From the Departments of Medicine and Physiology,
University of Michigan, Ann Arbor, Michigan 48109 and the
§ Department of Cell Biology, Pfizer Global Research and
Development, Ann Arbor, Michigan 48105
Received for publication, December 4, 2000
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ABSTRACT |
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In adipose and muscle, insulin stimulates
glucose uptake and glycogen synthase activity. Phosphatidylinositol
3-kinase (PI3K) activation is necessary but not sufficient for these
metabolic actions of insulin. The insulin-stimulated translocation of
phospho-c-Cbl to lipid rafts, via its association with CAP, comprises a
second pathway regulating GLUT4 translocation. In 3T3-L1 adipocytes, overexpression of a dominant negative CAP mutant (CAP Insulin stimulates the storage of glucose as glycogen in muscle
and fat through a coordinate increase in glucose uptake and glycogen
synthesis (1). Glycogen synthase
(GS),1 the rate-limiting
enzyme for glycogen synthesis, is regulated by covalent and allosteric
modification (2). Insulin promotes the net dephosphorylation of GS
through the activation of glycogen-targeted protein-phosphatase-1 (PP1)
and inhibition of glycogen synthase kinase-3 (GSK-3). This action of
insulin is facilitated by the stimulation of glucose uptake, since the
resulting increase in glucose-6-phosphate leads to the allosteric
activation of GS and increased susceptibility to PP1-mediated
dephosphorylation (3, 4).
The stimulation of glucose disposal by insulin requires the activation
of phosphatidylinositol 3-kinase (PI3K), which leads to the
increased activity of PIP3-dependent kinases
such as Akt and protein kinase C The PI3K-independent pathway might involve the tyrosine phosphorylation
of the proto-oncogene c-Cbl (15). The phosphorylation of c-Cbl by
insulin requires the c-Cbl-associated protein (CAP), an adapter
molecule that recruits c-Cbl to the insulin receptor (16). CAP
expression correlates with c-Cbl phosphorylation and insulin
sensitivity. Moreover, expression of the CAP gene is increased by insulin-sensitizing activators of the nuclear receptor PPAR Upon phosphorylation, Cbl translocates to a Triton-insoluble lipid raft
microdomain (18), where CAP binds to the caveolar protein flotillin
(19). In 3T3-L1 adipocytes, expression of a CAP mutant in which the SH3
domains have been deleted (CAP Materials--
All cell culture reagents were purchased from
Life Technologies, Inc. with the exception of serum, which was
obtained from Summit Biotechnology (Ft. Collins, CO). Insulin,
2-deoxy-glucose, and differentiation agents were supplied by Sigma.
UDP-[U-14C]glucose (308 mCi/mmol) was from by ICN, and
D-[14C]glucose (3.4 mCi/mmol) and
2-deoxy-D-[14C]glucose (323 mCi/mmol) were
obtained from PerkinElmer Life Sciences. Anti-phospho-Akt (serine 473)
and anti-phospo-GSK3 (serine 21/serine 9) antibodies were
purchased from New England Biolabs, and horseradish peroxidase goat
anti-mouse and goat anti-rabbit IgGs were from Bio-Rad. CAP and
CAP Cell Culture and Experimental Treatment--
3T3-L1 fibroblasts
were maintained and differentiated into adipocytes as previously
reported (20). Electroporation of adipocytes was performed as described
(21). Prior to insulin stimulation, cells were washed two times with
low serum medium (Dulbecco's modified Eagle's medium containing 5 mM glucose, 0.5% fetal bovine serum, 25 mM
Hepes (pH 7.4), 100 units/ml penicillin, 100 units/ml streptomycin, and
0.29 mg/ml glutamine) and incubated in the same medium for 3 h.
Enzymatic and Metabolic Assays--
Glucose transport
measurements were done as described previously (22) with minor
modifications. Briefly, following serum starvation, adipocytes were
washed three times with PBS and placed in 0.5 ml/well Krebs-Ringer
buffer with 30 mM Hepes (pH 7.4) and 0.5% bovine serum
albumin in the absence and presence of 100 nM insulin.
