Tetsuya Sujishi

Lipopolysaccharides accelerate hepatic steatosis in the development of nonalcoholic fatty liver disease in Zucker rats

Nonalcoholic fatty liver disease (NAFLD) can develop into end-stage disease that includes cryptogenic cirrhosis and hepatocellular carcinoma. Bacterial endotoxin, for example lipopolysaccharide (LPS), plays an important role in the pathogenesis of NAFLD. The aim of this study was to assess the role of LPS in the development of NAFLD. Twenty-one male Zucker (fa/fa) rats were divided into three groups: rats fed for twelve weeks on a diet rich in disaccharide (D12 group), rats similarly managed but treated with LPS (LPS group), and those on the same diet for 24 weeks (D24 group). Histological examination demonstrated that this protocol induced hepatic steatosis in the LPS and D24 groups. Significant, marked accumulation of lipid droplets was observed in the LPS group, compared with the D24 group. Rats from the LPS group showed a decrease in plasma adiponectin levels, an increase in plasma leptin levels, and greater expression of FAS and SREBP-1c mRNA in the liver, compared with rats from the D24 group. These finding coincided with histological findings. We therefore suggest that LPS may accelerate the progression of hepatic steatosis.

Sitagliptin can inhibit the development of hepatic steatosis in high-fructose diet-fed ob/ob mice.

The beneficial effect of dipeptidyl peptidase-4 inhibition on diet-induced extra-pancreatic effects, especially on liver tissue remains poorly understood. Thus, we made the experimental designs as follows; five-week-old male ob/ob mice, which develop type 2 diabetic mellitus and nonalcoholic fatty liver disease by taking a high-carbohydrate diet (HCD), were divided into a group in which a HCD was given for 8 weeks as control, and another in which a HCD added with 0.0018% sitagliptin was given for 8 weeks. Hepatic steatosis was seen in all mice, but the mean grade of steatosis in the sitagliptin-administrated mice was significantly decreased. The acetyl-CoA concentrations were lower in sitagliptin-administrated mice, although the differences were not significant. However, the malonyl-CoA concentrations were significantly lower in sitagliptin-administrated mice. The expression of acetyl-CoA carboxylase 1 was inhibited in sitagliptin-administrated mice, irrespective of expressions of carbohydrate responsive element-binding protein (ChREBP) or sterol regulatory element-binding protein (SREBP)-1c. In conclusion, sitagliptin may affect the development of nonalcoholic fatty liver disease by inhibiting the production of malonyl-CoA and thus synthesis of fatty acids in the liver.

Is administrating branched-chain amino acid-enriched nutrition achieved symptom-free in malnourished cirrhotic patients?

Administration of branched-chain amino acids (BCAA) has been reported to improve liver function, quality of life (QOL). However, in some malnourished patients, serum albumin levels do not improve in response to BCAA granules. In this study, we examined the effects of BCAA-enriched enteral nutrition in patients unresponsive to BCAA granules. Thirty-two decompensated cirrhotic patients at Osaka Medical College were enrolled in this study. Since all patients showed no improvement in serum albumin levels despite 3 months of BCAA granule administration, they were administered 50 g of a flavored BCAA-enriched enteral nutrient twice daily, i.e., during the daytime and late evening. Serum albumin levels and major cirrhotic symptoms were examined 1, 3, and 5 months after treatment initiation. Serum albumin levels improved significantly 3 months after treatment initiation (3.14 ± 0.32 g/dl vs 3.5 ± 0.31 g/dl, p<0.01), and Child–Pugh scores decreased significantly (p<0.01). In the majority (53–80%) of patients, muscles cramps, fatigue, fatigability, edema, and sleep disturbance improved within 3 months after therapy initiation. Moreover, approximately 90% of the patients became symptom-free 5 months after treatment initiation. These results indicate that switching to BCAA-enriched nutrients improves QOL of cirrhotic patients unresponsive to BCAA granules.

