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Keywords:

  • chronic hepatitis C;
  • diabetes mellitus;
  • FoxO1;
  • insulin resistance;
  • PGC1α

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The maintenance of glucose homeostasis is a complex process in which the insulin signalling pathway plays a major role. Disruption of insulin-regulated glucose homeostasis is frequently observed in chronic hepatitis C (CHC) infection and might potentially contribute to type 2 diabetes mellitus (T2DM) development. Presently, the mechanism that links HCV infection to insulin resistance remains unclear. Previously, we have reported that HCV protein expression in HCV transgenic mice (B6HCV) leads to an overexpression of protein phosphatase 2A (PP2A) through an ER stress response. In the present work, we describe an association of FoxO1 hypophosphorylation and upregulation of both PGC-1α and G6Pase to phenotypic hyperglycaemia and insulin resistance in B6HCV mice. In vitro, we observed that PGC1α is concomitantly induced with PP2A. Moreover, we show that the enhanced PP2A expression is sufficient to inhibit insulin-induced FoxO1 phosphorylation via blockade of insulin-mediated Akt activation or/and through direct association and dephosphorylation of pS-FoxO1. Consequently, we found that the gluconeogenic gene glucose-6-phosphatase is upregulated. These observations were confirmed in liver biopsies obtained from CHC patients. In summary, our results show that HCV-mediated upregulation of PP2A catalytic subunit alters signalling pathways that control hepatic glucose homeostasis by inhibiting Akt and dephosphorylation of FoxO1.


Abbreviations
CHC

chronic hepatitis C

CMV

cytomegalovirus

FLD

fatty liver disease

G6Pase

glucose-6-phosphatase

HA

haemagglutinin protein

HCV

Hepatitis C virus

IR

insulin resistance

IRS-1

insulin receptor substrate-1

PP2A

protein phosphatase 2A

T2DM

type 2 diabetes mellitus

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The liver has a central role in the regulation of glucose homeostasis. Indeed, it has the capacity to remove high glucose content from the blood through storage as glycogen or to produce and release new glucose molecules when low blood glucose is detected [1]. The maintenance of glucose metabolism is a complex process in which insulin, a hormone secreted by pancreatic beta cells, plays a major role. In hyperglycaemic condition, insulin represses the expression of gluconeogenic genes such as glucose-6-phosphatase (G6Pase), whereas in hypoglycaemic condition, it upregulates the gluconeogenic genes [2]. The association of insulin to its tyrosine kinase receptor leads to an autophosphorylation of the intracellular domain of the receptor which then rapidly recruits and phosphorylates phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2) by phosphoinositide-3-kinase (PI3K) [3-5]. PtdIns(4,5)P2 is an important second messenger in the insulin signalling pathway that has as an effector partner protein kinase B (PKB) also termed Akt. The binding of Akt to PtdIns(4,5)P2 occurs at the plasma membrane because of the subcellular localization of PtdIns(4,5)P2. Once recruited to the membrane, Akt is activated through phosphorylation on threonine 308 and serine 473 by protein kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) and mammalian target of rapamycin complex 2 (mTORC2), respectively [6]. Activated Akt then dissociates from the plasma membrane and phosphorylates numerous cytoplasmic and nuclear proteins that are important in glucose homeostasis. FoxO1 and PGC1α are two transcriptional components that are essential to initiate gluconeogenesis in the liver. The transcriptional activity of FoxO1 is negatively regulated by Akt-mediated serine phosphorylation leading to a nuclear exclusion of FoxO1. PGC1α expression is independent from the insulin signalling pathway [7, 8]. It has been reported that PGC1α associates to FoxO1 and behaves as a transcriptional co-activator of FoxO1 resulting in an enhanced transcription of insulin-induced gluconeogenic genes [9]. This canonical pathway of insulin-regulated hepatic glucose homeostasis has recently been questioned. Indeed, it was reported that in vivo Akt is a dispensable regulator of insulin-regulated glucose homeostasis in the absence of FoxO1 [10]. Nevertheless, disruption of insulin signalling pathway is frequently observed in metabolic syndrome (MS) diseases and predisposes to T2DM development [11].

Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases. Interestingly, extrahepatic manifestations can be observed in chronic HCV infection (CHC) that is considered as a risk factor for T2DM [12]. Heretofore, the molecular mechanisms that link HCV infection to insulin resistance (IR) and thus to T2DM remain unclear. Several studies were performed during the previous years, attempting to clarify the mechanism of HCV-mediated IR. For instance, it has been shown that HCV core-induced serine phosphorylation on insulin receptor substrate-1 (IRS-1) results in an impairment of insulin-mediated IRS-1 tyrosine phosphorylation and thus inhibits the insulin signalling pathway [13]. Using an HCV infectious system, another study reported an HCV core-mediated inhibition of IRS-1 phosphorylation enhancing the mTOR/S6K1 pathway and thus resulting in an impairment of the insulin signalling pathway [14]. Finally, we have demonstrated an inhibition of the Akt pathway in liver biopsies from CHC patients, suggesting a role of HCV infection in IR [15].

The major serine/threonine protein phosphatase 2A is ubiquitously expressed in all cell types. This trimeric enzyme is composed of a scaffolding subunit A, a regulatory subunit B and a catalytic C subunit [16]. The C and A subunits associate together to form the catalytic core [17]. PP2A expression is maintained constant within the cell through an autoregulatory mechanism, making unsuccessful attempts to overexpress the fully active PP2A catalytic subunit (PP2Ac) [18]. Nevertheless, addition of a peptide sequence derived from the influenza haemagglutinin protein (HA) at the N-terminal end of the PP2Ac sequence has been reported to increase the phosphatase activity [19]. This autoregulatory mechanism is disrupted in CHC and chronic hepatitis B (CHB) virus infection leading to an overexpression of the catalytic subunit [20, 21]. The regulatory B subunit determines the subcellular localization of the heterotrimeric enzyme as well as the substrate specificity through specific sequence recognition on the A subunit [22]. Several post-translational modifications on the C subunit are described and have been shown to modulate the activity of PP2A. For instance, the phosphorylation on tyrosine 307 inhibits PP2A activity [23], and the carboxymethylation on leucine 309 controls the interaction of the catalytic subunit with the Bα subunit [24]. PP2A has been shown to form a complex with FoxO1 and acts as a physiological phosphatase [25]. Previously, we have reported that HCV-mediated PP2Ac upregulation impairs insulin-induced Akt phosphorylation [15]. In the present work, we describe the molecular mechanism by which HCV-mediated PP2Ac upregulation modulates gluconeogenic genes expression and therefore alters glucose homeostasis. We found that PP2Ac and PGC1α are overexpressed in HCV transgenic mice and in HCVcc system. Furthermore, PP2A blocks insulin-mediated FoxO1 phosphorylation via reduction in Akt activation or/and through direct association and dephosphorylation of FoxO1 resulting in an enhanced G6Pase expression. Finally, we show that these molecular changes induced by PP2Ac upregulation in HCV transgenic mice lead to hyperglycaemia.

Experimental Procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Reagents, antibodies and plasmids

Human insulin, okadaic acid and actin antibody were from Sigma (Fluka Chemie, GmbH; Buchs, Switzerland). Purified PP2Ac and PP2Ac antibodies were from Upstate (LucernaChem, Luzern, Switzerland). Antibodies against pS473Akt, Akt, pS256FoxO1 and FoxO1 were from Cell Signalling (Bioconcept, Allschwil, Switzerland).

Human liver biopsies

Liver biopsy samples from control, CHC and nonviral hepatitis metabolic syndrome diseases were collected according to the protocol approved by the local ethics committee of the Canton Basel-Stadt (Table 1). Written informed consent was obtained from all patients. Liver biopsy extracts were prepared as described [26]. After removal of a 20–25 mm long biopsy specimen for routine histopathological workup, the remaining biopsy specimens were immediately incubated in 100-nm insulin or kept in 25 mm HEPES for 5 min. Samples were then used for total protein extracts and Western blotting as previously described [27].