After 30 min at 37 °C, 20 µM
2-deoxy-D-[14C]glucose (~20 cpm/pmol) was
added to all wells. After 5 min at room temperature, the assay was
terminated by the adding 50 µl of 200 mM 2-deoxyglucose
and washing the cells 3 times with PBS on ice. Adipocytes were
collected in 0.5 ml of distilled water. 10 µl of the cell suspension
was retained to determine protein concentration and the remainder was
subject to scintillation counting.
Glycogen synthase assays were performed as described (22). In some
experiments, the cells were washed and incubated in glucose-free, low
serum media immediately prior to insulin treatment. Measurement of PP1 activity was performed as described previously (23), with 1-2
µg of cell extract assayed for 2 min at 30 °C. Glycogen synthesis
rate was measured in 6-well dishes as reported (22). Briefly, cells
were treated in the absence and presence of 100 nM insulin
for 15 min, and then 1 µCi of [14C]glucose (20 cpm/pmol) was added to all wells. After 45 min, cells were washed on
ice with PBS, and glycogen was precipitated with 30% potassium
hydroxide and quantitated by scintillation counting.
Other Procedures--
Immunoblotting (22) and
immunoprecipitations (15) were performed as described previously.
Protein measurements were determined by the method of Bradford.
CAP Inhibition of GS Activation by CAP
The PI3K inhibitor wortmannin completely blocked the regulation of GS,
PP1, and GSK-3 by insulin (10, 11). In contrast, pretreatment of 3T3-L1
adipocytes with 200 nM wortmannin did not significantly
change the insulin-stimulated tyrosine phosphorylation of c-Cbl (Fig.
3A). However, wortmannin
pretreatment of transfected cells incubated in glucose-free
media completely blocked the residual activation of GS by
insulin (Fig. 3B). Thus, a PI3K-dependent pathway is likely to trigger the glucose-independent activation of
GS.
CAP Insulin stimulates glycogen synthesis in muscle and fat by a
coordinate increase in GLUT4 vesicle translocation and covalent modification of glycogen metabolizing enzymes (1). Insulin promotes the
dephosphorylation of glycogen synthase and phosphorylase, resulting in
enzymatic activation and inactivation, respectively. Both phosphatase
activation and kinase inactivation contribute to the dephosphorylation
of both proteins in response to insulin. Additionally, increased
glucose-6-phosphate levels allosterically activate GS, overriding
inhibition caused by phosphorylation (2). Finally, glucose metabolites
induce the translocation of cytosolic GS to glycogen-containing
fractions in primary hepatocytes (25), skeletal muscle (26), and 3T3-L1
adipocytes (20). Insulin therefore activates GS through covalent
modification, allosteric activation, and enzymatic translocation.
However, the complex interplay between insulin signaling
cascades, elevated levels of glucose, and its metabolites and glycogen
stores in the regulation of GS activity and glycogen synthesis rates
remains unclear.
The activation of PI3K and downstream serine/threonine kinases plays an
important role in the hormonal regulation of glucose uptake and
storage. Pharmacological inhibition of PI3K blocked the stimulation of
glucose transport (7) and the enzymes involved in glycogen metabolism,
such as GS, PP1, and GSK-3 (10, 11). Further, overexpression of PI3K or
downstream enzymatic effectors in cells partially increased glucose
uptake (27, 28). However, PI3K activation is not sufficient to mediate
changes in glucose metabolism produced by insulin (12-14), implicating
a second signaling pathway.
