Molecular pathways for glucose homeostasis, insulin signaling and autophagy in hepatitis C virus induced insulin resistance in a cellular model

Virology 434 (2012) 5–17

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Molecular pathways for glucose homeostasis, insulin signaling and autophagy in hepatitis C virus induced insulin resistance in a cellular model Gokul C. Das n, F. Blaine Hollinger Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, United States

a r t i c l e i n f o

abstract

Article history: Received 28 June 2012 Accepted 1 July 2012 Available online 3 August 2012

Chronic HCV infection induces insulin resistance (IR). We studied this in a persistently infected cell line with defects in glucose homeostasis resulting from the phosphorylation of glycogen synthase (GS Ser641) and GS kinase isoform 3b (GSK 3bSer9). Reversal of these effects in cells cured of HCV with interferon supports viral specificity. Insulin signaling was disrupted by IRS-1 Ser312 phosphorylation and dysregulation of the Akt pathway. In infected cells, active autophagy was revealed by the formation of LC3 puncta or by increased levels (50–200%) of the markers Beclin 1 and conjugated Atg5/Atg12. Inhibition of autophagy by 3-methyl-adenine (3-MA) reduced Beclin1 levels, inhibited IRS-1 Ser312 or GS Ser641 phosphorylation and decreased viral load. Furthermore, IRS-1 Ser312 and Beclin1 were co-immunoprecipitated suggesting that they interact. It thus appears that HCV infection disturbs glucose homeostasis or insulin signaling to induce IR and also elicits autophagy that may contribute to this process. & 2012 Elsevier Inc. All rights reserved.

Keywords: Hepatitis C virus Glucose homeostasis Insulin signaling Autophagy GSK 3 phosphorylation IRS-1 phosphorylation Atg conjugation

Introduction More than 200 million people worldwide are infected with hepatitis C virus (HCV). From 60–80% of these will become chronically infected and are at high risk for developing chronic hepatitis, cirrhosis and hepatocellular carcinoma. In addition, recent epidemiological studies indicate that the incidence of type 2 diabetes mellitus (T2DM) is about 3 times higher in HCV-infected individuals when compared to those who have spontaneously cleared their infection, to those with chronic hepatitis B or to an uninfected population (Mehta et al., 2000). In patients with chronic hepatitis C, the virus is known to induce insulin resistance (IR), and IR is associated with the development of T2DM, steatosis, progression of fibrosis and non-responsiveness to conventional interferon therapy, but the molecular basis is not understood at present (D’Souza et al., 2005; Romero-Gomez et al., 2005). When bound to its receptor (IRc), insulin activates intrinsic tyrosine (Tyr) kinase activity to induce Tyr phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2). These phosphorylated substrates bind to the IRc and serve as the binding sites for the p85 subunit of phosphatidylinositol 3-kinase (PI-3K) downstream (Summers et al., 1999). Although the mechanism is complex, Tyr phosphorylation of IRS-1 serves as a positive signal (Zick, 2001). The impairment of postreceptor signaling generally involves phosphorylation of serine/threonine (Ser/Thr) residues of IRS-1 by potential IRS kinases including c-jun-N-terminal kinase n Corresponding author at: Baylor College of Medicine, One Baylor Plaza, BCM-385 Houston, TX 77030, United States. Fax: þ1 713 798 3490. E-mail address: [email protected] (G.C. Das).

0042-6822/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2012.07.003

(JNK), protein kinase C zeta (PKCx), mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK-3) (Aguirre et al., 2002; Gopal et al., 2006; Kanazawa et al., 2004; Liberman and Eldar-Finkelman, 2005). Ser residues in the phosphotyrosine binding (PTB) domain of IRS-1, such as Ser312, are of special interest as they can inhibit the binding of Tyr phosphorylated IRS-1 to the IRc or the PI-3K docking proteins downstream (Herschkovitz et al., 2007; Liu et al., 2004). This inhibitory Ser phosphorylation is part of a negative feedback to insulin signaling and serves as a mechanism for cross talks from other pathways that are induced or are affected by IR. Phosphorylation of Akt at either Thr308 or Ser473 downstream of the PI-3K/Akt pathway is critical for its activation and subsequent inactivation of glycogen synthase (GS). This is mediated through GS Ser641 phosphorylation by GSK-3 inhibiting glycogen synthesis (Cross et al., 1995; Roach, 2002; Skurat and Roach, 1995). Two different isoforms of GSK-3, GSK-3a and GSK3b, are active in a tissue-specific manner, b being more active in the muscle and in resting cells, and a in the liver (Patel et al., 2008). They are inactivated by Ser9 and Ser21 phosphorylation, respectively (Sutherland et al., 1993). The specific roles of the two GSK-3 isoforms are not well understood, but they are not functionally identical as initially described (Wang et al., 1994). More importantly, the status of GS and of individual GSK-3 isoforms in HCV infection has not been elucidated yet. In the past, GSK-3b has been the major focus of research in cellular growth, proliferation and metabolism (Jope et al., 2007; Patel et al., 2008). It reduced GS activity when over expressed in transgenic animals or in transfected cells suggesting that the active form of GSK-3b may be involved in inducing IR (Pearce et al., 2004; Summers et al., 1999). More recently, gene knock out

