Action of Insulin Receptor Substrate-3 (IRS-3) and IRS-4 to Stimulate Translocation of GLUT4 in Rat Adipose Cells
Lixin Zhou,
Hui Chen,
Pin Xu,
Li-Na Cong,
Salvatore Sciacchitano,
Yunhua Li,
David Graham,
Aviva R. Jacobs,
Simeon I. Taylor and
Michael J. Quon
Diabetes Branch (L.Z., P.X., S.S., D.G., A.R.J., S.I.T.)
National Institute of Diabetes and Digestive and Kidney Disease and
Hypertension-Endocrine Branch (H.C., L-N.C., Y.L., M.J.Q.) National
Heart, Lung, and Blood Institute National Institutes of Health
Bethesda, Maryland 20892-1754
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ABSTRACT
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The insulin receptor initiates insulin action by
phosphorylating multiple intracellular substrates. Previously, we have
demonstrated that insulin receptor substrates (IRS)-1 and -2 can
mediate insulins action to promote translocation of GLUT4 glucose
transporters to the cell surface in rat adipose cells. Although IRS-1,
-2, and -4 are similar in overall structure, IRS-3 is
50% shorter
and differs with respect to sites of tyrosine phosphorylation.
Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can
also stimulate translocation of GLUT4. Rat adipose cells were
cotransfected with expression vectors for hemagglutinin (HA)
epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human
IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface
GLUT4-HA in cells incubated in the absence of insulin; overexpression
of either IRS-3 or IRS-4 elicited a larger increase in cell surface
GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was
40% greater than the effect of a maximally stimulating
concentration of insulin in cells not overexpressing IRS proteins.
Because phosphatidylinositol (PI) 3-kinase is essential for
insulin-stimulated translocation of GLUT4, we also studied a mutant
IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all
four YXXM motifs (the phosphorylation sites predicted to bind to and
activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did
not promote translocation of GLUT4-HA, but actually inhibited the
ability of insulin to stimulate translocation of GLUT4-HA to the cell
surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating
PI 3-kinase-dependent metabolic actions of insulin in adipose cells,
and that IRS proteins play a physiological role in mediating
translocation of GLUT4.
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INTRODUCTION
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Tyrosine phosphorylation plays a necessary role in mediating the
biological actions of insulin (1, 2, 3, 4, 5). After insulin binds to its
receptor, the activated receptor tyrosine kinase phosphorylates
multiple proteins including insulin receptor substrates (IRS)-1, -2,
-3, and -4 (6, 7, 8, 9, 10), Grb2-associated binder-1 (Gab1) (11), Shc (12), and
pp120/HA4 (13). Phosphotyrosine residues in these substrates bind to
SH2 domains in proteins such as phosphatidylinositol (PI) 3-kinase,
resulting in activation of multiple intracellular signaling pathways.
There is apparent functional redundancy in the system because multiple
proteins share the ability to activate the same signaling pathway
(e.g. activation of PI 3-kinase). Nevertheless, several
observations suggest that each IRS may serve distinct functions in
mediating insulin action. Each substrate has a characteristic pattern
of expression in specific tissues. In addition, each substrate
possesses unique structural features that determine which downstream
signaling pathways will be triggered when the protein becomes
phosphorylated. For example, IRS-3 has a structure that is quite
distinct from the three other members of the IRS family. In contrast to
IRS-1, -2, and -4, IRS-3 is a smaller molecule and has fewer
phosphorylation sites (8, 9).
Although the necessary role of the insulin receptor tyrosine kinase in
insulin action is well established (1, 2, 3, 4, 5), it remains controversial
whether IRS proteins are absolutely required to mediate the metabolic
actions of insulin (14, 15). We have previously demonstrated that both
IRS-1 and IRS-2 are capable of mediating insulins action to stimulate
translocation of GLUT4 glucose transporters from intracellular vesicles
to the plasma membrane in rat adipose cells (16, 17). Furthermore,
expression of an antisense ribozyme directed against IRS-1 resulted in
a 4-fold decrease in the sensitivity of the dose-response curve for
insulin-induced translocation of GLUT4 in adipose cells (16). In
contrast, studies in 3T3-L1 cells have suggested that phosphorylation
of IRS-1 may not be required to mediate insulins action to stimulate
glucose transport (14, 15). However, in knockout mice lacking IRS-1,
adipose cells are partially resistant to the metabolic actions of
insulin, strongly suggesting that IRS-1 indeed participates in this
action of insulin in this physiological target cell (18, 19, 20, 21). Since
adipose cells from mice lacking IRS-1 retain some ability to respond to
insulin, albeit with a less sensitive dose-reponse curve, other
substrates may also contribute to this action of insulin in adipose
cells. When IRS-3 was identified as the major insulin-stimulated
phosphotyrosine-containing protein in adipose cells from mice lacking
IRS-1 (18, 19), this raised the question of whether IRS-3 can mediate
the metabolic actions of insulin.
