Hepatitis C Virus Activates the mTOR/S6K1 Signaling Pathway in
Inhibiting IRS-1 Function for Insulin Resistance
Sandip K. Bose,a,cShubham Shrivastava,bKeith Meyer,aRatna B. Ray,band Ranjit Raya,c
Departments of Internal Medicine,aPathology,band Molecular Microbiology & Immunology,cSaint Louis University, St. Louis, Missouri, USA
(17). Chronic HCV infection can lead to a spectrum of liver dis-
and hepatocellular carcinoma (16, 17, 27, 31, 32). Infection by
HCV often leads to insulin resistance and can predispose to the
infection (2, 13, 14, 38). Several mechanisms are likely to be in-
volved in the pathogenesis of HCV-related insulin resistance (1).
of insulin receptor substrate 1 (IRS-1) and IRS-2 (42, 45, 46).
Numerous defects in insulin signaling, including decreased acti-
vation of the insulin receptor ?-subunit and a reduced tyrosine
phosphorylation of insulin receptor substrate (IRS-1), have been
identified in type 2 diabetic patients. Reports suggest that HCV
upregulates suppressor of cytokine signaling 3 (SOCS3) expres-
sion (30) and increases tumor necrosis factor alpha (TNF-?) se-
cretion (42), thereby impairing the insulin signaling pathway.
Phosphorylation of Ser/Thr residues of IRS-1 blocks interactions
between positive IRS-1 Tyr phosphorylation and negative Ser
phosphorylation of IRS-1 in an insulin-resistant state (44).
ucts tuberin and hamartin, respectively (34). Cells lacking either
malian Akt phosphorylates TSC2 at two to four critical sites, in-
epatitis C virus (HCV) often causes chronic infection and
may lead to end stage liver disease (16). Approximately 200
expression of TSC1/TSC2 blocks S6K1 activation (34). TSC1/
til recently, when multiple investigators discovered that TSC1/
TSC2 functioned as a GTPase-activating protein (GAP), be-
longing to a conserved member of the Ras family, Rheb.
Overexpression of Rheb has effects on cell size and cell cycle that
expression of Rheb leads to high-level activation of S6K1, while
reduction in Rheb by small interfering RNA (siRNA) reduces
growth factor-induced S6K1 activation (23).
Previous reports have shown that HCV infection interferes
with normal glucose metabolism (35), and increased blood glu-
shown that HCV replication downregulates cell surface expres-
sion of GLUT2, thereby lowering glucose uptake by hepatocytes.
We have previously reported that HCV core protein upregulates
Ser-312 phosphorylation of IRS-1 and impairs the Akt signaling
pathway, which may in part be involved in the generation of in-
sulin resistance (11). Thus, we became interested in understand-
Received 9 January 2012 Accepted 20 March 2012
Published ahead of print 28 March 2012
Address correspondence to Ranjit Ray, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
June 2012 Volume 86 Number 11Journal of Virologyp. 6315–6322jvi.asm.org
ing the mechanism of HCV-mediated regulation of the down-
stream Akt signaling pathway. In this study, we have shown that
HCV infection of hepatocytes suppresses the expression of TSC-
downstream target S6K1 in inhibiting insulin signaling via IRS-1
degradation (Fig. 1). Ectopic expression of TSC-1/TSC-2 signifi-
cantly recovered IRS-1 levels in HCV protein-expressing hepato-
ing. HCV infection downregulates the expression of glucose
genic enzyme PCK2, both of which are known to increase blood
MATERIALS AND METHODS
Immortalized human hepatocytes, transfection, and generation of cell
erated by stable transfection of the HCV core (genotype 1a) genomic
region into primary hepatocytes as previously described (12, 39). IHH
exhibited a weak level of HCV core protein expression and were differen-
tiated (28). IHH and Huh7.5 cells were transfected with a plasmid DNA-
containing core gene from HCV genotype 2a under the control of a cyto-
megalovirus (CMV) promoter in a mammalian expression vector
(pcDNA3-Core) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).
