Calmodulin Antagonists Inhibit Insulin-Stimulated GLUT4 (Glucose Transporter 4) Translocation by Preventing the Formation of Phosphatidylinositol 3,4,5-Trisphosphate in 3T3L1 Adipocytes

Chunmei Yang, Robert T. Watson, Jeffrey S. Elmendorf, David B. Sacks and Jeffrey E. Pessin

Department of Physiology and Biophysics (C.Y., R.T.W., J.S.E., J.E.P.) The University of Iowa Iowa City, Iowa 52242
Department of Pathology (D.B.S.) Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts 02115


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It has been previously reported that calmodulin plays a regulatory role in the insulin stimulation of glucose transport. To examine the basis for this observation, we examined the effect of a panel of calmodulin antagonists that demonstrated a specific inhibition of insulin-stimulated glucose transporter 4 (GLUT4) but not insulin- or platelet-derived growth factor (PDGF)-stimulated GLUT1 translocation in 3T3L1 adipocytes. These treatments had no effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, IRS1 or phosphotyrosine antibody immunoprecipitation of phosphatidylinositol (PI) 3-kinase activity was not affected. Despite the marked insulin and PDGF stimulation of PI 3-kinase activity, there was a near complete inhibition of protein kinase B activation. Using a fusion protein of the Grp1 pleckstrin homology (PH) domain with the enhanced green fluorescent protein, we found that the calmodulin antagonists prevented the insulin stimulation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] formation in vivo. Similarly, although PDGF stimulation increased PI 3-kinase activity in in vitro immunoprecipitation assays, there was also no significant formation of PI(3,4,5)P3 in vivo. These data demonstrate that calmodulin antagonists prevent insulin-stimulated GLUT4 translocation by inhibiting the in vivo production of PI(3,4,5)P3 without directly affecting IRS1- or phosphotyrosine-associated PI 3-kinase activity. This phenomenon is similar to that observed for the PDGF stimulation of 3T3L1 adipocytes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It is well established that insulin stimulation results in increased glucose uptake in adipose and muscle cells primarily from the recruitment of the glucose transporter 4 (GLUT4) protein to the cell surface (1, 2, 3, 4, 5, 6). This event is triggered by the initial activation and autophosphorylation of the insulin receptor tyrosine-specific protein kinase and subsequent tyrosine phosphorylation of downstream effectors, most notably insulin receptor substrates 1 and 2 (IRS1 and IRS2) (7, 8, 9). The tyrosine phosphorylation of IRS1/2 provides docking sites for the SH2 domains of several effectors and in particular, the p85 regulatory subunit of the phosphatidylinositol 3-kinase (PI 3-kinase) (9). Although numerous studies have clearly demonstrated that PI 3-kinase function is necessary for insulin-stimulated GLUT4 translocation, the identification of the subsequent targets remains controversial with evidence both for and against the involvement of protein kinase B (PKB/Akt) and the atypical protein kinase C (PKC) isoforms {zeta} and {lambda} (10, 11, 12, 13, 14, 15, 16).

