From the Departments of ¶ Pathology and Laboratory Medicine,
Surgery, and ** Pediatrics, University of
Rochester School of Medicine and Dentistry, Rochester,
New York 14642
Received for publication, October 18, 2002, and in revised form, January 28, 2003
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ABSTRACT |
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Interleukin-6 (IL-6) is one of several
pro-inflammatory cytokines implicated in insulin resistance during
infection, cachexia, and obesity. We recently demonstrated that
IL-6 inhibits insulin signaling in hepatocytes (Senn, J. J., Klover,
P. J., Nowak, I. A., and Mooney, R. A. (2002) Diabetes
51, 3391-3399). Members of the suppressors of cytokine
signaling (SOCS) family associate with the insulin receptor (IR), and
their ectopic expression inhibits IR signaling. Since several SOCS
proteins are induced by IL-6, a working hypothesis is that
IL-6-dependent insulin resistance is mediated, at least in
part, by induction of SOCS protein(s) in insulin target cells. To
examine the involvement of SOCS protein(s) in
IL-6-dependent inhibition of insulin receptor signaling,
HepG2 cells were treated with IL-6 (20 ng/ml) for periods from 1 min to
8 h. IL-6 induced SOCS-3 transcript at 30 min with a maximum effect at 1 h. SOCS-3 protein levels were also markedly elevated at 1 h. Transcript and protein levels returned to near basal
levels by 2 h. SOCS-3 induction by IL-6 paralleled
IL-6-dependent inhibition of IR signal transduction.
Ectopically expressed SOCS-3 associated with the IR and suppressed
insulin-dependent receptor autophosphorylation, insulin receptor
substrate-1 (IRS-1) tyrosine phosphorylation, association of IRS-1 with
the p85 subunit of phosphatidylinositol 3-kinase, and activation of
Akt. SOCS-3 was also a direct inhibitor of insulin receptor
autophosphorylation in vitro. In mice exposed to IL-6 for
60-90 min, hepatic SOCS-3 expression was increased. This was
associated with inhibition of hepatic insulin-dependent receptor autophosphorylation and IRS-1 tyrosine phosphorylation. These data suggest that induction of SOCS-3 in liver may be an important mechanism of IL-6-mediated insulin resistance.
Insulin resistance is a critical component in the pathogenesis of
type 2 diabetes. Several cellular lesions have been associated with
insulin resistance, including decreased insulin receptor tyrosine
kinase activity and decreased phosphorylation of target proteins such
as insulin receptor substrate-1
(IRS-1)1 (1, 2). Elevated
levels of cytokines such as IL-6, IL-1, and TNF SOCS proteins are a recently discovered family of proteins, identified
simultaneously by several groups that were screening for negative
regulators of cytokine signaling, particularly, inhibitors of JAK/STAT
signal transduction (13-15). There are currently eight members of this
family (CIS and SOCS1-7) that share a similar three domain structure.
These proteins are defined by an N-terminal variable length region
followed by a central Src homology domain 2 (SH2) and a highly
conserved C-terminal domain of 40-50 amino acids termed the SOCS box
(16). Because SOCS proteins inhibit cytokine-induced signaling pathways
and because the expression of some SOCS genes is induced by the
corresponding cytokines, SOCS proteins are believed to play a role in
the negative feedback control of cytokine signaling. SOCS proteins
appear to employ several mechanisms to inhibit cytokine signaling (17).
SOCS-1 inhibits several cytokine receptor signaling pathways by binding directly to and inhibiting the associated Janus kinases (JAKs) (14-20). In contrast, SOCS-3 binds more weakly to JAK kinases and inhibits several cytokine receptor signaling pathways by binding to
phosphotyrosine residues on these receptors. This association allows it
to interact with and inhibit the receptor-bound JAKs (21-23). The JAK
inhibitory activity of SOCS-1 has been mapped to a motif it shares with
SOCS-3 in the N-terminal regions of these proteins, which is distinct
from the phosphotyrosine binding determinants (18-20). Additionally,
it has been shown that the SOCS proteins can inhibit signaling by
targeting their binding partners for proteosomal degradation (24-27).
This activity appears to be mediated by the SOCS box, which contains a
functional BC box similar to that in Elongin A and in the von-Hippel
Lindau protein (24).
Recent reports describe the interaction of several of the SOCS family
members with both the IR and the insulin-like growth factor receptors
in vivo, in vitro, and in the yeast two-hybrid system (10, 11, 28, 29). Since SOCS proteins are induced by cytokines
and hormones associated with insulin resistance, the SOCS proteins are
excellent candidates for mediating cytokine-induced insulin resistance.
Our laboratory has reported that SOCS-1 and SOCS-6 can associate with
the insulin receptor in HepG2 cells and inhibit insulin receptor signal
transduction (11). These SOCS proteins can also inhibit in
vitro receptor kinase activity (11). Emanuelli et al.
