Keishi Kanno

Advanced glycation end products enhance the proliferation and activation of hepatic stellate cells.

BackgroundAdvanced glycation end products (AGEs), final reaction products of protein with sugars, are known to contribute to diabetes-related complications. We have recently demonstrated high levels of serum AGEs in patients with nonalcoholic steatohepatitis (NASH). However, direct evidence for the participation of AGEs in hepatic infl ammation and fibrosis has not been shown. To explore the pathogenesis of NASH, we examined the biological infl uence of AGEs on hepatic stellate cells (HSCs) in vitro.MethodsAn established human HSC line, LI90, was exposed to a glyceraldehyde-derived-AGE (glycer-AGE), and the phenotypical changes of the LI90 cells were investigated. Intracellular formation of reactive oxygen species (ROS) was measured using a fl uorescent probe. Cell proliferation was examined by MTS assay. Fibrogenic marker gene expression was analyzed by quantitative real-time polymerase chain reaction. The production of monocyte chemoattractant protein 1 (MCP-1) was assessed by enzyme-linked immunosorbent assay.ResultsThe expression of AGE receptor was confirmed in LI90 cells at the mRNA and protein levels. In addition to increasing intracellular ROS generation, glycer-AGE upregulated fibrogenic genes such as those encoding for α-smooth muscle actin, transforming growth factor-β1, and collagen type Iα2. The expression of MCP-1 mRNA in LI90 cells as well as its secretion into the culture medium was significantly increased in response to AGEs. These changes were attenuated by treatment with the antioxidant N-acetylcysteine.ConclusionsThese data indicate that AGEs induce ROS generation and intensify the proliferation and activation of HSCs, supporting the possibility that antioxidants may represent a promising treatment for prevention of the development of hepatic fibrosis in NASH.

AT1A-deficient mice show less severe progression of liver fibrosis induced by CCl4

The renin-angiotensin system has been shown to contribute to fibrogenesis in varieties of organs, including the liver. Here, we investigated whether the angiotensin II type 1A receptor (AT1A) is implicated in the development of liver fibrosis, using AT1A-deficient and wild-type (WT) mice. After single dose of carbon tetrachloride (CCl(4)), there were no significant differences between two groups with regard to hepatic inflammation and necrosis. After 4 weeks of treatment with CCl(4), histological examination revealed that AT1A-deficient mice showed less infiltration of inflammatory cells and less severe progression of liver fibrosis compared with WT mice. These findings were accompanied by the hepatic content of hydoxyproline and the expression of alpha-smooth muscle actin (alpha SMA). The level of transforming growth factor-beta 1 (TGF-beta 1) messenger RNA was markedly higher in WT mice when compared with AT1A-deficient mice. These results confirm that signaling via AT1A plays a pivotal role in hepatic fibrogenesis.

Angiotensin II Participates in Hepatic Inflammation and Fibrosis through MCP-1 Expression

In this study, we assessed the hypothesis that angiotensin (Ang) II could modulate inflammatory cell recruitment into the liver through hepatic expression of monocyte chemoattractant protein (MCP)-1 during liver injury. For in vivo study, Ang II type 1a knockout (AT1a KO) mice and wild-type (WT) mice were treated with CCl4 for 4 weeks. After CCl4 treatment, AT1a KO mice showed lower expression of MCP-1 and fewer CD68-positive cells in the liver compared with WT mice. For in vitro study, Ang II was added to LI90 cells. Ang II enhanced MCP-1 mRNA together with RhoA mRNA and also induced secretion of MCP-1 into the culture medium. This change was strongly blocked by Y-27632, a specific Rho-kinase inhibitor. These results suggest that Ang II modulates hepatic inflammation via production of MCP-1 by hepatic stellate cells, and the effect of Ang II on MCP-1 production is, at least partly, mediated by the Rho/Rho-kinase pathway.

Deletion of angiotensin II type I receptor reduces hepatic steatosis

BACKGROUND/AIMS A distinct subgroup of angiotensin II type 1 receptor (AT1R) blockers (ARBs) have been reported to suppress the development of hepatic steatosis. These effects were generally explained by selective peroxisome proliferator-activated receptor (PPAR) gamma modulating properties of ARBs, independent of their AT1R blocking actions. Here, we provide genetic evidence of the direct role for AT1R in hepatic steatosis. METHODS The effect of AT1R deletion on steatohepatitis was investigated in AT1a(-/-) mice. Furthermore, the influence of AT1R inhibition by telmisartan as well as gene silencing of AT1R by siRNA was assessed in an in vitro experiment using HepG2 cells. RESULTS Compared to wild-type (WT), AT1a(-/-) mice fed methionine-choline deficient (MCD) diet resulted in negligible lipid accumulation in the liver with marked induction of PPARalpha mRNA. In vitro experiments also demonstrated reduced cellular lipid accumulation by telmisartan and AT1R knockdown following exposure of long chain fatty acids. This is presumably explained by the observation that the expression of PPARalpha and its target genes were significantly up-regulated in specific siRNA treated HepG2 cells. CONCLUSIONS Our data indicate, in addition to pharmacological effect of ARBs on PPARgamma activation, a key biological role for AT1R in the regulation of hepatic lipid metabolism.