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experiments demonstrated that disruption of GSK-3b is lethal at an early embryonic stage, whereas GSK-3a knockout mice are viable and fertile, providing compelling evidence that GSK-3a does not compensate for the loss of GSK-3b function (Hoeflich et al., 2000). Also, inactivation of GSK-3a results in improved insulin signaling and glucose homeostasis compared to tissuespecific GSK-3b knock out mice thereby suggesting its important role in T2DM (MacAulay et al., 2007; Patel et al., 2008). Unlike GSK-3b which shuttles between the nucleus and the cytoplasm, GSK-3a is excluded from the nucleus controlled by its N-terminal region (Azoulay-Alfaguter et al., 2011). As mentioned earlier, GSK3 activation is mediated through phosphorylation of two Akt residues, Thr308 by protein kinase PDK1, and Ser473 by the mTORC2 complex involving Rictor (Alessi et al., 1997; Sarbassov et al., 2005a). Both active forms of Akt are capable of phosphorylating the inhibitory residues Ser21 and Ser9 on GSK-3a or -3b, respectively, and may act synergistically, or Akt phosphorylation at both sites is required for full activity (Vanhaesebroeck and Alessi, 2000). Also, GSK-3b regulates mTOR and Akt473 phosphorylation through phosphorylation of Rictor at Ser1235 in the mTORC2 complex (Chen et al., 2011). It is over expressed under insulin stimulation and at least 10 Ser residues can be phosphorylated by multiple kinases including protein kinase A and protein kinase C (Eldar-Finkelman et al., 1996). Thus a differential phosphorylation and dephosphorylation of upstream Akt and subsequently of GSK-3 isoforms in a cellular context seems to be critical for the development of IR. Additionally, GSK-3b also is capable of inducing Ser phosphorylation of IRS-1 which might be disruptive for insulin signaling (Eldar-Finkelman et al., 1996). Moreover, the presence of two isoforms of GSK-3, their distinct but overlapping roles in cellular function, and their differential phosphorylation or shuttling between nucleus and cytoplasm makes their functional characterization an important subject of investigation concerning the development of a GSK-3-based therapeutic strategy. A systematic investigation of the molecular basis of HCVinduced IR (HCV-IR) in chronic infection was not possible previously because there were no small animal models or cell culture systems available until recently. None of these models, however, have been utilized to characterize HCV-IR. The ability of insulin to lower the plasma glucose level was impaired in a transgenic HCV core protein mouse model similar to what has been observed in chronic hepatitis C patients (Shintani et al., 2004). Accordingly, the core protein has been found to up-regulate phosphorylation of IRS-1 at Ser312 and to regulate IRS-1 in a genotype-specific mechanism (Banerjee et al., 2008; Pazienza et al., 2007). Another viral protein, NS-5A can bind to PI-3K and activate Akt (Street et al., 2004). While these viral proteins potentially can lead to IR by impairing the downstream Akt/PKB signaling pathway, it is still of limited applicability to understand the overall mechanism of HCV-IR in the absence of testing for other viral proteins. In this respect, studies on the signaling pathways utilizing clinical specimens are limited. In HCV-infected liver specimens from nonobese and nondiabetic patients given insulin, a decrease in IRS-1 Tyr phosphorylation was accompanied by significant reductions in IRS-1/PI-3K activity suggesting a direct interaction between HCV and the insulin-signaling pathway (Aytug et al., 2003). Therefore, identifying and dissecting the pathways involved in IR by using a cell culture system could be of great benefit. Both HCV and dengue virus of the Flaviviridae family and several other viruses induce an autophagic response in infected cells as a possible mechanism to facilitate viral infection, replication or pathogenesis (Ait-Goughoulte et al., 2008; Lee et al., 2008; Sir et al., 2008). But its connection with IR is not known, although the emerging role of autophagy in the pathophysiology of diabetes mellitus has been recognized recently (Jung et al., 2008; Meijer and Codogno, 2007;

Miquilena-Colina et al., 2011). Generally, mTOR, a Ser/Thr kinase, integrates signals from nutrients and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (Alessi et al., 2009). When inhibited, mTOR leads to the induction of autophagy in which cytosolic proteins and organelles are sequestered by double-membraned vesicles known as autophagosomes. Its outer membrane fuses with lysozomes allowing access to the inner content of the vesicles for subsequent degradation (Codogno et al., 2012). Formation of this vesicle involves several steps including nucleation, expansion and completion or closure. The initial sequestering component is the membrane-derived phagophore from which the autophagosome originates. Most autophagy-related proteins (Atg) are co-localized at the phagophore assembly site (PAS) to participate in vesicle formation in a concerted manner (Codogno et al., 2012; Geng and Klionsky, 2008). For instance, Beclin-1, an ortholog of yeast Atg6, interacts with the class III PI-3K kinase complex in the nucleation step and also serves as a Bcl-2/Bcl-XL binding protein to regulate autophagy negatively (Sinha et al., 2008). Two conjugation systems of Atg proteins are involved in the expansion and closure of the autpophagosome formation; these are essential in autophagy as mutations of any member of the congjugates or of the components required for their formation results in a defect in autophagosome formation (He and Klionsky, 2009; Xie and Klionsky, 2007). First, Atg12, being activated by an E1-like enzyme, Atg7, is conjugated to Atg5 and formation of this Atg12– Atg5 conjugate is functionally necessary for the end product of the second conjugation system, microtubule-associated protein1 light chain 3 (LC3,Atg8) conjugated to phosphatidylethanolamine (PE). The Atg12–Atg5 conjugates not only facilitate the lipidation of LC3 (LC3-II or LC3-PE), but also direct its correct localization in the autophagosomal membrane (Codogno et al., 2012; Geng and Klionsky, 2008). Interestingly, Atg12–Atg5 conjugation occurs constitutively and virtually there is no free Atg5 present in cells (Lee et al., 2008; Mizushima et al., 2001); this conjugation is irreversible, whereas lipidation of LC3 is reversible and cell line dependent (Mizushima et al., 1998; Mizushima et al., 2001). Therefore, the protein levels and redistribution of Atg12–Atg5 and/or LC3-II conjugates are considered as hallmarks of autophagy and used to measure autophagic activity (Kabeya et al., 2000). Several studies report their up regulation during this process (Kim et al., 2006; Park et al., 2012; Wang et al., 2012; Yang et al., 2010b; Zou et al., 2012). Currently, the study of autophagic response under different physiological or pathological conditions is under intense investigation. An inducer of autophagy, rapamycin, also elicits Ser phosphorylation of IRS-1 at Ser312 through the Rapto-mTORS6K1-dependent pathway (Huynh et al., 2007; Shah and Hunter, 2006). Although suggestive, a possible role for autophagy in HCV infected cells during IR development has not been systematically investigated in a cellular model by key biochemical criteria (Blumer et al., 2008; Meijer and Codogno, 2007; Sarbassov et al., 2005b). Toward this goal, we report the characterization of a cell-based model that mimics chronically infected HCV and exhibits IR as judged by critical parameters controlling both glucose homeostasis and insulin signaling pathways. Our results suggest that in addition to the disruption of glucose homeostasis and dysregulation of insulin signaling, autophagy in infected cells might have a contributory role in HCV-IR.

Results HCV infected liver cells in culture mimicking chronic infection have a reduced growth rate We used a hepatoma cell line (Huh 7) stably transfected with an HCV genome (genotype 2a) that produces and secretes continuously