The PI3K-independent arm of insulin action may result from the
phosphorylation of c-Cbl via its association with CAP. Insulin stimulated c-Cbl phosphorylation and translocation of the Cbl·CAP complex to lipid rafts, where CAP directly bound flotillin (15, 18,
19). The insulin-dependent localization of phospho-Cbl to
subdomains of the plasma membrane resulted in the generation of
signaling pathways involved in GLUT4 translocation. In 3T3-L1 adipocytes, overexpression of the dominant negative CAP mutant, CAP Overexpression of the dominant negative mutant CAP In primary adipocytes, insulin and glucose synergistically promote the
dephosphorylation and activation of GS (29). Removal of extracellular
glucose decreases insulin-stimulated GS activation by 40% in 3T3-L1
adipocytes (Fig. 2B) (20). Similarly, blockade of glucose
transport by CAPSH3) completely blocked the insulin-stimulated glucose transport and glycogen synthesis
but only partially inhibited glycogen synthase activation. In contrast,
CAP
SH3 expression did not affect glycogen synthase activation by
insulin in the absence of extracellular glucose. Moreover, CAP
SH3
has no effect on the PI3K-dependent activation of protein
phosphatase-1 or phosphorylation of glycogen synthase kinase-3. These
results indicate blockade of the c-Cbl/CAP pathway directly inhibits
insulin-stimulated glucose uptake, which results in secondary
inhibition of glycogen synthase activation and glycogen synthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
(5, 6). The PI3K inhibitor
wortmannin blocks many of the metabolic actions of insulin, including
the effects of the hormone on glucose transport and GS activities (7-9). Moreover, both the inactivation of GSK-3 and the activation of
PP1 are blocked by wortmannin (10, 11). However, the activation of PI3K
alone is not sufficient for insulin action, since other hormones and
adhesion molecules can stimulate PI3K activity without reproducing the
metabolic effects of insulin (12, 13). Furthermore, cell-permeable
derivatives of PIP3 do not fully mimic the effects of
insulin on glucose transport (14). Thus, although the PI3K pathway is
necessary, there is at least one additional pathway required for
glucose transport regulation by insulin.
(17).
These data suggest a potential link between CAP/Cbl and the metabolic
actions of insulin.
SH3) disrupted the formation of a
CAP·flotillin·Cbl complex in lipid rafts, blocked
translocation of the insulin-stimulated glucose transporter GLUT4, and
inhibited glucose transport, glycogen, and lipid synthesis (19).
However, it remained unclear whether CAP
SH3 directly blocked the
signaling pathways involving GS dephosphorylation that are independent
of glucose transport. We demonstrate here that the CAP/Cbl pathway
regulates GS activation exclusively through a
glucose-dependent mechanism.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SH3 expression vectors were constructed as previously described
(16, 19). ECL reagent was purchased from Amersham Pharmacia Biotech,
and GF/A filters were supplied by Whatman.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SH3 Expression Blocks Glucose Uptake and Glycogen Synthesis
in 3T3-L1 Adipocytes--
CAP
SH3 overexpression in 3T3-L1
adipocytes significantly inhibits the translocation of the
insulin-stimulated glucose transporter GLUT4 to the plasma membrane,
without affecting PI3K-dependent signals (19). To explore
the implications of this result in more detail, we examined the effects
of CAP
SH3 expression on glucose uptake and glycogen synthesis over a
range of insulin concentrations. In control LacZ-transfected 3T3-L1
adipocytes, insulin produced a 15-fold increase in glucose transport
(Fig. 1A) and a 10-fold
increase in glycogen synthesis (Fig. 1B). Overexpression of
CAP
SH3 decreased maximal glucose uptake by 40-50% (Fig.
1A). The construct blocked the responsiveness to insulin
without effecting the EC50 for the hormone. Not
surprisingly, CAP
SH3 expression also blocked insulin-stimulated
glycogen synthesis (Fig. 1B), which is dependent on
increased glucose uptake under these conditions. Extrapolating for an
average 50% transfection efficiency (data not shown), these results
demonstrate that expression of CAP
SH3 in 3T3-L1 adipocytes produces
a nearly complete inhibition of insulin-stimulated glucose uptake and
glycogen synthesis.
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Fig. 1.
CAP SH3 expression
inhibits insulin-stimulated glucose transport and glycogen
synthesis. 3T3-L1 adipocytes were electroporated with either LacZ
or constructs, replated, and allowed to recover for 36 h. After a
3-h incubation in low serum media containing 5 mM
glucose, basal and insulin-stimulated glucose transport (A)
and glycogen synthesis (B) rates were determined as
described under "Experimental Procedures." Results are the average
of three independent experiments, each performed in triplicate. *,
significant difference from LacZ control, p < 0.01, n = 3 by Student's t test. **, significant
difference from LacZ control, p < 0.05, n = 3, by Student's t test.