In the present study, we have demonstrated that overexpression of
either mouse (m)IRS-3 or human (h)IRS-4 (in the presence or absence of
insulin) leads to an increase in the number of GLUT4 molecules
recruited to the surface of rat adipose cells. Furthermore, this
ability of mIRS-3 requires the presence of one or more of the four YXXM
motifs in the molecule; thus, it is likely that activation of PI
3-kinase is required for the action of mIRS-3 to promote translocation
of GLUT4-containing vesicles. Finally, mutant mIRS-3 lacking all four
YXXM motifs inhibits this action of insulin. Since this mutant would be
predicted to compete with endogenous IRS proteins for binding to the
insulin receptor, our data support the hypothesis that phosphorylation
of IRS proteins plays a physiological role in the pathway that mediates
the ability of insulin to promote translocation of GLUT4.
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RESULTS
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Phosphotyrosine-Containing Proteins in Rat Adipose Cells
In freshly isolated rat adipose cells, insulin increased the
phosphotyrosine content of three major bands (data not shown). As
reported previously, these bands correspond to the insulin receptor
(Mr, 95,000), IRS-1 and -2 (Mr, 185,000), and
IRS-3 (Mr, 60,000) (8, 17). Because subcellular
localization of IRS proteins may contribute importantly to signal
specificity (17, 22, 23, 24, 25), we investigated the distribution of these
phosphoproteins between particulate and cytosolic fractions. As
expected, the insulin receptor (which is an integral membrane protein)
was located exclusively in the particulate fraction of the cell.
Similarly, the Mr 185,000 band (corresponding to IRS-1 and
-2) and the Mr 60,000 band (corresponding to IRS-3) were
also located in the particulate fraction, without any detectable
quantities in the cytosolic fraction (data not shown). Therefore, in
subsequent studies, we assayed for IRS proteins only in the particulate
fraction of the cell.
In our transfection studies, we incubated rat adipose cells overnight
after electroporation to allow for expression of the recombinant
proteins. Therefore, we assessed the effect of these experimental
interventions upon the expression of insulin-stimulated
phosphotyrosine-containing proteins. After electroporation and
overnight incubation, we observed a decrease in the intensity of
phosphotyrosine-containing bands (Mr 185,000 and
Mr 130,000) in the particulate fraction of cells incubated
in the absence of insulin (Fig. 1
, lanes
1 and 3). Acute stimulation with insulin (60 nM, 2 min)
increased the phosphotyrosine content of several bands. In cells
incubated overnight, insulin induced an increase in phosphorylation of
the IRS-1/2 band that was comparable to that seen in freshly isolated
cells. In contrast, insulin-stimulated phosphorylation of the insulin
receptor appeared to be increased and phosphorylation of IRS-3 appeared
to be decreased in cells cultured overnight (Fig. 1
, lanes 2 and 4). In
addition, there is a phosphotyrosine containing band at Mr
130,000, the identity of which is not certain. Overnight culture led to
a decrease in the basal phosphorylation of this band, without affecting
the insulin-stimulated level of phosphorylation. Consequently, insulin
appeared to increase phosphorylation of the Mr 130,000
protein in cultured cells, but not in freshly isolated cells.
Furthermore, there is a marked increase in the intensity of a band
corresponding to a Mr 75,000 phosphotyrosine-containing
protein in cultured cells after stimulation with insulin (Fig. 1
, lane
4).

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Figure 1. Tyrosine-Phosphorylated Proteins in Freshly
Isolated and Cultured Adipose Cells
Freshly isolated cells or adipose cells that had undergone transfection
with pCIS2 (empty expression vector) and overnight culture were treated
without or with insulin (60 nM, 2 min). Membrane fractions
were then prepared and 40 µg protein from each group were separated
by SDS-PAGE on a 7.5% gel followed by immunoblotting with the
antiphosphotyrosine antibody 4G10. A representative blot is shown from
an experiment that was repeated independently three times.
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Characterization of Overexpressed Recombinant IRS Proteins in Rat
Adipose Cells
We assessed the ability of recombinant IRS proteins to become
phosphorylated in response to insulin stimulation in transfected
adipose cells. In control cells transfected with the empty expression
vector pCIS2, insulin increased the phosphotyrosine content of bands
corresponding to IRS-1/2 (17), IRS-3 (8), and the insulin receptor
(Fig. 2
, lanes 1 and 2). Overexpression
of each IRS protein in rat adipose cells led to marked increases in the
intensities of bands corresponding to IRS-1, -2, -3, and -4 after
insulin stimulation (Fig. 2
, lanes 4, 6, 8, and 12, respectively).
Under our experimental condition, we obtain expression of recombinant
proteins in approximately 5% of the adipose cells (3). Therefore,
these observations are consistent with high-level overexpression of IRS
proteins in the small fraction of cells that are actually transfected.