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HCV in the culture media (Cai et al., 2005). We prepared three independent cell stocks after continuous culture over varying periods of time (12, 19 and 23 weeks). These cells show evidence of IR by a disruption of glucose homeostasis and the insulinsignaling pathway, as described below. The doubling time of this cell line (designated as Huh.HCV2a) was about 1.5-fold longer than the control cells (not shown). For a qualitative comparison of infected and uninfected cells and to determine whether the reduced growth rate was due to a reduced rate of proliferation, we measured new DNA synthesis by the incorporation of EdU, a nucleoside homologue, by immunofluorescence microscopy as described under the Materials and methods section (Salic and Mitchison, 2008). Our results show that HCV infected cells were larger in size than the uninfected control Huh 7 cells (Banerjee et al., 2008) and exhibit lower fluorescent intensity under the same experimental conditions and magnification indicating a lower rate of DNA replication (Fig. 1A). The HCV infected cell line used in this study exhibited a reduced growth rate and cellular proliferation compared to the parental uninfected cells. To investigate the ability of the HCV infected cells to continuously produce virus, as in chronic infection, we harvested the cell culture media after passing the cells every 3–4 day for 12 weeks. The virus particles were concentrated and fractionated through an iodixanol gradient, and the viral concentration determined for each fraction. The results show that the majority of the HCV RNA was concentrated in fractions 11 and 12 corresponding to a density of 1.14–1.15 g/mL (Fig. 1B). This is in good

agreement with those reported for HCV purified from infected cells in another laboratory and these particles were infectious (Lindenbach et al., 2005; Wakita et al., 2005). Taken together, the cell line Huh.HCV2a mimics persistently infected cells with reduced ability to proliferate and to secrete virus in the media for at least three months. Disturbance of glucose homeostasis in HCV infected cells As a test of IR, we examined how HCV infection influences glucose uptake from the media under insulin stimulation. Semiconfluent cells in serum-free media were stimulated with 100 nM of insulin for 2 h, the final 30 min being in the presence of 3H-2-deoxy-glucose. Our results show that glucose uptake under insulin stimulation was about 2- to 3-fold less in cells infected with HCV when compared to uninfected cells (Fig. 2A). To determine the effect on glucose metabolism, we next focused on two cellular proteins that play a key role in this process; one is a kinase, GSK-3, which is present in two isoforms, a and b, and was initially identified as a regulator of glycogen synthesis in response to insulin. They negatively regulate various pathways involved in cell growth and proliferation and are inactivated by Ser21 and Ser9 phosphorylation, respectively, to turn on genes involved in various pathways (Eldar-Finkelman et al., 1996; Sutherland et al., 1993; Welsh et al., 1996). The other cellular protein is an enzyme, GS, a substrate for GSK-3 that could be inactivated by Ser641 phosphorylation (Pearce et al., 2004).

Huh.HCV2a

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1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Fraction Number

Fig. 1. (A) Proliferation studies. Cells were permeabilized and labeled with a nucleoside analog (Edu) following the manufacturer’s protocol as described briefly in the Materials and methods section and green fluorescence was examined under a fluorescence microscope; Magnification:  63,000. (B) Viral load in the culture media. After 12 weeks of continuous passage, cell culture media was concentrated and loaded onto a 10–40% iodixanol gradient and 0.75 mL fractions were collected from the bottom of the gradient. The density of each fraction is in g/mL and the HCV RNA copy number per mL were determined and plotted.

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3H-deoxy-2-glucose (CPMXe-2)

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500 450 400 350 300 250 200 150 100 50 0 Huh

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Pos Huh Huh.HCV Cured 50 75 50 75 50 75 GSK-3α GSK-3 β GSK-3 α Ser 21 GSK-3 β Ser 9

Actin α/β:

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antibody, we detected high levels of phosphorylated species in infected cells but at a very low level, if any, in uninfected cells suggesting that Ser9 residues are differentially phosphorylated in these cells. These results, therefore, suggest that in infected cells GSK-3a Ser21 is not phosphorylated, but it is the GSK-3b that is phosphorylated at Ser9. Interestingly, in the cells cured of HCV by IFNa, GSK-3b Ser9 phosphorylation is not observed, similar to what is seen in the uninfected Huh 7 cells providing additional support for our observation and viral specificity. Thus, GSK-3 isoforms are differentially regulated in HCV infected cells in which GSK-3b is inactivated by phosphorylation while GSK-3a remains active. GS is regulated downstream from GSK-3 by multiple-site phosphorylation and plays a critical role in the disturbance of glucose homeostasis (Roach, 2002; Skurat and Roach, 1995). We measured the total GS level and its inactivating phosphorylation in HCV infected cells by Western blot analysis (Fig. 2C). The level of total GS is elevated by about 2-fold in infected cells compared to the uninfected control. We observed a low level of GS Ser641 phosphorylation in Huh cells which is increased by about 6-fold in the infected cells, thus reducing its ability drastically to incorporate glucose into glycogen. Curing of the HCV infection by IFNa inhibits phosphorylation, thus implying viral specificity. Since GSK-3b is not active in infected cells, it is probably GSK-3a that is involved in the inactivation of GS downstream. It also is possible that inactivation of GSK-3b releases inhibition of a pathway leading to the phosphorylation of GS (Cohen and Frame, 2001). Taken together, our results strongly suggest that the disturbance in glucose homeostasis seen in persistent HCV infection is generated both by a defect in glucose uptake and metabolism that are biochemical hallmarks for the development of IR. Disruption in insulin signaling pathway

GS: p GS:

0.3 0.4

0.8 2.2

0.4 0.1

Fig. 2. Disturbance in glucose homeostasis. (A) Glucose uptake. The ability of the HCV infected (Huh.HCV) and uninfected (Huh) cells to take up glucose was measured using 3H-2-deoxy-glucose under conditions of insulin stimulation (Ins; 100 nM, 2 h) compared with no insulin stimulation, (P o 0.05). (B) Detection of GSK-3 by Western blot analysis. Two different amounts of cellular proteins (50 mg and 75 mg) in lysates from HCV infected and uninfected cells were fractionated and subjected to Western blot analysis as described in the text. Actin was used as a loading control when appropriate. GSK-3a, GSK-3b and the phosphorylated species were detected with specific antibody as indicated in the Materials and methods section. A normalized average ratio of GSK-3a to GSK-3b (a/b) is shown at the bottom of the figure under each lane. Lysates from interferon-cured cells (cured) and Akt-induced Jurkat cells (Pos) were run in the same gel as controls. (C) Detection of glycogen synthase (GS) by Western blot analysis. Two different amounts (50 mg and 75 mg) of total proteins were run in parallel from uninfected, infected and interferon-cured cells and probed with specific antibodies. Total GS and GS Ser641 (pGS) are marked. Actin, loading control.

