Masatake Tanaka

Liver X receptor in cooperation with SREBP-1c is a major lipid synthesis regulator in nonalcoholic fatty liver disease

Aim:  Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of liver dysfunction and its incidence has increased markedly. However, the mechanisms involved in the pathogenesis of NAFLD in humans have not been thoroughly investigated. Sterol regulatory element binding protein (SREBP)‐1c and carbohydrate responsive element binding protein (ChREBP) are transcriptional factors that regulate the expression of lipogenic genes, including acetyl‐CoA carboxylases (ACCs) and fatty acid synthase (FAS). SREBP‐1c and ChREBP are transactivated by liver X receptor (LXR), a nuclear receptor that regulates the metabolism of cholesterol and fatty acids. To understand the mechanisms involved in the pathogenesis of NAFLD, we investigated the transcriptional factors and lipogenic genes activated in the liver with NAFLD.

Increased hepatic expression of dipeptidyl peptidase-4 in non-alcoholic fatty liver disease and its association with insulin resistance and glucose metabolism

Dipeptidyl peptidase-4 (DPP4) is a serine protease that degrades glucagon-like peptide-1 (GLP-1), an incretin hormone that stimulates insulin secretion from pancreatic β-cells. DPP4 is also involved in the regulation of T cell-mediated inflammatory processes. These properties of DPP4 suggest that it may play a role in the progression of non-alcoholic fatty liver disease (NAFLD). Hepatic DPP4 mRNA expression levels were analyzed by real-time PCR using liver biopsy samples from 17 NAFLD patients and 10 healthy subjects. In NAFLD patients, we also examined correlations between DPP4 expression levels and metabolic factors, including homeostasis model assessment-insulin resistance (HOMA-IR), body mass index (BMI), and serum cholesterol and triglyceride levels. To examine the potential effects of nutritional factors, DPP4 expression levels were analyzed in HepG2 cells subjected to various culture conditions. Hepatic DPP4 mRNA expression was significantly greater in NAFLD patients than in control subjects. DPP4 expression levels were negatively correlated with HOMA-IR and positively correlated with serum cholesterol levels. In HepG2 cells, high glucose significantly enhanced DPP4 expression, whereas insulin, fatty acids and cholesterol did not. Increased hepatic expression of DPP4 in NAFLD may be associated with metabolic factors, including insulin resistance, and may adversely affect glucose metabolism in this liver disease.

Potential role of branched‐chain amino acids in glucose metabolism through the accelerated induction of the glucose‐sensing apparatus in the liver

Branched‐chain amino acids (BCAAs) have a potential to improve glucose metabolism in cirrhotic patients; however, the contribution of liver in this process has not been clarified. To estimate the effect of BCAA on glucose metabolism in liver, we evaluated the mRNA expression levels of glucose‐sensing apparatus genes in HepG2 cells and in rat liver after oral administration of BCAA. HepG2 cells were cultured in low glucose (100 mg/dl) or high glucose (400 mg/dl) in the absence or presence of BCAA. The mRNA expression levels and protein levels of GLUT2 and liver‐type glucokinase (L‐GK) were estimated using RT‐PCR and immunoblotting. The expression levels of transcriptional factors, including SREBP‐1c, ChREBP, PPAR‐γm and LXRα, were estimated. The mRNA expression levels of transcriptional factors, glycogen synthase, and genes involved in gluconeogenesis were evaluated in rat liver at 3 h after the administration of BCAA. BCAA accelerated the expression of GLUT2 and L‐GK in HepG2 cells in high glucose. Expression levels of ChREBP, SREBP‐1c, and LXRα were also increased in this condition. BCAA administration enhanced the mRNA expression levels of L‐GK, SREBP‐1c, and LXRα and suppressed the expression levels of G‐6‐Pase in rat liver, without affecting the expression levels of glycogen synthase or serum glucose concentrations. BCAA administration enhanced the bioactivity of the glucose‐sensing apparatus, probably via the activation of a transcriptional mechanism, suggesting that these amino acids may improve glucose metabolism through the accelerated utility of glucose and glucose‐6‐phosphate in the liver. J. Cell. Biochem. 112: 30–38, 2011.