In addition, the phosphotyrosine content of the Mr 60,000
band in cells overexpressing mIRS-3 but incubated in the absence of
insulin (Fig. 2
, lane 7) was
2-fold greater than that seen in
control cells incubated in the presence of insulin (Fig. 2
, lane 2). We
also overexpressed a mutant mIRS-3 (denoted IRS-3-F4) in which Phe was
substituted for the four Tyr residues in Tyr-Xaa-Xaa-Met motifs
(Tyr341, Tyr350, Tyr361, and
Tyr390). While IRS-3-F4 underwent insulin-stimulated
tyrosine phosphorylation, the intensity of the band was markedly
reduced in comparison to mIRS-3. Note that the presence of the myc
epitope tag retarded the electrophoretic mobilities of both recombinant
mIRS-3 and IRS-3-F4 in comparison with endogenous rat IRS-3. Thus, in
this experiment it is possible to evaluate the phosphorylation of IRS-3
and IRS-3-F4 independent of endogenous IRS-3. Since IRS-4 is not
expressed endogenously in adipose cells, phosphorylation of
recombinant IRS-4 is also easily appreciated.

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Figure 2. Insulin-Stimulated Phosphorylation of Overexpressed
IRS Proteins in Rat Adipose Cells
Total membrane fractions were isolated from cells transfected (6 µg
DNA/cuvette) with expression vector pCIS2 (lanes 1 and 2), hIRS-1
(lanes 3 and 4), mIRS-2 (lanes 5 and 6), mIRS-3 (lanes 7 and 8),
mIRS-3-F4 (lanes 9 and 10), or hIRS-4 (lanes 11 and 12) after treatment
without or with insulin (60 nM, 2 min). The samples (50
µg protein) were separated by SDS-PAGE on a 7.5% gel and
immunoblotted with antiphosphotyrosine antibody 4G10. The intensity of
the Mr 185,000 band corresponding to phosphorylated IRS-1,
-2, and -4 was increased 2-fold in cells overexpressing IRS-1 (lane 4),
IRS-2 (lane 6), and IRS-4 (lane 12) as compared with the control cells
(lane 2). Relative to the intensity of the phosphorylated
Mr 60,000 band in the control cells (lane 2), transfection
led to a 6-fold increase in the level of IRS-3 in cells overexpressing
mIRS-3 (lane 8). In addition, the level of phosphorylated mIRS-3 in the
absence of insulin (lane 7) was approximately twice the level of
phosphorylated endogenous IRS-3 after insulin stimulation of control
cells (lane 2). Note also that the addition of a myc epitope tag to
both the mIRS-3 and IRS-3-F4 constructs resulted in a slower migration
on the gel so that these recombinant proteins can be distinguished from
the endogenous IRS-3.
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Several lines of evidence demonstrate that activation of PI 3-kinase
mediates insulins effect to stimulate translocation of GLUT4 to the
plasma membrane (19, 22, 23, 26, 27, 28, 29, 30). As a consequence of insulin
binding, insulin receptors phosphorylate Tyr-Xaa-Xaa-Met (YXXM) motifs
on IRS-1, -2, -3, and -4. These phosphorylated YXXM motifs bind to SH2
domains in the p85 regulatory subunit of PI 3-kinase, thereby
activating the p110 catalytic subunit. Therefore, we evaluated the
ability of mIRS-3 to bind to the p85 regulatory subunit of PI 3-kinase.
Recombinant myc-tagged mIRS-3 was expressed in rat adipose cells; the
particulate fraction of the cell was solubilized and subjected to
immunoprecipitation with anti-myc antibody followed by immunoblotting
with anti-p85 antibody. Even in the absence of insulin, there was a
detectable association between mIRS-3 and p85. Stimulation of cells
with insulin led to 4.5-fold increase in the quantity of p85
co-immunoprecipitated with mIRS-3 (Fig. 3
, lane 4). As expected, mutation of the
four Tyr-Xaa-Xaa-Met motifs abolished the association of p85 with
IRS-3-F4 (Fig. 3
, lanes 5 and 6). Comparable levels of p85 were
detected in the particulate fractions, irrespective of whether cells
were transfected with pCIS-2, mIRS-3, or IRS-3-F4 or whether or not the
cells were stimulated with insulin (Fig. 3
, lanes 712). Furthermore,
as judged by immunoblotting with antibody to the myc-epitope tag,
mIRS-3 and mIRS-3F4 were expressed at comparable levels (Fig. 3
, bottom panel, lanes 36).