Recently, several studies have implicated a calmodulin-dependent step in regulated exocytosis of synaptic vesicles in neurons and in vacuole fusion in yeast (17, 18, 19). Although it is generally believed that there is no specific regulatory role for calcium in the metabolic actions of insulin, it has been reported that calmodulin antagonists inhibit insulin-stimulated glucose transport activity in adipocytes and skeletal muscle (20, 21, 22, 23). Furthermore, calmodulin is capable of being phosphorylated by the insulin receptor both in vitro and in vivo and was found to directly interact with IRS1 and the PI 3-kinase in vitro (24, 25, 26). However, the signaling steps and potential mechanisms by which calmodulin antagonists inhibit insulin-stimulated glucose uptake have not been investigated. In this manuscript, we demonstrate that a panel of calmodulin antagonists specifically inhibit insulin-stimulated GLUT4 but not GLUT1 translocation. This apparently occurs through an inhibition of PI(3, 4, 5)P3 formation in vivo but does not result from the inhibition of PI 3-kinase activity as determined in phosphotyrosine and IRS1 immunocomplex assays in vitro. The effect of these antagonists is similar to that of platelet-derived growth factor (PDGF) stimulation, which also appears to have no effect on PI 3-kinase activity when analyzed in immunoprecipitates but which is also unable to induce the formation of PI(3, 4, 5)P3 in vivo.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Calmodulin Antagonists Inhibit Insulin-Stimulated GLUT4 Translocation
Previous studies have observed that pretreatment of 3T3L1 adipocytes with calmodulin antagonists inhibited insulin-stimulated glucose transport activity (20). To determine whether this was due to the effect of the inhibitors on GLUT4 translocation, 3T3L1 adipocytes were pretreated with a panel of calmodulin inhibitors (Fig. 1Go). As typically observed, insulin stimulation resulted in the translocation of GLUT4 to the cell surface membrane as detected by increased GLUT4 immunofluorescence in isolated plasma membrane sheets (Fig. 1Go, A and B, panels 1 and 8). Preincubation of the 3T3L1 adipocytes with trifluoperazine (TFP) had little effect on the basal state level of plasma membrane-localized GLUT4 but markedly inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner (Fig. 1AGo, panels 2, 3, 9, and 10). To further examine the specificity for calmodulin in this process, we next examined the effect of the highly selective inhibitor W13. Pretreatment with W13 also resulted in an inhibition of insulin-stimulated GLUT4 translocation (Fig. 1AGo, panels 6, 7, 13, and 14). In contrast, the less effective structural analog W12 had only a marginal inhibition of insulin-stimulated GLUT4 translocation (Fig. 1AGo, panels 4, 5, 11, and 12). Similarly, the calmodulin-specific antagonists Orphiobolin A (Orph) and W7 were both potent inhibitors of GLUT4 translocation whereas W5, the less effective structural analog of W7, had essentially no effect (Fig. 1BGo, panels 1–14).



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Figure 1. Calmodulin Antagonists Inhibit Insulin-Stimulated GLUT4 Translocation

A, 3T3L1 adipocytes were left untreated (panels 1 and 8) or pretreated for 20 min with either 40 µM or 70 µM trifluoperazine (TFP; panels 2, 3, 9, and 10), W12 (panels 4, 5, 11, and 12) and W13 (panels 6, 7, 13, and 14). B, 3T3L1 adipocytes were left untreated (panels 1 and 8) or pretreated for 20 min with either 25 µM or 50 µM Orphiobolin A (Orph; panels 2, 3, 9, and 10), 40 µM or 70 µM W5 (panels 4, 5, 11, and 12) and W7 (panels 6, 7, 13, and 14). The cells were then incubated in the absence (panels 1–7) or presence (panels 8–14) of 100 nM insulin for 30 min at 37 C. Plasma membrane sheets were prepared and processed for GLUT4 immunofluorescence as described in Materials and Methods. These are representative fields of plasma membrane sheets obtained from five independent experiments.

 
Calmodulin Antagonists Have No Effect on Insulin- or PDGF-Stimulated GLUT1 Translocation
In addition to GLUT4, 3T3L1 adipocytes express the GLUT1 glucose transporter isoform, which also displays insulin-stimulated translocation to the plasma membrane (27). As expected, insulin stimulation resulted in an increased amount of GLUT1 protein at the plasma membrane (Fig. 2Go, panels 1 and 8). Pretreatment of 3T3L1 adipocytes with trifluoperazine had no effect on the amount of basal or insulin-stimulated plasma membrane-associated GLUT1 protein (Fig. 2Go, panels 2 and 9). Similarly, none of the other calmodulin antagonists or control structural analogs had any effect on either the basal or insulin-stimulated translocation of GLUT1 (Fig. 2Go, panels 3–7, 10–14).



