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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 insulin’s 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 {approx}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 {approx}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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 insulin’s 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 insulin’s 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go, 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. 1Go, 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. 1Go, 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.

 
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. 2Go, 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. 2Go, 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. 2Go, lane 7) was {approx}2-fold greater than that seen in control cells incubated in the presence of insulin (Fig. 2Go, 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.

 
Several lines of evidence demonstrate that activation of PI 3-kinase mediates insulin’s 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. 3Go, lane 4). As expected, mutation of the four Tyr-Xaa-Xaa-Met motifs abolished the association of p85 with IRS-3-F4 (Fig. 3Go, 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. 3Go, lanes 7–12). Furthermore, as judged by immunoblotting with antibody to the myc-epitope tag, mIRS-3 and mIRS-3–F4 were expressed at comparable levels (Fig. 3Go, bottom panel, lanes 3–6).



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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 1–6). 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 7–12). 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).

 
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. 4Go). 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. 5Go).



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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).

 
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. 6Go). 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 insulin’s 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 (0–60 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 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).

 
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. 7Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 insulin’s 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 insulin’s 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 insulin’s 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. 1Go, 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. 1Go). 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. 2Go, 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. 2Go, 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. 2Go, lane 11) was lower than that of mIRS-3 (Fig. 2Go, 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. 1Go). 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 insulin’s 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 insulin’s 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Student’s 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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