(10) recently demonstrated that SOCS-3 is induced by insulin in
adipocytes and inhibits insulin-dependent activation of
STAT5. They propose that an association of SOCS-3 with Tyr-960 of the
insulin receptor antagonizes STAT5 activation based on yeast two-hybrid
studies. While this group could not demonstrate association between
SOCS-3 and the insulin receptor in vivo, they report an
inhibition of insulin-mediated IRS-1 tyrosine phosphorylation and p85
association by SOCS-3 in COS cells overexpressing IRS-1 and SOCS-3
(30). Emanuelli et al. (30) also report increased SOCS-3
expression in adipose tissue in ob/ob mice without changes in liver or
muscle. These results suggest that SOCS proteins may play a negative
regulatory role in insulin signal transduction in response to obesity
and other conditions promoting insulin resistance.
IL-6 is the primary pro-inflammatory cytokine affecting the liver. This
cytokine has also been shown to be elevated in the circulation of type
2 diabetics and to correlate most closely with insulin resistance (3,
31-34). IL-6 has recently been shown by our laboratory to inhibit
insulin signal transduction in hepatocytes and HepG2 cells (35). The
IL-6-induced inhibition is apparent after 1 h of exposure to the
cytokine. The current investigation posed the question of whether
SOCS-3 could play a role in mediating the observed
IL-6-dependent insulin resistance. The results indicate that IL-6 induces SOCS-3 in a temporal pattern that is consistent with
its role in inhibiting insulin receptor signaling. Additional data
demonstrate that SOCS-3 is a potent in vivo and in
vitro inhibitor of insulin receptor signaling. Together these
results provide evidence that SOCS-3 can be an important mediator of
IL-6-dependent insulin resistance in hepatocytes.
Reagents--
The isolation of the cDNA for human SOCS-3 has
been described previously (29). The pBIG-2i expression plasmid was a
gift from Dr. Craig Strathdee (The John P. Robarts Research Institute, London, Ontario) (36). Transient Expression of SOCS-3 in Cell Lines--
HepG2 human
hepatocarcinoma cells were grown to 75% confluence in a humidified
atmosphere of 5% CO2, 95% air at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were transfected with the pBIG-2i-FLAG-SOCS-3 constructs or
pBIG-2i-FLAG-null vector using FuGENE-6 transfection reagent according
to the manufacturer's instructions. FLAG-tagged SOCS-3 was also
transiently expressed in COS cells using the same expression plasmids
and protocols.
Coimmunoprecipitation of SOCS-3 with the Insulin
Receptor--
HepG2 cells were transiently transfected with the SOCS-3
construct as described above. After 6 h, the cells were placed in serum-free medium for 18 h. Cells were then treated with insulin at 10 In Vivo Insulin Receptor Autophosphorylation, Insulin-induced
IRS-1 Tyrosine Phosphorylation and p85 Association, and Activation of
Akt--
HepG2 cells transiently expressing SOCS-3 were harvested
after treatment with insulin for 5 min as described above. To analyze the phosphorylation of the insulin receptor, tyrosine-phosphorylated proteins were immunoprecipitated with In Vitro Interactions between the Insulin Receptor and
SOCS-3--
FLAG-tagged SOCS-3 was harvested from transiently
transfected COS cells by immunoprecipitation with anti-FLAG antibody.
COS cells were used for the isolation rather than HepG2 cells because the former express few insulin receptors, thus avoiding the possible in vivo complexing of SOCS proteins with endogenous insulin
receptors. Insulin receptors were isolated from HepG2 cells using wheat
germ agglutinin-agarose affinity chromatography. Briefly, 5 × 106 cells were lysed in a buffer containing 50 mM Tris, pH 7.4, 100 mM NaCl, 1% Triton X-100,
1 mg/ml bacitracin, 25 mM benzamidine, and 1 mM
phenylmethylsulfonyl fluoride. After homogenization in a Dounce
homogenizer and centrifugation, the supernatant was applied to a column
containing 1 ml of wheat germ agglutinin-conjugated agarose beads.
After 30 min at 4 °C, the column was drained dry and washed three
times with wash buffer (50 mM Tris, pH 7.4, and 0.1%
Triton X-100), and the IR was eluted with buffer containing 300 mM N-acetylglucosamine, 50 mM Tris,
pH 7.4, and 0.1% Triton X-100.
Insulin (10 In Vivo IL-6 Injection and Insulin Treatment of Mouse
Liver--
Male C57BL/6 mice (6-8-month-old) were injected with 1 µg/kg IL-6 or vehicle by subcutaneous administration. After 30, 60, and 90 min, livers were rapidly harvested. To examine the hepatic response to insulin, additional mice were anesthetized in a halothane chamber 90 min after IL-6 injections. The portal vein was exposed, 30 ng of insulin or vehicle were injected, and after 60 s the livers
were rapidly frozen in liquid nitrogen. Tissue extracts were prepared
in ice-cold 2× homogenization buffer containing no detergent. Three
10-s cycles of a Polytron homogenizer were used, chilling homogenates
between each cycle. The final 1× homogenization buffer contained 100 mM Hepes, 150 mM NaCl, 10% glycerol, 1%
Triton X-100, 2 mM EDTA, 2 mM EGTA, 100 mM NaF, and 1× protease inhibitor mixture set I
(Calbiochem). Extracts were kept on ice for 30 min prior to a 10-min
centrifugation at 10,000 × g at 4 °C. The
supernatant was then collected, protein content determined by the
Bradford method (38), and subjected to immunoprecipitation and Western blot analysis. All procedures involving laboratory animals were approved by the University of Rochester Committee on Animal Resources.