Stable colonies from transfected cells were selected, using neomycin as
previously described (39). Mock-transfected cells were used in parallel as
a control. HCV genotype 2a (clone JFH1) was grown in IHH or Huh7.5
cells as previously described (28). Cell culture supernatant was filtered
through a 0.45-?m-pore-size cellulose acetate membrane (Nalgene,
Rochester, NY). HCV RNA was quantified by real-time PCR (in an ABI
(ASRs) (Abbott Molecular) (Department of Pathology, Saint Louis Uni-
fluorescent focus-forming assay. HCV titer was calculated to be ?105
focus-forming units/ml. We have used a multiplicity of infection (MOI)
of 1 for virus infection of hepatocytes.
Antibodies. Commercially available antibodies to IRS-1, IRS-2, p-Ser-
1101 IRS-1, TSC1, TSC2, Rheb, mTOR, S6K1, PCK2 (Cell Signaling Tech-
nology, Danvers, MA), and GLUT4 (Santa Cruz Biotechnology, Inc., CA)
was kindly provided by Arvind Patel (University of Glasgow, United King-
Plasmid DNAs. Human TSC-1 and TSC-2 plasmid DNAs were ob-
tained from Addgene (Cambridge, MA).
Western blot analysis. Proteins from cell lysates in sample-reducing
buffer were resolved by SDS-PAGE. Proteins were transferred onto a ni-
trocellulose membrane and blocked with 3% nonfat dry milk. The mem-
brane was incubated with a primary antibody, followed by a secondary
antibody coupled to horseradish peroxidase to detect protein bands by
chemiluminescence (Amersham, Piscataway, NJ). Cellular actin was de-
tected, using a specific antibody, for comparisons of the protein load in
Real-time PCR analyses for PCK2 mRNA levels. Cellular RNA was
isolated by TRIzol (Invitrogen, Carlsbad, CA). cDNA synthesis was car-
transcriptase (Invitrogen). The mRNA statuses of IRS-1 and PCK2 were
determined by real-time PCR (Applied Biosystems, Foster City, CA) by
using specific oligonucleotide primers (assay identification number
Hs00356436_m1 PCK2; Applied Biosystems). The results were normal-
ized for 18S. All reactions were performed in triplicate in an ABI Prism
7500 Fast analyzer.
Immunofluorescence. IHH adapted in Dulbecco modified Eagle me-
dium (DMEM) was grown in an 8-well chamber slide to about 60% con-
fluence. On the next day, cells were infected with HCV2a (clone JFH1)
and grown in DMEM containing 2% FBS for 72 h. Cells were serum
starved for 3 h in DMEM containing 0.1% fetal bovine serum (FBS) and
were fixed with formaldehyde (3.7%) and permeabilized using 0.2% Tri-
ton X-100. Cells were stained for GLUT4 with a mouse monoclonal anti-
body and a secondary antibody conjugated with Alexa Fluor 488 (Molec-
ular Probes, CA). Cells were treated with ProLong Gold antifade reagent
with DAPI (4=6-diamidino-2-phenylindole) (Invitrogen, Carlsbad, CA)
for fluorescence microscopy (Olympus FV1000). HCV core protein was
also stained using a rabbit polyclonal antibody and a secondary antibody
conjugated with Alexa Fluor 594 (Molecular Probes, CA).
HCV genotype 2a infection impairs IRS-1 expression. IHH and
Huh7.5 cells were infected with HCV genotype 2a (clone JFH1),
and the IRS-1 status was determined by Western blot analysis
ined as an index of viral infection (Fig. 2A). IRS-1 protein was
undetectable in virus-infected hepatocytes (Fig. 2B). To deter-
FIG 1 Simplified schematic presentation of HCV genotype 2a-mediated
blocking of the insulin signaling pathway. We suggested previously that HCV
infection upregulates Ser-312 phosphorylation of IRS-1 and impairs the Akt
ing in an upregulation of Rheb. This results in downstream activation of
mTOR and S6K1, which in turn acts as a negative regulator of IRS-1, causing
regulation of specific signaling molecules.
