited homocysteine‐induced glucose‐regulated protein 78 (GRP78) and C/EBP‐homologous protein (CHOP) and homocysteine‐induced cell death. A betaine treatment protected primary mouse hepatocytes from a homocysteine‐induced increase in GRP78 and cell death but not a tunicamycin‐induced increase. Homocysteine induced greater CHOP expression (2.7‐fold) in BHMT small interfering RNA (siRNA)–transfected cells than in a control (1.9‐fold). Homocysteine‐induced cell death was increased by 40% in the siRNA‐treated cells in comparison with the control. Apolipoprotein B (apoB) expression was higher and triglycerides and cholesterol were lower in HepG2 expressing BHMT. In primary mouse hepatocytes, homocysteine induced the accumulation of triglycerides and cholesterol, which was reduced in the presence of betaine. Betaine partially reduced homocysteine‐induced sterol regulatory element binding protein 1 expression in HepG2 cells and increased S‐adenosylmethionine in primary mouse hepatocytes. Conclusion: The BHMT/betaine system directly protects hepatocytes from homocysteine‐induced injury but not tunicamycin‐induced injury, including an endoplasmic reticulum stress response, lipid accumulation, and cell death. This system also exhibits a more generalized effect on liver lipids by inducing ApoB expression and increasing S‐adenosylmethionine/S‐adenosylhomocysteine. (HEPATOLOGY 2007.)

Differences in betaine‐homocysteine methyltransferase expression, endoplasmic reticulum stress response, and liver injury between alcohol‐fed mice and rats

Chronic ethanol infusion resulted in greater serum alanine aminotransferase elevation, lipid accumulation, necroinflammation, and focal hepatic cell death in mice than rats. Mice exhibited a remarkable hyperhomocysteinemia but no increase was seen in rats. Similarly, a high‐methionine low‐folate diet (HMLF) induced less steatosis, serum alanine aminotransferase increase, and hyperhomocysteinemia in rats than in mice. Western blot analysis of betaine homocysteine methyltransferase (BHMT) expression showed that rats fed either ethanol or HMLF had significantly increased BHMT expression, which did not occur in mice. Nuclear factor‐κB p65 was increased in mouse in response to alcohol feeding. The human BHMT promoter was repressed by homocysteine in mouse hepatocytes but not rat hepatocytes. BHMT induction was faster and greater in primary rat hepatocytes than mouse hepatocytes in response to exogenous homocysteine exposure. Mice fed ethanol intragastrically exhibited an increase in glucose‐regulated protein 78 and inositol‐requiring enzyme 1, which was not seen in the rat, and sterol regulatory element binding protein 1 was increased to a greater extent in mice than rats. Thus, rats are more resistant to ethanol‐induced steatosis, endoplasmic reticulum stress, and hyperhomocysteinemia, and this correlates with induction of BHMT in rats. Conclusion: These findings support the hypothesis that a critical factor in the pathogenesis of alcoholic liver injury is the enhanced ability of rat or impaired ability of mouse to up‐regulate BHMT which prevents hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury. (HEPATOLOGY 2010.)

Effect of Transgenic Extrahepatic Expression of Betaine-Homocysteine Methyltransferase on Alcohol or Homocysteine-Induced Fatty Liver