Table 1. Patients' characteristics. PP2Ac, PGC1α and G6Pase values are expressed relative to GAPDH
DiagnosisGTViral loadCoding # PP2Ac PGC1α G6Pase
  1. n.d., not determined; G6Pase, glucose-6-phosphatase; HCV, Hepatitis C virus; PPA2c, PP2A catalytic subunit.

Drug toxicity  B7330.02130.02600.0560
Normal liver  B6520.01700.07880.1703
Drug Toxicity  B7320.02780.06670.0987
Normal, no T2DM  A7710.01670.01100.0565
HCV1a1389686B3920.04460.18220.2867
HCV11698243B7650.03340.05730.2112
HCV22818383B9320.04380.03470.0769
HCV1a720851B5230.01570.07050.1453
HCV1a7585775B6090.01060.03520.0623
HCV1an.d.B6790.02810.09330.1184
HCV1a455164B7280.01380.32330.0976
HCV1an.d.B7540.03310.07980.1214
HCV1a69713B7780.04410.11410.2313
HCV1an.d.B7840.04290.05580.1064
HCV1a5815142B8060.03780.03400.0514
HCV1a1348962B8760.01780.04590.0957
HCV1a32800B9180.02820.09290.1409
HCV1a/b941000B7360.01780.06410.1784
HCV1b530221B8070.02390.02240.0390
HCV1b2398832B8190.03370.04510.2283
HCV1b2940128B8750.01320.09630.2238
HCV31445439B7570.02110.03050.1015
HCV3474333B7860.03410.04590.1614
HCV4139013B6690.01630.10270.2602
HCV4439039B6730.01700.07970.4130
HCV41380384B6780.01740.02270.0840
HCV43162278B8230.01880.04110.0523
HCV3a2398833B5850.01710.08890.0598
HCV3a11988B6110.02150.03380.0465
HCV3a2818383B6310.01910.07400.1996
HCV3a6025596B6580.04280.21760.2320
HCV3a672811B6750.02740.07500.0719
HCV3an.d.B7800.05270.10000.2617
HCV3an.d.B8930.01240.06090.1882
HCV3a802225B9160.01810.02640.2577
NAFLD  B7200.03460.07770.2483
ASH/NASH  B6220.06340.02220.1442
NAFLD  B7050.03240.05900.1939
Steatosis  B6800.04010.04070.3438
NAFLD  B7350.03300.05190.2699
ASH/NASH  B6330.02780.03040.1444
Steatohepatitis  B4470.03360.03530.0452

B6HCV and C57BL6 Mice

Animals were maintained in the animal facility of the Department of Biomedicine at the University Hospital Basel, in a specific-pathogen-free environment on a 12-hour light and 12-h dark schedule. Food and drinking water were provided ad libitum. Experiments were approved by the veterinary office of the Canton Basel-Stadt.

Cell lines

Huh7.5.1, Huh7 and HA-PP2Ac cells are described elsewhere [27]. Cells were grown in DMEM medium containing 10% FCS and 25 mm glucose.

SDS-PAGE, immunoblotting and immunoprecipitation

Cells were lysed in whole cells lysis buffer containing 50 mm Tris-HCL, pH 7.5, 100 mm NaCl, 1 mm EDTA, 0.1% Triton X-100, 10 mm NaF, 1 mm PMSF and 1 mm orthovanadate. Lysates were cleared by centrifugation and quantified by Bradford assay (Bio-Rad Protein Assay; Bio-Rad Laboratories AG, Reinach, Switzerland). Proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membrane and probed with specific antibodies. Immunoprecipitation was performed as described previously [26].

Signal intensity of protein bands was analysed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).

Hepatitis C virus infectious in cell culture particles production and infection of Huh7.5.1 cells

RNA preparation and electroporation were performed as described [28]. Huh7.5.1 cells were infected with 1MOI of hepatitis C virus infectious in cell culture (HCVcc) strain JC1 particles, and replication was analysed by quantitative PCR (qPCR) [28]. HCV primers were 5′-AGGAGGCCCGCACTGCCATA-3′ and 5′-CTGGCGCGGCAACGTCTGTA-3′.