Bose et al.
jvi.asm.orgJournal of Virology
mine whether HCV core protein is involved in regulating IRS-1,
we transfected IHH and Huh7.5 cells with HCV core plasmid
of IRS-1 in both cell types (Fig. 2C). The level of IRS-2 was also
determined in HCV 2a-infected cells to examine if this effect was
specific for IRS-1. HCV 2a infection did not significantly alter
IRS-2 protein level in IHH or Huh7.5 cells (Fig. 2D). The results
from this set of experiments suggested that total IRS-1 expression
is markedly reduced in HCV genotype 2a-infected hepatocytes as
well as in hepatocytes transiently transfected with HCV core plas-
HCV infection suppresses TSC-1/TSC-2 and increases Rheb
expression. We previously reported that HCV impairs Akt acti-
sor complex TSC-1/TSC-2, which lies downstream in the IRS-1/
treated or infected with HCV genotype 2a. The status of TSC-1/
TSC-2 was determined by Western blot analysis using specific
control. TSC-1/TSC-2 expression could not be detected in HCV
protein was separately determined by transfecting into IHH or
Huh7.5 cells. Core from HCV 2a suppressed TSC-1/TSC-2 ex-
pression (Fig. 3B). Since TSC-1/TSC-2 acts directly to suppress
and Huh7.5 cells. Rheb was highly upregulated in IHH and
Huh7.5 cells infected with HCV 2a compared to that in mock-
2a increased Rheb expression in both IHH and Huh7.5 cells (Fig.
3D). Thus, our results suggested that HCV genotype 2a activates
Rheb via repression of the tumor suppressor complex TSC-1/
TSC-2 and HCV core protein plays a key role in this process.
we determined the expression status of mTOR and its activated
phosphorylated moiety, a downstream signaling component of
Rheb. For this, IHH was infected with HCV genotype 2a. An in-
crease in both total and phosphorylated mTOR (phospho-Ser-
2448) expression was observed in infected hepatocytes (Fig. 4A).
Insulin-induced anabolic responses, most notably ribosome bio-
genesis and protein synthesis, rely in part on S6K1, a member of
pho-S6K1 (Thr-389) status in HCV 2a-infected hepatocytes. For
this, IHH and Huh7.5 cells were separately infected with HCV
genotype 2a. The phospho-S6K1 level was upregulated in HCV
genotype 2a-infected IHH or Huh7.5 cells (Fig. 4B and C), sug-
gesting a novel mechanism for IRS-1 inhibition. Transient trans-
fection of HCV core in IHH and Huh7.5 cells also increased the
p-S6K1 level, suggesting the involvement of core protein in mod-
ulating the expression of p-S6K1 (Fig. 4D). We also measured the
sion levels in HCV 2a-infected and core 2a-transfected cells and
the control (Fig. 4E and F). These results suggested that HCV
genotype 2a infection enhances both mTOR and phospho-S6K1
status and the involvement of core protein in modulating expres-
sion in this signaling pathway.
wanted to determine how IRS-1 and TSC-1/TSC-2 expression is
FIG 2 IRS-1 expression is markedly reduced upon HCV infection or HCV
core protein expression. (A) Expression status of HCV core protein in HCV
genotype 2a-infected IHH or Huh7.5 cells is shown. (B) IRS-1 status in cell
culture-grown HCV genotype 2a-infected IHH or Huh7.5 cells. The expres-
sion status of actin in each lane was determined as a loading control. Mock-
status in IHH and Huh7.5 cells following transient transfection of HCV core.
(D) IRS-2 expression status in HCV genotype 2a-infected IHH and Huh7.5
FIG 3 HCV genotype 2a infection or introduction of core gene inversely
correlates with the TSC1/TSC2 and Rheb expression status. (A) Mock- or
HCV genotype 2a-infected IHH or Huh7.5 cells were analyzed for TSC-1/
in each lane was determined as a loading control. (B) The TSC-1/TSC-2 ex-
(C) IHH and Huh7.5 cells infected with HCV genotype 2a were similarly
lane was used as a loading control. (D) The expression status of Rheb from
HCV core 2a-transfected IHH or Huh7.5 cells is shown.