Northern Blot Analysis of SOCS-3 Expression in HepG2 Cells and
Mouse Liver--
Total RNA was extracted from HepG2 cells and mouse
livers described above using the guanidium thiocyanate
phenol/chloroform method. mRNA was purified from the total RNA
preparation using poly(A) Quik mRNA isolation kit (Stratagene). RNA
electrophoresis was then performed using 10 µg of RNA or 1 µg of
mRNA per lane on a 1% agarose gel containing 2.2 M
formaldehyde and then transferred to a Zeta-Probe GT membrane
(Bio-Rad). RNA integrity was confirmed by ethidium bromide staining of
the gel. An [ Statistical Analysis--
Densitometry of autorads determined by
using the ChemImager (Alpha Innotech, San Leandro, CA). Statistical
analysis was performed using the Student's t test in the
Stataquest statistical software package.
IL-6 Induces SOCS-3 mRNA and Protein Expression in the HepG2
Cell Line--
The ability of IL-6 to induce expression of SOCS
mRNA in HepG2 cells was examined at several time points during an
8-h incubation with the cytokine. Total cellular RNA was isolated, and
the presence of SOCS-1, SOCS-2, SOCS-3, and SOCS-6 mRNA were
examined by Northern blot analysis. Only SOCS-3 expression was elevated
in response to IL-6. SOCS-3 mRNA levels increased after 15 min of
IL-6 treatment and were maximal by 1 h followed by a nadir at
2 h and then a small 2-3-fold increase at 4 h (Fig.
1A). Following an identical time course of IL-6 treatment, SOCS-3 protein expression was detectable following 1 h of IL-6 treatment and then declined rapidly. No induction of SOCS-1, SOCS-2, or SOCS-6 was observed under these conditions (data not shown). These results indicate that IL-6 is
capable of specifically inducing SOCS-3 mRNA and protein in HepG2
cells. Importantly, this induction closely correlates with our
observations that IL-6 can inhibit insulin receptor signal transduction
in hepatocytes and HepG2 cells after 1 h of pretreatment (35).
Transiently Expressed SOCS-3 Associates with the Endogenous Insulin
Receptor in HepG2 Cells--
We have recently reported that SOCS-1 and
6 can be co-immunoprecipitated with the insulin receptor, and this
association is markedly enhanced by insulin (11). It has been suggested
by yeast two-hybrid and co-localization experiments that SOCS-3 may also interact with the insulin receptor (10, 29). To determine if
SOCS-3 was capable of directly interacting with the insulin receptor, a
FLAG epitope-tagged SOCS-3 was transiently expressed in HepG2 cells.
Insulin receptor immunoprecipitates were examined by Western blot
analysis for the presence of FLAG-tagged SOCS-3. As indicated in Fig.
2, SOCS-3 associated with the insulin
receptor in the presence or absence of insulin. This differs from
experimental results with SOCS-1 and SOCS-6, which required insulin for
maximum association. Additionally, this association was weak when
compared with total SOCS-3 expression (Fig. 2, lower panel)
and when compared with identical experiments performed with SOCS-1 and
SOCS-6 (data not shown). While SOCS-3 can directly associate with the
insulin receptor, suggesting that it may be involved in insulin signal transduction, the mechanism of interaction appears to differ from that
of SOCS-1 and SOCS-6.
Ectopic Expression of SOCS-3 Inhibits Insulin Signal Transduction
in HepG2 Cells--
Since SOCS-3 can directly interact with the IR
when ectopically expressed, the following experiments examined whether
SOCS-3 was capable of altering insulin signal transduction. We have
recently shown that ectopic expression of SOCS-1 and SOCS-6 proteins
can inhibit insulin signal transduction without effecting IR
autophosphorylation (11). Interestingly, ectopic expression of SOCS-3
was found to inhibit insulin receptor autophosphorylation in response
to 100 nM insulin by ~40-50% (Fig.
3). Ectopic expression of SOCS-3 appeared
to elevate basal autophosphorylation; however, this increase was not
statistically significant. SOCS-3 expression did not affect the total
cellular mass of the insulin receptors in HepG2 cells. These data
provide further evidence that the interaction of SOCS-3 with the
insulin receptor differs from that of SOCS-1 and SOCS-6.
SOCS-3 expression also inhibited insulin-induced tyrosine
phosphorylation of IRS-1 by a similar 40-50% (Fig.
