Tomoji Nishioka

Hepatitis C Virus Core Protein Modulates Fatty Acid Metabolism and Thereby Causes Lipid Accumulation in the Liver

We studied the roles of hepatitis C virus (HCV) core protein in hepatic steatosis and changes in hepatic lipid metabolism. HCV core protein expression plasmid was transfected in HepG2 . Triacylglyceride (TG) and mRNA level associated with lipid metabolism were measured. Male C57BL/6 mice were infected with HCV core recombinant adenovirus and used for lipids and mRNA studies. In HCV core protein-expressing cells, peroxisome proliferator-activated receptor (PPAR)α, multidrug resistance protein (MDR) 3, and microsomal triglyceride transfer protein (MTP) were down-regulated 48 hr after transfection. In HCV core protein-expressing mice, hepatic TG content and hepatic thiobarbituric acid-reactive substances increased. PPARα, MDR2, acyl-CoA oxidase (AOX), and carnitine palmitoyl transferase-1 (CPT-1) were down-regulated. HCV core protein down-regulated lipid metabolism-associated gene expression, Mdr2, CPT, and AOX, accompanied by down-regulation of PPARα. There findings may contribute to the understanding of HCV-related steatosis, induction of reactive oxygen species, and carcinogenesis.

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.

Orlistat treatment increases fecal bilirubin excretion and decreases plasma bilirubin concentrations in hyperbilirubinemic Gunn rats.

OBJECTIVE To determine whether serum levels of unconjugated bilirubin (UCB) can be decreased by enhancing fecal fat excretion. STUDY DESIGN Gunn rats were fed a high-fat diet (control) or the same diet mixed with the lipase inhibitor orlistat. At regular intervals, plasma UCB concentrations were determined and 72-hour fat balances were performed. RESULTS Orlistat treatment decreased plasma UCB concentrations (at 3 weeks; 100 mg/kg, -33%+/-8%, P<.05; 200 mg/kg, -46%+/-10%, P<.01). Within days of treatment, orlistat treatment increased fecal excretion of UCB (at day 3; +220%, P<.05). During 24 weeks of orlistat treatment (200 mg/kg diet), the plasma bilirubin concentrations were continuously approximately 35% lower than in control rats. Plasma UCB concentrations were inversely correlated with the amount of fecal fat excretion (n=12, r=-0.87, P<.001). CONCLUSIONS In Gunn rats, orlistat treatment increases the fecal excretion of fat and enhances the disposal of UCB. This approach could lead to novel strategies for prevention and treatment of unconjugated hyperbilirubinemia in patients.

Unique inhibition of bile salt-induced apoptosis by lecithins and cytoprotective bile salts in immortalized mouse cholangiocytes

Bile duct epithelium is physiologically exposed to high concentrations of bile salts, suggesting the presence of a cytoprotective mechanism(s). The aim of this study was to clarify whether bile salts cause bile duct cell damage and to elucidate the mechanism(s) providing protection against such an action of bile salts. Immortalized mouse cholangiocytes were incubated with taurocholate, taurochenodeoxycholate, glycochenodeoxycholate (GCDC), taurodeoxycholate, and tauroursodeoxycholate (TUDC), followed by flow-cytometric analysis and caspase activity assay to evaluate the induction of apoptosis. GCDC time-dependently induced caspase 3 (3.4-fold)- and caspase 9 (1.4-fold)-mediated apoptosis of cholangiocytes, but this was inhibited by lecithins and TUDC. Further, expression of cholangiocyte bile salt transporters (apical sodium-dependent bile salt transporter [Asbt] and multidrug resistance protein 3 [Mrp3]) was examined by RT-PCR and western blotting, and cholangiocyte bile salt uptake was determined using radiolabeled bile salts. Expression of cholangiocyte Asbt and Mrp3 was increased by bile salts, whereas lecithins interestingly reduced bile salt uptake to inhibit cholangiocyte apoptosis. In conclusion, bile salts themselves cause cholangiocyte apoptosis when absorbed by and retained inside the cell, but this is inhibited by washing out cytotoxic bile salts according to Mrp3, a rescue exporting molecule. Biliary lecithin is seemingly another cytoprotective player against cytotoxic bile salts, reducing their uptake, and this is associated with a reduced expression of Mrp3.

A Combination Therapy With Simvastatin and Ursodeoxycholic Acid Is More Effective for Cholesterol Gallstone Dissolution Than Is Ursodeoxycholic Acid Monotherapy

Inhibitors of 3-hydroxy,3-methylglutaryl coenzyme A (HMG-CoA) reductase have been reported to decrease the cholesterol saturation index (CSI) in duodenal bile in humans and to prevent formation of cholesterol gallstones in animal studies. We performed a prospective study to evaluate the role of HMG-CoA reductase inhibitors as gallstone-dissolving agents. Fifty patients with radiolucent gallstones in a gallbladder opacifying at drip infusion cholecystography were treated with either 10 mg/day simvastatin plus 600 mg/day ursodeoxycholic acid (group 1, n=26) or 600 mg/day ursodeoxycholic acid alone (group 2, n=24) for 12 months. The ratio of solitary to multiple gallstone cases was 21:29. Plasma lipid levels were assessed and ultrasonographic examination of the gallbladder was performed at baseline and at 3-month intervals during treatment. Duodenal bile sampling was performed in five patients in each group at baseline and after 12 months of treatment. Plasma cholesterol decreased significantly in group 1 but not in group 2. In solitary gallstone cases, no significant difference in dissolution rates was observed between groups 1 (3 of 9, 33%) and 2 (4 of 12, 33%). In contrast, the dissolution rate in multiple gallstone cases was significantly higher in group 1 (12 of 17, 71%) than in group 2 (3 of 12, 25%) (p < 0.01). Bile cholesterol saturation index was significantly decreased (p < 0.01) but did not significantly differ between the two groups. These results suggest that combination therapy with simvastatin and ursodeoxycholic acid is more effective for cholesterol gallstone dissolution than ursodeoxycholic acid monotherapy in patients with multiple gallstones.