HCV and Insulin Resistance
June 2012 Volume 86 Number 11jvi.asm.org 6317
downregulated in HCV genotype 2a-infected or HCV core plas-
mid DNA-transfected hepatocytes. For this, we infected IHH and
Huh7.5 cells with the virus. After 72 h of infection, the cells were
mock treated or incubated with 10 ?M MG132 (proteasome in-
ence of MG132 (Fig. 5A and B). A similar recovery of TSC-1/
TSC-2 expression was also observed in IHH and Huh7.5 cells in-
ubiquitin proteasome pathway in HCV-infected hepatocytes.
Phosphorylation of IRS-1 at Ser-1101 via the mTOR-S6K1
at multiple serine residues, including Ser-1101, which leads to
degradation of IRS-1 (49). We wanted to determine if activated
could recover IRS-1 expression from MG132-treated HCV 2a-
was phosphorylated by activated p-S6K1. Interestingly, we found
that there was phosphorylation of Ser-1101 of IRS-1 in HCV ge-
notype 2a-infected IHH and Huh7.5 cells (Fig. 5E and F). This
result indicated that IRS-1 is degraded specifically via the activa-
tion of S6K1 in HCV-infected hepatocytes.
ectopic expression of TSC-1/TSC-2. Plasmid DNAs encoding
TSC-1 and TSC-2 were cotransfected into prior HCV core 2a-
blot analysis (Fig. 6A). A significant recovery of IRS-1 was ob-
served in a similar experiment upon introduction of TSC-1/
difference in the rescue level of IRS-1 could be due to the level of
viral protein expression in transiently versus virus-infected hepa-
the role of TSC-1/TSC-2 in the regulation of IRS-1 through the
mTOR/S6K1/IRS-1 signaling pathway.
HCV downregulates GLUT-4 expression level. GLUT-4 is
normally an adipose- and muscle-specific glucose transporter.
However, expression of GLUT4 has also been observed in liver-
specific cells (HepG2, hepatic stellate cells [HSC], and IHH).
(GLUT-4), a protein involved in glucose uptake from hepatocyte
surface. Western blot analysis suggested that the GLUT-4 level is
significantly downregulated in HCV genotype 2a-infected IHH
(Fig. 7A). Treatment with MG132 (10 ?M) did not rescue the
radation process (Fig. 7B). Immunofluorescence microscopy also
suggested a lower level of GLUT-4 expression in HCV genotype
2a-infected IHH compared to control cells (Fig. 7C). Downregu-
lation of GLUT-4 results in decreased uptake of glucose from
blood via cells which in turn causes the level of blood glucose to
rise. We also used a cell surface marker (CD81) to see the cellular
localization of GLUT-4 in the presence of insulin and found that
of insulin. But this surface expression was reduced upon HCV 2a
infection (data not shown). The results from this set of experi-
FIG4 HCV genotype 2a infection enhances expression of mTOR and p-S6K1. (A) Mock- and HCV genotype 2a-infected IHH cells were analyzed for total and
Ser-2448 phosphorylation status of mTOR. The tubulin level in each lane was determined as a loading control. (B and C) Activated phosphorylated-S6K1
wasusedforcomparisonofproteinloads.(D)p-S6K1 expression status from HCV core 2a-transfected IHH or Huh7.5 cells is shown. (E) Total S6K1 status
in mock-infected or HCV genotype 2a-infected IHH or Huh7.5 cells were analyzed by Western blotting. (F) Total S6K1 expression status from HCV core
2a-transfected IHH or Huh7.5 cells is also shown.
Bose et al.
jvi.asm.org Journal of Virology
hepatocyte surface, which results in a higher blood glucose level.
HCV infection enhances gluconeogenic PCK2 expression.