Lentiviral particles production and transduction

pSIH1-puro-control shRNA and pSIH1-puro-shPP2Ac were cotransfected with packaging vectors into HEK293T cells using Lipofectamine 2000 according to the manufacturer's instructions. Supernatants containing lentiviral particles were cleared by centrifugation and stored at −70 °C. Huh7.5.1 cells were transduced with 1MOI of lentiviral particles for 3 days, and PGC1α and G6Pase expression was analysed by qPCR.

Analysis of Gene expression

Total RNA extraction, cDNA synthesis and SYBR-based qPCR were performed as described elsewhere [27]. Primers (Microsynth AG, Balgach, Switzerland) were designed across exon–exon sequences to avoid genomic DNA amplification (Table 2).

Table 2. Human and mouse primers for quantitative PCR (qPCR)
 ForwardReverse
  1. G6Pase, glucose-6-phosphatase; PPA2c, PP2A catalytic subunit.

Human
PGC1α 5′-TTTTCTCGACACAGGTCGTG-3′5′-CGTGATCTCACATACAAGGG-3′
G6Pase 5′-TTGTGGTTGGGATTCTGG-3′5′-TTCTCCAAAGTCCACAGGAG-3′
PP2Ac 5′-CCACAGCAAGTCACACATTGG-3′5′-CAGAGCACTTGATCGCCTACAA-3′
GAPDH 5′-GCTCCTCCTGTTCGACAGTCA-3′5′-ACCTTCCCCATGGTGTCTGA-3′
Mouse
PGC1α 5′-TTTTCTCGACACAGGTCGTG-3′5′-CGTGATCTCACATACAAGGG-3′
G6Pase 5′-TTGTGGTTGGGATTCTGG-3′5′-TTCTCCAAAGTCCACAGGAG-3′
RPL19 5′-ATCCGCAAGCCTGTGACTGT-3′5′-TCGGGCCAGGGTGTTTTT-3′

Blood glucose and insulin measurement

Mice were fasted overnight (o/n). Glucose was measured from tail vein blood by Ascensia Contour blood glucose monitoring system (Bayer AG Health Care, Zurich, Switzerland). Insulin was measured from 5 μL serum using Mercodia Ultrasensitive Mouse Insulin ELISA (Mercodia, Uppsala, Sweden). HOMA-IR was calculated by the formula: fasting glucose (mm) × fasting insulin (mIU/L)/22.5 [29].

Glucose and insulin tolerance tests

For the glucose tolerance test, mice were fasted o/n and a glucose solution in saline was injected intraperitoneally at 2 g/kg of body weight. Glucose level was measured from tail vein blood before and 15, 30, 45, 60, 90 and 180 min after injection using Ascensia Contour blood glucose monitoring system (Bayer, Switzerland).

For the insulin tolerance test, mice were fasted for 4 h and an insulin solution was injected at 0.8 IU/kg body weight. Glucose level was measured from tail vein blood before and 15, 30, 45, 60 and 90 min after injection.

Immunoblotting and gene expression analysis from mice

Phosphorylated proteins were analysed by Western Blot. Mice were starved o/n and then injected intraperitoneally with insulin at 0.8 IU/kg body weight or 25 mm HEPES buffer as negative control for 40 min. Livers were harvested, snap-frozen and stored in liquid nitrogen until use. Densitometry analysis of protein bands was performed with ImageJ software (NIH Image). For gene expression analysis, mice were starved for 8 h.

In vitro dephosphorylation assay

Whole cell lysate from Huh7 cells stimulated with 100 nm insulin for 15 min was prepared and then incubated with or without purified PP2Ac (Upstate, Millipore AG, Zug, Switzerland) at 37°C for 5 min. The reaction was then stopped by heating at 100°C for 5 min and then loaded on a SDS-PAGE. pS-FoxO1 was then visualized with specific antibody.