Su1693 The Clinical Benefit of L-Carnitine Supplement on Muscle Cramp and Peripheral Blood Cell Abnormality in Patients With Liver Cirrhosis

Background: L-carnitine (4-N-trimethyl ammonium 3-hydroxybutyric acid) is a naturally occurring amino acid that functions to transfer long-chain fatty acids across the mitochondrial membrane, enabling oxidative release of energy. The primary sources of l-carnitine are endogenous synthesis by the liver and kidney and dietary intake. It has been shown that cirrhotic patients become gradually l-carnitine deficient because of poor synthesis and reduction in dietary intake. An administration of l-carnitine is an accepted treatment for mitochondrial myopathy and encephalomyopathy, as well as other states of primary and secondary l-carnitine deficiency. In this study, we examined the effect of l-carnitine on muscle cramping and peripheral blood cell abnormality in patients with liver cirrhosis. Methods: A total of 23 cirrhotic patients at Osaka Medical College from December 2011 to October 2012 were enrolled in this study (13 male, mean age 69±9 Y/O, mean Child Pugh score 7.2±1.2). All patients have diagnosed as liver cirrhosis by laboratory data, imaging test or liver biopsy. Patients were administrated a 300mg of l-carnitine tablet twice a day. The evaluation of muscles cramp was investigated by using an original questionnaire and serum creatine kinase (CK) level. Also, peripheral blood cells profile and other liver function parameters were examined 1, 3, 6 months after treatment. Results: Muscle crump was significantly improved in the majority (90%) of patients 1 month after treatment (p , 0.01). Serum CK levels were also significantly decreased after l-carnitine administration (186±121 vs. 106±70 mg/dl, p , 0.05). Blood platelet count was significantly increased 3 months after treatment (10.0±4.5 vs. 11.7±4.0 x104 /dl, p , 0.01). Moreover, serum ammonia levels were significantly decreased 1 month after treatment (136 ±92 μg/dl vs. 64±32 μg/ dl, p, 0.01). Prothrombin time was significantly increased 3 months after treatment (79±14 vs. 84±18, p, 0.05). Serum albumin, bilirubin level and Child-Pugh score were not different after treatment. No serious adverse events have been occurred in this study. Discussion: Currently, l-carnitine supplementation in dialysis patients has been used for the treatment of muscle cramping. On the other hands, l-carnitine has been proposed as a potential adjuvant treatment to improve anemia, thrombocytopenia, leukopenia and immunological function. Thrombocytopenia occurs up to 70% of patients with liver cirrhosis and increases the risk of bleeding. Frequent muscle crump is worsen a QOL of cirrhotic patients. The lcarnitine supplement may offer the possibility of improving the QOL and prognosis in patients with liver cirrhosis.

Effects of Oral L-Carnitine on Liver Functions after Transarterial Chemoembolization in Intermediate-Stage HCC Patients.

Transarterial chemoembolization (TACE) is usually followed by hepatic dysfunction. We evaluated the effects of L-carnitine on post-TACE impaired liver functions. Methods. 53 cirrhotic hepatocellular carcinoma patients at Osaka Medical College were enrolled in this study and assigned into either L-carnitine group receiving 600 mg oral L-carnitine daily or control group. Liver functions were evaluated at pre-TACE and 1, 4, and 12 weeks after TACE. Results. The L-carnitine group maintained Child-Pugh (CP) score at 1 week after TACE and exhibited significant improvement at 4 weeks after TACE (P < 0.01). Conversely, the control group reported a significant CP score deterioration at 1 week (P < 0.05) and 12 weeks after TACE (P < 0.05). L-carnitine suppressed serum albumin deterioration at 1 week after TACE. There were significant differences between L-carnitine and control groups regarding mean serum albumin changes from baseline to 1 week (P < 0.05) and 4 weeks after TACE (P < 0.05). L-carnitine caused prothrombin time improvement from baseline to 1, 4 (P < 0.05), and 12 weeks after TACE. Total bilirubin mean changes from baseline to 1 week after TACE exhibited significant differences between L-carnitine and control groups (P < 0.05). The hepatoprotective effects of L-carnitine were enhanced by branched chain amino acids combination. Conclusion. L-carnitine maintained and improved liver functions after TACE.