SH3 Is a Result of Reduced
Glucose Uptake--
We next examined the effects of CAP
SH3
expression on the regulation of GS activity by insulin. Electroporation
of 3T3-L1 adipocytes with CAP
SH3 resulted in a 30% decrease in the
activation of GS by insulin, compared with both LacZ- and
CAP-expressing cells (Fig.
2A). In these experiments the
electroporation efficiency was ~50-60% (data not shown), indicating
that CAP
SH3 overexpression reduced GS activation by half. Previous
studies in 3T3-L1 adipocytes indicated that removal of extracellular
glucose also decreased the insulin-stimulated GS activity by 40-50%
(20), suggesting that the effect of CAP
SH3 on GS activation may be
secondary to inhibition of glucose transport. To evaluate this
possibility directly, the electroporated adipocytes were incubated in
glucose-free medium during the insulin treatment, and GS activity was
assayed in vitro. Removal of extracellular glucose decreased
insulin-stimulated GS activity by 40% in LacZ- and CAP-expressing
cells (Fig. 2B; compare with Fig. 2A). However,
CAP
SH3 expression had no further inhibitory effect on GS activation
by insulin. Thus, the glucose-independent pathway that mediates the
dephosphorylation and subsequent activation of GS by insulin appears
not to involve the CAP/Cbl pathway.
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Fig. 2.
CAP SH3 inhibits the
glucose-dependent activation of glycogen synthase.
3T3-L1 adipocytes were electroporated with the indicated constructs and
allowed to recover for 36 h. A, after a 3-h incubation
in low serum media containing 5 mM glucose, cells
were treated in the absence and presence of 100 nM insulin
for 15 min. Cells were washed on ice with PBS and harvested, and GS
activity was measured in vitro in the absence and presence
of 10 mM glucose-6-phosphate (G6P).
B, after a 3-h serum starvation, all wells were washed twice
and incubated in low serum media lacking glucose immediately
prior to the insulin treatment. Results are an average of three
independent experiments done in duplicate. *, significant difference
from LacZ control, p < 0.01, n = 3 by
Student's t test.
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Fig. 3.
Wortmannin inhibits the glucose-independent
activation of glycogen synthase. A, 3T3-L1 adipocytes
were serum starved for 3 h and then preincubated for 30 min with
either 200 nM wortmannin (Wort.) or an equal
volume of Me2SO (DMSO). Cells were
stimulated for 2 min in the absence or presence of 100 nM
insulin. Cells were harvested, and anti-c-Cbl immunoprecipitations
(I.P.) were preformed. Samples were then analyzed by
anti-phosphotyrosine and c-Cbl immunoblotting (I.B.).
B, 3T3-L1 adipocytes were electroporated with the indicated
constructs and treated as in Fig. 2B, except the indicated
wells were pretreated for 30 min with 200 nM wortmannin
(Wort). After a 15 min treatment with 100 nM
insulin (Ins), glycogen synthase activity was determined
in vitro. Results are representative of three independent
experiments (A) or the average of three independent
experiments performed in duplicate (B). *, significant
difference from LacZ control, p < 0.01, n = 3 by Student's t test.
SH3 Has No Effect on the Regulation of PP1 and GSK-3 by
Insulin--
Insulin promotes the activation of GS by decreasing the
phosphorylation state of the enzyme. Both the activation of
glycogen-targeted PP1 and the inactivation of GSK-3 have been proposed
to mediate this effect of the hormone (5). We next examined the effect of CAP
SH3 expression on the regulation of these enzymes by insulin. Electroporation of LacZ, CAP, or CAP
SH3 into 3T3-L1 adipocytes had
no effect on the insulin-stimulated phosphorylation of GSK3 (Fig.
4A), which results in enzymatic
inactivation (24). Insulin treatment of LacZ-electroporated cells also
caused a 50% increase in PP1 activity (Fig. 4B).
Overexpression of either CAP or CAP
SH3 had no effect on PP1
activation by insulin in 3T3-L1 adipocytes (Fig. 4B). These
results cumulatively indicate that the CAP/Cbl pathway is not necessary
for the insulin-mediated regulation of enzymes that covalently modify
GS.