Bach1 gene ablation reduces steatohepatitis in mouse MCD diet model

Bach1 is a transcriptional repressor of heme oxygenase-1 (HO-1, a.k.a. HSP-32), which is an inducible enzyme and has anti-oxidation/anti-inflammatory properties shown in various models of organ injuries. Since oxidative stress plays a pivotal role in the pathogenesis of nonalcoholic steatohepatitis (NASH), HO-1 induction would be expected to prevent the development of NASH. In this study, we investigated the influence of Bach1 ablation in mice on the progression of NASH in methionine-choline deficient (MCD) diet model. Bach1 ablation resulted in significant induction of HO-1 mRNA and its activity in the liver. When fed MCD diet, Bach1−/− mice exhibited negligible hepatic steatosis compared to pronounced steatohepatitis in wild type mice with 6-fold increase in hepatic triglyceride content. Whereas feeding of MCD diet decreased mRNA expressions of peroxisome proliferator-activated receptor (PPAR) α and microsomal triglyceride transfer protein (MTP) in wild type mice, there were no change in Bach1−/− mice. In addition, hepatic concentration of malondialdehyde (MDA), a biomarker for oxidative stress as well as plasma alanine aminotransferase (ALT) was significantly lower in Bach1−/− mice. These findings suggest that Bach1 ablation exerts hepatoprotective effect against steatohepatitis presumably via HO-1 induction and may be a potential therapeutic target.

Phospholipid alterations in hepatocyte membranes and transporter protein changes in cholestatic rat model.

Biliary components are transported by hepatic adenosine triphosphate-binding cassette (ABC) transporters that are located in canalicular membranes. Physiological transporter function is related to membrane fluidity, which is modulated by the phospholipid composition of the lipid bilayer. We hypothesized that cholestasis may alter transporter function by modifying phospholipid species to protect the cell from cholestatic damage. Therefore, we examined the expression of ABC transport proteins and their mRNA levels in canalicular membrane vesicles isolated from rat liver 6 hr or three days after bile duct ligation. Membrane lipid composition and membrane fluidity of both sinusoidal and canalicular membrane vesicles were also examined. By 6 hr after bile duct ligation, we found a clear increase of mdr2 and bsep mRNA. These changes were associated with an increase of mdr-Pgp and with a clear decrease of mrp2 protein, and small decrease of bsep protein. In addition, mdr1b mRNA showed a strong increase by three days after bile duct ligation. Canalicular membrane fluidity decreased in a marked time-dependent manner, whereas sinusoidal membranes showed biphasic changes: increased fluidity at 6 hr and a decrease at three days. These changes were closely related to the changes of membrane lipid constitution; the saturated/unsaturated fatty acid ratio increased for phosphatidylcholine in canalicular membrane and the reverse occurred in sinusoidal membrane, and those for sphingomyelin showed the opposite pattern. We conclude that cholestasis causes modulation of ABC transporters as well as that of the lipid constitution in lipid bilayer. These may confer cytoprotective resistance to hepatocytes against cholestatic stress.

Spontaneously hypertensive rats develop pronounced hepatic steatosis induced by choline‐deficient diet: Evidence for hypertension as a potential enhancer in non‐alcoholic steatohepatitis

Aim:  Patients with non‐alcoholic steatohepatitis (NASH) frequently have many co‐morbidities including essential hypertension, which is reported to increase vascular production of reactive oxygen species (ROS) and alter the hepatic anti‐oxidant defense system. Since ROS play a role in the pathogenesis of NASH, it is hypothesized that hypertension modulates the hepatic oxidative status and influences the development of NASH. The aim of this study was to investigate the potential effects of hypertension on the progression of NASH.

Lack of adiponectin promotes formation of cholesterol gallstones in mice.

Plasma adiponectin levels are reduced in obese people, and hypoadiponectinemia is recently reported to associate with cholesterol gallstone formation in human. The aim of this study was to examine the role of adiponectin in gallstone formation using adiponectin knockout mice. We analyzed male knockout and C57BL6J mice fed normal or lithogenic diet for 6 weeks. On lithogenic diet, the prevalence rate of gallstone was significantly greater in knockout mice than C57BL6J mice. The molar percentages of beta and omega-muricholic acid were significantly higher and hepatic sterol 12 alpha-hydroxylase expression (cyp8b1) was significantly lower in knockout mice than C57BL6J mice fed normal diet. The bile apolipoprotein A-I protein levels were decreased in knockout mice. Histological examination showed gallbladder wall thickening and accumulation of glycoprotein in the gallbladder of knockout mice. Gallbladder phospholipase A2-IVA expression was significantly higher in knockout mice than in C57BL6J mice fed lithogenic diet. Our results indicate that lack of adiponectin promotes gallstone formation in mice.

Unique reciprocal changes of hepatocellular membrane transporter expression and fluidity in rats with selective biliary obstruction

BACKGROUND AND AIMS: The localized occlusion of bile ducts by intrahepaticlithiasis and intrahepatic neoplasms dose not cause severe jaundice, although the reason is unclear. The aim of this study was to clarify the molecular change of the liver in partial cholestasis, especially with respect to transporter expression (Mrp2, Mrp3, Bsep, P-gps) and membrane fluidity. METHODS AND RESULTS: After 3 days of selective bile duct ligation in Sprague-Dawley rats, the obstructed lobe (OL) and non-OL were analyzed. Canalicular and sinusoidal membrane fluidity were decreased in the OL compared with the non-OL. Mrp2 protein expression was significantly lower in the OL (by 62.9+/-6.9%) when compared with the non-OL. Mdr1b and Mdr2 mRNA were up-regulated in the OL when compared with the non-OL. Interestingly, Mrp3 protein and mRNA were induced in both the non-OL and OL of rats without hyperbilirubinemia. CONCLUSIONS: It seems that the reduction of membrane fluidity in the OL is a local response to localized cholestasis, while the induction of Mrp3 observed in both the OL and non-OL may be a response of the whole liver to localized impairment of bile secretion, although the regulatory mechanism is yet to be established.

Report on the 2013 national cholelithiasis survey in Japan.

The Academic Committee of the Japan Biliary Association conducted a national survey of cholelithiasis and analyzed its current management centering on epidemiology.