We carried out Western blot analyses using lysate from Jurkat cells treated with calyculin A to induce Akt as the positive control. Our results show that the GSK-3a level is about 30% more than the GSK-3b level in HCV infected cells, whereas GSK-3b was about 30% reduced in Huh.HCV cells, resulting in an elevation of the GSK-3a/GSK-3b ratio by about 60–90% in different experiments (Fig. 2B). Curing infected cells by IFNa resulted in a partial shift of this ratio to about 30–40% when compared in uninfected cells. When probed with an antibody that recognizes GSK-3a Ser21, we could not detect bands in any of the lanes except in the positive control. In a subsequent analysis with GSK-3b Ser9 specific

Since IRS-1 and IRS-2 play important roles in postreceptor insulin signaling, we investigated whether and how the IRSmediated signaling pathway is involved in defective glucose homeostasis in HCV infected cells. To accomplish this, the expression of IRS-1 and IRS-2 and their phosphorylation status were examined in the infected cells. A representative Western blot for IRS-1 and its phosphorylated product, IRS-1 Ser312 is shown in Fig. 3A. We used two different time points (48 h and 72 h) to identify the effect of viral growth on phosphorylation, if any, in chronic infection. The figure shows that the IRS-1 level is increased 2–3 fold in HCV infected cells compared to the control Huh 7 cells (left). IRS-1 is predominantly phosphorylated at Ser312 with respect to uninfected cells which have a relatively low level of that product (right). This site is localized to the PTB domain that also contains phosphorphylated Tyr residues required for the interaction of IRc or downstream docking of proteins for positive regulation. Thus IRS-1 Ser312 phosphorylation can lead to the inhibition of IRS-1 binding to the IRc or the PI3K docking protein. The ratio of hyperphosphorylated IRS-1 Ser312 to IRS-1 protein in HCV infected cells varied between 75 and 200% compared to uninfected control cells in different experiments. Other Ser residues within and outside the PTB domain, such as at positions 302, 612 and 789, were not phosphorylated and thus serve as a negative control (data not shown). To further validate our observation, we determined the status of IRS-1 Ser312 phosphorylation in three independent Huh.HCV2a cell lines at three different time intervals spanning 12–23 weeks of infection. When normalized with respect to the uninfected Huh internal control, we observed a 3–6 fold increase in IRS-1 Ser312 phosphorylation in the HCV infected cells (Fig. 3B). The level of IRc in the whole cell lysate remained virtually unaltered in both HCV infected and control Huh 7 cells

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9

Fig. 3. Disruption in insulin signaling. (A) Representative Western blot for detection and quantification of IRS-1 and of phosphorylated IRS-1 Ser312. Biological duplicates were used for HCV infected cells at 48 h and 72 h; 75 mg of total proteins was loaded. Tubulin, loading control. (B) Detection and comparison of IRS-1 Ser312 in three independent Huh. HCV cell stocks prepared at different time points (12, 19 and 23 weeks). Fold changes with respect to the internal control is indicated beneath each lane.

200–300% in infected cells to make the band visible. We interpret these results to mean that IRS-1 Ser312 phosphorylation leads to the modulation of Akt expression and to the differential phosphorylation of Akt Thr308 and Akt Ser473 in infected cells through the PI-3K-Akt pathway dysregulating normal insulin signaling. It also supports our contention that there is a disturbance of glucose homeostasis in chronic HCV infection. Induction of autophagy in chronic HCV infection

Fig. 4. Dysregulation of PI-3K/Akt pathway. Detection and quantification of total Akt (Akt-T), Akt Thr308 and Akt Ser473 by Western blot analysis. Two different amounts (50 mg and 75 mg) of lysate prepared were loaded. Actin, loading control.

(not shown). Our results suggest that over expression and phosphorylation of IRS-1 Ser312 in HCV infected cells is not a clonal artifact. Increased expression of IRS-1 may have a protective effect facilitating viral growth as observed in many cancer cells, and IRS-1 Ser312 phosphorylation may be involved in negative regulation of postreceptor insulin signaling leading to IR in persistently infected cells. To identify the defect in downstream signaling resulting from IRS-1 Ser312 phosphorylation, we focused on the status of Akt, a kinase that is regulated by PI-3K in many cell signaling pathways and is activated by phosphorylation at Thr308 and Ser473 residues by PDK1 and the mTORC2 complex, respectively (Alessi et al., 1997; Jacinto et al., 2006). A representative Western blot with a positive control from Akt-induced Jurkat cell lysate is shown in Fig. 4. In HCV infected cells, the total Akt level (Akt-T) when normalized with respect to the internal control is about 50% less than that seen in the uninfected cells, whereas phosphorylation of Akt Thr308 is increased to about 50–300% in different experiments (Fig. 4). In contrast, phosphorylation of Akt Ser473 is barely detectable in uninfected cells, but its level is increased by

Since insulin signaling regulates autophagy negatively, and HCV infection induces an autophagic response, we examined whether autophagy may serve as a contributory factor in IR development. We explored three hallmarks of autophagy, i.e., Beclin 1 expression, LC3 lipidation and Atg12–Atg5 conjugation by biochemical analysis and immunofluorescence microscopy. It is well known that during formation of autophagosomes, LC3 is converted from a more diffuse state to an aggregated state and is localized to the autophagic vacuoles. When fused to green fluorescent protein (GFP-LC3) and over expressed in cells undergoing autophagy, these protein aggregates are visible as puncta (green dots) under the immunofluorescence microscope (Fig. 5A–D). We transfected the HCV infected and the uninfected cells with the LC3 recombinant plasmid or the empty vector. We observed clear GFP-LC3 expression in both infected (Huh.HCV) and uninfected Huh 7 cells, but LC3-II puncta was predominantly formed only in the former (Fig. 5B). In control Huh 7, cells with puncta were very rare (Fig. 5D). When tranfected with empty vector, neither of the infected nor uninfected cells showed any puncta (Fig. 5A and C). These analyses support the presence of autophagy in infected cells. For further analysis, we examined the status of two other hallmarks of autophagy involved in the nucleation and expansion stage of autophagosome formation (Geng and Klionsky, 2008; Mizushima et al., 1998). This process could be associated with an increased level or modification of Beclin-1 or of Atg12–Atg5 conjugate thus regulating the level of autophagy (Geng and Klionsky, 2008; Liu et al., 2008; Park et al., 2012; Zhao et al., 2010). Our Western blot analyses show that Beclin1 is over expressed in HCV infected cells in different experiments by about 50–150% at 48 h and 72 h post-culture (Fig. 6A). The conjugation

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Fig. 5. (A)–(D) Induction of autophagy. Formation of LC3 puncta (green dots) when HCV infected cells were transfected with a an empty vector (A) and a plasmid (GFP-LC3) containing LC3 fused to GFP protein (B); Huh 7 cells transfected with empty vector (C) and GFP-LC3 (D). The bar represents 25 um. Nuclei were stained blue with 4’,6diamidino-2-phenyl indole dihydrochloride (DAPI). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of Atg12–Atg5 in the maturation of autophagosome is important in the autophagic pathway as it regulates the last stage of LC3-PE (LC3-II) conjugation followed by their recruitment to the autophagosomal membrane. We identified a band of about 53 kDa with Atg5 antibody which corresponds to the molecular weight of the Atg12–Atg5 conjugate, and the intensity of this band increased from 50% to 200% with increasing time (24 h to 72 h) in culture (Fig. 6B). The Atg12 antibody recognized this complex relatively poorly, but a comparable increase in the level of the conjugate was observed (Fig. 6C). When the whole blot was probed, no free Atg5 (33 kda) or Atg12 (16 kda) was detected, as shown in Fig. 6D (Supplemental data). In an over loaded (200 mg) and overexposed blot of the whole gel probed with Atg5 antibody, we could not detect individual Atg5 (Fig. 6E, Supplemental data). These results suggest the participation of Atg5 and Atg12 as a conjugate in autophagy during IR development. Taken together, the formation of LC3 puncta together with the Western blot analyses of marker proteins involved in the autophagic pathway strongly support the

concept that autophagy is active in infected cells with a disturbed glucose homeostasis and dysregulated insulin signaling.

