Elsevier

Journal of Hepatology

Volume 49, Issue 3, September 2008, Pages 429-440
Journal of Hepatology

Virus-induced over-expression of protein phosphatase 2A inhibits insulin signalling in chronic hepatitis C

https://doi.org/10.1016/j.jhep.2008.04.007Get rights and content

Background/Aims

Hepatitis C virus (HCV) infection disturbs glucose and lipid metabolism contributing to the development of liver steatosis, insulin resistance and type 2 diabetes mellitus. On the other hand, insulin resistance and steatosis have been found to be associated with increased rates of fibrosis progression and lower rates of response to interferon therapy in chronic hepatitis C (CHC). The molecular mechanisms contributing to insulin resistance in CHC are not well understood. We have shown previously that protein phosphatase 2A (PP2A) is over-expressed in biopsies from patients with CHC. In this study, we tested if PP2A over-expression leads to insulin resistance.

Methods

We studied insulin signalling in cell lines that allow the regulated over-expression of HCV proteins and of the PP2A catalytic subunit (PP2Ac). Insulin signalling and PP2Ac expression were also studied in HCV transgenic mice and in liver biopsies from patients with CHC.

Results

Over-expression of PP2Ac in cells inhibited insulin signalling by dephosphorylation of PKB/Akt. PP2Ac over-expression and impaired insulin signalling were found in the liver of HCV transgenic mice and in liver biopsies of patients with CHC.

Conclusions

HCV-induced over-expression of PP2A in the liver contributes to the pathogenesis of insulin resistance in patients with CHC.

Introduction

Chronic hepatitis C virus (HCV) infection is associated with liver steatosis, insulin resistance and type 2 diabetes mellitus. There is growing evidence that the virus directly causes insulin resistance through interference with the insulin signalling pathway and that HCV-induced dysregulation of important regulators of lipid metabolism such as sterol regulatory element binding protein (SREBP-1c) contributes to liver steatosis [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. On the other hand, insulin resistance and steatosis have been found to be associated with increased rates of fibrosis progression and lower rates of response to interferon therapy in chronic hepatitis C (CHC) [5], [11], [12], [13], [14], [15], [16].

Insulin exerts its effects through binding to the insulin receptor, a receptor tyrosine kinase. The activated receptor then phosphorylates tyrosine residues on at least nine intracellular substrates including insulin-receptor substrate 1 (IRS-1), IRS-2 and Cbl [17], [18]. The phosphorylated tyrosines in these substrates provide “docking sites” for proteins that contain src-homology-2 (SH2) domains, such as Grb2, SHP2, Fyn and the p85 regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). PI(3)K has a central role in the metabolic actions of insulin [19]. PI(3)K catalyses the generation of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), which activates phosphoinositide-dependent kinase 1 (PDK1). PDK1 together with mTORC2 complex activates the important serine/threonine kinase Akt/PKB [20], [21]. Akt/PKB is activated by phosphorylation on threonine 308 and serine 473 [22]. Akt/PKB is a central regulator of the function of many cellular proteins that are involved in metabolism, survival, apoptosis, differentiation and proliferation [23]. Amongst these proteins are mTOR (mammalian target of rapamycin), S6K1 (S6 kinase 1) and GSK-3β (glycogen synthase kinase-3) [24], all of which are important for the control of glucose metabolism. In general, the activation of these proteins leads to enhanced glucose transport and the synthesis of glycogen and proteins.

Contrary to the activating effect of tyrosine phosphorylation, serine phosphorylation of IRS proteins inhibits insulin signalling. This inhibitory phosphorylation is part of a negative feedback to insulin signalling, because insulin activates a number of serine/threonine kinases including mTOR, S6K1 and atypical isoforms of protein kinase C (PKCζ and PKCλ) that mediate serine phosphorylation of IRS proteins [18], [25]. Moreover, serine phosphorylation of IRS serves as a mechanism for cross-talk from other pathways that induce insulin resistance. Excess nutrients, fatty acids, pro-inflammatory cytokines (for example tumor necrosis factor α, TNFα, and interleukin-6, IL-6), the adipocyte secreted hormone resistin, and endosplasmic reticulum stress could at least in part exert their negative effect on insulin signalling through the activation of serine/threonine kinases such as c-Jun NH2-terminal kinase (JNK), stress-activated protein kinases, and PKCθ [25], [26], [27], [28], [29], [30].

Impaired IRS-1 and PI(3)K signalling has also been implicated in the pathogenesis of HCV associated insulin resistance and type 2 diabetes [31]. Insulin-induced IRS-1 tyrosine phosphorylation (but not IRS-1 content) was found to be decreased by 2-fold in liver biopsies from patients with CHC compared to controls [31]. Another group reported a decrease in IRS-1 and IRS-2 expression in liver biopsies from HCV patients and in HCV core transgenic mice [32], and more recently, that successful treatment of patients with CHC improves insulin sensitivity and increases IRS-1 and IRS-2 expression in the liver [33].