BACKGROUND Chronic alcohol feeding induces hyperhomocysteinemia (HHcy). Previously, we reported a protective role of betaine-homocysteine methyltransferase (BHMT) in homocysteine-induced injury in cultured hepatocytes. In this study, we investigated the direct role of BHMT in alcohol or homocysteine-induced liver injury. METHODS Betaine-homocysteine methyltransferase transgenic (Tg) mice were generated. Comparisons were made between the Tg and wild type (WT) mice in their response to intragastric alcohol infusion or to oral feeding of a high methionine low folate diet (HMLF). RESULTS Expression of the Tg BHMT was increased in organs peripheral to the liver. The alcohol infusion for 4 weeks increased: plasma ALT by 5-fold in WT mice and 2.7-fold in Tg mice; plasma homocysteine by 7-fold in WT mice and 2-fold in Tg mice; liver triglycerides by 4-fold in WT mice and 2.5-fold in Tg mice. The alcohol-induced fatty liver was more severe in WT than in Tg mice based on H&E staining. The HMLF feeding for 4 weeks increased plasma ALT by 2-fold in WT mice and 1-fold in Tg mice; plasma homocysteine by 21-fold in WT mice and 3.3-fold in Tg mice; liver triglycerides by 2.5-fold in WT mice and 1.5-fold in Tg mice. HMLF induced accumulation of macro fat droplets in WT but not Tg mice. Betaine supplementation decreased partially the alcohol or HMLF-induced increase of ALT, homocysteine and liver lipids in WT mice. However, Tg mice were normal when fed both HMLF and betaine. In WT mice, both alcohol and HMLF induced moderate increase of sterol regulatory element binding protein 1 (SREBP1) protein which was partially reduced by betaine supplementation. In Tg mice, alcohol but not HMLF increased SREBP1. Carbohydrate responsive element-binding protein was increased by alcohol in either WT or Tg mice which was not affected by betaine supplementation. Ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) was reduced by 50% in WT and by 20% in Tg mice fed alcohol. Ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was reduced in WT but not Tg mice fed alcohol. Changes in PE methyltransferase activities were not detected in response to alcohol or HMLF feeding but were increased by betaine. CONCLUSIONS The BHMT Tg mice are resistant to alcohol or HMLF-induced HHcy and liver steatosis indicating that peripheral metabolism of homocysteine protected the liver without a direct effect of BHMT in the liver. Multiple mechanisms are involved in protection by betaine including increased SAM/SAH and PC/PE ratios.

Human immunodeficiency virus protease inhibitors modulate Ca2+ homeostasis and potentiate alcoholic stress and injury in mice and primary mouse and human hepatocytes†

A portion of human immunodeficiency virus (HIV)‐infected patients undergoing protease inhibitor (PI) therapy concomitantly consume or abuse alcohol leading to hepatic injury. The underling mechanisms are not known. We hypothesize that HIV PIs aggravate alcohol‐induced liver injury through an endoplasmic reticulum (ER) stress mechanism. To address this, we treated mice, primary mouse hepatocytes (PMHs), and primary human hepatocytes (PHHs) with alcohol and the HIV PIs ritonavir (RIT) and lopinavir (LOP). In mice, RIT and LOP induced mild ER stress and inhibition of sarco/ER calcium‐ATPase (SERCA) without significant increase in serum alanine aminotransferase (ALT) levels. However, a single dose of alcohol plus the two HIV PIs caused a more than five‐fold increase in serum ALT, a synergistic increase in alcohol‐induced liver lipid accumulation and ER stress response, and a decrease of SERCA. Mice treated with chronic HIV PIs and alcohol developed moderate liver fibrosis. In PMHs, the HIV drugs plus alcohol also inhibited SERCA expression and increased expression of glucose‐regulated protein 78, C/EBP homologous protein, sterol regulatory element‐binding protein 1c, and phosphorylated c‐Jun N‐terminal kinase 2, which were accompanied by a synergistic increase in cell death compared with alcohol or the HIV drugs alone. In PHHs, treatment with RIT and LOP or alcohol alone increased messenger RNA of spliced X box‐binding protein 1 and decreased SERCA, which were accompanied by reduced levels of intracellular calcium. Alcohol combined with the HIV drugs significantly reduced intracellular calcium levels and potentiated cell death, which was comparable to the cell death caused by the SERCA inhibitor thapsigargin. Conclusion: Our findings suggest the possibility that HIV PIs potentiate alcohol‐induced ER stress and injury through modulation of SERCA and maintaining calcium homeostasis could be a therapeutic aim for better care of HIV patients. (HEPATOLOGY 2012;)