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Figure 3. Insulin-Stimulated Association between Recombinant
IRS-3 Proteins and PI 3-Kinase
Membrane fractions were obtained from adipose cells transfected with
pCIS2, mIRS-3, or IRS-3-F4 (6 µg DNA/cuvette) after overnight
incubation and treatment without or with insulin (60 nM, 2
min). Aliquots of each sample containing 130 µg protein were
subjected to immunoprecipitation (ippt) with an anti-myc antibody
followed by immunoblotting with either an antibody against the p85
subunit of PI 3-kinase or an antibody against the myc epitope (lanes
16). Aliquots of the same membrane fractions (20 µg protein) were
also immunoblotted with the antibody against p85 without prior
immunoprecipitation to demonstrate that all samples contained
comparable amounts of p85 (lanes 712). Insulin treatment caused a
4.5-fold increase in the amount of p85 coimmunoprecipitated with the
mIRS-3 construct (lanes 3 and 4). In contrast, there was no detectable
association of p85 with IRS-3-F4 either in the absence or presence of
insulin (lanes 5 and 6).
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Effect of Overexpression of mIRS-3 and hIRS-4 on Translocation of
GLUT4
When rat adipose cells were cotransfected with pCIS2 and GLUT4-HA,
insulin treatment (60 nM) caused a 2.5-fold increase in
cell surface GLUT4-HA (Fig. 4
).
Furthermore, as shown previously (16, 17), overexpression of hIRS-1 in
adipose cells stimulated translocation of GLUT4-HA to the cell surface.
In the absence of insulin, recombinant hIRS-1 led to a 2-fold increase
in cell surface GLUT4-HA; in the presence of a maximally effective
concentration of insulin (60 nM), the level of cell surface
GLUT4-HA in cells overexpressing IRS-1 exceeded by 25% the maximal
recruitment observed in insulin-stimulated control cells.
Interestingly, recombinant mIRS-3 exerted a larger effect upon
translocation of GLUT4-HA to the cell surface. In the absence of
insulin, recombinant mIRS-3 led to a 3.5-fold increase in cell surface
GLUT4-HA. Moreover, the level of cell surface GLUT4-HA was greater in
cells overexpressing mIRS-3 (incubated in the absence of insulin) than
was observed in control cells (i.e., not overexpressing
recombinant IRS proteins) that had been incubated in the presence of
insulin. Addition of insulin did not elicit a further increase in the
content of GLUT4-HA on the cell surface. Similarly, even in the absence
of insulin, overexpression of hIRS-4 led to a supramaximal increase in
recruitment of GLUT4-HA to the cell surface (
30% greater than the
stimulation of control cells in response to insulin), which was not
increased further upon addition of insulin (Fig. 5
).

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Figure 4. Effects of Overexpression of hIRS-1 or mIRS-3 on
Translocation of GLUT4 in Rat Adipose Cells
Cells were cotransfected with GLUT4-HA (3 µg/cuvette) and pCIS2
(control), hIRS-1, or mIRS-3 (6 µg/cuvette). We measured the amount
of GLUT4-HA at the cell surface in the basal state or in response to
insulin (0.07 or 60 nM). Data are expressed as a percentage
of cell surface GLUT4-HA in the presence of a maximally effective
insulin concentration for the control group (pCIS2). Results are the
means ± SEM of four independent experiments. The
actual value for the specific cell-associated radioactivity for the
control group at 60 nM insulin was 1620 ± 80 cpm. In
the absence of insulin (basal state), cells overexpressing either
hIRS-1 or mIRS-3 had significantly higher levels of GLUT4-HA at the
cell surface than the corresponding control (P <
0.001). At 60 nM insulin, the level of GLUT4-HA at the
surface of cells overexpressing mIRS-3 was significantly greater than
for cells overexpressing hIRS-1 (P < 0.007). These
studies were performed using an mIRS-3 construct without the myc
epitope tag.
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Figure 5. Effects of Overexpression of hIRS-4 on
Translocation of GLUT4 in Rat Adipose Cells
Cells were cotransfected with GLUT4-HA (3 µg/cuvette) and either
pCIS2 (control), or hIRS-4 (6 µg/cuvette). We measured the amount of
GLUT4-HA at the cell surface in the basal state or in response to
insulin (0.024, 0.072, 0.3, or 60 nM). Data are expressed
as a percentage of cell surface GLUT4-HA in the presence of a maximally
effective insulin concentration for the control group (pCIS2). In the
absence of insulin (basal state), cells overexpressing hIRS-4 had
levels of GLUT4-HA at the cell surface that were significantly higher
than those seen in the control cells stimulated with 60 nM
insulin (P < 0.04). For cells overexpressing
hIRS-4, treatment with insulin did not result in a further increase in
cell surface GLUT4-HA. Results are the means ± SEM of
four independent experiments. By MANOVA, the two dose-response curves
are significantly different (P <
10-9).
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Effect of Overexpression of mIRS-3-F4 on Translocation of GLUT4
To evaluate the role of PI 3-kinase in the action of IRS-3, we
investigated the effect of overexpressing the IRS-3-F4 mutant.
mIRS-3-F4 inhibited insulin-stimulated translocation of GLUT4 in
transfected rat adipose cells (Fig. 6
).
