- Email: [email protected]
The metabolic regulator PGC-1α links hepatitis C virus infection to hepatic insulin resistance
Research Article
The metabolic regulator PGC-1a links hepatitis C virus infection to hepatic insulin resistance Amir Shlomai1, Maya Mouler Rechtman2, Ela Olga Burdelova1, Alona Zilberberg2, Sarit Hoffman3, Irit Solar3, Sigal Fishman1, Zamir Halpern1, Ella H. Sklan2,⇑ 1 The Research Center for Digestive Tract and Liver Diseases, Tel-Aviv Sourasky Medical Center and The Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel; 2Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel; 3The Institute of Pathology and Cancer Research, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel
Background & Aims: Chronic hepatitis C virus (HCV) infection is strongly associated with insulin resistance and diabetes mellitus. Peroxisome proliferator-activated receptor-gamma co-activator 1a (PGC-1a) is a transcriptional co-activator involved in the initiation of gluconeogenesis in the liver. Increased hepatic expression of PGC-1a has been implicated in insulin resistance. We investigated whether modulation of PGC-1a levels following HCV infection underlies HCV-associated hepatic insulin resistance. Methods: HCV genomes were expressed in hepatoma cells followed by analysis of PGC-1a and gluconeogenesis levels. Results: PGC-1a was robustly induced in HCV infected cells. PGC-1a induction was accompanied by an elevated expression of the gluconeogenic gene glucose-6 phosphatase (G6Pase) and increased glucose production. The induction of gluconeogenesis is HCV dependent, since interferon treatment abolishes PGC-1a and G6Pase elevation and decreases glucose output. Moreover, PGC-1a knockdown resulted in a significant reduction of G6Pase levels in HCV full length replicon cells, emphasizing the central role of PGC-1a in the exaggerated gluconeogenic response observed in HCV patients. Treatment of HCV replicon cells with the antioxidant N-acetylcysteine resulted in reduction of PGC1a levels, suggesting that HCV-induced oxidative stress promoted PGC-1a upregulation. Finally, both PGC-1a and G6Pase RNA levels were significantly elevated in liver samples of HCV infected patients, highlighting the clinical relevance of these results.
Keywords: Hepatitis C virus; Peroxisome proliferator-activated receptor gamma co-activator 1a; Diabetes; Insulin resistance; Oxidative stress. Received 17 December 2011; received in revised form 19 May 2012; accepted 14 June 2012; available online 23 June 2012 ⇑ Corresponding author. Tel.: +972 3 6408197; fax: +972 3 6409160. E-mail address: [email protected] (E.H. Sklan). Abbreviations: HCV, hepatitis C virus; PGC-1a, peroxisome proliferator-activated receptor gamma, co-activator 1a; G6Pase, glucose-6 phosphatase; RNA, ribonucleic acid; PI3, phosphatidylinositol 3-kinase; FLRP, full length replicon; SGR, subgenomic replicon; NS5A, non-structural protein 5A; SEAP, secreted alkaline phosphatase; NS, non-structural; SDS, sodium dodecyl sulfate; RIPA, radio immunoprecipitation assay; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; HPRT, hypoxanthine phosphoribosyltransferase; HA, hemagglutinin; ECL, enhanced chemiluminescence; IFN, interferon; SOCS, suppressors of cytokine signaling; FOXO1, forkhead box protein O1; ER, endoplasmic reticulum; JNK, c-Jun N terminal kinase; NAC, N-acetylcysteine; IRS-1, insulin receptor substrate-1.
Conclusions: PGC-1a is robustly induced following HCV infection, resulting in an upregulated gluconeogenic response, thereby linking HCV infection to hepatic insulin resistance. Our results suggest that PGC-1a is a potential molecular target for the treatment of HCV-associated insulin resistance. Ó 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Chronic hepatitis C virus (HCV) infection is a major global health problem. Chronically infected patients may develop cirrhosis and liver cancer that are implicated in substantial morbidity and mortality [1]. Although the liver is the target organ for HCV infection, extrahepatic manifestations of HCV, including insulin resistance and diabetes mellitus, are frequently encountered in the clinical setting [2]. Insulin resistance was found to be a specific feature of chronic HCV associated with genotypes 1 and 4 and with high serum viral RNA levels [3]. Insulin resistance and diabetes mellitus also correlated with poor response to anti-HCV therapy, while eradication of the virus correlates with a marked improvement in insulin resistance [4]. The molecular mechanism by which HCV promotes insulin resistance is still undetermined. HCV infection was found to cause a postreceptor defect in the insulin signaling cascade [5]. In addition, the activation of proinflammatory mediators, such as nuclear factor-kappa-B and tumor necrosis factor-a, caused by the chronic infection state, interferes with insulin signaling and contributes to both hepatic and peripheral insulin resistance [6,7]. In agreement with the evidence suggesting a central role for reactive oxygen species in the development of insulin resistance [8], HCV infection is known to promote cellular oxidative stress through multiple mechanisms, including chronic inflammation, iron overload, and liver injury. Some of the HCV proteins were reported to directly contribute to this process [9,10]. Peroxisome proliferator-activated receptor gamma co-activator 1a (PGC-1a) is a transcription co-activator and a master regulator of gluconeogenesis that functions via interaction with transcription factors located on the promoters of gluconeogenic genes [11–13]. Although PGC-1a is scarcely detectable in the
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Research Article liver under basal conditions, it is robustly induced in response to starvation, an effect that is mediated by the counter-regulatory hormonal response in which glucagon and glucocorticoids are involved [14,15]. PGC-1a has been shown to be overexpressed in livers of obese-hyperglycemic (Ob/Ob) mice [15], most probably reflecting the amplified hepatic gluconeogenesis that results in insulin resistance and diabetes mellitus. Interestingly, reducing hepatic oxidative stress in diabetic (db/db) mice resulted in a significant improvement in insulin resistance that was accompanied by a marked reduction of hepatic PGC-1a levels [16]. These data suggest that PGC-1a might be a central molecular player in the interplay between hepatic oxidative stress and insulin resistance. We hypothesized that the increased oxidative stress produced during HCV infection might lead to elevated liver PGC-1a expression, thereby promoting hepatic insulin resistance and diabetes mellitus. Here, the role of PGC-1a in HCV-induced insulin resistance was investigated. By analyzing PGC-1a levels and the gluconeogenic response in HCV infected cells, as well as in HCV-infected patients, we show that PGC-1a has a central role in HCV-promoted exaggerated gluconeogenesis. These findings further clarify the molecular pathways leading to HCV induced insulin resistance and might have therapeutic implications.