We had reported previously that HCV induces the over-expression of the serine/threonine phosphatase PP2A (protein phosphatase 2A) through an endoplasmatic reticulum (ER) stress response pathway [34], and that PP2A over-expression inhibits interferon α signalling in liver cells [35], [36]. Interestingly, PP2A dephosphorylates and inactivates Akt/PKB in mouse fibroblasts [37]. Furthermore, PP2A was shown to negatively regulate insulin signalling in 3T3-L1 adipocytes [38]. We therefore investigated if HCV-induced over-expression of PP2A could be a molecular pathway linking HCV infection to insulin resistance.

Section snippets

Reagents and antibodies

Human insulin, okadaic acid and wortmannin were from Sigma (Fluka Chemie, GmbH, Buchs, Switzerland). Purified PP2Ac and PP2Ac-, p85PI3K- and IRS-1- antibodies were from Upstate (LucernaChem, Luzern, Switzerland). Antibodies against pS473Akt, Akt, pT308Akt, pS9GSK3β, pThr172AMPKα and AMPKα were from Cell Signalling (Bioconcept, Allschwil, Switzerland), β-Actin from Sigma and p-Y-99 from Santa Cruz (LabForce AG, Nunningen, Switzerland). Secondary antibodies IRDye 800CW and IRDye 680 were from

Expression of HCV proteins or PP2Ac in cells inhibits insulin signalling

To test if HCV protein expression inhibits insulin signalling, we made use of UHCV-57.3 cells that allow the regulated expression of HCV proteins [42]. Insulin-induced tyrosine phosphorylation of IRS-1 and IRS-1 association with PI3K were normal in cells expressing HCV proteins, but serine phosphorylation of Akt/PKB and GSK3β were impaired (Fig. 1A).

We had previously shown that the inhibitory effects of HCV protein expression on interferon α signalling were caused by HCV-induced PP2A

Discussion

We had previously reported that HCV infection induces an over-expression of the important cellular phosphatase PP2A, and the PP2A over-expression per se inhibits IFNα signalling through the Jak-STAT pathway [34], [35], [36], [40], [48]. Here we show that PP2A over-expression also inhibits insulin signalling through the Akt/PKB signalling pathway. PP2A directly or indirectly dephosphorylates Akt/PKB on serine 473, and thereby lowers the kinase activity of Akt/PKB, with consequences on both

Acknowledgements

The authors thank Nicola La Monica and Marco Tripodi for the B6HCV mice, Darius Moradpour for UHCV cells and Michelangelo Foti for help and advice.

The study was supported by the Swiss National Science Foundation Grants 3200B0-103958, 320000-116106 and 3200B0-103727/1, by the Leenaards Foundation and Swiss Cancer League Grants OCS-01475-02-2004 and KLS-01832-02-2006.

References (53)

  • Y. Li et al.

    Protein kinase C Theta inhibits insulin signaling by phosphorylating IRS1 at Ser(1101)

    J Biol Chem

    (2004)
  • S. Aytug et al.

    Impaired IRS-1/PI3-kinase signaling in patients with HCV: a mechanism for increased prevalence of type 2 diabetes

    Hepatology

    (2003)
  • T. Kawaguchi et al.

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

    Am J Pathol

    (2004)
  • F.H. Duong et al.

    Hepatitis C virus inhibits interferon signaling through up-regulation of protein phosphatase 2A

    Gastroenterology

    (2004)
  • P. Bedossa et al.

    An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group

    Hepatology

    (1996)
  • A. Blindenbacher et al.

    Expression of hepatitis c virus proteins inhibits interferon alpha signaling in the liver of transgenic mice

    Gastroenterology

    (2003)
  • J. Schmidt-Mende et al.

    Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase

    J Biol Chem

    (2001)
  • B.B. Kahn et al.

    AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism

    Cell Metab

    (2005)
  • D. Carling

    AMP-activated protein kinase: balancing the scales

    Biochimie

    (2005)
  • T. Poynard et al.

    Effect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis in patients infected with hepatitis C

    Hepatology

    (2003)
  • S.H. Mehta et al.

    Prevalence of type 2 diabetes mellitus among persons with hepatitis C virus infection in the United States

    Ann Intern Med

    (2000)
  • A.L. Mason et al.

    Association of diabetes mellitus and chronic hepatitis C virus infection

    Hepatology

    (1999)
  • T. Asselah et al.

    Steatosis in chronic hepatitis C: why does it really matter?

    Gut

    (2006)
  • A.I. Su et al.

    Genomic analysis of the host response to hepatitis C virus infection

    Proc Natl Acad Sci USA

    (2002)
  • K. Moriishi et al.

    Critical role of PA28gamma in hepatitis C virus-associated steatogenesis and hepatocarcinogenesis

    Proc Natl Acad Sci USA

    (2007)
  • G. Waris et al.

    Hepatitis C virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress

    J Virol

    (2007)
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    The authors declare that they do not have anything to disclose regarding funding from industries or conflict of interest with respect to this manuscript.

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