Statistical analysis

Box plot diagrams, Wilcoxon's test, t-test and Spearman's rank correlations were performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

High blood glucose production and insulin resistance in HCV transgenic mice

We analysed the effect of HCV-mediated PP2Ac upregulation on the disturbance of gluconeogenesis by fasting HCV transgenic mice and control animals and then measured the blood glucose. Fasting glucose levels were significantly higher in B6HCV mice than in C57BL6 mice (Fig. 1a). We measured a significantly higher level of insulin in B6HCV mice than in control animals and thus an elevated HOMA-IR in HCV transgenic mice (Fig. 1b,c). These observations suggest an inability of insulin to regulate glucose homeostasis in transgenic animals. We validated this observation by performing a glucose tolerance test. We observed a higher peak of blood glucose 15 min after intraperitoneal injection in B6HCV mice compared with C57BL6 mice, followed by a decrease to basal values reached at 90 min, confirming a glucose metabolism disturbance in HCV mice (Fig. 1d). Next, we performed an insulin tolerance test on fasted animals and showed that insulin was less efficient in reducing blood glucose level in HCV transgenic animals than in control mice (Fig. 1e).

image

Figure 1. Glucose metabolism disturbance in HCV transgenic mice. HCV transgenic (n = 6) and C57BL6 (n = 6) mice were fasted overnight. Blood glucose content (a) and insulin level (b) were measured. (c) HOMA-IR was determined according to the formula described by Matthews et al. [29]. (d) Glucose tolerance test was performed using blood from B6HCV mice (n = 19, open circle) and C57BL6 mice (n = 19, full circle). (e) Insulin tolerance test was performed with B6HCV mice (n = 21, open circle) and C57BL6 mice (n = 14, full circle).

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PGC1α and G6Pase are upregulated in HCV transgenic mice

The maintenance of glucose homeostasis is a complex process that requires several interacting partners. Among them, PGC1α and G6Pase are essential in regulating hepatic gluconeogenesis. We therefore measured the expression of PGC1α and G6Pase in the liver of HCV transgenic and control mice fasted for 8 h. We show a significant upregulation of hepatic expression of PGC1α and G6Pase in B6HCV mice compared with control mice (Fig. 2a,b), suggesting an upregulation of gluconeogenesis and a possible alteration of the insulin signalling pathway.

image

Figure 2. Alteration of PGC1α and G6Pase expression in HCV mice. Mice (B6HCV, n = 7; C57BL6, n = 8) were fasted for 8 h, and the expression levels of PGC1α (a) and G6Pase (b) were quantified by quantitative PCR.

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Modulation of PP2A activity in human hepatoma cells alters gluconeogenic gene expression

We have previously reported that expression of HCV proteins in B6HCV mice leads to an upregulation of PP2Ac [26]. Because of the lack of an animal model that overexpresses PP2Ac, we were not able to distinguish if the alteration of PGC1α and G6Pase expression was due solely to an upregulation of PP2Ac or also requires the presence of HCV proteins. We therefore decided to investigate further the role of PP2A by transfecting human hepatoma cells with a HA-PP2Ac plasmid that allows a constitutive phosphatase activity of PP2A. We found that the expression of HA-PP2Ac did not upregulate PGC1α (Fig. 3a). However, we demonstrated a significant increase in G6Pase expression in PP2Ac overexpressing cells (Fig. 3a). Next, we treated Huh7 cells with OA, a specific PP2A inhibitor, or silenced PP2Ac using siRNA. We found that PGC1α expression was not altered (Figs 3b,c) by PP2Ac knockdown or by PP2Ac inhibition. G6Pase was significantly downregulated in OA-treated and in PP2Ac-silenced cells (Figs 3b,c).

image

Figure 3. Modulation of PP2Ac expression alters G6Pase expression. Cells (n = 5 independent experiments) were grown in medium containing 25 mm glucose. PGC1α and G6Pase levels were measured by quantitative PCR in cells overexpressing PP2Ac (a), cells treated with okadaic acid (b) and PP2Ac silenced cells (c). Statistical analysis was performed using t-test.