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Figure 2. Calmodulin Antagonists Do Not Inhibit Insulin-Stimulated GLUT1 Translocation

3T3L1 adipocytes were left untreated (panels 1 and 8) or pretreated for 20 min with 70 µM trifluoperazine (TFP; panels 2 and 9), 70 µM W12 (panels 3 and 10), 70 µM W13 (panels 4 and 11), 50 µM Orphiobolin A (Orph; panels 5 and 12), 70 µM W5 (panels 6 and 13) and 70 µM W7 (panels 7 and 14). The cells were then incubated in the absence (panels 1–7) or presence (panels 8–14) of 100 nM insulin for 30 min at 37 C. Plasma membrane sheets were prepared and processed for GLUT1 immunofluorescence as described in Materials and Methods. These are representative fields of plasma membrane sheets obtained from four independent experiments.

 
To examine the specificity of the calmodulin antagonists on the signaling events elicited by another growth factor receptor, we examined the ability of the PDGF tyrosine kinase receptor to induce GLUT4 and GLUT1 translocation (Fig. 3Go). Previously, several studies have observed that PDGF and/or epidermal growth factor stimulation can result in GLUT4 translocation while other studies have reported the opposite, i.e. PDGF does not induce GLUT4 translocation in 3T3L1 adipocytes (28, 29, 30). Although controversial, we have also observed that PDGF stimulation does not induce the translocation of GLUT4 whereas in the same population of cells insulin was an effective stimulator (Fig. 3AGo, panels 1–3). As previously observed, pretreatment of the cells with W13 prevented the insulin stimulation of GLUT4 translocation (Fig. 3AGo, panels 4–6). However, both insulin and PDGF were capable of inducing GLUT1 translocation (Fig. 3BGo, panels 1–3). Furthermore, W13 pretreatment was unable to inhibit either the insulin or PDGF stimulation of GLUT1 translocation (Fig. 3BGo, panels 4–6). Similar results were also obtained with W7 and trifluoperazine treatments (data not shown). Together, these data demonstrate that these calmodulin antagonists are effective inhibitors of insulin-stimulated GLUT4 translocation but do not block general exocytosis, at least for those pathways mediating GLUT1 translocation.



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Figure 3. Calmodulin Antagonist Inhibit Insulin-Stimulated GLUT4 Translocation but Not Insulin- or PDGF-Stimulated GLUT1 Translocation

3T3L1 adipocytes were either untreated (panels 1–3) or pretreated for 20 min with 70 µM W13 (panels 4–6). The cells then incubated in the absence (panels 1 and 4) or in the presence of 100 nM insulin (panels 2 and 5) or 3 nM PDGF (panels 3 and 6) for 30 min at 37 C. Plasma membrane sheets were then prepared and processed for either GLUT4 (A) or GLUT1 (B) immunofluorescence as described in Materials and Methods. These are representative fields of plasma membrane sheets obtained from four independent experiments.

 
Insulin Receptor Autophosphorylation and IRS1 Tyrosine Phosphorylation Are Not Affected by Calmodulin Antagonists
To investigate the insulin-mediated signaling step(s) inhibited by the calmodulin antagonists, we first examined both insulin receptor autophosphorylation and IRS1 tyrosine phosphorylation by phosphotyrosine immunoblotting (Fig. 4Go). As typically observed, insulin stimulation resulted in the tyrosine phosphorylation of the approximately 185-kDa IRS1 protein and the approximately 95-kDa insulin receptor ß-subunit (Fig. 4Go, lanes 1 and 2). Pretreatment of 3T3L1 adipocytes with the nonfunctional structural analog W12 had no effect on the insulin stimulation of either the insulin receptor or IRS1 tyrosine phosphorylation (Fig. 4Go, lanes 3 and 4). Similarly, pretreatment with the specific calmodulin antagonist, W13, also did not affect the insulin stimulation of insulin receptor or IRS1 tyrosine phosphorylation (Fig. 4Go, lanes 5 and 6). Identical results were obtained when we examined the effects of trifluoperazine, W5, and W7 (data not shown).