4A). This
SOCS-dependent inhibition was produced without affecting
IRS-1 protein levels. As expected, insulin-induced p85 association with
IRS-1 was also decreased by ~50% (Fig. 4B), although this
change did not reach statistical significance. As with IR
autophosphorylation, SOCS-3 alone appeared to modestly elevate IRS-1
tyrosine phosphorylation and its association with p85, but this
observation was quite variable and was not statistically significant.
More distal components in the insulin signal transduction pathway did
not show this pattern. A 100 nM insulin concentration also
produced a 3.5-fold increase in phosphorylation of Akt on Ser-473. In
the presence of SOCS-3, however, this increase was reduced to ~2-fold
(Fig. 5). These experiments illustrate
that SOCS-3 can directly inhibit insulin receptor signal transduction.
Additionally, it is not anticipated that the SOCS-3 construct was being
expressed in all cells following transient expression. Therefore, the
40-50% inhibition of insulin signaling is probably an underestimation
of the effectiveness of SOCS-3 in inhibiting signaling.
In Vitro Inhibition of Insulin Receptor Autophosphorylation by
SOCS-3--
Ectopic expression of SOCS-3 inhibited IR
autophosphorylation and signal transduction in transfected HepG2 cells.
This suggests but does not prove that SOCS-3 is a direct inhibitor of
the IR. In the following experiments, an in vitro kinase
assay was used to examine whether SOCS-3 is a direct inhibitor of the
IR kinase activity. IR was isolated from HepG2 cells using wheat germ
agglutinin affinity chromatography. Receptors were pre-activated with
unlabeled ATP and insulin (10 IL-6 Suppresses Hepatic Insulin Receptor Signaling in the
Mouse--
To examine the relationship between IL-6 and SOCS-3
expression and inhibition of insulin signaling in the liver of mice,
IL-6 (1 µg/kg) was injected subcutaneously into mice that had been fasted overnight. SOCS-3 expression increased severalfold at 90 min in
the liver of animals injected with IL-6 (Fig.
7A). This is consistent with
the IL-6-dependent induction of SOCS-3 expression that was
observed in HepG2 cells after 60 min of IL-6 treatment (Fig. 1). After
90 min, insulin at a relatively low dose of 30 ng/mouse was injected
into the portal vein, and livers were harvested 1 min later.
Importantly, IL-6 caused a marked suppression of insulin receptor
autophosphorylation under these conditions while receptor mass was
unchanged (Fig. 7B). Insulin-dependent tyrosine phosphorylation of IRS-1 was also suppressed in IL-6-treated mice. However, tyrosine phosphorylation of IRS-1 in the mice that did not
receive insulin was substantial, rendering the effect of exogenous insulin less pronounced (Fig. 7B). Nonetheless, these
results demonstrate that IL-6 exerts an in vivo inhibitory
effect on hepatic insulin receptor signaling, which corresponds with
induction of SOCS-3 expression.
The recent observation that circulating IL-6 levels and adipose
tissue secretion of IL-6 correlate with insulin resistance suggests
that this cytokine could be a link between obesity and insulin
resistance (3, 31-34, 41). IL-6 is the major cytokine mediator in the
liver. As such, it may play an important role in hepatic insulin
resistance. Its effect on skeletal muscle is less clear since IL-6
receptor content in skeletal muscle appears to be very low (42). The
results of the current study demonstrate that acute exposure to IL-6
rapidly induces a transient expression of SOCS-3 in HepG2 cells and in
the liver of mice. We also show that SOCS-3 is a potent inhibitor of
insulin receptor signal transduction in HepG2 cells. In
vitro kinase assays confirm that SOCS-3 directly inhibits insulin
receptor autophosphorylation. These results complement other
investigations of IL-6 by our laboratory that have shown that this
cytokine inhibits insulin receptor signal transduction in primary mouse
hepatocytes and HepG2 cells (35). The temporal effects of acute IL-6 on
SOCS-3 expression and inhibition of insulin receptor signaling are
tightly correlated including the marked effects at 30-90 min followed
by a rapid decrease at 2 h. Similarly, we now demonstrate that
IL-6 not only induces SOCS-3 expression in the livers of mice but also
inhibits hepatic insulin receptor signaling in these animals. Together,
these observations strongly suggest a role for SOCS-3 in mediating
IL-6-dependent insulin resistance in the liver.
SOCS-3 is induced in liver by growth hormone, insulin, and TNF The hypothesis that members of the SOCS family play a role in mediating
insulin resistance is accumulating experimental support. Our current
data and that of Emanuelli et al. (30) indicate that SOCS-3
can inhibit insulin receptor signal transduction. The precise mechanism
is not yet defined. We demonstrate here that SOCS-3 can associate with
the insulin receptor and can be co-immunoprecipitated when ectopically
expressed in cultured cells. This has not been previously demonstrated.
We have also observed association between the insulin receptor and
endogenously expressed SOCS-3 in HepG2 cells after IL-6 treatment, but
this association has been weak and inconsistent (data not shown).