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The presence of HCV proteins is required to upregulate PGC1α

Because we have observed a significant increased expression of PGC1α in B6HCV mice but not in cells overexpressing PP2Ac, we tested whether the alteration of PGC1α expression requires HCV proteins. Indeed, it has been reported that PGC1α transcription is mediated by CREB [30]. Furthermore, we have shown previously that HCV proteins' expression leads to an ER stress response that activates CREB, resulting in an upregulation of PP2Ac [21]. This ER stress is absent in HA-PP2Ac cells. Therefore, to clarify this issue, we have infected Huh7.5.1 cells with HCVcc and then quantified PGC1α and G6Pase expression. As expected, infection of Huh7.5.1 cells induced PP2Ac upregulation (Fig. 4a) and a significant increase expression of PGC1α (Fig. 4b). We also measured more G6Pase expression (Fig. 4c). These data clearly suggest that the presence of HCV proteins is required to modulate the expression of PGC1α.

image

Figure 4. HCV-induced PP2Ac upregulation increases PGC1α and G6Pase expression. Huh7.5.1 cells (n = 3 independent experiments) were infected with 1 MOI HCVcc for 6 and 48 h. The expression levels of PP2Ac and HCV (a), PGC1α (b) and G6Pase (c) were then measured by quantitative PCR. Statistical analysis was performed using t-test.

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HCV-induced PP2Ac upregulation impairs hepatic FoxO1 phosphorylation

PGC1α and FoxO1 regulate gluconeogenesis. FoxO1 is phosphorylated by activated Akt in response to insulin stimulation [31]. Previously, we have shown that PP2A blocks insulin-mediated Akt phosphorylation. Therefore, it is likely that the impairment of FoxO1 phosphorylation is caused by a reduction in pS-Akt. However, because FoxO1 is phosphorylated on serine residue and PP2A is a major serine/threonine phosphatase, we hypothesize that PP2A may also directly inhibits pS-FoxO1 in B6HCV mice. Indeed, purified PP2A catalytic subunit has been previously reported to directly associate and dephosphorylate FoxO1 [25]. We therefore performed a co-immunoprecipitation analysis of PP2Ac and FoxO1 using mouse liver homogenates. Our data show an overexpression of PP2Ac, and more FoxO1 bound to PP2Ac in B6HCV mice than in control animals (Fig. 5a). Next, we analysed FoxO1 phosphorylation signal in HCV transgenic mice compared with control animals. The mice were injected intraperitoneally with insulin, and pS-FoxO1 signal was quantified by immunoblotting. As shown on Fig. 5b, insulin strongly induces FoxO1 phosphorylation in C57BL6 mice but not in HCV transgenic mice demonstrating an inhibitory effect of PP2A on pS-FoxO1. We confirmed our results obtained from B6HCV mice by stimulating liver biopsies from CHC or from control patients ex vivo with insulin and then analysed pS-FoxO1 signal. We have previously reported that PP2A is overexpressed in CHC patients [26]. We demonstrated here that insulin-induced Akt phosphorylation was impaired in CHC patients (Figure S1), and thus consequently, we observed a blockade of insulin-induced FoxO1 phosphorylation in CHC samples (Fig. 5c). Finally, we performed an in vitro dephosphorylation assay using purified PP2Ac and showed that PP2A directly dephosphorylates pS-FoxO1 (Fig. 5d). Taken together, our results demonstrate that PP2A reduces insulin-mediated FoxO1 phosphorylation through the inhibition of insulin-induced Akt phosphorylation and/or through direct binding and dephosphorylation of FoxO1.

image

Figure 5. PP2Ac associates with FoxO1 and dephosphorylates pS-FoxO1. (a) 1 μg of liver homogenates from B6HCV and C57BL6 mice was used to immunoprecipitate PP2Ac. FoxO1 was then visualized by immunoblotting. Shown is a representative blot of two independent experiments. (b) B6HCV (n = 3) and C57BL6 (n = 3) mice were injected intraperitoneally with 0.8 IU/kg insulin for 40 min. Serine phosphorylation on FoxO1 was then visualized by immunoblotting using specific antibody. (c) Liver biopsies from control (n = 4) and CHC (n = 4) patients were incubated ex vivo for 15 min with 100 nm insulin at 37°C and phosphorylation on FoxO1 was detected by immunoblotting. (d) Huh7 cells were stimulated with 100 nm insulin for 15 min and whole cell lysates prepared. Lysates were then incubated at 37°C with purified PP2Ac for 5 min. PP2Ac and phosphoserine FoxO1 were then visualized by immunoblotting. Shown is a representative blot from two independent experiments.