Possible role of autophagy in HCV-induced IR To investigate whether induction of IR has a direct relationship with the genesis of autophagy, we inhibited autophagy in HCV infected cells by 3-methyl adenine (3-MA), a known inhibitor. Bands corresponding to Beclin1 are present in HCV infected cells (lanes 1 and 2) that are not treated with 3-MA (Fig. 7A). Upon treatment, Beclin 1 level was reduced by 2–3 fold. Interestingly, repression of Beclin1 was associated with a dephosphorylation of IRS-1 Ser312 and GS Ser641. These data, taken together, suggest the possibility of a role for autophagy in the development of IR. However, it is also possible that a decrease in the viral load by 3-MA is responsible, at least partially, for reduced level of IRS-1 Ser312 or GS Ser641 phosphorylation, as shown below.

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1.7

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3.0

11

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0.51 0.52 0.90 0.88 Atg5 Antibody

0.28 0.43 0.48 Atg12 Antibody

Fig. 6. Expression of autophagic markers. Detection and quantification of autophagic marker proteins in HCV infected cells by Western blot analysis. Equal amounts of total protein (50 mg) in whole cell lysate at each time point was fractionated and analyzed, as described in the Materials and methods section. Fold change with respect to the internal control is indicated beneath each lane. (A) Beclin 1 (62 kda) at two different time points (B) Conjugate of Atg5/Atg12 (53 kda) (using Atg5 antibody) at different time points of post culturing; (C) conjugated complex of Atg5/Atg12 at 48 h recognized individually by Atg5 and Atg12 antibody at two different protein concentrations. Blots of the whole gels are shown in the Supplemental data (Figs. 6D and E).

2

HCV RNA (copies/mL x 105)

1.8 1.6 1.4 1.2 1 Fig. 8. Coimmunopreciptation of IRS-1 Ser312 and Beclin 1. Two different amounts of each lysate (100, 150 mg) from infected and uninfected cells were immunoprecipitated either with IRS-1 Ser312 antibody (A) or with Beclin 1 antibody (B), fractionated on a 10% PAGE and probed with IRS-1 Ser312 and Beclin 1 antibody separately. Respective cell lysates were run in parallel (input) for direct Western blot to identify bands. Specific bands for IRS-1 Ser312 and Beclin 1 and that for IgG heavy chain are marked.

0.8 0.6 0.4 0.2 0 0

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Concentration in mM Fig. 7. Inhibition of autophagy by 3-MA. (A) Down-regulation of Beclin 1 along with reversal of IRS-1 Ser312 and GS Ser641 phosphorylation. Two different amounts of lysate were probed. Actin, loading control. (B) Semi-confluent cells were treated with increasing concentrations of 3-MA for 2 h and allowed to grow. Viral load in the media was determined 24 h later as shown by a histogram.

If autophagy is associated with IR, it raises a question as to its role in viral replication. To analyze this, autophagy was inhibited in HCV infected cells by treating the cultures with 3-MA and then allowing

the cells to grow and secrete virus for another 72 h. Our results show that even at 2.5 mM, the viral load in the media was reduced by about 60% (Fig. 7B). A further increase in the 3-MA concentration decreased the viral load progressively resulting in an 80% reduction at 7.5 mM. We also determined the number of live cells. The percentage of viable cells, as measured by trypan blue staining, were comparable (88–90%) in the treated and untreated control cells at each concentration of 3-MA (not shown). By electron microscopy, we observed autophagosome formation characterized by double-membraned structures that were frequently distributed in the cytoplasm of the infected cells but not in the control cells (results not shown).

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These structures are known to facilitate virus replication and thus supports our observation of a reduced virus load when autophagy is inhibited by 3MA. To identify whether there might be an interaction between IRS-1 Ser312 and autophagic proteins, we immunoprecipitated lysates with an antibody specific for IRS-1 Ser312. The immunoblot was probed with both IRS-1 Ser312 and Beclin 1 antibody (Fig. 8A). We observed that both IRS-1 Ser312 and Beclin 1 bands were clearly present in lysates prepared from HCV infected and uninfected cells when compared with input control and control IgG lane. A similar pattern was obtained when the same lysate was immunoprecipitated with Beclin 1 antibody and probed with IRS-1 Ser312 and Beclin 1 specific antibodies (Fig. 8B). IgG lane shows a non-specific band of low intensity migrating faster than IRS-1 Ser312 band. Since IRS-1 Ser312 phosphorylation and Beclin 1 are present in uninfected cells, corresponding bands are also present in Huh lanes. Thus, both IRS-1 Ser312 and Beclin 1 are present in the same immunocomplex, strongly suggesting that they interact.

Discussion It is widely accepted that chronic HCV infection induces IR and that IR not only accelerates the progression of liver disease but also makes treatment of chronically infected patients with IFN-based therapy more difficult. The molecular basis of IR development and the basis for non-response to conventional therapy have generated much current interest from a therapeutic perspective. In a cell culture system that mimics persistent infection, we have demonstrated a disturbance in glucose homeostasis through reduced glucose uptake, by a differential activation of GSK-3 isoforms, and by subsequent inactivation of GS by phosphorylation downstream. This is further accompanied by a disruption in insulin signaling through a dysregulated IRS-1/PI-3K/Akt pathway, the biochemical hallmark for IR. Curing infected cells with interferon treatment reverses, at least partially, critical parameters of IR implying viral specificity of these effects. Furthermore, our results suggest that virus-induced autophagy might be a contributory factor in the development of IR. Although uptake of glucose in our study was reduced only about 2-fold in Huh 7 cells infected with HCV genotype 2a, this effect may be amplified in one of the five other genotypes that may be capable of inducing IR more strongly (Duseja et al., 2009). One of the most critical factors in glucose metabolism could be the rate-limiting GS pathway which is the last step in glycogen synthesis. Four different NH2-terminal sites of GS are the targets for GSK-3-mediated phosphorylation. Phosphorylation of at least one of these sites, GS Ser641, reduces GS activity to inhibit glycogen synthesis (Skurat and Roach, 1995). The two GSK-3 isoforms, a and b, are constitutively active and usually regulate pathways negatively. They display both overlapping and distinct roles in different tissues with GSK-3a more active in the liver, and their specificity for GS Ser641 phosphorylation is not currently understood (Matsuda et al., 2008; Patel et al., 2008). We have observed that GSK-3a remains active in both infected and uninfected cells, whereas GSK-3b is predominantly inactivated in infected cells through Ser9 phosphorylation. As other kinases and phosphatases can participate in the phosphorylation of GS, the level of GS Ser641 phosphorylation may not strictly correlate with Ser9 or Ser21 phosphorylation (Roach, 2002). Expression and inactivation of GSK 3 isoforms also may be regulated by the proliferation status of the infected and uninfected cells. However, it has been proposed that Wnt protein of the Wnt-b catenine pathway can inactivate GSK-3 isoforms at the same site as Akt, and this may lead to dephosphorylation of GS through a distinct mechanism (Ding et al., 2000; Yuan et al., 1999). It remains to be