Emanuelli et al. (10) propose that SOCS-3 associates with
the insulin receptor at Tyr-960 based on cellular co-localization
studies and yeast two-hybrid analyses. We too have identified SOCS-3
along with SOCS-1, SOCS-2, and SOCS-6 as binding partners for the
insulin receptor by the yeast two-hybrid technique. The interaction of SOCS-3 with the insulin receptor displays some differences from that of
SOCS-1 and SOCS-6 (11). The association of SOCS-3 with the insulin
receptor does not require insulin. SOCS-3 inhibits insulin-dependent receptor autophosphorylation when
ectopically expressed in HepG2 cells. SOCS-3 also inhibits insulin
receptor autophosphorylation in vitro. This has not been
previously reported. In contrast, SOCS-1 and SOCS-6 require insulin for
maximum association with the insulin receptor and do not inhibit
insulin receptor autophosphorylation when ectopically expressed in
HepG2 cells. They do exert an inhibitory effect on insulin receptor
autophosphorylation in vitro. Interestingly, SOCS-3 as well
as the other SOCS proteins only associate with kinase active insulin
receptor in the yeast two-hybrid system (data not shown). The nature of
the SOCS-3-insulin receptor association in HepG2 cells in the absence
of insulin is unclear. One explanation is that SOCS-3 binds to
otherwise short-lived, insulin-independent phosphotyrosine residues on
the insulin receptor. This would be expected to increase
phosphorylation of the insulin receptor through trapping of the bound
tyrosine residues in a phosphorylated state. In fact, tyrosine
phosphorylation of insulin receptors is increased in HepG2 cells in the
absence of insulin when the cells are transfected with SOCS-3 (Fig. 3). Insulin receptors from livers of mice exposed to IL-6 for 90 min also
display a modest increase in receptor tyrosine phosphorylation in the
absence of insulin when compared with the IL-6-free controls (Fig.
7B). Further investigations will be required to define the mechanism of association between the insulin receptor and SOCS-3.
We observe that ectopic SOCS-3 expression in HepG2 cells inhibits
in vivo insulin receptor autophosphorylation. Subcutaneous IL-6 injections in mice also lead to suppression of hepatic
insulin-dependent insulin receptor autophosphorylation.
Interestingly, IL-6 does not inhibit insulin receptor
autophosphorylation in HepG2 cells (data not shown), although
insulin-dependent IRS-1 tyrosine phosphorylation and Akt
activation are inhibited. Thus, inhibition of autophosphorylation may
not always be a component of IL-6-dependent suppression of insulin receptor signal transduction. Inhibition of autophosphorylation may depend upon the relative concentrations of SOCS-3 and insulin receptor. This is supported by a set of experiments in which HepG2 cells were transfected with increasing levels of SOCS-3 construct. Insulin-dependent activation of Akt was suppressed at lower
SOCS-3 levels than were necessary for inhibition of IR
autophosphorylation (data not shown). It has also been suggested by
Emanuelli et al. (10) that SOCS-3 and insulin receptor
substrates compete for association with the activated insulin receptor
at Tyr-960. This competitive association would lead to inhibition of
insulin receptor substrate phosphorylation. This may be a mechanism of
inhibition of insulin receptor signal transduction that is observed at
lower SOCS-3/insulin receptor ratios where autophosphorylation is not impaired. At high levels of expression, SOCS-3 may associate with the
insulin receptor at an additional site(s) and directly antagonize autophosphorylation. This step probably requires kinase activity and
basal levels of autophosphorylation. Perhaps, this interaction involves
the kinase inhibitory domain of SOCS-3 acting similarly to this domain
in SOCS-1, which has been shown to be essential for mediating
inhibition of JAKs and to participate in the association of this SOCS
protein to the cytokine receptor complex (18-20). Inhibition is
apparently mediated through an N-terminal peptide sequence of SOCS-1
that acts as a pseudosubstrate for JAKs. A similar mechanism with the
insulin receptor may be in effect at high SOCS-3 expression in the
current studies.
It cannot be ruled out that SOCS-3 also mediates an effect distal to
the insulin receptor. Complexing of SOCS-3 with IRS-1/2, for example,
is an additional mechanism to explain SOCS-3-dependent inhibition of insulin receptor signal transduction. Kawazoe et al. (12) have reported that SOCS-1 but not SOCS-3 binds to IRS-1 when each is expressed in 293T cells. This group did not observe association of either SOCS-1 or SOCS-3 with the insulin receptor when
both were ectopically expressed in these cells. However, we have not
observed IRS-1/2 in immunoprecipitates of SOCS-3 under conditions in
which the insulin receptor is co-immunoprecipitated. It has been
proposed that SOCS proteins play a role in proteosomal targeting of
associated proteins by complexing with Elongins B and C (24-27). We
can exclude a mechanism by which SOCS-3 promotes IRS-1 degradation in
HepG2 cells since IRS-1 levels are unaffected by ectopic expression of
SOCS-3 (Fig. 4A). In summary, our current observations,
particularly the in vitro kinase assay and the
co-immunoprecipitation study, plus those reported by Emanuelli et
al. (10, 30) are most consistent with a direct interaction between
the insulin receptor and SOCS-3. No stable interaction between SOCS-3
and IRS-1/2 has been observed in the current study, but an association between these proteins cannot be ruled out.