Reduction of fatty acid oxidation and responses to hypoxia correlate with the progression of de-differentiation in HCC

The prognosis of patients with hepatocellular carcinoma (HCC) may be improved by novel treatments focusing on the characteristic metabolic changes of this disease. Therefore, we analyzed the biological interactions of metabolic features with the degree of tumor differentiation and the level of malignant potential in 41 patients with completely resectable HCC. The expression levels in resected samples of mRNAs encoded by genes related to tumor metabolism and metastasis were investigated, and the correlation between these expression levels and degrees of differentiation was analyzed. Of the 41 patients, 2 patients had grade I, 27 had grade II, and 12 had grade III tumors. Reductions in the levels of 3-hydroxyacyl-CoA dehydrogenase (HADHA) and acyl-CoA oxidase (ACOX)-2 mRNAs, and increases in pyruvate kinase isoenzyme type M2 (PKM2) mRNA were significantly correlated with the progression of de-differentiation. Analysis of partial correlation coefficients showed that the level of PKM2 mRNA expression was significantly correlated with those of pro-angiogenic genes, vascular endothelial growth factor (VEGF) and ETS-1. Moreover, the levels of VEGF-A and ETS-1 mRNA expression were independently correlated with that of the epithelial-mesenchymal transition (EMT)‑related gene SNAIL. These findings suggest that reductions in fatty acid oxidation and responses to hypoxia may affect the progression of malignant phenotypes in HCC.

Sphincter of Oddi motor activity in patients with stones in gall‐bladder, common bile duct or intrahepatic duct and the effect of morphine

Abstract Sphincter of Oddi phasic motor activity and common bile duct pressure were investigated in controls (seven patients) and in patients with gall‐bladder stones (five patients), common bile duct stones (15 patients), or intrahepatic stones (13 patients). There were no significant differences in amplitude and frequency of the phasic activity or the common bile duct pressure between the controls and disease groups. Basal pressure of the phasic contraction, however, was significantly lower in patients with common bile duct or intrahepatic stones than in the controls or gall‐bladder stone group. The administration of morphine, known to cause spasm of the sphincter of Oddi, increased the basal pressure and frequency of the phasic waves in all groups, while the amplitude remained unchanged. Response to morphine in patients with common bile duct or intrahepatic stones was similar to the controls. However, the basal pressure in these latter groups was lower than in the controls, even after stimulation by morphine. The high incidence of bacterial growth in bile from these patients hitherto reported may be attributable to ascending infection possibly resulting from the low basal pressure of the sphincter of Oddi.

Exenatide improves hepatic steatosis by enhancing lipid use in adipose tissue in nondiabetic rats

AIM To investigate the metabolic changes in skeletal muscle and/or adipose tissue in glucagon-like peptide-1-induced improvement of nonalcoholic fatty liver disease (NAFLD). METHODS Male Wistar rats were fed either a control diet (control group) or a high-fat diet (HFD). After 4 wk, the HFD-fed rats were subdivided into two groups; one group was injected with exenatide [HFD-Ex(+) group] and the other with saline [HFD-Ex(-) group] every day for 12 wk. The control group received saline and were fed a control diet. Changes in weight gain, energy intake, and oxygen consumption were analyzed. Glucose tolerance tests were performed after 8 wk of treatment. Histological assessments were performed in liver and adipose tissue. RNA expression levels of lipid metabolism related genes were evaluated in liver, skeletal muscle, and adipose tissue. RESULTS Exenatide attenuated weight gain [HFD-Ex(-) vs HFD-Ex(+)] and reduced energy intake, which was accompanied by an increase in oxygen consumption and a decrease in the respiratory exchange ratio [HFD-Ex(-) vs HFD-Ex(+)]. However, exenatide did not affect glucose tolerance. Exenatide reduced lipid content in the liver and adipose tissue. Exenatide did not affect the expression of lipid metabolism-related genes in the liver or skeletal muscle. In adipose tissue, exenatide significantly upregulated lipolytic genes, including hormone-sensitive lipase, carnitine palmitoyltransferase-1, long-chain acyl-CoA dehydrogenase, and acyl-CoA oxidase 1 [HFD-Ex(-) vs HFD-Ex(+)]. Exenatide also upregulated catalase and superoxide dismutase 2 [HFD-Ex(-) vs HFD-Ex(+)]. CONCLUSION In addition to reducing appetite, enhanced lipid use by exenatide in adipose tissue may reduce hepatic lipid content in NAFLD, most likely by decreasing lipid influx into the liver.

Antithrombin III injection via the portal vein suppresses liver damage

AIM To investigate the effects of antithrombin III (AT III) injection via the portal vein in acute liver failure. METHODS Thirty rats were intraperitoneally challenged with lipopolysaccharide (LPS) and D-galactosamine (GalN) and divided into three groups: a control group; a group injected with AT III via the tail vein; and a group injected with AT III via the portal vein. AT III (50 U/kg body weight) was administrated 1 h after challenge with LPS and GalN. Serum levels of inflammatory cytokines and fibrin degradation products, hepatic fibrin deposition, and hepatic mRNA expression of hypoxia-related genes were analyzed. RESULTS Serum levels of alanine aminotransferase, tumor necrosis factor-α and interleukin-6 decreased significantly following portal vein AT III injection compared with tail vein injection, and control rats. Portal vein AT III injection reduced liver cell destruction and decreased hepatic fibrin deposition. This treatment also significantly reduced hepatic mRNA expression of lactate dehydrogenase and heme oxygenase-1. CONCLUSION A clinically acceptable dose of AT III injection into the portal vein suppressed liver damage, probably through its enhanced anticoagulant and anti-inflammatory activities.