In the absence of insulin, the level of cell surface GLUT4-HA in cells
overexpressing mIRS-3-F4 was approximately 30% lower than that of the
control cells. Furthermore, at an insulin concentration of 0.3
nM, there was no significant increase in cell surface
GLUT4-HA in cells overexpressing mIRS-3-F4, although this concentration
of insulin elicited a near-maximal effect in cells expressing only
pCIS2. Nevertheless, at a maximally effective concentration of insulin
(60 nM), mutant mIRS-3-F4 decreased the content of GLUT4-HA
on the cell surface by only 30% in comparison to control cells. It is
likely that mIRS-3-F4 exerts its inhibitory action by competing with
the ability of the insulin receptor to phosphorylate endogenous IRS-1,
-2, and -3. As a result, mutant mIRS-3-F4 would inhibit activation of
PI 3-kinase, thereby inhibiting insulins action to stimulate
translocation of GLUT4.

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Figure 6. Inhibitory Effect of mIRS-3-F4 on
Insulin-Stimulated Translocation of GLUT4
Cells were cotransfected with GLUT4-HA (3 µg/cuvette) and either
pCIS2 (control) or mIRS-3-F4 (6 µg/cuvette). We measured the amount
of GLUT4-HA at the cell surface in the basal state or in response to
insulin (060 nM). Data are expressed as a percentage of
cell surface GLUT4-HA in the presence of a maximally effective insulin
concentration for the control group (pCIS2). Results are the means
± SEM of four independent experiments. The actual value
for the specific cell-associated radioactivity for the control group at
60 nM insulin was 2280 ± 380 cpm. In the absence of
insulin (basal state), cells overexpressing mIRS-3-F4 had significantly
lower levels of GLUT4-HA at the cell surface than the corresponding
control (P < 0.03). At 60 nM insulin,
the level of GLUT4-HA at the surface of cells overexpressing mIRS-3-F4
was also significantly lower than for control cells
(P < 0.02). By MANOVA, the two dose-response
curves are significantly different (P <
10-9).
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In separate experiments, we verified that overexpression of mIRS-3-WT,
mIRS-3-F4, and hIRS-1 did not significantly affect the level of
expression of GLUT4-HA (Fig. 7
). Thus,
the effects of recombinant IRS proteins are due to their effects upon
the subcellular localization of GLUT4-HA, rather than altering the
level of expression of the GLUT4-HA reporter molecule.

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Figure 7. Expression of Recombinant GLUT4-HA
Cells were cotransfected with GLUT4-HA (3 µg/cuvette) and either
pCIS2 (control) or various expression vectors for IRS proteins (6
µg/cuvette). Triton X-100 extracts of total membrane fractions (100
µg protein) were subjected to SDS-PAGE, followed by immunoblotting
with anti-HA antibody.
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DISCUSSION
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Necessary Role of IRS Proteins in Recruitment of GLUT4
Considerable evidence suggests that IRS proteins play a necessary
role in mediating metabolic actions of insulin such as the recruitment
of GLUT4. For example, absence of IRS-1 in knockout mice leads to
insulin resistance in the whole animal and also in adipose cells
incubated in vitro (18, 19). Furthermore, mice lacking IRS-2
are also insulin resistant although studies of isolated fat cells have
not yet been reported (31). In addition to these studies of the effect
of chronic deficiencies of IRS molecules, we have previously reported
that expression of an antisense ribozyme directed against IRS-1 mRNA in
rat adipose cells led to a rightward shift in the dose-response curve
for insulin action (16). Nevertheless, some reports suggest that IRS
proteins may not be necessary to mediate the effect of insulin to
recruit GLUT4 in 3T3-L1 cultured adipocytes (14, 15). In those reports,
microinjection of various reagents (including anti-IRS-1 antibodies,
isolated phosphotyrosine binding domains, and SAIN domains of IRS-1)
blocked the mitogenic actions of insulin but failed to inhibit
metabolic actions.
Our experiments using overexpression of IRS-3 and -4 directly
demonstate that these substrates are capable of mediating metabolic
actions of insulin such as recruitment of GLUT4. Furthermore, in
contrast to studies in 3T3-L1 cells, our present studies with IRS-3-F4
demonstrated a striking inhibition of insulins ability to promote
translocation of GLUT4. The observation that IRS-3-F4 inhibits the
cells metabolic response to insulin suggests that this mutant
molecule interferes with the endogenous signaling pathway and is
consistent with the hypothesis that IRS molecules play a necessary role
in mediating insulins action to stimulate translocation of GLUT4. It
is likely that mIRS-3-F4 binds to pTyr960 in the
juxtamembrane domain of the insulin receptor and thereby inhibits the
phosphorylation of other substrates containing phosphotyrosine
binding domains (e.g. IRS-1, -2, -3) (32, 33, 34, 35). Thus,
IRS-3-F4 is probably inhibiting signaling mediated by IRS-1 and -2 in
addition to IRS-3. However, our experimental design does not permit us
to test this hypothesis directly. Because only 5% of cells become
transfected under our experimental conditions (3), it is not possible
to quantitate phosphorylation of endogenous IRS proteins in the small
number of transfected cells because this is obscured by the presence of
a much larger number of nontransfected cells. Furthermore, we have
attempted to use various methods to separate transfected from
nontransfected cells. However, because of the fragility of the adipose
cells after electroporation followed by overnight tissue culture, these
efforts have not been successful (M. J. Quon, unpublished
observations). In any case, it is noteworthy that high concentrations
of insulin can partially overcome the inhibition due to mIRS-3-F4. This
might be explained if mIRS-3-F4 is less than 100% effective at
inhibiting phosphorylation of endogenous substrates such as IRS-1, -2,
and -3. However, we cannot completely rule out the possibility that
there may also be IRS-independent pathways that contribute to some
extent to the ability of insulin to promote GLUT4 translocation.