Materials and methods Cell culture, transfections, and treatments HuH7 and 7.5 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (Biological Industries, Israel). Non-essential amino acids (Biological Industries) were added to HuH7.5 cells. Human interferon-a (IFN) B2 was purchased from PBL (Piscataway, NJ), and N-acetyl-L-cysteine was purchased from Sigma. Luciferase reporter assays Cells were co-transfected with PGC-Luc reporter (a gift from B.M. Spiegelman (Dana-Farber, Harvard) [18]) and pM1-secreted alkaline phosphatase (SEAP, transfection efficiency control; Roche, Palo Alto, CA). Luciferase activity was determined 48 h post-transfection. SEAP activity was determined in the culture media using the Phospha-Light System (Applied Biosystems, Carlsbad, CA). RNA analysis
ScaI linearized plasmids encoding HCV genotype 1b (Bart79I, [17]) replicon were in vitro transcribed using Ribomax RNA production kit (Promega) under nucleasefree conditions. The plasmid encoding the full-length chimeric HCV J6/JFH1 genome was linearized with XbaI, followed by mung bean nuclease treatment. The linearized template was transcribed using a MEGAscript T7 kit (Ambion, Austin, TX). The DNA template was digested by DNase. RNA was extracted using TRIReagent. In vitro transcribed RNAs were electroporated into HuH7.5 cells, as described [19]. The pulsed cells were left to recover for 15 min and then plated in 6-well plates for further treatment. PGC-1a knockdown HIV-1-based lentiviral pGIPZ vectors containing shPGC-1a (clone V2LHS_71173) and scrambled shRNAs (clone RHS_4346) were purchased from Open Biosystems (Huntsville, AL). shRNAs harboring viruses were produced by transfection of 293T cells with 20 lg of shRNA plasmid together with the helper plasmids pCMVDR8.2 (15 lg) and pVSVG (5 lg). Three days post-transfection, the culture medium was collected and filtered. HuH7 and FLRP cells in 6-well plates were incubated with 1 ml of infectious medium supplemented with polybrene (8 lg/ml; Sigma) for 8 h. The medium was replaced and cells were then incubated for additional 48 h. Glucose production The production of glucose was measured by an AmplexÒ Red Glucose/Glucose oxidase assay kit (Invitrogen). One million HuH7 or FLRP cells were seeded in a 6-well plate. The next morning, the culture medium was replaced with PBS (Biological Industries) supplemented with 20 mM sodium lactate. Following a 2-h incubation, media were collected and glucose levels were determined. The readings were normalized to cell viability determined by Alamar blue (Invitrogen). Gluconeogenesis levels were calculated by subtracting glucose levels in the presence of the gluconeogenic substrates from baseline glucose levels. RNA extraction from formalin-fixed paraffin-embedded liver tissue Paraffin-embedded liver tissues were taken from historical patients who were infected with HCV genotype 1, controls were HCV negative patients whom liver biopsies were taken for evaluation of elevated liver function tests from various other etiologies. The study was approved by the Tel-Aviv Sourasky Medical Center Ethics Committee (#0133-12-TLV), and was conducted in accordance with the principles of the Declaration of Helsinki. RNA extraction from formalin fixed paraffin embedded liver tissues was performed as described [20]. Briefly, tissue sections were deparaffinized using xylene and ethanol, air dried and incubated at 55 °C for 16 h in RNA lysis buffer (20 mM Tris pH 7.5, 20 mM EDTA, 1% SDS and 0.5 lg/ll proteinase K). RNA was extracted using Tri-Reagent (Sigma). Real-time PCR was preformed as described above.
Results
RNA was extracted from cells by Tri-reagent (Sigma). After treatment with RNasefree DNase I (Promega), the RNA was subjected to reverse transcription using the Verso cDNA kit (Thermo, Waltham, MA). Real-time PCR was performed on the resulting cDNA to quantify the amounts of HCV, PGC-1a, and G6Pase mRNAs using the primers listed in Supplementary Table 1. Results were normalized to the mRNA levels of the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) (Supplementary Table 1). Protein analysis Proteins were extracted from cells using radio immunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitors (Sigma) and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The following primary antibodies were used for Western blot: monoclonal mouse anti-HCV NS5A (Virostat, Portland, ME), mouse anti-PGC-1a antibody (Calbiochem, La Jolla, CA), mouse anti-tubulin antibody (Sigma) or mouse anti-actinin (Santa Cruz Biotechnology, Santa Cruz, CA). A goat anti-mouse antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody. Proteins were detected by chemiluminescence.
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PGC-1a is elevated in HCV infected cells We hypothesized that HCV replication induces PGC-1a. To verify this hypothesis, PGC-1a mRNA levels were first examined in the context of a newly established HCV infection, namely, electroporation of a genotype 1b subgenomic replicon (SGR, Bart 79I [17]). RNA was extracted 72 h following electroporation and analyzed for HCV and PGC-1a mRNA levels. HCV mRNA levels dramatically increased following electroporation (138-fold). A replicon with a lethal mutation in the RNA-dependent RNA polymerase (pol-) served as a negative control [17]. In agreement with our hypothesis, PGC-1a mRNA levels were significantly increased (7-fold) in these replicon cells (Fig. 1A). Since this replicon does not contain the viral structural genes (see scheme in Fig. 1), this results also indicates that the structural genes are not essential for PGC-1a elevation. Because our hypothesis links PGC-1a overexpression
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To determine whether PGC-1a induction is HCV-dependent, FLRP cells were treated with interferon a (IFNa) for 72 h, after which PGC-1a mRNA and protein levels were analyzed. IFNa treatment dramatically decreased HCV RNA levels (Fig. 2A, left) and attenuated PGC-1a induction to levels similar to those of the HuH7 controls (Fig. 2A, right). In contrast, IFNa treatment did not affect PGC-1a mRNA levels in the control HuH7 cells. Western blot analysis of lysates from the IFNa-treated cells using PGC-1a antibodies confirmed these results (Fig. 2A, bottom). Next, PGC-1a was knockeddown in FLRP and HuH7 cells. Following knockdown, PGC-1a mRNA levels decreased significantly (2- to 3-folds) in FLRP cells to levels comparable to parental HuH7 cells, while HCV RNA levels were not significantly altered (Fig. 2B). These data suggest that while HCV expression enhances PGC-1a expression, PGC-1a itself has no effect on HCV RNA levels.