understood whether and how differential activation of GSK-3 isoforms participate either directly or indirectly in GS phosphorylation or glucose homeostasis (Mora et al., 2005); it could be speculated that if GSK-3a is involved, a second factor might be needed to phosphorylate GS in HCV infected cells that could be supplied by GSK-3b Ser9-mediated gene activation. In HCV infected cells cured by IFNa, dephosphorylation of GSK-3b Ser9 occurred similar to what was seen in the Huh 7 control cells implying viral specificity of Ser9 phosphorylation (Fig. 2B). This phosphorylation may be induced by either or both of Akt Thr308 and Akt Ser473, where phosphorylation at both sites simultaneously may be required for full activity (Cross et al., 1995). The total Akt level under our assay conditions is reduced by about 2-fold in infected cells, while both Akt Thr308 and Akt Ser473 are increased by about 50–300% in different experiments. Since Akt is phosphorylated by two different kinases, the differential Akt phosphorylation of GSK-3 seems to be a complex one and will require further study. Interestingly, recent studies suggest that GSK-3b can serve upstream from Akt as well, reducing the mTORC2-mediated Akt Ser473 phosphorylation through phosphorylation and dissociation of Rictor from the mTOR. Rictor complex (Alessi et al., 1997; Chen et al., 2011; Sarbassov et al., 2005b) and inactivation of GSK-3b may therefore increase Akt Ser473 phosphorylation. On the other hand, mTORC1 activated S6K1 phosphorylates Rictor at Thr1135 to stimulate mTORC2mediated Akt phosphorylation at Ser473, implicating a cross-talk between GSK-3b, mTOR and Akt (Julien et al., 2010). This suggests the presence of a complex feed-back with regulatory loops, one involving Akt and GSK-3 and the other involving mTOR and Akt or mTOR and GSK-3. In the type II diabetic rat, a reduced Akt Ser473 phosphorylation (30%) has been observed when compared to the nondiabetic rat even after insulin administration, but the status of Akt Thr308 was not examined in the same experiment which seems to be an equally important prognostic factor in human pathogenesis (Rondinone et al., 1999). An up-regulation of Akt Ser473 by HCV core protein or by transfected HCV RNA in Huh 7 cells, as observed by other investigators, supports our observation (Banerjee et al., 2008). We observed both an in increase in IRS-1 level as well as an increased phosphorylation at Ser312 by about 2–4 fold in HCV infected cells. In hepatocellular carcinoma cells, IRS-1 over expression is frequently observed that prevents apoptosis through the TGFb1 pathway (Furusaka et al., 1994; Nehrbass et al., 1998; Tanaka and Wands, 1996). We observed IRS-1 Ser312 phosphorylation in three independent cell preparations harvested after continuous culturing for 12, 19 and 23 weeks (Fig. 3B), strengthening our conclusion that IRS-1 Ser312 may indeed serve as a critical disruption point for postreceptor insulin signaling. It is not known how early this impairment may occur. In one study, IRS-1 Ser312 phosphorylation occurred in Huh 7 cells when transfected either with genotype 2 HCV RNA or with an expression vector for HCV core protein suggesting that it might be an early event (Banerjee et al., 2008). Among other Ser sites located juxtaposed to the PTB domain, Ser302, Ser612 and Ser789 were not phosphorylated, suggesting that Ser312 phosphorylation of IRS-1 may be at the center of dysregulation of insulin signaling. Both a protective effect of increased IRS-1 expression against cell death and a disruption of insulin signaling by increased Ser312 phosphorylation are possibly important in HCV induced IR. Our preliminary data as well as those from other laboratories indicate that when Huh 7 liver cells are transfected with HCV RNA or infected with infectious virions, virus production decreases after several passages probably due to massive cell death (Wakita et al., 2005; Zhong et al., 2005). This limited time span may not allow the establishment of chronic infection and IR to develop fully as opposed to our system of a stable cell line producing and

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secreting virus continuously in the media mimicking persistent infection (Cai et al., 2005). Although there are limitations in both approaches, IRS-1 Ser312 phosphorylation, reduced glucose uptake and increased levels of Beclin 1 are shared by both systems (Banerjee et al., 2008; Waris et al., 2007). Autophagic response in our HCV infected cell culture system is in agreement with those reported earlier (Ait-Goughoulte et al., 2008; Lee et al., 2008; Mizui et al., 2009; Sir et al., 2008). Although modification of autophagy proteins are a common occurrence in the process of autophagy, their over expression is also reported in the literature. In our study, Beclin-1 levels were increased is about 50–100% in infected cells and the level of Atg12–Atg5 conjugates varied by about 50–200% in infected cells between 24 h and 72 h post culture. Although Atg12 antibody recognized this conjugate relatively poorly, the level of this conjugate in the infected cells was comparable (Fig. 6C). Increased levels of these marker proteins may suggest the presence of increased levels of autophagy or number of autophagosomes over the basal level and is, in general, in agreement with other reports (Kim et al., 2006; Liu et al., 2008; Mai et al., 2012; Park et al., 2012; Wang et al., 2012; Zhao et al., 2010). It is reported that stimulation of the Atg12– Atg5 conjugate may also be caused by ribosomal RNA, and that the two systems being involved in protein metabolism may communicate in this fashion (Shao et al., 2007). As shown in the Supplemental data (Fig. 6D and E), we were unable to detect free Atg5 even by overloading the gel. This is in agreement with previous reports showing that there is virtually no free Atg5 in cells and almost all Atg5 exists as an Atg12–Atg5 conjugate (Liu et al., 2008; Mizushima et al., 2001). The Atg12 antibody, however, recognizes the epitope poorly in the conjugated form. Since sequential conjugation of Atg proteins are involved in the expansion and completion of autophagosome formation, the process of conjugation might be more important than the level of the individual proteins. A faint band of about 140 kda was present in the overexposed Atg5 blot that possibly could be an intermediate in the conjugation process of Atg proteins or a nonspecific band and is outside the scope of the current work (Supplemental data, Fig. 6E). Recently, the emerging role of autophagy in the pathophysiology of diabetes mellitus has been recognized (Gonzalez et al., 2011). We report here that all three processes, the disturbance of glucose homeostasis, dysregulated insulin signaling or induction of autophagy are contributory factors in the development of IR. Our following observations and those recently reported from others suggest a possible contributory role of autophagy in HCV-IR. Inhibition of autophagy by 3-MA not only inhibits the expression of Beclin 1 in infected cells, but it also inhibits phosphorylation of IRS-1 Ser 312 and GS Ser641 (Fig. 7); also, our immunoprecipitation experiment shows the presence of IRS-1 Ser312 and Beclin 1 in the same immunocomplex suggesting that they interact and this interaction possibly plays a role in the development of IR. It has been reported recently that both dengue virus and HCV induce autophagy of lipids in virus infected cells to alter lipid metabolism (Heaton and R et al., 2010; Vescovo et al., 2012). Free fatty acid (FFA) released in this process can contribute to IR directly (Boden, 2003; Dong and Czaja, 2011; Heaton and Randall, 2010; Vescovo et al., 2012). Also, generation of FFA induces beta oxidation in the mitochondria resulting in an increased ATP production to stimulate viral replication. Since inhibition of autophagy by 3-MA reduces virus yield, autophagy may also facilitate viral replication utilizing double-membraned closed structures at the early stage of autophagosome formation that are visible under the electron microscope (our unpublished observation). We cannot, however, rule out the possibility that reduced viral replication by 3-MA treatment also has a contributory role in the reduced phosphorylation of IRS-1 Ser312 and GS