Evaluation of Hemodynamics in Focal Steatosis and Focal Spared Lesion of the Liver Using Contrast-Enhanced Ultrasonography with Sonazoid

We aim to investigate the hemodynamics in focal steatosis and focal spared lesion of the liver using contrast-enhanced ultrasonography (CEUS) with Sonazoid. The subjects were 47 patients with focal steatosis and focal spared lesion. We evaluated enhancement patterns (hyperenhancement, isoenhancement, and hypoenhancement) in the vascular phase and the presence or absence of a hypoechoic area in the postvascular phase for these lesions using CEUS. Of the 24 patients with focal steatosis, the enhancement pattern was isoenhancement in 19 and hypoenhancement in 5. Hypoechoic areas were noted in the postvascular phase in 3 patients. Of the 23 patients with focal spared lesions, the enhancement pattern was isoenhancement in 18 and hyperenhancement in 5. No hypoechoic areas were noted in the postvascular phase in any patient. The hemodynamics in focal steatosis and focal spared lesions in nondiffuse fatty liver can be observed using low-invasive procedures in real-time by CEUS. It was suggested that differences in the dynamics of enhancement in the vascular phase of CEUS were influenced by the fat deposits in the target lesion, the surrounding liver parenchyma, and the third inflow.

726 Type 1 IFN-Alpha Receptor Expression by Peripheral Blood Monocytes During Triple Therapy Influences Rapid Virological Response in Genotype 1b-Infected Patients With Chronic Hepatitis C and High Viral Load

BACKGROUND/AIM: We previously reported that type I IFN receptor alpha-2 (INFAR-2) expression in peripheral blood monocytes (Mo) is related to early virological response (EVR) in patients with chronic hepatitis C (CHC) infected with genotype 1b and having high viral loads (Intervirology 53: 105-110, 2010). The aim of this study was to clarify whether IFNAR2 expression by peripheral blood Mo influences rapid virological response (RVR) in CHC patients infected with genotype 1b, having high viral loads and treated with triple therapy comprising teraprevir (TPV), pegylated (PEG)-interferon (IFN) and ribavirin (RBV). PATIENTS AND METHODS: Twenty-nine adult patients with biopsy-proven CHC were studied. Enrollment criteria included 26 to 82 (median: 58) years of age, baseline serum HCV-RNA quantified by RT-PCR between 5.2 and 7.2 log copies/ml, and infection with HCV genotype 1b. Twenty-four patients (15 males and 9 females) were treated with triple therapy. As a pilot study, TPV alone was administered to 5 patients (4 males and 1 female) for 7 days in order to investigate the effects of TPV for IFNAR-2 expression by peripheral blood Mo. A negative result for serum HCV-RNA on RT-PCR at 4 weeks after starting therapy was defined as RVR. IFNAR-2 expression by peripheral blood Mo was determined using flow cytometry by measuring the mean fluorescence intensity before and up to 14 days after starting triple therapy. IFNAR-2 expression was also determined before and up to 7 days after starting TPV alone. All 29 patients consented to genetic investigation for polymorphisms in the interleukin (IL)-28B gene at rs8099917. Twenty-four patients had genotype TT, 5 patients had genotype TG, and none had genotype GG. RESULTS: RVR was achieved in 19 patients (15 patients with genotype TT and 4 patients with genotype TG) and was not achieved in 5 patients (non-RVR) with genotype TT. Genotype at rs8099917 was not associated with RVR (P=0.65, by χ2 test). IFNAR-2 expression by peripheral blood Mo was not altered at 3 and 7 days after starting therapy, as compared to before therapy in patients receiving TPV alone (4 patients with genotype TT and 1 patient with genotype TG). IFNAR-2 expression by peripheral blood Mo before starting therapy did not differ between RVR patients and non-RVR patients. However, IFNAR-2 expression by peripheral blood Mo after starting therapy was always higher in RVR patients than in non-RVR patients, and IFNAR-2 expression by peripheral bloodMo 3 days after starting therapy was significantly (P , 0.05 on Mann-Whitney test) higher in RVR patients than in non-RVR patients. CONCLUSION: This study showed that IFNAR-2 expression by peripheral blood Mo at day 3 of triple therapy influences RVR in patients infected with genotype 1b and having high viral loads.