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Figure 4. Calmodulin Antagonists Do Not Affect Insulin Receptor Autophosphorylation or IRS1 Tyrosine Phosphorylation

3T3L1 adipocytes were either untreated (lanes 1 and 2) or pretreated for 20 min with 70 µM W12 (lanes 3 and 4) or 70 µM W13 (lanes 5 and 6). The cells were then incubated in the absence (lanes 1, 3, and 5) or in the presence of 100 nM insulin (lanes 2, 4, and 6) for 5 min at 37 C. Whole-cell detergent lysates were generated and immunoblotted using the PY20 phosphotyrosine antibody as decribed in Materials and Methods. This is a representative immunoblot from five independent experiments.

 
Immunoprecipitation of IRS Protein-Associated PI 3-Kinase Activity Is Not Affected by Calmodulin Antagonists
Previous studies have established an essential requirement for PI 3-kinase activity for insulin-stimulated GLUT4 translocation (10, 11). In addition, the majority of insulin-stimulated PI 3-kinase is associated with the IRS proteins (31). As typically observed, there was a marked increase in the amount of IRS1-immunoprecipitated PI 3-kinase activity after insulin stimulation (Fig. 5AGo, lanes 1 and 2). Neither W12 nor W13 pretreatment had any effect on the subsequent insulin stimulation of PI 3-kinase in the IRS1 immunoprecipitates (Fig. 5AGo, lanes 3 and 4). Since the PDGF receptor does not tyrosine phosphorylate the IRS proteins but instead directly associates with the PI 3-kinase after activation, we examined PI 3-kinase activity in phosphotyrosine immunoprecipitates (Fig. 5BGo). As expected, both insulin and PDGF treatment resulted in a strong increase in the amount of PI 3-kinase activity associated with tyrosine-phosphorylated proteins (Fig. 5BGo, lanes 1–3). Consistent with the IRS1 immunoprecipitation results, W12 and W13 had no effect on the insulin stimulation of PI 3-kinase activity in the phosphotyrosine immunoprecipitates (Fig. 5BGo, lanes 4 and 5). Similarly, trifluoperazine, W5, and W7 were also ineffective in inhibiting insulin-stimulated PI 3-kinase activity in phosphotyrosine immunoprecipitates (data not shown). Together, these data demonstrated that the calmodulin antagonists do not inhibit the insulin-stimulated association of the PI 3-kinase with IRS proteins. In addition, although PDGF does not stimulate GLUT4 translocation, it is perfectly capable of inducing the association of PI 3-kinase activity with the PDGF receptor.



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Figure 5. Calmodulin Antagonists Have No Effect on Insulin-Stimulated Immunoprecipitable PI 3-Kinase Activity

A, 3T3L1 adipocytes were either untreated (lanes 1 and 2) or pretreated for 20 min with 70 µM W12 (lane 3) or 70 µM W13 (lane 4). The cells were then incubated in the absence (lane 1) or in the presence of 100 nM insulin (lanes 2–4) for 5 min at 37 C. Whole cell detergent lysates were prepared, immunoprecipitated with the IRS1 antibody and assayed for the presence of PI 3-kinase activity as described under Materials and Methods. B, 3T3L1 adipocytes were either untreated (lanes 1–3) or pretreated for 20 min with 70 µM W12 (lane 4) or 70 µM W13 (lane 5). The cells were then incubated in the absence (lane 1) or in the presence of 3 nM PDGF (lane 2) or 100 nM insulin (lanes 3–5) for 5 min at 37 C. Whole-cell detergent lysates were prepared, immunoprecipitated with the PT66 phosphotyrosine antibody, and assayed for the presence of PI 3-kinase activity as described in Materials and Methods. IP, Immunoprecipitation; PIP, phosphatidylinositol phosphate; Ori, origin; C, control; I, insulin; P, PDGF. These are representative results from four independent experiments.