While SOCS-3 has been linked most directly with insulin resistance in
insulin responsive tissues, the potential roles of SOCS-1 and SOCS-6
have also been investigated. We have identified these SOCS proteins as
binding partners for the insulin receptor and both inhibit insulin
receptor signal transduction (11). Interestingly, these SOCS proteins
require insulin for maximum association with the insulin receptor but
do not inhibit receptor autophosphorylation. As with SOCS-3, SOCS-1 is
induced in liver in response to IL-6, though we report here and others
have also observed (51) that IL-6 does not induce SOCS-1 in HepG2
cells. It has been suggested that the phenotype of the SOCS-1 knockout
mouse indicates a possible role for this protein in glucose homeostasis
and insulin action (12). SOCS-1-null mice at postnatal day 7-10 have
lower plasma glucose levels than matched controls despite comparable
insulin levels. Glucose and insulin tolerance tests were not performed nor were the animals examined using the euglycemic clamp technique. Importantly, SOCS-1-null mice have a 40% decrease in body weight on
postnatal day 9 compared with controls and do not survive beyond 3 weeks (52). It is not clear if this growth retardation and brief
survival, which accompanies altered lymphopoiesis, is impacting glucose
homeostasis independent of insulin action. Interestingly, a SOCS-3
deletion results in embryonic lethality at 12-16 days with marked
erythrocytosis (53).
Until very recently, little was known about the physiological
significance of SOCS-6. We have reported that ectopically expressed SOCS-6 associates with the insulin receptor and inhibits signal transduction in vivo (11). The insulin receptor kinase is
also directly inhibited by SOCS-6 in vitro. Krebs et
al. (54) now report that SOCS-6 is ubiquitously expressed and has
binding affinity toward IRS-2 and IRS-4. Nonetheless, SOCS-6-null mice
do not exhibit defects in glucose homeostasis and the only apparent
phenotype is a 10% decrease in body weight. Further investigations
will be necessary to determine the role of SOCS-6 and the significance of its association with members of the IRS family.
While considerable evidence points to a role for SOCS proteins in
mediating cytokine-dependent insulin resistance, the
precise mechanism and relative contribution of these proteins is yet to be determined. The pro-inflammatory cytokines have pleiotropic effects
on target tissues. Our current report plus other recent observations
suggest that SOCS protein expression may be one of several mechanisms
working in concert to mediate cytokine-dependent insulin
resistance in the liver and other insulin-responsive tissues.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and hormones such as
growth hormone and insulin have been linked to insulin resistance
(3-6). The mechanisms for these effects, however, have not been
clearly defined. One proposed mechanism, which is currently being
actively investigated, is the serine phosphorylation of IRS-1 and its
subsequent direct inhibitory effect on insulin receptor kinase activity
(7-9). An alternate hypothesis is that cytokines and hormones induce the expression of cellular proteins that inhibit IR signal
transduction. Our laboratory and others have proposed that the
suppressors of cytokine signaling (SOCS) family of regulatory proteins
may indeed fulfill this role (10-12).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-IR-1 (37) was a gift of Dr. S. Jacobs (Wellcome Research Laboratories, Research Triangle Park, NC). The
antibodies to phospho-Akt (Ser-473) were purchased from Cell Signaling
Technology (Beverly, MA). The anti-FLAG antibodies M2 and M5 were from
Sigma. The anti-phosphotyrosine antibody (4G10), recombinant rat IRS-1,
anti-IRS-1 antibody, and anti-p85 antibody were from Upstate
Biotechnology Inc., Lake Placid, NY. Anti-SOCS-3 antibody (M20)
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
transfection reagent FuGENE 6 was purchased from Roche Molecular
Biochemicals. Other reagents were obtained from commercial sources as
indicated in the text and figure legends.
7 M for 1 min. After two washes with
cold phosphate-buffered saline, cells were lysed in Lysis Buffer A
containing 50 mM Tris, pH 7.4,140 mM NaCl, 1%
Triton X-100, 50 mM NaF, 10 mM tetrasodium
pyrophosphate, 25 mM benzamidine, protease inhibitor
mixture (Calbiochem), 2.5 mM pervanadate, 2 mM
phenylmethylsulfonyl fluoride, and 10% glycerol. Lysates were passed
10 times through an 18-gauge needle, centrifuged at 10,000 × g for 10 min, and then adjusted to contain equal amounts of
protein as determined by the Bradford method (38). Insulin receptors
were immunoprecipitated using an anti-human insulin receptor antibody
(
-IR-1) bound to protein G-Sepharose. Immune complexes were washed
four times with wash buffer (1% Triton X-100, 100 mM
Tris-HCl, pH 7.4, and 150 mM NaCl) and separated by
SDS-PAGE, and associated FLAG-tagged SOCS-3 was detected by Western
blot analysis using anti-FLAG antibody.
