Shinichiro Hanada

Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3

The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3(-/-) mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination.

Clearance of HCV improves insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate 1 and 2.

OBJECTIVES:Hepatitis C virus (HCV) infection is linked to greater insulin resistance. Although HCV itself is a candidate for the development of insulin resistance, the effects of antiviral treatment on impaired glucose metabolism remain unclear. The aim of this study is to examine the effects of clearance of HCV on insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate (IRS)1/2, central molecules for insulin signaling.METHODS:We analyzed 89 biopsy-proven patients with chronic HCV infection. Patients received interferon-α or interferon-α plus ribavirin for 6 months and were classified into three groups at 6 months after the conclusion of antiviral therapy according to their response to antiviral therapy: sustained responders (N = 29), relapsers (N = 12), and nonresponders (N = 48). Insulin resistance and beta-cell function were assessed by the homeostasis model assessment method (HOMA-IR and HOMA-%B, respectively). Hepatic expression of IRS1/2 was evaluated by immunoblotting and immunostaining in 14 sustained responders.RESULTS:In nonresponders and relapsers, there were no significant changes in HOMA-IR and HOMA-%B values after antiviral therapy. On the other hand, in sustained responders, HOMA-IR values significantly decreased to 1.7 ± 0.8 from 3.1 ± 1.1 (P < 0.05) after antiviral therapy. Similarly, HOMA-%B values significantly decreased to 90.6 ± 10.0 from 113.7 ± 15.3 (P < 0.05). Immunoblotting showed a threefold increase in IRS1/2 expression after clearance of HCV. Immunostaining revealed that greater IRS1/2 expression was seen in hepatocytes.CONCLUSIONS:We showed that clearance of HCV improves insulin resistance, beta-cell function, and hepatic IRS1/2 expression.

Keratin Variants Predispose to Acute Liver Failure and Adverse Outcome: Race and Ethnic Associations

BACKGROUND & AIMS Keratins 8 and 18 (K8/K18) provide anti-apoptotic functions upon liver injury. The cytoprotective function of keratins explains the overrepresentation of K8/K18 variants in patients with cirrhosis. However, K8/K18 variant-associated susceptibility to acute liver injury, which is well-described in animal models, has not been studied in humans. METHODS We analyzed the entire coding regions of KRT8 and KRT18 genes (15 total exons and their exon-intron boundaries) to determine the frequency of K8/K18 variants in 344 acute liver failure (ALF) patients (49% acetaminophen-related) and 2 control groups (African-American [n = 245] and previously analyzed white [n = 727] subjects). RESULTS Forty-five ALF patients had significant amino-acid-altering K8/K18 variants, including 23 with K8 R341H and 11 with K8 G434S. K8 variants were significantly more common (total of 42 patients) than K18 variants (3 patients) (P < .001). We found increased frequency of variants in white ALF patients (9.1%) versus controls (3.7%) (P = .01). K8 R341H was more common in white (P = .01) and G434S was more common in African-American (P = .02) ALF patients versus controls. White patients with K8/K18 variants were less likely to survive ALF without transplantation (P = .02). K8 A333A and G434S variants associated exclusively with African Americans (23% combined frequency in African American but none in white controls; P < .0001), while overall, K18 variants were more common in non-white liver-disease subjects compared to whites (2.8% vs 0.6%, respectively; P = .008). CONCLUSIONS KRT8 and KRT18 are important susceptibility genes for ALF development. Presence of K8/K18 variants predisposes to adverse ALF outcome, and some variants segregate with unique ethnic and race backgrounds.

The Wilson Disease Protein ATP7B Resides in the Late Endosomes with Rab7 and the Niemann-Pick C1 Protein

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease.

Proteasome inhibition induces inclusion bodies associated with intermediate filaments and fragmentation of the Golgi apparatus

The ubiquitin-proteasome system is involved in a variety of biological processes. Inclusion bodies associated with intermediate filaments (IFs) and ubiquitin are observed in various diseases; however, the precise mechanisms of formation and the pathological significance of inclusion bodies have not been fully understood. We examined the effect of proteasome inhibitors on the structure of IF using anti-cytokeratin antibodies or transfection of green fluorescent protein-fused cytokeratin 18 in a hepatoma cell line, Huh7. Intracellular organelles were visualized by immunofluorescent and electron microscopies. Proteasome inhibitors induced IF inclusions associated with ubiquitin. Electron microscopic examination revealed inclusion bodies surrounded by filamentous structures. Autophagic vacuoles and lysosomes were frequently observed, and the organization of the Golgi apparatus was disrupted in these cells. After the removal of the proteasome inhibitors, the IF network and organization of the Golgi apparatus were restored. The IF inclusions could be induced by inhibition of the proteasome function. IF inclusions induced fragmentation of the Golgi apparatus and might inhibit the function of this important station of membrane traffic. The IF inclusions disappeared by restoring proteasome function, and autophagy and lysosomal degradation might be, at least in part, associated with the elimination of inclusion bodies.

Incidence of Sjögren's syndrome in Japanese patients with hepatitis C virus infection

Background and Aim: Hepatitis viruses induce not only chronic liver diseases but also the impairment of other organs and tissues as extrahepatic manifestations. In particular, hepatitis C virus (HCV) is involved in various extrahepatic manifestations. The purpose of the present study was to evaluate Sjögrens syndrome (SS) and lichen planus (LP) involvement, which are various extrahepatic manifestations in patients with liver diseases related to hepatitis B virus (HBV) or HCV.

Switching in discoid domain receptor expressions in SLUG‐induced epithelial‐mesenchymal transition

Acquired features of cells under epithelial‐mesenchymal transition (EMT) have not yet been fully identified. The current study was conducted to assess alterations in both the proliferative potential and the responsiveness to extracellular matrices (ECMs) in EMT.

Copper incorporation into ceruloplasmin is regulated by Niemann–Pick C1 protein

Aim:  Wilson disease is a genetic disorder of copper metabolism characterized by impaired biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. Our previous study showed the late endosome localization of ATP7B and described the copper transport pathway from the late endosome to trans‐Golgi network (TGN). However, the cellular localization of ATP7B and copper metabolism in hepatocytes remains controversial. The present study was performed to evaluate the role of Niemann–Pick type C (NPC) gene product NPC1 on intracellular copper transport in hepatocytes.

Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides.

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.

Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation.

Differential expression and activity of the cellular energy regulators GAPDH and NDPK underlie reactive oxygen species–induced damage in the mouse liver and may contribute to human liver disease progression.