 
Calmodulin Antagonists Inhibit Insulin Stimulation of PKB Activation
One major downstream target of the PI 3-kinase is the serine/threonine kinase PKB (13). This kinase becomes activated upon the binding of the PKB PH domain to PI(3, 4, 5)P3 and by the subsequent phosphorylation on serine 473 and threonine 308 by the phosphoinositide-dependent protein kinases, PDK1 and PDK2 (13, 32, 33). We therefore next examined the phosphorylation of PKB to assess PDK activation and production of PI(3, 4, 5)P3 in vivo (Fig. 6Go). Insulin stimulation resulted in a marked increase in serine 473 phosphorylation compared with the control unstimulated cells (Fig. 6AGo, lanes 1 and 3). Pretreatment with the noneffective calmodulin antagonist W12 did not affect the subsequent insulin stimulation of PKB serine 473 phosphorylation (Fig. 6AGo, lanes 4 and 5). However, W13 markedly blunted the insulin-stimulated PKB serine 473 phosphorylation (Fig. 6AGo, lanes 6 and 7). The insulin stimulation of serine 473 phosphorylation in the presence of W13 was similar to the weak phosphorylation induced by PDGF treatment (Fig. 6AGo, lane 2). Similarly, the insulin-stimulated phosphorylation of threonine 308 was completely inhibited by pretreatment with W13 but was unchanged in the presence of W12 (Fig. 6BGo, lanes 4–7). Furthermore, PDGF was also completely ineffective in stimulating PKB threonine 308 phosphorylation (Fig. 6BGo, lanes 1–3).



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Figure 6. Calmodulin Antagonists Inhibit Insulin-Stimulated Phosphorylation of PKB

A, 3T3L1 adipocytes were either untreated (lanes 1–3) or pretreated for 20 min with 70 µM W12 (lanes 4 and 5) or 70 µM W13 (lanes 6 and 7). The cells were then incubated in the absence (lanes 1, 4, and 6) or in the presence of 3 nM PDGF (lane 2) or 100 nM insulin (lanes 3, 5, and 7) for 5 min at 37 C. Whole-cell detergent lysates were prepared and immunoblotted with the phosphoserine 473-specific (A) and phosphothreonine 308-specific (B) PKB antibodies. These are representative immunoblots from five independent experiments.

 
Insulin-Stimulated PI(3, 4, 5)P3 Formation in Vivo Is Inhibited by Calmodulin Antagonists
Since PKB is an established target of PI 3-kinase activation, these data suggest that either there was an inhibition of PDK1/2 activities and/or a block of PI(3, 4, 5)P3 formation in vivo. Previous studies have demonstrated that the pleckstrin homology (PH) domain of Grp1 has a high degree of specificity and affinity for PI(3, 4, 5)P3 (34, 35). Therefore, to assess the in vivo production of PI(3, 4, 5)P3, we took advantage of this property and generated a fusion protein consisting of the enhanced green fluorescent protein (EGFP) fused to the PH domain of Grp1 (EGFP-PH/Grp1). In the absence of insulin, expression of EGFP-PH/Grp1 resulted in its predominant localization into the nucleus with a smaller amount distributed throughout the cell cytoplasm (Fig. 7AGo, panel 1). The accumulation of the EGFP-PH/Grp1 fusion protein in the nucleus is a property of EGFP in 3T3L1 adipocytes as expression of just EGFP itself also results in a predominant nuclear localization (data not shown). In any case, insulin stimulation resulted in the accumulation of the EGFP-PH/Grp1 fusion protein at the cell surface membrane indicative of PI(3, 4, 5)P3 formation at the plasma membrane (Fig. 7AGo, panel 3). In contrast, PDGF stimulation was unable to induce a significant increase in PI(3, 4, 5)P3, at least as detected by the EGFP-PH/Grp1 fusion protein (Fig. 7AGo, panel 2). Although insulin was fully capable of stimulating the plasma membrane accumulation of PI(3, 4, 5)P3 in the presence of W12, this was substantially inhibited by the specific calmodulin antagonist W13 (Fig. 7AGo, panels 4 and 5). In addition, the apparent extent of plasma membrane fluorescence was substantially reduced even in those cells that still displayed a translocation of the EGFP-PH/Grp1 fusion protein.



