Exendin­4, a glucagon­like peptide­1 receptor agonist, modulates hepatic fatty acid composition and Δ­5­desaturase index in a murine model of non­alcoholic steatohepatitis.

Glucagon­like peptide­1 (GLP­1) is involved in the development of non­alcoholic steatohepatitis (NASH), which is characterized by fatty acid imbalance. The aim of this study was to investigate the effects of the GLP­1 receptor (GLP­1R) agonist, exendin­4 (Ex­4), on hepatic fatty acid metabolism and its key enzyme, Δ­5­desaturase, in a murine model of NASH. NASH was induced in db/db mice fed a methionine­choline deficient (MCD) diet. Ex­4 (n=4) or saline [control (CON); n=4] was administered intraperitoneally for 8 weeks. Steatohepatitis activity was evaluated by non­alcoholic fatty liver disease (NAFLD) activity score. Hepatic fatty acid composition and Δ­5­desaturase index were analyzed by gas chromatography. Ex­4 treatment significantly reduced body weight and the NAFLD activity score. Hepatic concentrations of long­chain saturated fatty acids (SFAs) were significantly higher in the Ex­4 group compared to the CON group (23240±955 vs. 31710±8436 µg/g•liver, P<0.05).Ex­4 significantly reduced hepatic n­3 polyunsaturated fatty acid (PUFA)/n­6 PUFA ratio compared to the CON group (13.83±3.15 vs. 8.73±1.95, P<0.05). In addition, the hepatic Δ­5­desaturase index was significantly reduced in the Ex­4 group compared to the CON group (31.1±12.4 vs. 10.5±3.1, P<0.05). In conclusion, the results showed that Ex­4 improved steatohepatitis in a murine model of NASH. Furthermore, Ex­4 altered hepatic long­chain saturated and PUFA composition and reduced the Δ­5­desaturase index. Thus, Ex­4 may improve NASH by regulating hepatic fatty acid metabolism.

Dipeptidyl peptidase-4: a key player in chronic liver disease.

Dipeptidyl peptidase-4 (DPP-4) is a membrane-associated peptidase, also known as CD26. DPP-4 has widespread organ distribution throughout the body and exerts pleiotropic effects via its peptidase activity. A representative target peptide is glucagon-like peptide-1, and inactivation of glucagon-like peptide-1 results in the development of glucose intolerance/diabetes mellitus and hepatic steatosis. In addition to its peptidase activity, DPP-4 is known to be associated with immune stimulation, binding to and degradation of extracellular matrix, resistance to anti-cancer agents, and lipid accumulation. The liver expresses DPP-4 to a high degree, and recent accumulating data suggest that DPP-4 is involved in the development of various chronic liver diseases such as hepatitis C virus infection, non-alcoholic fatty liver disease, and hepatocellular carcinoma. Furthermore, DPP-4 occurs in hepatic stem cells and plays a crucial role in hepatic regeneration. In this review, we described the tissue distribution and various biological effects of DPP-4. Then, we discussed the impact of DPP-4 in chronic liver disease and the possible therapeutic effects of a DPP-4 inhibitor.

Decreased expression of insulin and increased expression of pancreatic transcription factor PDX-1 in islets in patients with liver cirrhosis: a comparative investigation using human autopsy specimens.