Su2039 Changes of Cytokines in Cirrhosis Patients With Advanced Hepatocellular Carcinoma Treated by Sorafenib

PURPOSE:Sorafenib is multi-kinase inhibitor against RAF, involved in the growth of cancer cells and VEGFR(Vascular Endotherial Growth Factor Receptor), involved in angiogenesis around cancer. It is known that tumor stain is reduced by the administration of sorafenib. We retrospectively evaluated whether the decrease of blood flow after the administration of sorafenib affect the overall survival (OS) or not. METHODS:From May 2009 to November 2011, 127 patients out of 201 patients with advanced hepatocellular carcinoma (HCC) were treated with sorafenib and included in the present study. Patients received CE-CT or GdEOB-DTPA-MRI before treatment and every 4~6 weeks were evaluated to find the therapeutic effect. Patients were divided into 3 groups (No change group, partially decrease group, partially disappearance group) of tumor vascularity. We calculated OS of these 3 groups. RESULT:In the decrease group (85 cases, partially decrease group and partially disappearance group), the median OS was 19.6 months (95%C.I. 14.7-24.9). In the no change group (42 cases), the median OS was 8.6 months (95%C.I. 5.6-12.0). There were statistically significant differences between the two groups (p<0.001). In the partially disappearance group (51 cases), the median OS was 19.9 months (95%C.I. 11.3-28.6). In the partially decrease group (34 cases), the median OS was 22.0months (95%C.I. 12.2-32.0). There were no statistically significant differences between the two groups (p=0.59). CONCLUSION:In the treatment of sorafenib for advanced HCC, even if there is no necrosis, decrease of blood flow improves the OS.

Su2043 Serum VEGF Level and the Response to Combined Intra-Arterial Chemotherapy in Patients With Advanced Hepatocellular Carcinoma

A S L D A b st ra ct s 40.1±19.9kPa, p=0.01) and patients with nodules in the right liver lobe compared to the left lobe (46±20.4kPa vs 27.3±10.8kPa, p 15), patients with nodules >50mm had a significantly higher LSM value (71.8 ±4.8kPa vs 42.2±16.1kPa, p=0.001). Conclusion: There is a good correlation between liver stiffness assessed by Fibroscan® and HCC nodule size, in cirrhotic patients. Our data suggests that higher LSM values are registered in the presence of nodules >50mm in patients with low MELD score (< 15). Aggressive screening for HCC in cirrhotic patients with low MELD score and high LSM is mandatory, in order to early detect patients within Milan criteria for liver transplantation.

Su2041 Sorafenib Cancels Mechanisms to Escape From the Host Immune System in Cirrhosis Patients With Advanced Hepatocellular Carcinoma

A S L D A b st ra ct s 40.1±19.9kPa, p=0.01) and patients with nodules in the right liver lobe compared to the left lobe (46±20.4kPa vs 27.3±10.8kPa, p 15), patients with nodules >50mm had a significantly higher LSM value (71.8 ±4.8kPa vs 42.2±16.1kPa, p=0.001). Conclusion: There is a good correlation between liver stiffness assessed by Fibroscan® and HCC nodule size, in cirrhotic patients. Our data suggests that higher LSM values are registered in the presence of nodules >50mm in patients with low MELD score (< 15). Aggressive screening for HCC in cirrhotic patients with low MELD score and high LSM is mandatory, in order to early detect patients within Milan criteria for liver transplantation.