It is not clear why our studies in rat adipose cells reached different
conclusions from those reported in studies of 3T3-L1 adipocytes (14, 15). Both studies employed reagents that are thought to inhibit insulin
action by similar mechanisms (i.e. competitively inhibiting
phosphorylation of endogenous IRS molecules and Shc). It is possible
that the different effects on insulin-stimulated recruitment of GLUT4
might be explained by differences in cell type, differences in the
inhibitory reagents, or other differences in experimental methods
employed. For example, the full-length mIRS-3-F4 might be more
effective than isolated phosphotyrosine binding or SAIN domains at
competitively inhibiting activation of PI 3-kinase.
Relative Roles of IRS Proteins in Rat Adipocytes
Previously, we have presented evidence that both IRS-1 and IRS-2
are capable of mediating insulins action to stimulate glucose
transport in rat adipose cells (16, 17). Studies in mice lacking IRS-1
have demonstrated that IRS-3 is the major phosphotyrosine-containing
protein that binds to PI 3-kinase in insulin-treated adipose cells from
the knockout mice (18, 19). The present study provides direct evidence
that both IRS-3 and IRS-4 can also mediate the action of insulin to
promote translocation of GLUT4 glucose transporters in adipose cells.
Thus, phosphorylation of any of these four substrates by the insulin
receptor tyrosine kinase (i.e. IRS-1, -2, -3, or -4) is
sufficient to trigger translocation of GLUT4 to the cell surface. This
raises the question as to the relative importance of the four IRS
proteins in mediating insulin action in rat adipocytes. Unlike IRS-1,
-2, and -3, which are all endogenously expressed in rat adipocytes,
IRS-4 is not expressed at detectable levels in this cell type (36).
Previously, we (17) and others (37) have estimated that IRS-1
contributes approximately twice as much as IRS-2 to the pathway leading
to the activation of PI 3-kinase. In the present study, the level of
expression of IRS-3 appears roughly comparable to the sum total of
IRS-1 plus IRS-2, as judged by the phosphotyrosine content of these
proteins after insulin stimulation in freshly isolated rat adipocytes
(Fig. 1
, lane 2). However, several considerations dictate caution in
interpreting the results of immunoblotting studies with
antiphosphotyrosine antibodies. For example, the total phosphotyrosine
content does not necessarily reflect the phosphorylation of a specific
tyrosine phosphorylation site. In addition, it is possible that the
amino acid sequence flanking a phosphotyrosine residue may alter that
avidity for binding of antiphosphotyrosine antibodies. Furthermore, the
literature contains conflicting reports with respect to the intrinsic
activities of various IRS proteins to activate PI 3-kinase. Previously,
Smith-Hall et al. (18) suggested that IRS-3 may be more
effective than IRS-1 at activating PI 3-kinase in rat adipose cells.
Specifically, they reported that the time course for the association of
PI 3-kinase with IRS-3 was faster and the extent of the association was
greater than for IRS-1. In contrast, Kaburagi et al. (19)
reached the opposite conclusion that IRS-1 is the more potent activator
in murine adipose cells. In comparing the activities of recombinant
IRS-1 with IRS-3, we noted that recombinant IRS-3 appeared to be more
effective than recombinant IRS-1 in promoting translocation of GLUT4 to
the plasma membrane (Fig. 1
). In fact, the phosphotyrosine content of
mIRS-3 in transfected cells incubated in the absence of insulin
appeared less than the phosphotyrosine content of hIRS-1 in transfected
cells incubated in the presence of insulin. Nevertheless, the
relatively low levels of phosphorylated mIRS-3 in basal cells (Fig. 2
, lane 7) led to a greater increase in the number of GLUT4 molecules on
the cell surface than was induced by the higher levels of
phosphorylated hIRS-1 in insulin-stimulated cells (Fig. 2
, lane 4).