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Figure 7. Calmodulin Antagonists Inhibit Insulin-Stimulated Formation of PI(3 4 5 )P3 in Intact 3T3L1 Adipocytes

A, 3T3L1 adipocytes were transfected with the EGFP-PH/Grp1 cDNA. After 24 h, the cells were either untreated (panels 1–3) or pretreated for 20 min with 70 µM W12 (panel 4) or 70 µM W13 (panel 5). The cells were then incubated in the absence (panel 1) or in the presence of 3 nM PDGF (panel 2) or 100 nM insulin (panels 3–5) for 30 min at 37 C. The cells were then fixed in 2% paraformaldehyde and visualized by confocal fluorescence microscopy as described in Materials and Methods. B, Quantitation of the number of cells displaying EGFP-PH/Grp1 cell surface fluorescence was determined from counting of 50 cells from 3 independent experiments. Each bar represents the average number of cells displaying cell surface fluorescence ±SD.

 
Quantitation of these data demonstrated that in the basal state approximately 18% of the transfected 3T3L1 adipocyte cell population displayed a cell surface EGFP-PH/Grp1 fluorescence that was not significantly different after PDGF stimulation (Fig. 7BGo). In contrast, insulin stimulation resulted in greater than 80% of the cells with a strong cell surface fluorescence. Although W12 pretreatment resulted in a small decrease in the number of cells displaying an insulin-stimulated plasma membrane fluorescence (60%), this was reduced to less than 40% by preincubation of the cells with W13. Thus, these data demonstrate that the calmodulin antagonists do not directly inhibit PI 3-kinase activity but instead prevent the in vivo formation of PI(3, 4, 5)P3, thereby accounting, at least in part, for the lack of insulin-stimulated PKB activation and GLUT4 translocation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It is well established that insulin stimulation results in increased glucose uptake in striated muscle and adipose tissue through a mechanism that requires the translocation of intracellular compartmentalized GLUT4 protein to the plasma membrane (1, 2, 3, 4, 5, 6). However, the signal transduction pathway(s) responsible for this event is poorly understood. At present, only two signaling proteins are universally accepted as essential for this process, the insulin receptor itself and the Type 1 PI 3-kinase (7, 10, 11). In attempts to identify additional regulatory proteins involved in this process, several laboratories have examined the effect of various inhibitors of signaling pathways. In this regard, it has recently been reported that calmodulin directly interacts with the PI 3-kinase and can increase PI 3-kinase activity in vitro (26). Although numerous studies have established that insulin does not regulate intracellular calcium concentrations, several studies have suggested a potential role for calmodulin in insulin action (20, 21, 22, 23, 24). For example, insulin has been reported to induce the tyrosine phosphorylation of calmodulin (24). In addition, treatment of both adipocytes and muscle with calmodulin antagonists inhibited insulin-stimulated glucose transport activity (20, 21, 22, 23). More recently, a role for calmodulin in membrane trafficking events has been suggested, including vacuole fusion in yeast, exocytosis of synaptic vesicles, endocytosis of the serotonin 5-HT1A receptor, and the recycling of the transferrin receptor (17, 18, 19, 36, 37). Based upon these data, we examined the effect of a series of calmodulin antagonists on the GLUT4 and GLUT1 vesicle translocation process. Our data demonstrate that specific calmodulin antagonists abrogate insulin-stimulated GLUT4 translocation. However, it is important to recognize that although effects of these agents have the appropriate dose and specificity as calmodulin antagonists, they do not directly prove the involvement of calmodulin per se. Nevertheless, the effect of these antagonists is relatively specific for GLUT4 trafficking as they had no effect on insulin or PDGF stimulation of GLUT1 translocation. Thus, whether these agents are functioning through calmodulin or an as yet unidentified effector protein, they appear to specifically block an essential step in the GLUT4 translocation process.

To address this issue, we have determined that these calmodulin antagonists have no significant effect on insulin receptor autophosphorylation or tyrosine phosphorylation of IRS1. In contrast, these agents prevented the activation of PKB by inhibiting its insulin-stimulated serine/threonine phosphorylation. This is consistent with a necessary role of calmodulin in the serum stimulation of PKB activity in neuroblastoma cells (38). Surprisingly, however, despite the inhibition of insulin-stimulated PKB phosphorylation, there was no effect on the association of the PI 3-kinase with the IRS proteins or on the catalytic activity of the coimmunoprecipitated PI 3-kinase. This phenomenon was similar to that observed for PDGF stimulation of 3T3L1 adipocytes, which resulted in the in vitro induction of PI 3-kinase without any significant effect on PKB phosphorylation.