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Fig. 1. PGC-1a is elevated in HCV replicon cells. (A) HuH7.5 cells were electroporated with the HCV subgenomic replicon (SGR) or a replicon with a lethal polymerase mutation (Pol-). Total RNA was harvested after 72 h and used to determine HCV and PGC-1a mRNA levels by real-time RT-PCR. The results were normalized to HPRT levels. Values are mean ± standard deviations of at least three independent experiments and are expressed as fold increase. (B) FLRP and HuH7 cells were transfected with a PGC-Luc reporter. Luciferase activity in cell lysates was determined 48 h post-transfection. Total RNA was extracted from HuH7 and FLRP cells and subjected to real-time RT-PCR to determine PGC-1a mRNA levels. The results were normalized to HPRT levels and analyzed as described above. ⁄ p = 0.002 (Student’s t test). HuH7 cells were either transfected with PGC-HA [35], treated with dexamethasone (Dex) and forskolin (Forsk) or left untreated. Posttransfection (24 h), the cells were lysed and analyzed by Western blot together with FLRP cells. The graph shows a quantification of the band intensities. (C) HuH7.5 cells were electroporated with 5 lg of J6/JFH RNA or a pol- control. Total RNA was harvested after 72 h and used to determine HCV and PGC-1a mRNA levels by real-time PCR. The results were normalized to HPRT levels and analyzed as mentioned above.
PGC-1a induction following HCV infection can occur via a direct interaction with a viral component or via an indirect mechanism. Given that PGC-1a is elevated in the context of both full length and subgenomic replicons, the viral structural proteins were clearly not responsible for this induction. Among the non-structural (NS) proteins, NS5A would be a natural candidate since two recent studies either physically or mechanistically linked PGC-1a with NS5A [21,22]. In our hands, overexpression of genotype 1b NS5A in HuH7 cells did not significantly alter PGC-1a levels (Fig. 3A). Since the hepatic oxidative stress known to occur during HCV infection [9] was previously linked to insulin resistance [16] as well as to alterations in PGC-1a levels [23], we investigated whether oxidative stress is involved in PGC-1a induction promoted by HCV. HCV replicon cells were treated with the antioxidant N-acetyl-cysteine (NAC) after which PGC-1a levels were determined. NAC significantly reduced PGC-1a induction, while having an insignificant effect on HCV RNA levels (Fig. 3B), suggesting a role for oxidative stress as a mediator of HCV-promoted PGC-1a induction.
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Fig. 2. PGC-1a elevation is HCV dependent. HuH7 and FLRP cells were treated with 50 units/ml of interferon-a (IFNa) for 72 h or left untreated (A). Total RNA was extracted and subjected to real-time RT-PCR to determine HCV and PGC-1a mRNA levels. The results were normalized to HPRT RNA levels. HuH7 and FLRP cells from a similar experiment were analyzed by Western blot using PGC-1a and NS5A antibodies (bottom). (B) HuH7 and FLRP cells were infected with lentiviruses expressing PGC-1a or scrambled shRNAs. After 48 h, total RNA was extracted and subjected to real-time RT-PCR in order to determine PGC-1a and HCV RNA levels. ⁄p <0.005 compared to FLRP cells expressing scrambled shRNAs (Student’s t test). Values as mean ± standard deviation of at least two independent experiments are expressed as fold increase in all of the above experiments.
PGC-1a upregulation in HCV infected cells enhances gluconeogenesis G6Pase hydrolyzes glucose-6-phosphate to free glucose and a phosphate group. This catalytic action is the final step in gluconeogenesis and therefore plays a key role in the regulation of blood glucose levels [24]. PGC-1a plays a crucial role in the transcriptional regulation of G6Pase [14,15]. We analyzed G6Pase mRNA levels in HCV-expressing cells in order to further understand the role of PGC-1a upregulation in HCV-infected cells, and to investigate whether this elevation affects gluconeogenesis. G6Pase levels were significantly increased (6-fold) in HuH7 cells 72 h post-electroporation of a HCV subgenomic replicon (Fig. 4A, left). G6Pase levels were elevated by 2.5-fold in FLRP cells as well (Fig. 4A, right). IFNa treatment blocked G6Pase elevation in both HCV electroporated and stable replicon cells, confirming its dependency on the presence of HCV. To further illustrate that G6Pase induction is PGC-1a dependent, PGC-1a was knockeddown using specific shRNAs (Fig. 4B). As expected, PGC-1a depletion decreased G6Pase mRNA levels in both HuH7 and FLRP cells. G6Pase mRNA levels in PGC-1a-depleted cells were reduced to its levels in the parental HuH7 cells. Similarly,
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Fig. 3. A general antioxidant can abrogate PGC-1a induction following infection. (A) HuH7 cells in 6 wells (80–90% confluent) were transfected with pEF6-NS5A [36] or an empty vector. After 48 h, total RNA was harvested and subjected to real-time RT-PCR to determine PGC-1a mRNA levels. The results were normalized to HPRT RNA levels. Cells similarly transfected were lysed and analyzed by Western blot using PGC-1a and NS5A antibodies. Controls were similar to Fig. 1B. (B) HuH7 and FLRP cells were treated with 5 mM NAC for 4 h. Following treatment, total RNA was extracted and subjected to real-time RT-PCR to determine PGC-1a and HCV RNA levels. The results were normalized to HPRT RNA levels. ⁄p = 0.005 compared to non-treated FLRP cells, n.s. = not significant (Student’s t test). Values are mean ± standard deviation of two independent experiments expressed as fold increase.
NAC treatment reduced G6Pase mRNA levels to levels comparable to the HuH7 controls (Fig. 4C). To further assess the physiological relevance of PGC-1a and G6Pase upregulation in HCV replicon cells, we compared the levels of glucose output, reflecting endogenous gluconeogenesis, between FLRP and HuH7 cells. In agreement with our hypothesis, gluconeogenesis was significantly elevated in FLRP cells compared to HuH7 controls (7-fold, Fig. 4D). This elevated gluconeogenesis was HCV-dependent since IFNa treatment significantly abrogated it. PGC-1a and G6Pase are elevated in HCV infected patients To substantiate our results, liver biopsy samples from HCV infected patients and non-infected patients (controls) were analyzed for PGC-1a and G6Pase mRNA levels. In agreement with our results, a significant 3-fold elevation in the mRNA levels of both PGC-1a and G6Pase was detected in HCV patients as compared to controls. Furthermore, the degree of both PGC-1a and G6Pase inductions tended to correlate with each other and with the hepatic HCV RNA levels (Supplementary Fig. 1).