-IR-1, and autophosphorylation was detected by Western blot analysis using anti-phosphotyrosine antibody 4G10. To examine the effects of SOCS proteins on the insulin-dependent tyrosine phosphorylation of IRS-1 and p85
association, cell lysates obtained from insulin-treated (5 min) HepG2
cells transiently expressing SOCS-3 were immunoprecipitated using
anti-IRS-1 (
-IRS-1) antibody bound to protein A-Sepharose.
Immunoprecipitates were then subjected to SDS-PAGE and Western blot
analysis using anti-phosphotyrosine antibody 4G10 or anti-p85 antibody.
Lysates from the same HepG2 cells were also examined by Western blot
analysis for the presence of activated Akt kinase using
anti-phosphoserine 473 Akt antibody.
7 M) and ATP (10 µM)
were added to aliquots of the wheat germ agglutinin-purified receptors
and incubated at room temperature for 30 min. The receptor preparations
were then added to the SOCS protein immunoprecipitates and incubated
for an additional 30 min. Finally, 10 µM
[
-32P]ATP was added, and the reaction mixtures were
incubated for an additional 30 min at room temperature. The reactions
were stopped with Laemmli (39) sample buffer, and the constituents were
separated by SDS-PAGE. The SDS-polyacrylamide gel was treated with 1 N NaOH for 60 min at 55 °C, fixed, and dried. Analysis
was by autoradiography.
-P32]dCTP-labeled probe was created with
the Random Primers DNA labeling system (Invitrogen) using the 650-bp
human SOCS-3 cDNA as a template. The probe was then purified by
ethanol precipitation. Hybridization was performed using the
Express-Hyb solution (Clontech) according to the
manufacturer's protocol. The relative intensity of SOCS-3 message was
determined using a PhosphorImager (Storm 840, Molecular Dynamics) and autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
IL-6 induces SOCS-3 expression in HepG2
cells. HepG2 cells were exposed to IL-6 (20 ng/ml) for 15 min to
8 h before harvesting. A, mRNA (2 µg) from each
sample was separated, transferred, and blotted with a
32P-labeled probe. Quantitation was performed on a
phosphorimager. Plotted data represent the mean ± S.D. of four
experiments. B, Western blot of SOCS-3 protein expression at
time points after IL-6 treatment.
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Fig. 2.
Ectopically expressed SOCS-3 associates with
the endogenous insulin receptor. HepG2 cells at 50-75%
confluence were transfected with either vector containing FLAG-tagged
SOCS-3 cDNA or control vector using FuGENE 6 transfection reagent
(Roche Molecular Biochemicals). Cells were then serum-starved overnight
in Dulbecco's modified Eagle's medium with the addition of 1% bovine
serum albumin. Following treatment with 100 nM insulin for
5 min, cells were harvested and immunoprecipitated using the anti-IR
antibody, ( -IR-1). Immunoprecipitates (IP) were separated
using SDS-PAGE, and the presence of FLAG-tagged SOCS-3 was detected by
Western blot analysis (IB). Cell lysates were similarly
probed for total SOCS-3 expression.
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Fig. 3.
Expression of SOCS-3 inhibits insulin
receptor autophosphorylation. HepG2 cells at 50-75% confluence
were transfected with SOCS-3 construct or controls and treated with
insulin as in Fig. 2 except for a 2-min exposure to insulin. Insulin
receptors were immunoprecipitated (IP) using the -IR-1
antibody, and precipitates were separated using SDS-PAGE.
Autophosphorylation was determined by Western blot analysis
(IB) using anti-phosphotyrosine antibody (4G10). The plotted
data represent the average ± S.D. of three independent
experiments. Brackets and asterisks indicate bars
that differ at p < 0.05. SOCS-3 alone had no
statistically significant effect on basal IR autophosphorylation
(comparison of gray bars, p > 0.1). Results
of a representative experiment are also displayed.
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Fig. 4.
Expression of SOCS-3 inhibits insulin-induced
tyrosine phosphorylation of IRS-1 and formation of p85/IRS-1
complexes. HepG2 cells were treated as in Fig. 2 except for
exposure to insulin for 3 min. IRS-1 was immunoprecipitated
(IP) using -IRS-1 antibody (UBI), and precipitates were
separated using SDS-PAGE. A, tyrosine phosphorylation was
determined by Western blot analysis (IB) using
anti-phosphotyrosine antibody (4G10). IRS-1 mass was determined by
stripping and reprobing the blot with an anti-IRS-1 antibody.
B, association of the p85 subunit of phosphatidylinositol
3-kinase with IRS-1 immunoprecipitates was determined by Western blot
analysis of the IRS-1 immunoprecipitates with an anti-p85 antibody.
Data plots represent the average ± S.D. of three independent
experiments. Brackets and asterisks indicate bars
that differ at **, p < 0.01 and *, p = 0.06. SOCS-3 alone had no statistically significant effect on basal
levels (comparison of gray bars, p > 0.1).