13

Insulin

Insulin

Glut

Plasma Membrane

IRc

Glucose uptake

PI-3K

IRc

IRS-1 IRS-2 GSK-3 α GSK-3 β

Glycogen Synthase (GS)

Glycogen Synthesis

Akt308 Akt473 mTORC1 mTORC2 Glut Vesicle

Autophagy

Beclin 1

Bcl-2 Bcl-XL

Apoptosis

Fig. 9. Schematic diagram representing potential pathways for glucose homeostasis and insulin signaling in HCV infected cells. The downstream targets affecting glucose synthesis, glucose transport, autophagy and apoptosis are outlined. IRc, insulin receptor; IRS-1, insulin receptor substrate-1; mammalian target of rapamycin (mTOR); glycogen synthase kinase (GSK); glycogen synthase (GS); phosphatidylinositol 3-kinase (PI-3K); protein kinase B (Akt/PKB) are shown.

Ser641. Several other studies indirectly support a contributory role of autophagy in IR. An autophagy inducer, rapamycin, induces IRS-1 Ser312 phosphorylation through the mTOR/S6K1 pathway suggesting a connection between autophagy and IRS-1 phosphorylation (Shah and Hunter, 2006). Inhibition of GSK-3b activity by chemical inhibitors in the IRS-1–GSK-3–GS pathway also induces autophagic cell death by the Bif-1 dependent pathway implicating GSK-3b upstream from the autophagic pathway (Yang et al., 2010a). While preparing this manuscript, a requirement of Bcl-2 regulated autophagy in muscle glucose homeostasis was also reported that support our study (He et al., 2012). A potential pathway linking the IRS-1–GS axis with autophagy through mTOR signaling is shown schematically in Fig. 9. In addition to the Jak–Stat pathway, IFNa also can use the IRS-1/ Akt pathway (Lok and McMahon, 2001; Uddin et al., 1995; Uddin et al., 1997; Uddin et al., 2000). As a result, IRS-1 Ser312 phosphorylation could become a contributory factor in the development of a poor response to IFN-based therapy by the disruption of normal insulin signaling. Using this cell culture model, cross-communication of insulin signaling with IFN and the autophagic pathway may provide valuable insight into how IR can be minimized to control viral infection and the occurrence of T2DM.

Materials and methods Cell lines, plasmids and reagents The development of the hepatoma cell line, Huh 7, supporting HCV replication has been described (Lindenbach and Rice, 2005). The cell line Huh.HCV2a was derived from these cells and harbors a chromosomally integrated cDNA of full length genotype 2a HCV genomic RNA as previously described (Cai et al., 2005). The cells (designated as Huh.HCV2a) were selected by a very low concentration of blasticidin (from 5 mg/mL to 2.5 mg/mL). These cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum, nonessential amino acids in HEPES buffer (pH 7.2), and 5000 units of penicillin and streptomycin per mL. The cells were split every 3–4 day and the virus load in the media measured periodically by RT-PCR as described

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below. For many of the protein expression experiments, we harvested cells at different time points to evaluate the status of the proteins of interest. The hepatoma cell line, Huh 7, was a gift from Dr. Charles Rice, Rockefeller University, New York. For the in vivo labeling experiment, Edu labeling kit was purchased from Invitrogen (Carlsbad, CA). Iodixanol was purchased from Sigma (Saint Louis, MO). Mouse, rabbit or goat antibodies to IRS-1, Atg5, Atg12, GS and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA; Cell Signaling, Danvers, MA); IRS-1 Ser312 from Abcam (Cambridge, MA); and Beclin1, GSK-3a, GSK-3b, GSK-3aSer21, GSK-3bSer9, IRS-1 Ser302, Ser612, Ser789 and GS Ser641, Atg5, Atg12 and tubulin from cell signaling (Danvers, MA) including conformation specific secondary antibody and anti-mouse HRP for detecting secondary antibody in IP experiments.

Cell growth and proliferation assay For the cell proliferation assay, we used a Click-iTs EdU cell proliferation assay kit (Invitrogen, Carlsbad, CA). This assay offers a direct and accurate detection of new DNA synthesis that not only measures the proliferation of individual cells, but also allows detection of cells on any platform. The assay is based on a twostep click reaction involving copper-catalyzed triazole formation of an azide dye and an alkyne. Both azides and alkynes are biologically unique, inert, stable and extremely small molecules. The assay uses a modified nucleoside, EdU (5-ethynyl-20 -deoxyuridine), that is incorporated during DNA synthesis (Salic and Mitchison, 2008). The EdU contains both the alkyne and the fluorescent dye used to detect the new DNA that contains the azide. We grew cells on cover slips to 60–70% confluency. Cells were washed and exposed to EdU according to the manufacturer’s protocol. The cells were fixed, permeabilized and examined under the immunofluorescence microscope. The fluorescent intensity of the cells was compared under the same experimental condition and the lower the intensity, the slower is the rate of proliferation.