Discussion PGC-1a is a central metabolic regulator that is robustly induced in the liver upon starvation, to initiate gluconeogenesis through
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Fig. 4. PGC-1a upregulation in HCV infected cells enhances gluconeogenesis. (A) HuH7.5 cells were electroporated with the HCV subgenomic replicon (SGR) or a pol-, control replicon. Cells were treated with 50 units/ml of IFNa for 72 h or left untreated. HuH7 and FLRP cells were treated similarly. Total cellular RNA was extracted from the cells and subjected to real-time RT-PCR to determine G6Pase mRNA levels. The results were normalized to HPRT RNA levels. ⁄p <0.002 compared to non-treated FLRP (Student’s t test). (B) HuH7 and FLRP cells were infected with lentiviruses expressing PGC-1a or scrambled shRNAs. After 48 h, total cellular RNA was extracted from the cells and subjected to real-time RT-PCR to determine G6Pase mRNA levels. The results were normalized to HPRT RNA levels. (C) HuH7 and FLRP cells were treated with 5 mM NAC for 4 h. Total cellular RNA was extracted from the cells and subjected to real-time RT-PCR to determine G6Pase mRNA levels. The results were normalized to HPRT RNA levels. Values are (mean ± standard error) from four independent experiments expressed as fold increase. (D) HuH7 and FLRP cells were treated with 50 units/ml of IFNa for 72 h or left untreated. After 72 h, extracellular glucose production was measured, the results were normalized to cell viability. p <0.001 compared to non-treated FLRP (Student’s t test). Values are (mean ± standard deviation) from at least two independent experiments expressed as fold increase. (E) RNA extracted from formalin-fixed paraffin-embedded liver biopsies of HCV infected patients or controls was subjected to real-time RT-PCR. HCV, PGC-1a, and G6Pase mRNA levels were determined. The results were normalized to HPRT RNA levels. Values are mean ± standard deviation of 8 controls and 16 HCV patients. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.0001 (Student’s t test). (F) Schematic presentation of the model suggesting PGC-1a as the mediator of HCV-induced dysregulated hepatic gluconeogenesis.
co-activation of key gluconeogenic enzymes [15,18,25]. Here, we show that PGC-1a is strongly induced following HCV infection, in a HCV dependent manner. Most importantly, both IFNa treatment and PGC-1a knockdown resulted in a significant abolishment of hepatic gluconeogenesis, as reflected by reduced G6Pase expression and glucose production, suggesting a major role for PGC-1a in the development of HCV-associated diabetes mellitus and insulin resistance. Hepatic oxidative stress is a prominent feature of chronic HCV infection [9,10]. Interestingly, PGC-1a, which is a central generator of hepatic gluconeogenesis [15], and which was shown to be elevated in livers of mice with insulin resistance, is also induced upon oxidative stress [23]. Our results confirmed our contention that PGC-1a links HCV to insulin resistance and imply that HCV-induced cellular oxidative stress mediates this link. Initial studies in search of the molecular mechanism of HCVassociated insulin resistance used human liver samples and indicated that insulin signaling is impaired in HCV-infected livers [5,26]. HCV core protein was the first viral protein implicated in HCV-associated insulin resistance [27–30]. Among other mechanisms, core expression was shown to increase proteasome-mediated IRS-1 degradation, this increased degradation was shown to be mediated by activation of suppressors of cytokine signaling
(SOCS) family members [28]. Core proteins from different HCV genotypes can impair insulin signaling using different mechanisms [28]. Although core apparently plays a major role in the impaired insulin signaling during HCV infection, additional viral proteins most likely contribute to insulin resistance and diabetes in infected cells. Deng et al. [22] recently showed that NS5A increased the production of mitochondrial reactive oxygen species and decreased the phosphorylation of forkhead box protein O1 (FOXO1), via JNK activation. Decreased phosphorylation of FOXO1 resulted in its accumulation in the nucleus where it binds and activates gluconeogenic genes, thereby enhancing gluconeogenesis. Similar to our current results, neither core nor the other viral structural proteins were shown to be involved in this process, as evidenced by the similar enhanced gluconeogenesis levels in cells harboring subgenomic or full-length HCV replicons. However, in contrast to Deng et al., our results attributed the enhanced hepatic gluconeogenesis induced by HCV to PGC-1a that co-activates gluconeogenic genes through FOXO1 as well as through other transcription factors, such as HNF4a. In addition, our results do not attribute the induction of PGC-1a and the enhanced gluconeogenesis to NS5A, since overexpression of NS5A did not affect PGC-1a mRNA or protein levels (Fig. 3). One possible explanation for this discrepancy is the difference
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Research Article in the genotype used: most of the experiments by Deng et al. were performed using genotype 2a while we used a genotype 1b replicon. Indeed, changes in the phosphorylation status of FoxO1 and JNK as reported by Deng et al. [22] were less significant in replicon-harboring cells compared to infected cells. They attributed those changes to the long cultivation of the stable replicon cells in a selection medium. Since insulin resistance is known to develop in chronically HCV-infected patients and is more prevalent in patients infected with genotype 1b [31], our model, which more closely mimics chronic infection, may more reliably reflect the mechanism for insulin resistance in HCVinfected patients. Our results were validated in chronically infected HCV patients by analyzing their hepatic PGC-1a and G6Pase levels. As predicted by our in vitro studies, both hepatic PGC-1a and G6Pase levels were elevated in these patients as compared to controls. Taking into account the fact that liver biopsies were taken after an overnight fasting, a condition that induces hepatic PGC-1a, the relatively moderate elevation observed might underestimate the actual differences between control and infected individuals during steady state. Taken together, our results suggest that PGC-1a induction, which leads to elevated expression of gluconeogenetic genes and increased glucose production in HCV-infected cells, is a central mechanism of insulin resistance associated with HCV infection. This would make PGC-1a a major target for therapeutic interventions for treatment of HCV-associated diabetes and insulin resistance. PGC-1a has also been associated with mitochondrial biogenesis and cellular energy homeostasis, two elements underlying carcinogenesis [32,33]. Given the strong association between HCV induced metabolic alterations and hepatocellular carcinoma [34], further studies that focus on the role of PGC-1a in the triad of chronic HCV infection, metabolic derangements and liver cancer are warranted.
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Financial support This study was supported by research grants from the Ministry of Justice Public Trustee Maria Rosi Ascholi Fund and by a young researcher grant from the Israeli association for the Study of the Liver to A.S and E.H.S. Acknowledgements The authors thank Esther Eshkol for editorial assistance.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2012. 06.021.
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JOURNAL OF HEPATOLOGY [25] Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P. Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature 2005;434:113–118. [26] Kawaguchi T, Ide T, Taniguchi E, Hirano E, Itou M, Sumie S, et al. Clearance of HCV improves insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate 1 and 2. Am J Gastroenterol 2007;102:570–576. [27] Kawaguchi T, Yoshida T, Harada M, Hisamoto T, Nagao Y, Ide T, 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;165:1499–1508. [28] Pazienza V, Clément S, Pugnale P, Conzelman S, Foti M, Mangia A, et al. The hepatitis C virus core protein of genotypes 3a and 1b downregulates insulin receptor substrate 1 through genotype-specific mechanisms. Hepatology 2007;45:1164–1171. [29] Miyamoto H, Moriishi K, Moriya K, Murata S, Tanaka K, Suzuki T, et al. Involvement of the PA28{gamma}-dependent pathway in insulin resistance induced by hepatitis C virus core protein. J Virol 2007;81:1727–1735.