FoxO1 through activation of genes, including G6PC. We have
previously shown upregulation of G6PC mRNA in HCV-infected
cells (10). Here, we examined how HCV genotype 2a infection
impairs the gluconeogenic process by looking at the gluconeo-
genic enzyme PCK2. For this, we determined the PCK2 mRNA
expression level in IHH and Huh7.5 cells infected with HCV ge-
notype 2a. We observed an increased expression of PCK2 mRNA
infection also increased PCK2 protein levels in both IHH and
Huh7.5 cells. (Fig. 7F). Increased PCK2 levels indicate an in-
creased rate of gluconeogenesis. Hence more production of glu-
cose from noncarbohydrate substrates results in an increased
sis in blood.
In this study, we have shown that HCV genotype 2a infection of
hepatocytes activates the mTOR/S6K1 signaling pathway and in-
hibits IRS-1 function. HCV perturbs glucose metabolism via
downregulation of the glucose transporter (GLUT4) and upregu-
lation of key gluconeogenic enzyme PCK2. Tyrosine phosphory-
lation of IRS molecules by insulin receptor kinase is an important
step for insulin action on target cells. Activation of PI3K and one
effects of insulin. Therefore, a defect at any point in this pathway
may lead to insulin resistance.
in cells increased the phosphorylated Ser-312–IRS-1 protein level
although the kinetics of phosphorylation and degradation is not
clear due to a complexity involving multiple phosphorylation
sites. In this study, we have observed that total IRS-1 is degraded
upon HCV genotype 2a infection of IHH or Huh7.5 cells. The
of IRS-1 Ser-1101 in mock- or HCV 2a-infected IHH or Huh7.5 cells with or without MG132 treatment. The actin level was determined as a loading control.
FIG 6 Ectopic expression of TSC-1/TSC-2-rescued IRS-1 protein expression.
ectopic expression of TSC-1/TSC-2 in IHH transfected with HCV core 2a or
a loading control.
HCV and Insulin Resistance
June 2012 Volume 86 Number 11jvi.asm.org 6319
in kinetics in cell lines and/or an altered level of virus protein
suggest that HCV infection results in insulin resistance via IRS-1
modulation. IRS proteins are key players in propagating insulin
signaling and are therefore subjected to feedback regulatory sys-
tems that inhibit their action. Feedback regulation involves phos-
phatase-mediated dephosphorylation (20) or Ser/Thr phosphor-
ylation of functionally active Tyr-phosphorylated IRS proteins
teins from the insulin receptor or from downstream effectors or
could lead to their degradation (47). Identification of IRS-1 Ser-
study focused on a Ser-1101, which upon phosphorylation may
interfere with the association between IRS-1 and the insulin re-
ceptor (22, 49). Here, we have shown that Ser-1101 phosphoryla-
tion of IRS-1 results in IRS degradation. On the other hand, Ser-
eventual degradation resulting in insulin resistance.
We have shown previously that HCV core protein expression
decreases glucose uptake in human hepatocytes (11). Therefore,
we determined the status of the glucose transporter (GLUT4),
responsible for uptake of glucose from the cell surface, and found
that GLUT4 was significantly downregulated in HCV-infected
hepatocytes. Since HCV infection is associated with T2DM and is
characterized by a high blood glucose level, we wanted to deter-
mine how HCV affects glucose metabolism at the gluconeogenic
tein levels of the gluconeogenic enzyme PCK2. Our results agree
with the observations from Deng et al. (19), suggesting that HCV
infection promotes hepatic gluconeogenesis.
HCV core protein does not modulate phosphorylation of Akt
at Thr-308, while Ser-473 phosphorylation is significantly in-
creased (11). This IRS-1 modulation could participate, in part, in
the generation of a state associated with insulin resistance. How-
IRS-1 for insulin resistance (30). Activation of Akt by insulin ap-
pears to require phosphorylation of Thr-308 and Ser-473 by
PDK1 and TORC2, respectively (5, 6, 21). Studies on insulin-
droxystaurosporine suggested that Thr-308 phosphorylation of
Akt is necessary for increased glucose uptake, whereas Ser-473
tein kinase Akt underlies the pathology of a number of common
human diseases, such as cancer and diabetes.