Sa1840 Effects of Oral L-Carnitine on Liver Functions After Transarterial Chemoembolization (TACE) in Intermediate Stage Hepatocellular Carcinoma (HCC) Patients.

Background :Transarterial chemoembolization (TACE) is the standard treatment for intermediate stage hepatocellular carcinoma (HCC) and usually followed by hepatic dysfunction that limits its efficacy. L-carnitine (4-N-trimethyl ammonium 3-hydroxybutyric acid) is recently studied as hepatoprotective agent. In this study, we evaluated L-carnitine effects against the deterioration in liver functions after TACE. Study design: 53 sequential patients with intermediate stage HCC at Osaka Medical College enrolled to this study. All patients were treated by TACE and assigned into two groups; L-carnitine group (26 patients) who received L-carnitine 300 mg tablet twice daily from 2 weeks before to 12 weeks after TACE and Control group (27 patients) without L-carnitine therapy. Liver functions were evaluated for all patients at 2 weeks before, 1, 4, 12 weeks after TACE. 28 of study patients received branched chain amino acids granules. Results: L-carnitine suppressed deterioration in serum albumin level at 1 week after TACE. There were significant differences between L-carnitine group and control group in mean serum albumin change from baseline to 1 week and 4 weeks after TACE (p < 0.05). L-Carnitine maintained Child-Pugh score at 1 week after TACE and exhibited significant improvement at 4 weeks after TACE (p < 0.01 compared to 1 week after TACE). Conversely, control group reported significant Child-Pugh score deterioration from baseline to 1 week after TACE (p < 0.05) and 12 weeks after TACE (p < 0.05). There were significant differences between L-carnitine and control groups in mean Child-Pugh score change from baseline to 4 weeks (p < 0.05) and 12 weeks after TACE (p < 0.05). Interestingly, L-carnitine displayed improvement in prothrombin time (PT) from baseline to 1 week, 4 weeks (p < 0.05) and 12 weeks after TACE. Contrariwise, PT in control group declined less than baseline along all follow up intervals. There were significant differences between L-carnitine and control groups in PT mean change from baseline to 1 week (p < 0.05) and 4 weeks after TACE (p < 0.05). Total bilirubin in L-carnitine group decreased at 1 week post TACE while in control group, total bilirubin at 1 week significantly increased (p = 0.01). Mean total bilirubin change from baseline to 1 week after TACE reported significant differences between L-carnitine and control groups (p < 0.05). Alanine transaminase and C-reactive protein elevation at 1 week after TACE were suppressed in Lcarnitine group. The hepatoprotective effects of L-carnitine were enhanced by concomitant use of branched chain amino acids. No side effects appeared by L-carnitine administration in all clinical courses. Conclusion: L-carnitine and BCAA combination therapy maintained and improved liver functions after TACE and may be offered as a new liver support tool in HCC patients.

Mo1050 The Liver Protection Effect of L-Carnitine Administration After TACE on Intermediate-Stage Hepatocellular Carcinoma Patients