[30] Banerjee S, Saito K, Ait-Goughoulte M, Meyer K, Ray RB, Ray R. Hepatitis c virus core protein upregulates serine phosphorylation of insulin receptor substrate-1 and impairs the downstream Akt/protein kinase b signaling pathway for insulin resistance. J Virol 2008;82:2606–2612. [31] Knobler H, Schihmanter R, Zifroni A, Fenakel G, Schattner A. Increased risk of type 2 diabetes in noncirrhotic patients with chronic hepatitis C virus infection. Mayo Clin Proc 2000;75:355–359. [32] Ristow M. Oxidative metabolism in cancer growth. Curr Opin Clin Nutr Metab Care 2006;9:339–345. [33] Warburg O. On respiratory impairment in cancer cells. Science 1956;124:269–270. [34] Koike K. Steatosis, liver injury, and hepatocarcinogenesis in hepatitis C viral infection. J Gastroenterol 2009;44:82–88. [35] Shlomai A, Paran N, Shaul Y. PGC-1alpha controls hepatitis B virus through nutritional signals. Proc Natl Acad Sci USA 2006;103:16003–16008. [36] Sklan EH, Staschke K, Oakes TM, Elazar M, Winters M, Aroeti B, et al. A RabGAP TBC domain protein binds hepatitis C virus NS5A and mediates viral replication. J Virol 2007;81:11096–11105.
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The metabolic regulator PGC-1a links hepatitis C virus infection to hepatic insulin resistance Amir Shlomai1, Maya Mouler Rechtman2, Ela Olga Burdelova1, Alona Zilberberg2, Sarit Hoffman3, Irit Solar3, Sigal Fishman1, Zamir Halpern1, Ella H. Sklan2,⇑ 1 The Research Center for Digestive Tract and Liver Diseases, Tel-Aviv Sourasky Medical Center and The Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel; 2Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel; 3The Institute of Pathology and Cancer Research, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel
Background & Aims: Chronic hepatitis C virus (HCV) infection is strongly associated with insulin resistance and diabetes mellitus. Peroxisome proliferator-activated receptor-gamma co-activator 1a (PGC-1a) is a transcriptional co-activator involved in the initiation of gluconeogenesis in the liver. Increased hepatic expression of PGC-1a has been implicated in insulin resistance. We investigated whether modulation of PGC-1a levels following HCV infection underlies HCV-associated hepatic insulin resistance. Methods: HCV genomes were expressed in hepatoma cells followed by analysis of PGC-1a and gluconeogenesis levels. Results: PGC-1a was robustly induced in HCV infected cells. PGC-1a induction was accompanied by an elevated expression of the gluconeogenic gene glucose-6 phosphatase (G6Pase) and increased glucose production. The induction of gluconeogenesis is HCV dependent, since interferon treatment abolishes PGC-1a and G6Pase elevation and decreases glucose output. Moreover, PGC-1a knockdown resulted in a significant reduction of G6Pase levels in HCV full length replicon cells, emphasizing the central role of PGC-1a in the exaggerated gluconeogenic response observed in HCV patients. Treatment of HCV replicon cells with the antioxidant N-acetylcysteine resulted in reduction of PGC1a levels, suggesting that HCV-induced oxidative stress promoted PGC-1a upregulation. Finally, both PGC-1a and G6Pase RNA levels were significantly elevated in liver samples of HCV infected patients, highlighting the clinical relevance of these results.
Keywords: Hepatitis C virus; Peroxisome proliferator-activated receptor gamma co-activator 1a; Diabetes; Insulin resistance; Oxidative stress. Received 17 December 2011; received in revised form 19 May 2012; accepted 14 June 2012; available online 23 June 2012 ⇑ Corresponding author. Tel.: +972 3 6408197; fax: +972 3 6409160. E-mail address: [email protected] (E.H. Sklan). Abbreviations: HCV, hepatitis C virus; PGC-1a, peroxisome proliferator-activated receptor gamma, co-activator 1a; G6Pase, glucose-6 phosphatase; RNA, ribonucleic acid; PI3, phosphatidylinositol 3-kinase; FLRP, full length replicon; SGR, subgenomic replicon; NS5A, non-structural protein 5A; SEAP, secreted alkaline phosphatase; NS, non-structural; SDS, sodium dodecyl sulfate; RIPA, radio immunoprecipitation assay; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; HPRT, hypoxanthine phosphoribosyltransferase; HA, hemagglutinin; ECL, enhanced chemiluminescence; IFN, interferon; SOCS, suppressors of cytokine signaling; FOXO1, forkhead box protein O1; ER, endoplasmic reticulum; JNK, c-Jun N terminal kinase; NAC, N-acetylcysteine; IRS-1, insulin receptor substrate-1.
Conclusions: PGC-1a is robustly induced following HCV infection, resulting in an upregulated gluconeogenic response, thereby linking HCV infection to hepatic insulin resistance. Our results suggest that PGC-1a is a potential molecular target for the treatment of HCV-associated insulin resistance. Ó 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Chronic hepatitis C virus (HCV) infection is a major global health problem. Chronically infected patients may develop cirrhosis and liver cancer that are implicated in substantial morbidity and mortality [1]. Although the liver is the target organ for HCV infection, extrahepatic manifestations of HCV, including insulin resistance and diabetes mellitus, are frequently encountered in the clinical setting [2]. Insulin resistance was found to be a specific feature of chronic HCV associated with genotypes 1 and 4 and with high serum viral RNA levels [3]. Insulin resistance and diabetes mellitus also correlated with poor response to anti-HCV therapy, while eradication of the virus correlates with a marked improvement in insulin resistance [4]. The molecular mechanism by which HCV promotes insulin resistance is still undetermined. HCV infection was found to cause a postreceptor defect in the insulin signaling cascade [5]. In addition, the activation of proinflammatory mediators, such as nuclear factor-kappa-B and tumor necrosis factor-a, caused by the chronic infection state, interferes with insulin signaling and contributes to both hepatic and peripheral insulin resistance [6,7]. In agreement with the evidence suggesting a central role for reactive oxygen species in the development of insulin resistance [8], HCV infection is known to promote cellular oxidative stress through multiple mechanisms, including chronic inflammation, iron overload, and liver injury. Some of the HCV proteins were reported to directly contribute to this process [9,10]. Peroxisome proliferator-activated receptor gamma co-activator 1a (PGC-1a) is a transcription co-activator and a master regulator of gluconeogenesis that functions via interaction with transcription factors located on the promoters of gluconeogenic genes [11–13]. Although PGC-1a is scarcely detectable in the
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Research Article liver under basal conditions, it is robustly induced in response to starvation, an effect that is mediated by the counter-regulatory hormonal response in which glucagon and glucocorticoids are involved [14,15]. PGC-1a has been shown to be overexpressed in livers of obese-hyperglycemic (Ob/Ob) mice [15], most probably reflecting the amplified hepatic gluconeogenesis that results in insulin resistance and diabetes mellitus. Interestingly, reducing hepatic oxidative stress in diabetic (db/db) mice resulted in a significant improvement in insulin resistance that was accompanied by a marked reduction of hepatic PGC-1a levels [16]. These data suggest that PGC-1a might be a central molecular player in the interplay between hepatic oxidative stress and insulin resistance. We hypothesized that the increased oxidative stress produced during HCV infection might lead to elevated liver PGC-1a expression, thereby promoting hepatic insulin resistance and diabetes mellitus. Here, the role of PGC-1a in HCV-induced insulin resistance was investigated. By analyzing PGC-1a levels and the gluconeogenic response in HCV infected cells, as well as in HCV-infected patients, we show that PGC-1a has a central role in HCV-promoted exaggerated gluconeogenesis. These findings further clarify the molecular pathways leading to HCV induced insulin resistance and might have therapeutic implications.