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Protein phosphatase 2A expression level correlates with G6Pase expression in CHC patients

We have previously reported that HCV protein expression induces CREB transcriptional activity leading to an upregulation of PP2Ac [21]. Indeed, measurement of PP2Ac protein expression level showed a significantly higher amount in CHC than in control patients (Fig. 6a). We then analysed the expression of PP2Ac in samples from control, CHC and fatty liver disease (FLD). Our results showed that PP2Ac is upregulated in CHC and FLD patients (Fig. 6b). Because CREB activation enhances PGC1α expression, we therefore measured PGC1α level in liver biopsies from CHC patients. Although the upregulation of PGC1α did not achieve statistical significance, PGC1α was numerically upregulated in CHC samples versus control samples (Fig. 6c). This increased level of PGC1α was absent in FLD. Confirming our in vitro data, we showed that liver biopsies from CHC patients treated ex vivo with insulin displayed an inhibition of Akt phosphorylation because of an upregulation of PP2Ac (Figure S1). These results, together with data on insulin-induced FoxO1 phosphorylation inhibition, clearly suggest an alteration of gluconeogenic gene expression in CHC patients. Indeed, analysis of G6Pase level showed that the expression was enhanced in CHC and FLD samples (Fig. 6d). All together, we show here that HCV upregulates PGC1α and that HCV-mediated PP2Ac upregulation utilized two distinct mechanisms to inhibit insulin-induced FoxO1 tyrosine phosphorylation. These alterations lead to an enhanced G6Pase expression and thus disrupted glucose homeostasis.

image

Figure 6. Glucose-6-phosphatase (G6Pase) is upregulated in chronic hepatitis C (CHC) patients and correlates with PP2Ac expression. (a) Whole cell lysates from liver biopsies of control (n = 13) and CHC (n = 18) patients were prepared. PP2Ac protein expression level was measured by immunoblotting, and signal intensity of the bands was quantified using ImageJ software. Results are reported as box-plots. (b), (c) and (d) PP2Ac, PGC1α and G6Pase expression levels were quantified qPCR using control (n = 4), CHC (n = 31) and FLD (n = 7) samples. Results are shown as box-plots, and statistical analysis were performed with Mann–Whitney test.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

During the previous years, several studies have attempted to clarify the impact of HCV infection on T2DM development. In the present work, we add new information regarding the role of HCV-mediated PP2A upregulation in IR and thus potentially in T2DM initiation. Indeed, we demonstrate that the presence of HCV proteins resulted in an upregulation of PP2Ac and PGC1α. Furthermore, PP2Ac blocks insulin-mediated FoxO1 phosphorylation through the inhibition of Akt activation or/and via direct interaction and dephosphorylation of pS-FoxO1 (Figure S2). All together, these alterations lead to aberrant insulin signalling and abnormal G6Pase expression as a key enzyme of gluconeogenesis. Finally, we report that HCV transgenic mice that overexpress PP2Ac display IR and hyperglycaemia.

The notion of IR and glucose metabolism disturbance in HCV core expressing mice and in HCV expressing cells have been already described [32, 33]. However, to our knowledge, this is the first report that states PP2Ac overexpression as an important player in this process. Our findings that PP2A affects at different levels of the gluconeogenic pathway leading to glucose homeostasis dysregulation, is of great interest for future therapeutic strategies and designates this phosphatase as a potential target. Heretofore, only two natural molecules, cantharidin and fostriecin, that specifically associate and block PP2A activity have been described. These molecules were tested and showed potent antitumor activity on hepatoma and oesophageal carcinomas [34, 35]. However, despite their antitumor properties, their clinical use raises severe side effects presumably caused by the inhibition of PP2A that is the major phosphatase in the cell. Therefore, knowing that the phosphatase activity and the substrate specificity of PP2A are mediated by binding to specific regulatory B subunits, future studies should focus on the analysis of B subunits that are associated with the PP2A core structure and disrupt specifically the insulin signalling pathway.