Background: Hepatocellular (HCC) and Cholangiocellular carcinomas (CCA) are the two most common primary liver malignancies affecting the population worldwide and associated with dismal outcomes. Venous thrombosis is often present in patients with liver malignancies and complicates clinical management. It is not clear whether HCC and CCA are associated with different patterns of venous thrombosis since HCC is usually associated with cirrhosis which by itself promotes hypercoagulability. The true frequency and distribution of venous thrombosis in the patients with HCC and CCA is also poorly described. Aim: The goal of the study was to assess the differences in frequency and distribution of portal and nonportal venous thrombosis (PVT and NPVT) in patients with HCC and CCA. Methods: We conducted the retrospective review of the medical records of the 1185 consecutive patients with pathology proven HCC and 559 with pathology proven CCA seen at a tertiary care medical center (Mayo Clinic, Rochester, MN, USA) between 2000 and 2013. The frequency of the venous thrombosis and baseline demographic characteristics were analyzed. Results: Among patients with HCC, 45.6% (540/1185) had associated venous thrombosis. This was comparable to patients with CCA (35.4%; 198/559). Portal venous thrombosis was present in 31% of patients with HCC and 23.4% of patients with CCA. Non-portal venous thrombosis was observed twice less frequently as compared to portal VT and was present in 14.2% of patients with HCC and 12% of patients with CCA. The average age of patients with HCC was similar in portal and non-portal venous thrombosis groups (60 versus 59.5 years respectively) and was comparable to those patients with HCC who did not have venous thrombosis (62 years). This was also true for the patients with CCA where average age of patients with portal venous thrombosis was 61.5 years and non-portal venous thrombosis 62.5 years as compared to average age of 61 years in patients with CCA without venous thrombosis. Across all patient groups (HCC: PVT; HCC:NPVT; CCA:PVT; and CCA:NPVT) male to female ratio was approximately 1:2. Interestingly, females with non-portal venous thrombosis were slightly younger in the HCC group as compared to the CCA group (59 and 65 years respectively). Conclusions: The frequencies of portal and non-portal venous thrombosis are similar in patients with HCC and CCA making radiological and clinical differentiation of these malignancies based only on the distribution of venous thrombosis difficult. The similarity in the venous thrombotic pattern in HCC and CCA points toward a common pathogenesis, and is not likely to be increased by the underlying cirrhosis in HCC.

Mo1772 Lipopolysaccharides Can Accelerate the Development of Nonalcoholic Fatty Liver Disease, Caused by the Up-Regulation of Lipogenic Gene Expressions

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.

Mo1774 Sitagliptin Can Inhibit the Acceleration of the Development in NAFLD Caused by Lipopolysaccharides Through Inhibiting Expression of MYD88 and NfκB

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.

Mo1991 Sitagliptin Inhibits the Development of Hepatic Steatosis by Down-Regulated Expressions of Hepatic Acetyl-CoA Carboxyrase (ACC) and Fatty Acid Synthase (Fas)

Aims; Hepatic steatosis is a hallmark of nonalcoholic fatty liver disease (NAFLD) and a key factor of obesity-related metabolic dysfunctions featuring dyslipidemia, insulin resistance, and loss of glycemic control. Although sitagliptin (dipeptidyl peptidase-4 inhibitior) is a treatment drug used for type 2 diabetic mellitus (T2DM) patients, the role of sitagliptin in the development NAFLD has not yet been well known. It has yet to be completely understood how much dysregulated de novo lipogenesis contributes to the pathogenic development of hepatic steatosis. We investigated whether the treatment of sitagliptin is metabolically connected to hepatic steatosis. Methods: Five-week-old male ob/ob mice, which develop T2DM and NAFLD by taking a high-carbohydrate diet (HCD), were divided into a group in which a HCD was given for 8 weeks (Control group; n=6) as controls, and another in which a HCD added with 0.0018% sitagliptin was given for 8 weeks (Sitagliptin group; n=6). Results: Histological comparison between the liver of Control group and that of Sitagliptin group showed significantly more inhibition of hepatic steatosis than in Sitagliptin group. It was suggested that sitagliptin inhibited the development of hepatic steatosis in the mice induced by histologically. The enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) regulate the synthesis of fatty acids. Sitagliptin reduced the hepatic expressions of ACC and FAS, markedly inhibited hepatic de novo lipogenesis, and protected against hepatic steatosis in ob/ob mice. Conclusion: These findings suggested that sitagliptin has effects of downregulated hepatic ACC and FAS in lipid metabolism. Therefore, sitagliptin may serve as a potential target to treat NAFLD and T2DM.