Determination of viral load in the culture media of insulin-resistant cells Virions secreted in the media were concentrated by centrifuging at 27,000 rpm for 5 h in an SW28 rotor over a 20% iodixanol cushion. The concentrated crude virion preparation was layered over a 10–40% iodixanol gradient and centrifuged at 40,000 rpm for 16 h in an SW41 rotor at 8 1C. following which 0.75 mL fractions were collected from the bottom. The density of each fraction and corresponding viral load were determined, and the peak HCV RNA fraction was examined by electron microscopy for the presence of virions. To determine virus load, viral RNA was isolated using a kit from Qiagen (Qiagen Science, MD). The RNA was amplified by RTPCR using a Light Cycler III from Roche with SYBR Green as the fluorescent probe. A 226 bp segment in the 50 -noncoding region was amplified by a forward and reverse primer for amplification: (forward) 50 -GTCTAGCCATGGCGTTAGTATGAG-30 (np 77–100); (reverse) 50 -ACCCTATCAGGCAGTACCACAAG-30 (np 302–280). The melting peak for the viral RNA differs by about 8–10 1C from that of the primers. RNA was quantified from a standard curve drawn with a known concentration of HCV.2a RNA synthesized by a coupled in vitro transcription-translation system using a genomic clone pJFH.1 (Promega, Madison, WI) and was expressed as copies/mL.

Curing of HCV infection To cure cells from HCV infection, we treated cultured cells with a physiological concentration of IFN alpha (100 pg/mL) for 72 h in three consecutive passages, as described (Erickson et al., 2008). The level of viral load was measured in the supernatant, and the absence of detectable viral RNA of the concentrated media was taken as a measure of cure. Cell lysates were prepared from these cells and used as a negative control in the Western blot experiments. Measurement of glucose uptake For glucose uptake experiments, HCV infected Huh 7 cells and the uninfected control cells were plated in 12-well plates. At 24 h, cells were treated with insulin (100 nM) for 2 h, the last 30 min in the presence of 1 mCi of 3H-2-deoxy glucose (6 Ci/mmole). At the end of the incubation time, cells were washed with PBS and lysed with 1N NaOH. Cell lysate was transferred to scintillation vials and counted. The percentage incorporation of 3H glucose is compared for the two cell lines as described in the text (Ciaraldi et al., 1995). Inhibition of autophagy To inhibit autophagy, semiconfluent cells were exposed for 4 h to 2.5 nM of 3-methyl-adenine (3-MA), washed and allowed to grow for another 16–20 h. Cells were harvested and autophagic proteins were analyzed by Western blotting as described below. Virus load was determined by quantifying viral RNA in the media by RT-PCR from cell cultures exposed to increasing concentrations of 3-MA up to 10 nM. Immunofluorescence microscopy For immunofluorescence studies, Huh 7 cells were grown on round cover slips in a 24-well plate. After 24 h, cells were transfected with 100 ng of pEGFP-LC3 or pEGFP-C1. The dot formation by GFP-LC3 was detected after another 24 h under a fluorescence microscope. All the cells expressing GFP were counted and cells with Z5 dots were defined as autophagy positive cells. Therefore, the percentage of cells showing significant dot formation is equal to the number of autophagic cells divided by total number of GFP-expressing cells (Kabeya et al., 2000). Western blots (WB) and immunoprecipitation Cells were washed with ice cold, calcium-free, phosphate buffered saline and 70–100 million cells were suspended in 1 mL of a lysis buffer containing 20 mM Tris HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerol phosphate, 1 mM sodium orthovandate, 1 mM PMSF and one-tenth volume of an anti-protease cocktail (Sigma, Saint Louis, MO). The lysate was passed through an 18-gauge hypodermic needle and incubated on ice for 10 min. It was then centrifuged at 4 1C at 14,000  g for 30 min. The lysate was aliquoted and stored at  80 1C. For Western blot analysis, equal amounts of protein (50– 100 mg) for HCV-producing cells and uninfected control Huh 7 cells were fractionated on an SDS-polyacrylamide gel electrophoresis apparatus. The proteins were then transferred to a polyvinylidene difluoride membrane. The membrane was then blocked with 5% Blotto for nonspecific proteins and reacted overnight with mouse, rabbit or goat antibody against target proteins. After repeated washings, the immunoreactive bands

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were reacted with horseradish peroxidase conjugated anti-rabbit, – mouse or – goat secondary antibody. For immunoprecipitation experiments, about 100–125 mg of total proteins were treated with primary antibody for 1 h at room temperature and an additional hour in the presence of 20 mL of Protein agarose beads. The immunoprecipitates were collected by low speed centrifugation and washed thrice with the lysis buffer by repeated resuspension and centrifugation, and finally solubilized in 25 mL of SDS electrophoresis buffer. Proteins in the supernatant were sizefractionated by SDS-PAGE and transferred to membranes for analysis of proteins that interact with IRS-1 Ser312 (Shivakumar and Das, 1996). The bands were visualized by an enhanced chemiluminescence detection system, ECL plus (Amersham Biosciences, Piscataway, NJ), and quantified by a phosphor imager. The membrane was rinsed at 60 1C for 30 min with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS and 62.5 mM Tris– HCl, pH 6.7) to treat with another antibody of interest when required. The presence of the Beclin 1 band was also confirmed by using an IgG light chain-specific secondary antibody (Jackson Laboratory, Bar Harbor, ME). When appropriate biological duplicates were made intentionally by carrying out measurement at two different time points of cell growth; otherwise two different amounts of proteins for each cell lysate were analyzed as indicated in the figure legends. In experiments to analyze Akt, GS and GSK-3, we used as a positive control lysates from Jurkat cells in which PI-3K/AKT pathway was stimulated by calyculin A. Cell lysates from IFN-cured cells were used as negative control. Both tubulin and actin were interchangeably used as a loading control. Statistical significance For cell growth (n¼4), glucose uptake (n¼ 3) and IRS-1 expression (n¼3) experiments, data obtained in multiple experiments were statistically evaluated by the Student t-test. Po0.05 (two-tailed) was considered as statistically significant.

Acknowledgment This work was partially supported by a grant from the Gulf Coast Regional Blood Center, Houston and by funding from the Eugene B. Casey Foundation and the Sonya and William Carpenter Fund. The authors are thankful to Dr. Charles Rice, Rockefeller University, for the cell line Huh 7, to Dr. G. Luo, University of Kentucky School of Medicine, for the stable Huh 7 cell line producing HCV, to Dr. T. Eissa, Baylor College of Medicine, Houston for the construct expressing GFP-LC3 protein, to Mr. G. Cai and Ms. Wei Chen, for technical assistance and to Dr. J. Broughman for help in immunofluorescence microscopy.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2012.07.003.

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