Materials and methods Cell culture, transfections, and treatments HuH7 and 7.5 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (Biological Industries, Israel). Non-essential amino acids (Biological Industries) were added to HuH7.5 cells. Human interferon-a (IFN) B2 was purchased from PBL (Piscataway, NJ), and N-acetyl-L-cysteine was purchased from Sigma. Luciferase reporter assays Cells were co-transfected with PGC-Luc reporter (a gift from B.M. Spiegelman (Dana-Farber, Harvard) [18]) and pM1-secreted alkaline phosphatase (SEAP, transfection efficiency control; Roche, Palo Alto, CA). Luciferase activity was determined 48 h post-transfection. SEAP activity was determined in the culture media using the Phospha-Light System (Applied Biosystems, Carlsbad, CA). RNA analysis
ScaI linearized plasmids encoding HCV genotype 1b (Bart79I, [17]) replicon were in vitro transcribed using Ribomax RNA production kit (Promega) under nucleasefree conditions. The plasmid encoding the full-length chimeric HCV J6/JFH1 genome was linearized with XbaI, followed by mung bean nuclease treatment. The linearized template was transcribed using a MEGAscript T7 kit (Ambion, Austin, TX). The DNA template was digested by DNase. RNA was extracted using TRIReagent. In vitro transcribed RNAs were electroporated into HuH7.5 cells, as described [19]. The pulsed cells were left to recover for 15 min and then plated in 6-well plates for further treatment. PGC-1a knockdown HIV-1-based lentiviral pGIPZ vectors containing shPGC-1a (clone V2LHS_71173) and scrambled shRNAs (clone RHS_4346) were purchased from Open Biosystems (Huntsville, AL). shRNAs harboring viruses were produced by transfection of 293T cells with 20 lg of shRNA plasmid together with the helper plasmids pCMVDR8.2 (15 lg) and pVSVG (5 lg). Three days post-transfection, the culture medium was collected and filtered. HuH7 and FLRP cells in 6-well plates were incubated with 1 ml of infectious medium supplemented with polybrene (8 lg/ml; Sigma) for 8 h. The medium was replaced and cells were then incubated for additional 48 h. Glucose production The production of glucose was measured by an AmplexÒ Red Glucose/Glucose oxidase assay kit (Invitrogen). One million HuH7 or FLRP cells were seeded in a 6-well plate. The next morning, the culture medium was replaced with PBS (Biological Industries) supplemented with 20 mM sodium lactate. Following a 2-h incubation, media were collected and glucose levels were determined. The readings were normalized to cell viability determined by Alamar blue (Invitrogen). Gluconeogenesis levels were calculated by subtracting glucose levels in the presence of the gluconeogenic substrates from baseline glucose levels. RNA extraction from formalin-fixed paraffin-embedded liver tissue Paraffin-embedded liver tissues were taken from historical patients who were infected with HCV genotype 1, controls were HCV negative patients whom liver biopsies were taken for evaluation of elevated liver function tests from various other etiologies. The study was approved by the Tel-Aviv Sourasky Medical Center Ethics Committee (#0133-12-TLV), and was conducted in accordance with the principles of the Declaration of Helsinki. RNA extraction from formalin fixed paraffin embedded liver tissues was performed as described [20]. Briefly, tissue sections were deparaffinized using xylene and ethanol, air dried and incubated at 55 °C for 16 h in RNA lysis buffer (20 mM Tris pH 7.5, 20 mM EDTA, 1% SDS and 0.5 lg/ll proteinase K). RNA was extracted using Tri-Reagent (Sigma). Real-time PCR was preformed as described above.
Results
RNA was extracted from cells by Tri-reagent (Sigma). After treatment with RNasefree DNase I (Promega), the RNA was subjected to reverse transcription using the Verso cDNA kit (Thermo, Waltham, MA). Real-time PCR was performed on the resulting cDNA to quantify the amounts of HCV, PGC-1a, and G6Pase mRNAs using the primers listed in Supplementary Table 1. Results were normalized to the mRNA levels of the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) (Supplementary Table 1). Protein analysis Proteins were extracted from cells using radio immunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitors (Sigma) and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The following primary antibodies were used for Western blot: monoclonal mouse anti-HCV NS5A (Virostat, Portland, ME), mouse anti-PGC-1a antibody (Calbiochem, La Jolla, CA), mouse anti-tubulin antibody (Sigma) or mouse anti-actinin (Santa Cruz Biotechnology, Santa Cruz, CA). A goat anti-mouse antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody. Proteins were detected by chemiluminescence.