BACKGROUND: Glucose intolerance in patients with liver cirrhosis (LC), known as hepatogenous diabetes, is thought to be distinct from type 2 diabetes (T2DM) in some aspects. Hyperinsulinemia and/or insulin resistance in liver disease is associated with hepatocarcinogenesis, growth of hepatocellular carcinoma, and poor prognosis. However, the pathophysiological processes in islets that are responsible for hyperinsulinemia in LC are still not precisely known. Therefore, we investigated the histopathological differences in islets of Langerhans cells between LC and T2DM. METHODS: A total of 35 human autopsy pancreatic tissue samples were used in this study (control, n = 18; T2DM, n = 6; LC, n = 11). The expression of insulin, glucagon, somatostatin, pancreatic duodenal homeobox-1 (PDX-1), proliferating cell nuclear antigen (PCNA), and Ki-67 was examined using immunohistochemistry and quantitated by image analysis. RESULTS: Islet hypertrophy and a significant increase in PCNA-positive cells in islets were observed in the tissues from LC cases. The insulin-positive areas in islets were significantly decreased in LC cases compared with control and T2DM cases (P = 0.001, P = 0.035, respectively), whereas the PDX-1-positive area was significantly increased in LC cases (P = 0.001) compared with the control. Furthermore, disorganization of pancreatic endocrine cells and nucleocytoplasmic translocation of PDX-1 were both seen in the LC subjects. CONCLUSIONS: In LC, islets undergo hypertrophy and exhibit paradoxical expression of insulin and PDX-1. In the subjects autopsied, insulin expression was decreased, whereas expression of the pancreatic transcription factor PDX-1 was increased in LC. These results point to important distinctions between LC and T2DM.

Hepatitis C virus core protein upregulates the expression of vascular endothelial growth factor via the nuclear factor-κB/hypoxia-inducible factor-1α axis under hypoxic conditions.

AIM: Hepatitis C virus (HCV) core protein critically contributes to hepatocarcinogenesis, which is often observed in liver cirrhosis. Since the liver cirrhosis microenvironment is affected by hypoxia, we focused on the possible driving force of HCV core protein on signal relay from hypoxia-inducible factor (HIF)-1α to vascular endothelial growth factor (VEGF). METHODS: Human hepatocellular carcinoma cells stably overexpressing HCV core (Core cells) and NS5A (NS5A cells) were established; empty vector-transfected (EV) cells were used as controls. Hypoxia was induced by oxygen deprivation or by using cobalt chloride (CoCl(2) ). YC-1 was used to inhibit HIF-1α expression. Protein analyses for cultured cells and liver tissues obtained from CoCl(2) -treated HCV core-transgenic (Core-Tg) mice were performed by western blot and/or immunocytochemistry. Cellular mRNA levels were evaluated by quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Under hypoxia, the sustained expression of HIF-1α, but not HIF-2α, was profoundly observed in Core cells but, was faint in EV and NS5A cells. Immunocytochemistry revealed increased HIF-1α in the nucleus. HIF-1α mRNA levels were significantly higher in Core cells than in EV cells under both normoxia and hypoxia. The HIF-1α-targeted VEGF and Bcl-xL expressions were increased in Core cells under hypoxia and abolished by YC-1 treatment. Hypoxic liver samples of Core-Tg mice indicated significant increases in both HIF-1α and VEGF expression compared with the wild type. CONCLUSIONS: Hepatitis C virus core protein has the distinct potential to transcriptionally upregulate and sustain HIF-1α expression under hypoxia, thereby contributing to increased VEGF expression, a key regulator in the hypoxic milieu of liver cirrhosis.

Interaction of endothelial progenitor cells expressing cytosine deaminase in tumor tissues and 5-fluorocytosine administration suppresses growth of 5-fluorouracil-sensitive liver cancer in mice.

The drug delivery system to tumors is a critical factor in upregulating the effect of anticancer drugs and reducing adverse events. Recent studies indicated selective migration of bone marrow-derived endothelial progenitor cells (EPC) into tumor tissues. Cytosine deaminase (CD) transforms nontoxic 5-fluorocytosine (5-FC) into the highly toxic 5-fluorouracil (5-FU). We investigated the antitumor effect of a new CD/5-FC system with CD cDNA transfected EPC for hepatocellular carcinoma (HCC) in mice. We used human hepatoma cell lines (HuH-7, HLF, HAK1-B, KYN-2, KIM-1) and a rat EPC cell line (TR-BME-2). Escherichia coli CD cDNA was transfected into TR-BME-2 (CD-TR-BME). The inhibitory effect of 5-FU on the proliferation of hepatoma cell lines and the inhibitory effect of 5-FU secreted by CD-TR-BME and 5-FC on the proliferation of co-cultured hepatoma cells were evaluated by a tetrazolium-based assay. In mouse subcutaneous xenograft models of KYN-2 and HuH-7, CD-TR-BME was transplanted intravenously followed by 5-FC injection intraperitoneally. HuH-7 cells were the most sensitive to 5-FU and KYN-2 cells were the most resistant. CD-TR-BME secreted 5-FU and inhibited HuH-7 proliferation in a 5-FC dose-dependent manner. CD-TR-BME were recruited into the tumor tissues and some were incorporated into tumor vessels. Tumor growth of HuH-7 was significantly suppressed during 5-FC administration. No bodyweight loss, ALT abnormality or bone marrow suppression was observed. These findings suggest that our new CD/5-FC system with CD cDNA transfected EPC could be an effective and safe treatment for suppression of 5-FU-sensitive HCC growth.