Therapeutic Effect of Repeated Natural Killer T Cell Stimulation in Mouse Cholangitis Complicated by Colitis

Primary sclerosing cholangitis is often complicated by ulcerative colitis. Recently, we reported on Th1-dominant cholangitis associated with experimental colitis, and natural killer T (NKT) cells might play an important role in this model. The aim of this study was to clarify the immunopathogenic role of NKT cells in this model using α-galactosylceramide. CD-1 mice were administered 2.0% dextran sulfate sodium for 29 days and injection of α-galactosylceramide was performed every 5 days, then inflammation was assessed. Mononuclear cells from the liver were analyzed with respect to cytokine production and the surface marker. α -Galactosylceramide improved survival rate, weight gain, and inflammation score. Also, interferon-γ release from MNC, CD4/CD8 ratio, NKT cell population, and NK cell population were decreased by this treatment. These findings indicate that repeated stimulation of NKT cells modifies the Th1/Th2 balance to reduce Th1 dominance, and this may be a mechanism by which α -galactosylceramide has a therapeutic effect.

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.

Immune response in mouse experimental cholangitis associated with colitis induced by dextran sulfate sodium.

Background and Aim:  Primary sclerosing cholangitis is frequently complicated by inflammatory bowel disease. Although many colitis models have been reported, little information has been obtained about complicated cholangitis. The aim of the present study was to determine whether hepatobiliary disorders occur in mice experimental colitis, and to clarify the underlying mechanisms.

PARTIAL REPLACEMENT OF BILE SALTS CAUSES MARKED CHANGES OF CHOLESTEROL CRYSTALLIZATION IN SUPERSATURATED MODEL BILE SYSTEMS

Cholesterol crystallization is a key step in gallstone formation and is influenced by numerous factors. Human bile contains various bile salts having different hydrophobicity and micelle-forming capacities, but the importance of lipid composition to bile metastability remains unclear. This study investigated the effect of bile salts on cholesterol crystallization in model bile (MB) systems. Supersaturated MB systems were prepared with an identical composition on a molar basis (taurocholate/phosphatidylcholine/cholesterol, 152 mM:38 mM: 24 mM), except for partial replacement of taurocholate (10, 20, and 30%) with various taurine-conjugated bile salts. Cholesterol crystallization was quantitatively estimated by spectrophotometrically measuring crystal-related turbidity and morphologically scanned by video-enhanced microscopy. After partial replacement of taurocholate with hydrophobic bile salts, cholesterol crystallization increased dose-dependently without changing the size of vesicles or crystal morphology and the rank order of crystallization was deoxycholate>chenodeoxycholate>cholate (control MB). All of the hydrophilic bile salts (ursodeoxycholate, ursocholate and beta-muricholate) inhibited cholesterol precipitation by forming a stable liquid-crystal phase, and there were no significant differences among the hydrophilic bile-salt species. Cholesterol crystallization was markedly altered by partial replacement of bile salts with a different hydrophobicity. Thus minimal changes in bile-salt composition may dramatically alter bile lipid metastability.

Inhibition of cholesterol crystallization under bilirubin deconjugation: partial characterization of mechanisms whereby infected bile accelerates pigment stone formation.

Pigment gallstones have been reported to be closely associated with biliary tract infection. We previously reported that addition of unconjugated bilirubin (UCB), which is deconjugated by beta-glucuronidase in infected bile, could enhance cholesterol crystal formation in supersaturated model bile (MB). The present study evaluated the effect of beta-glucuronidase on the processes of pigment gallstone formation and cholesterol crystallization. Supersaturated MB (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 10.0 g/dl and a cholesterol saturation index (CSI) of 2.0) and native rat bile were mixed at a ratio of 3:1. Then, mixed bile was incubated with or without beta-glucuronidase and changes of the following parameters were investigated over time: (1) the UCB/total bilirubin ratio; (2) cholesterol crystal formation; (3) the precipitate weight and the cholesterol concentration in the precipitate and supernatant; and (4) the lipid distribution of vesicles in the supernatant. Compared with beta-glucuronidase-free bile, (1) beta-glucuronidase-containing bile showed a significant increase of the UCB/total bilirubin ratio, (2) as well as a significantly longer nucleation time (96+/-17.0 vs. 114+/-20.0) and fewer cholesterol crystals. (3) The precipitate weight and the cholesterol concentration in the precipitate were significantly increased, while the cholesterol concentration in supernatant was decreased. (4) When mixed bile was incubated with beta-glucuronidase, the cholesterol concentration in the vesicles was lower than in bile without beta-glucuronidase. The precipitate weight and the cholesterol concentration in the precipitate was increased by incubation with beta-glucuronidase, while cholesterol concentration was decreased in the supernatant (especially in the vesicles). This means that bile vesicles were more stable and it was more difficult for cholesterol crystals to form. Thus, the presence of beta-glucuronidase may inhibit the formation of pure cholesterol stones even in the presence of cholesterol supersaturation.