Interestingly, although IRS-4 is not endogenously expressed in adipose
cells, it resembles IRS-3 in that overexpression of either molecule led
to qualitatively similar results. In fact, although the phosphotyrosine
content of hIRS-4 in the absence of insulin (Fig. 2
, lane 11) was lower
than that of mIRS-3 (Fig. 2
, lane 7), overexpression of either IRS-3 or
IRS-4 led to supramaximal translocation of GLUT4. There are many
possible explanations that might account for different potencies of the
various IRS proteins to mediate the metabolic actions of insulin,
e.g. different subcellular localization, activation of
different downstream effector molecules, differential sensitivity to
inactivation by phosphotyrosine phosphatases, etc.
When isolated rat fat cells are cultured for prolonged periods in
vitro, they tend to lose differentiated functions. For example,
there is a progressive decrease in the level of expression of GLUT4
mRNA and protein (38), and a concomitant decrease of the insulin
response. Thus, another observation consistent with the hypothesis that
IRS-3 plays a role in mediating metabolic actions of insulin is the
fact that the level of endogenous IRS-3 decreased after 1 day in
culture. Interestingly, the levels of endogenous IRS-1 and -2 were
constant over the same period of time.
On balance, our data are consistent with the hypothesis that IRS-3 may
be an important IRS molecule mediating the metabolic actions of insulin
in rat adipocytes. However, to draw definitive conclusions about the
relative importance of various IRS molecules, it is necessary to
compare the roles of endogenous IRS-1, -2, and -3 in freshly prepared
adipocytes. Unfortunately, high quality antibodies to rat IRS-3 are not
yet available. If these antibodies were available, it would have been
possible to carry out immunoprecipitation experiments to determine the
relative quantities of PI 3-kinase associated with the various IRS
molecules. Nevertheless, uncertainty would still remain whether
activated PI 3-kinase is equally effective at stimulating translocation
of GLUT4 irrespective of whether PI 3-kinase is bound to IRS-1, -2, or
-3.
In addition to the known IRS molecules, we also detected at least two
other proteins (Mr 130,000 and Mr 75,000) in
cultured adipose cells that underwent tyrosine phosphorylation in
response to insulin (Fig. 1
). The Mr 75,000 phosphoprotein
was not detected in freshly isolated rat adipocytes. In contrast, there
was a constitutively phosphorylated protein band in the Mr
130,000 region of the gel in fresh cells. In the cultured cells, the
phosphotyrosine content of the Mr 130,000 band was
decreased, which facilitated detection of this band upon insulin
stimulation. Although the identities of these insulin-sensitive
phosphoproteins are not known, it is possible that they may play a role
in mediating insulin action in adipose cells.
Downstream from IRS Phosphorylation
In contrast to the apparent redundancy at the level of IRS, the
next step downstream in the metabolic signaling pathway
(i.e. activation of PI 3-kinase) is essential. For example,
when we expressed a mutant p85 that inhibits the activation of PI
3-kinase, the ability of insulin to promote translocation of GLUT4 in
rat adipose cells was essentially abolished (27). Similarly, agents
that inhibit the catalytic activity of PI 3-kinase also lead to
complete inhibition of this action of insulin (28). Our present studies
demonstrating an inhibitory effect of a mutant IRS-3-F4 that does not
associate with the p85 regulatory subunit of PI 3-kinase is also
consistent with this view. That is, since the regulatory subunit is
required for activation of PI 3-kinase, it is reasonable to conclude
that absence of p85 would correlate with absence of associated PI
3-kinase activity. It is generally accepted that activation of PI
3-kinase plays a necessary role in mediating insulin action to
stimulate translocation of GLUT4 to the plasma membrane. In addition,
while there is considerable evidence that activation of PI 3-kinase is
sufficient to promote translocation of GLUT4 (30, 39, 40), activated PI
3-kinase must be located in the correct subcellular compartment to
elicit this response (22, 23, 29, 41). Nevertheless, the cell also
possesses other mechanisms that can stimulate translocation of GLUT4,
even if these mechanisms do not appear to mediate insulins action
upon GLUT4. For example, although activation of Ras is capable of
triggering translocation of GLUT4, inhibitory mutants of Ras do not
block this action of insulin in rat adipose cells (27). Furthermore,
although wortmannin inhibits insulin-stimulated translocation of GLUT4,
wortmannin does not inhibit the effect of activated L61-ras (27). The
fact that mIRS-3 is capable of mediating translocation of GLUT4
provides additional evidence against a role of Ras in this pathway.
Because mIRS-3 does not contain an obvious binding site for the SH2
domain of Grb2 (9), it is unlikely that mIRS-3 would activate the Ras
pathway. Taken together, these data support the hypothesis that
activation of PI 3-kinase is both necessary and sufficient to mediate
insulins ability to stimulate translocation of GLUT4.
General Conclusions
Signals emanating from multiple cellular tyrosine kinases converge
upon an overlapping set of substrates for phosphorylation. Furthermore,
as we have shown in this and other studies (16, 17), the signals
emanating from IRS-1, -2, and -3 converge to activate the same
metabolic action of insulin to promote translocation of GLUT4. It is
likely that the various members of the IRS family of proteins mediate
activation of PI 3-kinase. However, in addition, there are divergent
signals that emanate from the IRS family of proteins resulting in
activation of other signaling pathways, e.g. Ras, SHP-2,
Fyn, etc. Interestingly, the various IRS proteins do not share all of
the same phosphorylation sites so that they are not predicted to
activate precisely the same set of diverging signaling pathways.