Quick and simple method for increasing the reduced albumin fraction in human serum albumin preparations by using stronger neo-minophagen C.

AIM: Serum albumin exists in both oxidized and reduced forms. Reduced albumin shows higher antioxidative effects; however, the percentage of reduced albumin is low in human serum albumin (HSA) preparation. Stronger neo-minophagen C (SNMC) containing cysteine, a nucleophilic amino acid, has the potential to control the redox state of albumin. The aim of this study was to develop a method for increasing the fraction of reduced albumin in HAS preparation using SNMC. METHODS: Human serum albumin preparations were purchased from four different manufacturers. As a reducing agent, SNMC (50, 100, or 200 µL) was added to 62.5 mg of each HSA preparation, and the mixture was incubated at room temperature for 10-480 min. The percentages of reduced albumin were determined by high-performance liquid chromatography. RESULTS: The percentage of reduced albumin in the HSA preparation significantly increased 15 min after treatment with 200 µL of SNMC (manufacturer A; 27.7 ± 0.18% vs. 78.7 ± 0.36%, P < 0.01), and a dose-dependent relationship was observed between the increase in the percentage of reduced albumin and dose of SNMC. Similar results were obtained with the other three HSA preparations. The percentage of reduced albumin reached its peak at 15 min after mixing SNMC with an HSA preparation, and then gradually decreased with duration, irrespective of the dose of SNMC. CONCLUSIONS: We devised a method for increasing the reduced albumin fraction in an HSA preparation by using SNMC. We also determined the time-and dose-differences in the effect of SNMC on redox state of HSA.

Insulin resistance and chronic liver disease.

Increased insulin resistance is frequently associated with chronic liver disease and is a pathophysiological feature of hepatogenous diabetes. Distinctive factors including hepatic parenchymal cell damage, portal-systemic shunting and hepatitis C virus are responsible for the development of hepatogenous insulin resistance/diabetes. Although it remains unclear whether insulin secretion from pancreatic beta cells is impaired as it is in type 2 diabetes, retinopathic and cardiovascular risk is low and major causes of death in cirrhotic patients with diabetes are liver failure, hepatocellular carcinoma and gastrointestinal hemorrhage. Hemoglobin A1c is an inaccurate marker for the assessment and management of hepatogenous diabetes. Moreover, exogenous insulin or sulfonylureas may be harmful because these agents may promote hepatocarcinogenesis. Thus, pathogenesis, cause of death, assessment and therapeutic strategy for hepatogenous insulin resistance/diabetes differ from those for lifestyle-related type 2 diabetes. In this article, we review features of insulin resistance in relationship to chronic liver disease. We also discuss the impact of anti-diabetic agents on interferon treatment and hepatocarcinogenesis.

Hybrid training of voluntary and electrical muscle contractions reduces steatosis, insulin resistance, and IL-6 levels in patients with NAFLD: a pilot study.