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PGC-1a is elevated in HCV infected cells We hypothesized that HCV replication induces PGC-1a. To verify this hypothesis, PGC-1a mRNA levels were first examined in the context of a newly established HCV infection, namely, electroporation of a genotype 1b subgenomic replicon (SGR, Bart 79I [17]). RNA was extracted 72 h following electroporation and analyzed for HCV and PGC-1a mRNA levels. HCV mRNA levels dramatically increased following electroporation (138-fold). A replicon with a lethal mutation in the RNA-dependent RNA polymerase (pol-) served as a negative control [17]. In agreement with our hypothesis, PGC-1a mRNA levels were significantly increased (7-fold) in these replicon cells (Fig. 1A). Since this replicon does not contain the viral structural genes (see scheme in Fig. 1), this results also indicates that the structural genes are not essential for PGC-1a elevation. Because our hypothesis links PGC-1a overexpression
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To determine whether PGC-1a induction is HCV-dependent, FLRP cells were treated with interferon a (IFNa) for 72 h, after which PGC-1a mRNA and protein levels were analyzed. IFNa treatment dramatically decreased HCV RNA levels (Fig. 2A, left) and attenuated PGC-1a induction to levels similar to those of the HuH7 controls (Fig. 2A, right). In contrast, IFNa treatment did not affect PGC-1a mRNA levels in the control HuH7 cells. Western blot analysis of lysates from the IFNa-treated cells using PGC-1a antibodies confirmed these results (Fig. 2A, bottom). Next, PGC-1a was knockeddown in FLRP and HuH7 cells. Following knockdown, PGC-1a mRNA levels decreased significantly (2- to 3-folds) in FLRP cells to levels comparable to parental HuH7 cells, while HCV RNA levels were not significantly altered (Fig. 2B). These data suggest that while HCV expression enhances PGC-1a expression, PGC-1a itself has no effect on HCV RNA levels.
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Fig. 1. PGC-1a is elevated in HCV replicon cells. (A) HuH7.5 cells were electroporated with the HCV subgenomic replicon (SGR) or a replicon with a lethal polymerase mutation (Pol-). Total RNA was harvested after 72 h and used to determine HCV and PGC-1a mRNA levels by real-time RT-PCR. The results were normalized to HPRT levels. Values are mean ± standard deviations of at least three independent experiments and are expressed as fold increase. (B) FLRP and HuH7 cells were transfected with a PGC-Luc reporter. Luciferase activity in cell lysates was determined 48 h post-transfection. Total RNA was extracted from HuH7 and FLRP cells and subjected to real-time RT-PCR to determine PGC-1a mRNA levels. The results were normalized to HPRT levels and analyzed as described above. ⁄ p = 0.002 (Student’s t test). HuH7 cells were either transfected with PGC-HA [35], treated with dexamethasone (Dex) and forskolin (Forsk) or left untreated. Posttransfection (24 h), the cells were lysed and analyzed by Western blot together with FLRP cells. The graph shows a quantification of the band intensities. (C) HuH7.5 cells were electroporated with 5 lg of J6/JFH RNA or a pol- control. Total RNA was harvested after 72 h and used to determine HCV and PGC-1a mRNA levels by real-time PCR. The results were normalized to HPRT levels and analyzed as mentioned above.
PGC-1a induction following HCV infection can occur via a direct interaction with a viral component or via an indirect mechanism. Given that PGC-1a is elevated in the context of both full length and subgenomic replicons, the viral structural proteins were clearly not responsible for this induction. Among the non-structural (NS) proteins, NS5A would be a natural candidate since two recent studies either physically or mechanistically linked PGC-1a with NS5A [21,22]. In our hands, overexpression of genotype 1b NS5A in HuH7 cells did not significantly alter PGC-1a levels (Fig. 3A). Since the hepatic oxidative stress known to occur during HCV infection [9] was previously linked to insulin resistance [16] as well as to alterations in PGC-1a levels [23], we investigated whether oxidative stress is involved in PGC-1a induction promoted by HCV. HCV replicon cells were treated with the antioxidant N-acetyl-cysteine (NAC) after which PGC-1a levels were determined. NAC significantly reduced PGC-1a induction, while having an insignificant effect on HCV RNA levels (Fig. 3B), suggesting a role for oxidative stress as a mediator of HCV-promoted PGC-1a induction.
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PGC-1a upregulation in HCV infected cells enhances gluconeogenesis G6Pase hydrolyzes glucose-6-phosphate to free glucose and a phosphate group. This catalytic action is the final step in gluconeogenesis and therefore plays a key role in the regulation of blood glucose levels [24]. PGC-1a plays a crucial role in the transcriptional regulation of G6Pase [14,15]. We analyzed G6Pase mRNA levels in HCV-expressing cells in order to further understand the role of PGC-1a upregulation in HCV-infected cells, and to investigate whether this elevation affects gluconeogenesis. G6Pase levels were significantly increased (6-fold) in HuH7 cells 72 h post-electroporation of a HCV subgenomic replicon (Fig. 4A, left). G6Pase levels were elevated by 2.5-fold in FLRP cells as well (Fig. 4A, right). IFNa treatment blocked G6Pase elevation in both HCV electroporated and stable replicon cells, confirming its dependency on the presence of HCV. To further illustrate that G6Pase induction is PGC-1a dependent, PGC-1a was knockeddown using specific shRNAs (Fig. 4B). As expected, PGC-1a depletion decreased G6Pase mRNA levels in both HuH7 and FLRP cells. G6Pase mRNA levels in PGC-1a-depleted cells were reduced to its levels in the parental HuH7 cells. Similarly,
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Fig. 3. A general antioxidant can abrogate PGC-1a induction following infection. (A) HuH7 cells in 6 wells (80–90% confluent) were transfected with pEF6-NS5A [36] or an empty vector. After 48 h, total RNA was harvested and subjected to real-time RT-PCR to determine PGC-1a mRNA levels. The results were normalized to HPRT RNA levels. Cells similarly transfected were lysed and analyzed by Western blot using PGC-1a and NS5A antibodies. Controls were similar to Fig. 1B. (B) HuH7 and FLRP cells were treated with 5 mM NAC for 4 h. Following treatment, total RNA was extracted and subjected to real-time RT-PCR to determine PGC-1a and HCV RNA levels. The results were normalized to HPRT RNA levels. ⁄p = 0.005 compared to non-treated FLRP cells, n.s. = not significant (Student’s t test). Values are mean ± standard deviation of two independent experiments expressed as fold increase.
NAC treatment reduced G6Pase mRNA levels to levels comparable to the HuH7 controls (Fig. 4C). To further assess the physiological relevance of PGC-1a and G6Pase upregulation in HCV replicon cells, we compared the levels of glucose output, reflecting endogenous gluconeogenesis, between FLRP and HuH7 cells. In agreement with our hypothesis, gluconeogenesis was significantly elevated in FLRP cells compared to HuH7 controls (7-fold, Fig. 4D). This elevated gluconeogenesis was HCV-dependent since IFNa treatment significantly abrogated it. PGC-1a and G6Pase are elevated in HCV infected patients To substantiate our results, liver biopsy samples from HCV infected patients and non-infected patients (controls) were analyzed for PGC-1a and G6Pase mRNA levels. In agreement with our results, a significant 3-fold elevation in the mRNA levels of both PGC-1a and G6Pase was detected in HCV patients as compared to controls. Furthermore, the degree of both PGC-1a and G6Pase inductions tended to correlate with each other and with the hepatic HCV RNA levels (Supplementary Fig. 1).