A high prevalence of extreme hyperferritinemia in acute hepatitis patients

Although the mechanism underlying acute liver failure (ALF) has not been clarified, recent reports indicate overactivation of macrophages is involved in its progression. In diseases in which activated macrophages participate in the progression, elevated serum ferritin concentration counts among the characteristic laboratory findings. If activated macrophages play a key role in the development of ALF, serum ferritin levels might reflect the severity of acute liver injury. To confirm this, we evaluated the correlation between the serum ferritin concentration and other laboratory measurements in patients with acute hepatitis including ALF. One hundred consecutive patients with acute liver injury were enrolled, of whom 19 fulfilled the criteria for ALF. Serum ferritin concentrations correlated with serum alanine transferase activity as a whole. Interestingly, the correlation was strong in patients infected by hepatitis viruses, but weak in others. Although most patients with ALF had high levels of serum ferritin, not a few patients without ALF showed similar results. The serum ferritin level was generally increased in acute hepatitis patients, probably reflecting the degree of macrophage activation in the liver. Overactivation of macrophages appears to be essential, but not sufficient, for the development of ALF.

Intrahepatic microcirculatory disorder, parenchymal hypoxia and NOX4 upregulation result in zonal differences in hepatocyte apoptosis following lipopolysaccharide- and D-galactosamine-induced acute liver failure in rats.

Although the mechanisms responsible for acute liver failure (ALF) have not yet been fully elucidated, studies have indicated that intrahepatic macrophage activation plays an important role in the pathogenesis of ALF through intrahepatic microcirculatory disorder and consequent parenchymal cell death. Intrahepatic microcirculatory disorder has been demonstrated in animal models using intravital microscopy; however, the limitations of this method include simultaneously evaluating blood flow and the surrounding pathological changes. Therefore, in this study, we devised a novel method involving tetramethylrhodamine isothiocyanate (TRITC)-dextran administration for the pathological assessment of hepatic microcirculation. In addition, we aimed to elucidate the mechanisms through which intrahepatic microcirculatory disorder progresses with relation to activated macrophages. ALF was induced in Wistar rats by exposure to lipopolysaccharide and D-galactosamine. Intrahepatic microcirculation and microcirculatory disorder in zone 3 (pericentral zone) of the livers of rats with ALF was observed. Immunohistochemical examinations in conjunction with TRITC-dextran images revealed that the macrophages were mainly distributed in zone 2 (intermediate zone), while cleaved caspase-3-positive hepatocytes, pimonidazole and hypoxia-inducible factor 1-α were abundant in zone 3. We also found that 4-hydroxy-2-nonenal and nicotinamide adenine dinucleotide phosphate oxidase (NOX)4-positive cells were predominantly located in the zone 3 parenchyma. The majority of apoptotic hepatocytes in zone 3 were co-localized with NOX4. Our results revealed that the apoptotic cells in zone 3 were a result of hypoxic conditions induced by intrahepatic microcirculatory disorder, and were not induced by activated macrophages. The increased levels of oxidative stress in zone 3 may contribute to the progression of hepatocyte apoptosis.

Acute liver failure caused by drug-induced hypersensitivity syndrome associated with hyperferritinemia

Drug-induced hypersensitivity syndrome (DIHS) is a severe reaction usually characterized by fever, rash, and multiorgan failure, occurring 2-6 wk after drug introduction. It is an immune-mediated reaction involving macrophage and T-lymphocyte activation and cytokine release. A 54-year-old woman was diagnosed with rheumatic arthritis and initiated salazosulfapyridine by mouth. About 10 d later, she had a high fever, skin rash and liver dysfunction. She was admitted to hospital and diagnosed with a drug eruption. She was treated with oral prednisolone 30 mg/d; however, she developed high fever again and her blood tests showed acute liver failure and cytopenia associated with hyperferritinemia. She was diagnosed with acute liver failure and hemophagocytosis caused by DIHS. She was transferred to the Department of Medicine and Bioregulatory Science, Kyushu University, where she was treated with arterial steroid injection therapy. Following this treatment, her liver function improved and serum ferritin immediately decreased. We hypothesized that an immune-mediated reaction in DIHS may have generated over-activation of macrophages and T-lymphocytes, followed by a cytokine storm that affected various organs. The measurement of serum ferritin might be a useful marker of the severity of DIHS.