Detailed studies of the role of the individual IRS proteins have the
potential to provide insights into the important contributions of both
redundancy and specificity in the complex network of signaling
pathways.
 |
MATERIALS AND METHODS
|
---|
Antibodies
Murine monoclonal antibodies directed against phosphotyrosine
and polyclonal antibodies directed against the p85 subunit of PI
3-kinase were obtained from Upstate Biotechnology, Inc. (Lake Placid,
NY). Monoclonal antibody against influenza HA epitope was obtained from
BAbCO (Berkeley, CA). Polyclonal antibodies against the myc epitope
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Expression Plasmids
In these studies, we used the expression vector pCIS2 (42) to
direct high level expression of various recombinant proteins in
transfected rat adipose cells. Expression vectors for GLUT4-HA (3),
hIRS-1 (16), and mIRS-2 (17) were constructed as previously described.
Murine IRS-3 cDNA (9) was ligated into the HpaI site of
pCIS2 expression vector by blunt-end ligation. IRS-3-F4 is a mutant
form of IRS-3 in which phenylalanines were substituted for the four
tyrosine residues (Tyr341, Tyr350,
Tyr361, and Tyr390) predicted to bind the p85
regulatory subunit of PI 3-kinase. Mutant IRS-3-F4 cDNA was ligated
into pCIS2 expression vector as described above. In addition, a myc
epitope tag was added at the C terminus of both the wild-type mIRS-3
and IRS-3-F4 constructs. Finally, the sequence at the translation start
site was modified to match the Kozak consensus sequence for
transcription start sites (43).
IRS4 was cloned by RT-PCR. Briefly, total RNA was isolated from 293
cells with TRIzol reagent (GIBCO BRL, Gaithersburg, MD). First-strand
cDNA was synthesized from 5 µg of total RNA using Superscript
II/RNase H-/MMLV reverse Transcriptase (GIBCO BRL) and a
gene-specific primer (5'-ccctaacactgtagactgtagcgcatcg-3'). Two
different fragments were generated by PCR. Using pfu DNA
polymerase (Stratagene, La Jolla, CA), we amplified two fragments of
cDNA with the following two pairs of primers:
5'-ggaaaccagtgctctagagatggcc-3' plus
5'-tatgggcccgacctcttttgggagagtcgaac-3'; and
5'-atagaattcgccaccatggcgagttgctccttcac-3' plus
5'-ccctggccatctctagagcactgg-3'. The cDNA encoding epitope-tagged IRS-4
was obtained by joining the two fragments of amplified cDNA and
ligating them into the pCDNA3.1 myc-his A vector. Full-length IRS-4
cDNA was excised from pCDNA3.1 myc-his A vector using EcoRI
and PmeI, blunt-ended, and subcloned into the
HpaI site of the pCIS2 vector. The nucleotide sequence of
IRS-4 cDNA was determined to be identical to the published sequence
(10) with two exceptions: substitution of T and A for C705
and C1203, respectively.
Experiments with Isolated Rat Adipose Cells
Rat adipose cells were isolated from epididymal fat pads of male
rats according to the method of Honnor et al. (44). Isolated
adipose cells were transfected by electroporation as described by Quon
et al. (3, 16, 27) with slight modifications (17).
Monoclonal anti-HA antibody was employed to measure the quantity of
epitope-tagged GLUT4 expressed on the cell surface (3). For some
immunoblotting experiments, freshly isolated adipose cells were treated
without or with insulin (60 nM, 2 min), and whole-cell
lysates were prepared as described (45). Whole-cell lysates were also
fractionated into particulate and cytosolic fractions by centrifugation
at 100,000 x g for 30 min as described (17). For
immunoblotting experiments with transfected cells, the particulate
fraction was isolated from the cells as described (17). Immunoblotting
and immunoprecipitation were performed according to previously
described methods (17, 45). Quantitation of immunoblots was performed
by scanning laser densitometry (Molecular Dynamics, Sunnyvale, CA).
Statistical Analysis
Paired Students t tests were used to compare
individual points where appropriate. Values of P <
0.05 were considered to indicate statistical significance. The
dose-response curves were fit using a nonlinear least squares method
and compared by multiple analysis of variance (MANOVA).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Michael J. Quon, M.D., Ph.D., National Institutes of Health, Building 10, Room 8C-103, 10 Center Drive MSC 1754, Bethesda, Maryland 20892-1754. E-mail:
quonm{at}gwgate.nhlbi.nih.gov
Received for publication July 31, 1998.
Revision received November 6, 1998.
Accepted for publication November 11, 1998.
 |
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