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Fig. 4.
Regulation of GSK-3 and PP1 by insulin is
unaffected by expression of CAP SH3.
3T3-L1 adipocytes were electroporated with the indicated constructs.
After a 36-h recovery, cells were treated for 15 min in the absence and
presence of 100 nM insulin. A,
anti-phospho-GSK-3 immunoblotting. 40 µg of cellular lysates were
separated by SDS-PAGE, transferred to nitrocellulose, and probed with
an anti-phospho-GSK-3 antibody. Antibody binding was visualized with
ECL and autoradiography. Results are representative of three
independent experiments. B, PP1 activity. After treatment,
cells were harvested, and PP1 activity was measured in vitro
using 32P-labeled phosphorylase as substrate. Results are
representative of four independent experiments, each performed in
triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SH3, specifically blocked the insulin-mediated translocation of
phospho-Cbl into lipid rafts and completely inhibited
insulin-stimulated GLUT4 translocation (19). Interestingly, the
insulin-mediated phosphorylation and translocation of Cbl were not
blocked by PI3K inhibitors (Fig. 3A, data not shown),
indicating that the Cbl·CAP·flotillin axis may comprise the
PI3K-independent pathway regulating glucose transport.
SH3 completely
blocked insulin-stimulated glucose transport and glycogen synthesis in
3T3-L1 adipocytes, correcting for transfection efficiency (Fig. 1,
A and B). Further, CAP
SH3 overexpression also
reduced insulin-mediated GS activation by 50% (Fig. 2A).
Interestingly, this effect was solely the result of reduced
glucose uptake, because CAP
SH3 did not block GS activation in the
absence of extracellular glucose (Figs. 2B and
3B). Moreover, the inhibition of GSK-3 and activation of PP1
by insulin were unaffected by CAP
SH3 expression (Fig. 4,
A and B), whereas the glucose-independent
activation of GS by insulin was completely suppressed by the PI3K
inhibitor wortmannin (Fig. 3B).
SH3 overexpression caused a 50% reduction in GS
activation by insulin. However, the regulation of GSK-3 and PP1 by
insulin was unaffected by CAP
SH3, indicating that these enzymes may
constitute the other 50% of GS activation. In agreement, the PI3K
inhibitor wortmannin, which completely blocks the effects of insulin on
GSK-3 and PP1 activities (10, 11), also blocked the glucose-independent
activation of GS (Fig. 3B). Cumulatively, these results
indicate that in 3T3-L1 adipocytes, insulin utilizes
glucose-dependent and independent pathways to regulate GS
activity. In parallel, insulin initiates PI3K-dependent and
-independent signaling pathways to mobilize GLUT4 translocation. Blockade of the PI3K-independent, CAP/Cbl-dependent insulin
pathway through CAP
SH3 expression inhibits GLUT4 translocation and
the glucose-dependent component of GS activation by
insulin. In contrast, pharmacological inactivation of PI3K blocks both
insulin-mediated stimulation of glucose transport and regulation of PP1
and GSK-3 activities, resulting in a complete inhibition of GS
activation. Whether insulin also utilizes a PI3K-independent pathway to
regulate PP1 and GSK-3 activities is presently under investigation.
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FOOTNOTES |
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* 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.
¶ These authors contributed equally to this work.
To whom correspondence should be addressed: University of
Michigan Medical Center, MSRB I, Rm. 4520, 1150 West Medical Center Dr., Ann Arbor, MI 48109. Tel.: 734-615-9787; Fax: 734-936-2888; E-mail: saltiel@umich.edu.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.C000856200
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ABBREVIATIONS |
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The abbreviations used are:
GS, glycogen
synthase;
PI3K, phosphatidylinositol 3-kinase;
PP1, type 1 protein
phosphatase;
GSK-3, glycogen synthase kinase-3;
CAP, c-Cbl-associating
protein;
PBS, phosphate-buffered saline;
LacZ, -galactosidase;
PIP3, phosphatidylinositol 1,4,5-trisphosphate;
SH3, Src 3 homology.
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