BACKGROUND: Physical inactivity is a risk factor for the development of non-alcoholic fatty liver disease (NAFLD). "Hybrid training", a training that involves both voluntary and electrical muscle contractions, causes beneficial alterations in muscles even after short durations of exercise. The aim of this study was to investigate the therapeutic efficacy of hybrid training in patients with NAFLD. METHODS: Thirty-five patients with NAFLD who were resistant to lifestyle counseling were assigned to a hybrid-training group (n = 12) or a control group (n = 23). In the hybrid-training group, quadriceps and hamstrings were contracted voluntarily or electrically for 19 min twice a week. In the control group, patients received lifestyle counseling. The therapeutic efficacy of the hybrid training was evaluated after 12 weeks of the intervention. RESULTS: Serum alanine aminotransferase (ALT) levels and hepatic steatosis grade were significantly decreased in the hybrid-training group compared to that of the control group (-14.1 ± 5.8 vs. 3.5 ± 5.4 IU/mL; P < 0.05, -0.67 ± 0.19 vs. 0.09 ± 0.06 grade; P < 0.01, respectively). No significant changes were seen between the two groups in skeletal muscle mass. The decreases in homeostasis model assessment of insulin resistance (HOMA-IR) value and in serum IL-6 levels were significantly greater in the hybrid-training group than in the control group (-6.2 ± 3.2 vs. 0.4 ± 0.6; P < 0.05, -3.1 ± 1.1 vs. 1.1 ± 0.5 pg/mL; P < 0.01, respectively). CONCLUSION: Hybrid training of voluntary and electrical muscle contractions improved hepatic steatosis and reduced insulin resistance and serum IL-6 levels in NAFLD patients who are resistant to lifestyle counseling.

Subjective global assessment is not sufficient to screen patients with defective hepatic metabolism.

BACKGROUND AND AIMS: Subjective global assessment (SGA) is useful for screening malnourished patients with several diseases, although it has been indicated to underestimate nutritional status for patients with liver disease. Accordingly, the aim of this study was to examine the usefulness of SGA as a nutritional screening tool for patients with liver disease, compared to patients with gastroenterological disease, without bias of personal ability and experience. METHODS: SGA was performed on 129 of hospitalized patients (86 with liver disease and 43 with gastroenterological disease). Nutritional status was categorized as well-nourished or malnourished status, based on nutritional indicators from laboratory data. RESULTS: The SGA screening ratio (sensitivity) for malnourished patients with liver disease was significantly lower than gastroenterological disease, while specificity or efficiency was not significantly different. In nutritional indicators from laboratory data, the difference between SGA-positive and SGA-negative patients with liver disease was significant but not so remarkable compared with the difference between those with other diseases. The positive number of SGA components per patient for the liver disease group was significantly less than gastroenterological disease group. CONCLUSIONS: SGA for patients with liver diseases was not sufficient as a nutritional screening tool because malnutrition induced by defective hepatic metabolism was not characterized fully.

The pathogenesis, complications and therapeutic strategy for hepatitis C virus-associated insulin resistance in the era of anti-viral treatment.

Recent experimental and clinical studies have shown that chronic hepatitis C virus (HCV) infection causes insulin resistance. Since insulin resistance decreases response to antiviral treatments, promotes inflammatory and fibrogenic reactions and increases a risk of hepatocellular carcinoma (HCC), amelioration of insulin resistance may be a novel therapeutic target, which could improve the prognosis in patients with HCV-related chronic liver disease. Despite the increased awareness of health risk of insulin resistance, there is no common therapeutic strategy for HCV-associated insulin resistance. Indeed, treatments with exogenous insulin or sulfonylureas may be rather harmful because these treatments are associated with the development of HCC in patients with HCV infection. Meanwhile, we, along with others, have found distinctive treatments which improve HCV-associated insulin resistance. Administration of branched-chain amino acids (BCAA), especially as a late evening snack, improves glucose metabolism by improving insulin-signal cascades in insulin resistance patients with HCV infection. In this paper, we discuss the pathogenesis and complications for HCV-associated insulin resistance and further review a recent clinical therapeutic strategy using these agents for the treatment of this devastating disorder. We also discuss therapeutic potentialities of incretin-based therapies, new anti-diabetic agents for HCV-associated insulin resistance and the significance of insulin resistance in the era of new anti-viral treatments.