Discussion PGC-1a is a central metabolic regulator that is robustly induced in the liver upon starvation, to initiate gluconeogenesis through
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Fig. 4. PGC-1a upregulation in HCV infected cells enhances gluconeogenesis. (A) HuH7.5 cells were electroporated with the HCV subgenomic replicon (SGR) or a pol-, control replicon. Cells were treated with 50 units/ml of IFNa for 72 h or left untreated. HuH7 and FLRP cells were treated similarly. Total cellular RNA was extracted from the cells and subjected to real-time RT-PCR to determine G6Pase mRNA levels. The results were normalized to HPRT RNA levels. ⁄p <0.002 compared to non-treated FLRP (Student’s t test). (B) HuH7 and FLRP cells were infected with lentiviruses expressing PGC-1a or scrambled shRNAs. After 48 h, total cellular RNA was extracted from the cells and subjected to real-time RT-PCR to determine G6Pase mRNA levels. The results were normalized to HPRT RNA levels. (C) HuH7 and FLRP cells were treated with 5 mM NAC for 4 h. Total cellular RNA was extracted from the cells and subjected to real-time RT-PCR to determine G6Pase mRNA levels. The results were normalized to HPRT RNA levels. Values are (mean ± standard error) from four independent experiments expressed as fold increase. (D) HuH7 and FLRP cells were treated with 50 units/ml of IFNa for 72 h or left untreated. After 72 h, extracellular glucose production was measured, the results were normalized to cell viability. p <0.001 compared to non-treated FLRP (Student’s t test). Values are (mean ± standard deviation) from at least two independent experiments expressed as fold increase. (E) RNA extracted from formalin-fixed paraffin-embedded liver biopsies of HCV infected patients or controls was subjected to real-time RT-PCR. HCV, PGC-1a, and G6Pase mRNA levels were determined. The results were normalized to HPRT RNA levels. Values are mean ± standard deviation of 8 controls and 16 HCV patients. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.0001 (Student’s t test). (F) Schematic presentation of the model suggesting PGC-1a as the mediator of HCV-induced dysregulated hepatic gluconeogenesis.
co-activation of key gluconeogenic enzymes [15,18,25]. Here, we show that PGC-1a is strongly induced following HCV infection, in a HCV dependent manner. Most importantly, both IFNa treatment and PGC-1a knockdown resulted in a significant abolishment of hepatic gluconeogenesis, as reflected by reduced G6Pase expression and glucose production, suggesting a major role for PGC-1a in the development of HCV-associated diabetes mellitus and insulin resistance. Hepatic oxidative stress is a prominent feature of chronic HCV infection [9,10]. Interestingly, PGC-1a, which is a central generator of hepatic gluconeogenesis [15], and which was shown to be elevated in livers of mice with insulin resistance, is also induced upon oxidative stress [23]. Our results confirmed our contention that PGC-1a links HCV to insulin resistance and imply that HCV-induced cellular oxidative stress mediates this link. Initial studies in search of the molecular mechanism of HCVassociated insulin resistance used human liver samples and indicated that insulin signaling is impaired in HCV-infected livers [5,26]. HCV core protein was the first viral protein implicated in HCV-associated insulin resistance [27–30]. Among other mechanisms, core expression was shown to increase proteasome-mediated IRS-1 degradation, this increased degradation was shown to be mediated by activation of suppressors of cytokine signaling
(SOCS) family members [28]. Core proteins from different HCV genotypes can impair insulin signaling using different mechanisms [28]. Although core apparently plays a major role in the impaired insulin signaling during HCV infection, additional viral proteins most likely contribute to insulin resistance and diabetes in infected cells. Deng et al. [22] recently showed that NS5A increased the production of mitochondrial reactive oxygen species and decreased the phosphorylation of forkhead box protein O1 (FOXO1), via JNK activation. Decreased phosphorylation of FOXO1 resulted in its accumulation in the nucleus where it binds and activates gluconeogenic genes, thereby enhancing gluconeogenesis. Similar to our current results, neither core nor the other viral structural proteins were shown to be involved in this process, as evidenced by the similar enhanced gluconeogenesis levels in cells harboring subgenomic or full-length HCV replicons. However, in contrast to Deng et al., our results attributed the enhanced hepatic gluconeogenesis induced by HCV to PGC-1a that co-activates gluconeogenic genes through FOXO1 as well as through other transcription factors, such as HNF4a. In addition, our results do not attribute the induction of PGC-1a and the enhanced gluconeogenesis to NS5A, since overexpression of NS5A did not affect PGC-1a mRNA or protein levels (Fig. 3). One possible explanation for this discrepancy is the difference
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Research Article in the genotype used: most of the experiments by Deng et al. were performed using genotype 2a while we used a genotype 1b replicon. Indeed, changes in the phosphorylation status of FoxO1 and JNK as reported by Deng et al. [22] were less significant in replicon-harboring cells compared to infected cells. They attributed those changes to the long cultivation of the stable replicon cells in a selection medium. Since insulin resistance is known to develop in chronically HCV-infected patients and is more prevalent in patients infected with genotype 1b [31], our model, which more closely mimics chronic infection, may more reliably reflect the mechanism for insulin resistance in HCVinfected patients. Our results were validated in chronically infected HCV patients by analyzing their hepatic PGC-1a and G6Pase levels. As predicted by our in vitro studies, both hepatic PGC-1a and G6Pase levels were elevated in these patients as compared to controls. Taking into account the fact that liver biopsies were taken after an overnight fasting, a condition that induces hepatic PGC-1a, the relatively moderate elevation observed might underestimate the actual differences between control and infected individuals during steady state. Taken together, our results suggest that PGC-1a induction, which leads to elevated expression of gluconeogenetic genes and increased glucose production in HCV-infected cells, is a central mechanism of insulin resistance associated with HCV infection. This would make PGC-1a a major target for therapeutic interventions for treatment of HCV-associated diabetes and insulin resistance. PGC-1a has also been associated with mitochondrial biogenesis and cellular energy homeostasis, two elements underlying carcinogenesis [32,33]. Given the strong association between HCV induced metabolic alterations and hepatocellular carcinoma [34], further studies that focus on the role of PGC-1a in the triad of chronic HCV infection, metabolic derangements and liver cancer are warranted.
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Financial support This study was supported by research grants from the Ministry of Justice Public Trustee Maria Rosi Ascholi Fund and by a young researcher grant from the Israeli association for the Study of the Liver to A.S and E.H.S. Acknowledgements The authors thank Esther Eshkol for editorial assistance.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2012. 06.021.
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