Here, we have shown that the tumor suppressor complex
ited by HCV genotype 2a. Rheb is a direct target of the tuberous
is significantly upregulated in HCV 2a-infected hepatocytes. The
signaling function of mTOR complex 1 is activated by Rheb-GTP
FIG 7 HCV genotype 2a infection represses GLUT-4 expression and enhances gluconeogenic enzyme PCK2 mRNA expression level. (A) Mock- and HCV
genotype 2a-infected IHH cells were analyzed for GLUT-4 protein expression by Western blotting. The actin levels were determined in each lane as a loading
?M). Actin levels were determined in each lane as a loading control. (C) Immunofluorescence displaying cellular localization of GLUT-4 in HCV genotype
2a-infected IHH. Mock- or HCV-infected cells were treated with insulin (200 nM) after 72 h of infection. Green indicates localization of GLUT4, and red
PCK2 mRNA expression from Huh7.5 cells is shown. The results were normalized to endogenous 18S rRNA. (F) The protein level of PCK2 was determined by
Western blotting in IHH and Huh7.5 cells infected with HCV genotype 2a.
Bose et al.
jvi.asm.orgJournal of Virology
(8) and stimulates phosphorylation of downstream component
S6K1 (37). We observed that HCV 2a activates mTOR and results
in activation of S6K1, which in turn causes IRS-1 downregulation
(Fig. 1). Ectopic expression of TSC-1/TSC-2 results in rescue of
ring via downregulation of TSC-1/TSC-2 by HCV and not medi-
ated by Akt itself. The effects of HCV on TSC, Rheb, and mTOR
may occur independently. However, upregulation of Rheb and
mTOR appears to be an event concurrent with inhibition of TSC.
We were curious to determine the possible mechanism by
which IRS-1 and TSC-1/TSC-2 are degraded, and hence we used
the proteasome inhibitor MG 132 to determine if we can prevent
IRS-1 and TSC-1/TSC-2 degradation. We observed that MG132
treatment of hepatocytes prevented degradation of IRS-1 and the
ubiquitin proteasome pathway. S6K1 phosphorylates IRS-1 at
by HCV genotype 2a infection, we wanted to determine if acti-
vated S6K1 was actually initiating the degradation of IRS-1. Since
we could recover IRS-1 expression in MG132-treated HCV 2a-
infected cells, we reprobed the same blot to see the status of
Ser-1101 of IRS-1. Interestingly, we found that IRS-1 was phos-
phorylated specifically at the Ser-1101 residue in HCV genotype
pic expression of TSC-1/TSC-2 significantly rescued IRS-1 pro-
tein expression, further suggesting their modulated role in the
mTOR/S6K1/IRS-1 signaling loop in HCV infection.
in the advanced stage. However, clinical and experimental work
suggests a direct role for HCV in glucose metabolism (35). HCV-
infected cirrhotic patients may present with T2DM more fre-
quently than patients with cirrhosis of other origins (7). A retro-
occurred in 21% of HCV-infected patients but only in 12% of
hepatitis B virus (HBV)-infected subjects (36). Multivariate anal-
ysis suggested that HCV infection and age were independent pre-
dictors of diabetes, and HCV genotype 2a was found in 29% of
HCV-infected diabetic patients but only in 3% of controls (25).
the insulin cascade via proteasomal degradation of the IRS-1 and
IRS-2 (30) and their functional impairment through increased
levels of proinflammatory cytokines, such as TNF-? (41) or an-
other postreceptor defect (9). Knobler and Schattner (32) have
suggested that HCV patients with more severe liver disease may
have an exaggerated intrahepatic TNF-? response, resulting in
insulin resistance and a higher risk of developing diabetes. In pa-
tients with HCV genotype 3a, the virus may alter intrahepatic
erator-activated receptor gamma (18). Other researchers have
shown that HCV downregulates IRS-1 and -2 through upregula-
tion of suppressor of cytokine signaling 3 (SOCS3) (30). Down-
and HCV core-transfected human hepatoma cells occurs via a
proteosomal degradation mechanism. Our study generated novel
naling pathway for IRS-1 suppression by HCV genotype 2a.
We thank Arvind Patel and Chen Liu for providing antibodies and Lin
Cowick for preparation of the manuscript.
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