Heretofore, the maintenance of glucose homeostasis is thought to require a fully functional insulin signalling pathway. However, Lu and colleagues have recently reported that Akt and FoxO1 are dispensable for insulin-mediated glucose metabolism in vivo [10]. Interestingly, they showed that the effect of FoxO1 on gluconeogenic genes expression is more pronounced in mice with IR. Therefore, our findings that PP2A can both impair the insulin signalling pathway via Akt inhibition and directly alter FoxO1 phosphorylation, suggesting that PP2A overexpression would provide optimal conditions for abnormal enhanced gluconeogenic gene expression.

Hepatic steatosis is a risk factor for diabetes development. The prevalence of hepatosteatosis is increased in a subset of CHC patients, and it has been reported that in HCV genotype 3, the accumulation of fat is due to the cytopathic effect of the virus, while in non-GT3, it is associated with IR [36, 37]. Our observation that G6Pase is highly expressed in FLD and correlates with PP2Ac expression level suggests that the upregulation of this phosphatase is sufficient to mediate G6Pase transcription, independently of the presence of HCV proteins. Therefore, we believe that the elevated G6Pase level observed in CHC samples resulted from a cytopathic effect of the virus and from an IR induced by ER stress response-mediated PP2Ac overexpression in the host cell. Further investigations are needed to clarify this issue.

Alteration of the expression of PP2A is not only restricted to HCV infection. We have previously reported that HBV also induces an upregulation of the PP2A catalytic subunit via ER stress response [20]. Additionally, it has been shown that PP1 and PP2A are overexpressed during cytomegalovirus (CMV) infection, demonstrating that the deregulation of the mechanism that maintains PP2Ac expression level constant is not solely limited to hepatotropic viruses [38]. Interestingly, the association of CMV infection with type 1 diabetes mellitus has been described since decades [39]. Moreover, the frequency of T2DM is higher in patients previously exposed to CMV infection, suggesting a relationship between T2DM development and CMV [40]. Our results showing that PP2Ac upregulation is an important player in IR and therefore in T2DM onset would provide an explanation of the missing link between CMV infection and diabetes mellitus. Finally, it is well known that IR is a prediabetic phase that is often observed in CHC and to a lesser extent to chronic hepatitis B (CHB) patients. Taken together, our data strongly suggest PP2Ac upregulation as a common mechanism used by these viruses to disrupt blood glucose homeostasis favouring T2DM development.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank all patients who consented to have a part of their liver biopsy analysed for research purpose. They further thank Nicola La Monica and Marco Tripodi for the B6HCV mice. We also thank Pere Puigserver for providing the mouse primers. This study was funded by grant from the Swiss National Science Foundation (Grant No. 3L0030_130243 to MHH).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
jvh12208-sup-0001-FigS1.pdfapplication/PDF622KFigure S1: Impairment of insulin-induced Akt phosphorylation in CHC liver biopsies. Control (n = 2) and CHC (n = 3) liver biopsies were incubated ex vivo with 100 nm insulin for 15 min. Phosphorylation of Akt was then visualized by immunoblotting.
jvh12208-sup-0002-FigS2.pdfapplication/PDF244KFigure S2: Proposed model of regulation of G6Pase transcription by HCV-mediated PP2Ac upregulation. HCV infection induces an ER stress response leading to the activation of CREB. Activated CREB upregulates PP2Ac and PGC1α. PGC1α interacts with FoxO1 to modulate the transcriptional activity of FoxO1. PP2Ac impairs insulin-mediated Akt activation and/or directly associates to FoxO1 preventing its phosphorylation. Consequently, FoxO1 phosphorylation is reduced and thus enhances G6Pase transcription.

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