Results of a representative experiment are also displayed.
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[in a new window]
Fig. 5.
SOCS-3 inhibits insulin-induced Akt
activation. HepG2 cells were treated as in Fig. 2 including
exposure to insulin for 5 min. Lysates were separated by SDS-PAGE. Akt
activation was determined by Western blot (IB) analysis
using anti-phospho-Ser-473 antibody (CST). The data represent the
average ± S.D. of three independent experiments.
Brackets and asterisks indicate bars that differ
at p < 0.05. SOCS-3 alone had no statistically
significant effect on basal Akt activity (comparison of gray
bars, p > 0.1). Results of a representative
experiment are also displayed.
7 M) before
being combined with radiolabeled ATP and SOCS-3 immunoprecipitated from
COS cells that had been transfected with the SOCS-3 construct. As shown
in Fig. 6, SOCS-3 inhibited
autophosphorylation of the IR in this assay by ~40%. In contrast to
the report by Peraldi et al. (40) no phosphorylation of
SOCS-3 was detected in this assay.
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Fig. 6.
SOCS-3 inhibits in vitro
insulin receptor autophosphorylation. Insulin receptors were
isolated from HepG2 cells by wheat germ agglutinin affinity
chromatography. Following addition of insulin and unlabeled ATP to
activate the receptors, the reactions were combined with SOCS-3
immunoprecipitates. Radiolabeled ATP was subsequently added to initiate
the reaction. Tyrosine-phosphorylated insulin receptors were visualized
by autoradiography following SDS-PAGE and treatment of the gel with
NaOH to hydrolyze serine/threonine phosphorylation. The data represent
the average ± S.D. of three independent experiments.
Brackets and asterisks indicate bars that differ
at p < 0.05. SOCS-3 alone had no statistically
significant effect on basal IR autophosphorylation (comparison of
gray bars, p > 0.1). Results of a
representative experiment are also displayed.
View larger version (47K):
[in a new window]
Fig. 7.
IL-6 induces hepatic SOCS-3 expression and
inhibits hepatic insulin receptor signal transduction in
vivo. A, male C127/BL6 mice were injected
subcutaneously with IL-6 at 1 µg/kg body weight or a saline control.
Livers were rapidly harvested at 90 min and placed in liquid nitrogen.
Total protein was isolated, and SOCS-3 expression was determined by
immunoprecipitation (IP) with SOCS-3 (M-20) antibody and
Western blot analysis using -SOCS-3. (IgG is an immunoglobulin
control). B, male C127/BL6 mice were injected subcutaneously
with IL-6 at 1 µg/kg body weight. At 90 min after IL-6 injection, the
portal vein was exposed, and 30 ng of insulin in 0.1 ml of saline or
saline alone was infused. Livers were rapidly harvested and placed in
liquid nitrogen. Insulin receptor was immunoprecipitated from
homogenates, separated by SDS-PAGE, and tyrosine phosphorylation
determined by Western blot analysis (IB). Blots were
reprobed for receptor mass. Tyrosine phosphorylation of IRS-1 was
similarly analyzed by Western blot analysis using an
anti-phosphotyrosine antibody and reprobed for IRS-1 mass with an
anti-IRS-1 antibody. Results are representative of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
as
well as IL-6 (30, 43-46). Each has been implicated in hepatic insulin
resistance. Thus, SOCS-3 may also contribute to hepatic insulin
resistance associated with these agents. TNF
has been investigated
most extensively as a potentially critical mediator of
obesity-dependent insulin resistance (3, 5, 47-49). Kern
et al. (3) have reported that human adipose tissue secretion
of TNF
correlates better than IL-6 secretion with obesity-related insulin resistance while plasma levels of IL-6 have a more significant correlation to insulin resistance than plasma TNF
. Emanuelli et al. (30) have demonstrated that TNF
induces SOCS-3
expression in insulin responsive tissues when administered in
vivo. Interestingly, TNF
induces IL-6 expression in the liver,
although it is controversial whether TNF
induction of SOCS-3
requires expression of IL-6 (46, 50). It is clear, however, that TNF
and other cytokines and hormones that have been linked to
obesity-dependent insulin resistance are capable of
inducing SOCS proteins in the liver.
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FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant R01-38138 (to R. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Member of the Graduate Program in Pharmacology and Physiology,
University of Rochester.
§ Member of the Graduate Program in Biochemistry, University of Rochester.
To whom correspondence should be addressed: Dept. of Pathology
and Laboratory Medicine, University of Rochester School of Medicine and
Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-7811;
Fax: 585-273-1101; E-mail: robert_mooney@urmc.rochester.edu.
Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M210689200
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ABBREVIATIONS |
---|
The abbreviations used are: IRS-1, insulin receptor substrate-1; IR, insulin receptor; IL, interleukin; TNF, tumor necrosis factor; SOCS, suppressor of cytokine signaling-3; STAT, signal transducers and activators of transcription; JAK, Janus kinase.
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