To further examine this discrepancy, we took advantage of the recently documented selectivity of specific PH domains for PI(3, 4, 5)P3 (34, 35, 39, 40, 41, 42). In this assay system, EGFP-PH fusion proteins have been successfully used to monitor the in vivo formation of PI(3, 4, 5)P3 (35, 39, 41, 42). In particular the PH domain of Grp1 has demonstrated that insulin stimulation results in the predominant production of PI(3, 4, 5)P3 at the plasma membrane of 3T3L1 adipocytes (35). Thus, based upon the selective affinity of the Grp1 PH domain for PI(3, 4, 5)P3, we have observed that the calmodulin antagonists prevented a significant increase in PI(3, 4, 5)P3 formation in vivo. Similarly, PDGF stimulation also was apparently ineffective in inducing the formation of PI(3, 4, 5)P3 at the plasma membrane. Although it remains possible that EGFP-PH/Grp1 reporter system is unable to detect specific PI(3, 4, 5)P3 subcompartments and/or displays affinity for an as yet identified product, the simplest interpretation of these data is that the calmodulin antagonists and PDGF stimulation uncouples PI 3-kinase activity from the steady-state accumulation of PI(3, 4, 5)P3 in vivo.

There are two possible mechanisms that can account for these findings. First, the calmodulin antagonists could induce the activation of a phosphatidylinositol phosphate phosphatase such as SHIP, thereby rapidly reducing any increase in PI(3, 4, 5)P3. Although formally possible, this is highly unlikely as PDGF would also have to sufficiently activate this phosphatase to prevent any measurable increase in PI(3, 4, 5)P3 formation. Alternatively, the calmodulin antagonists could alter the subcellular targeting of the PI 3-kinase, making it inaccessible to its substrate, PI(4, 5)P2. We favor this hypothesis as calmodulin appears to be responsible for the appropriate intracellular targeting of a number of molecules including the localization of p21Cip1 to the nucleus, calcium-calmodulin-dependent protein kinase II to postsynaptic densities, and Rad to the cell cytoskeleton (43, 44, 45). In this regard, PDGF recruits the PI 3-kinase to the PDGF receptor, whereas insulin targets the PI 3-kinase to the IRS proteins (9, 46, 47). This difference in PI 3-kinase targeting may reflect its signaling function in vivo without any intrinsic change in catalytic activity. In support of this model, it has been observed that the insulin-stimulated tyrosine phosphorylated IRS1 protein becomes localized to a low-density microsome cytoskeleton-enriched fraction (48). Similarly, insulin has also been reported to induce the redistribution of the PI 3-kinase to a similar low-density microsome fraction (48, 49). This is in marked contrast to PDGF, which targets the PI 3-kinase directly to the plasma membrane (48). Thus, we speculate that PDGF receptor activation in 3T3L1 adipocytes sequesters the PI 3-kinase into a subcellular compartment that is substrate inaccessible. Similarly, treatment of these cells with the calmodulin antagonists could prevent the appropriate subdomain targeting of the PI 3-kinase, thereby preventing the formation of PI(3, 4, 5)P3.

In any case, our data demonstrate that calmodulin antagonists specifically prevent insulin-stimulated GLUT4 translocation by inhibiting the in vivo formation of PI(3, 4, 5)P3. Similarly, the inability of PDGF to stimulate PI(3, 4, 5)P3 production also accounts for its ineffectiveness to induce GLUT4 translocation. At present the mechanism(s) that apparently uncouples PI 3-kinase activity as determined by in vitro kinase assays from PI(3, 4, 5)P3 production in vivo is an important issue that may provide the basis for receptor signaling specificity in adipocytes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Trifluoperazine, W12, W13, W5, W7, and the phosphotyrosine antibody (PT66) were purchased from Sigma(St. Louis, MO). Orphiobolin A was obtained from Calbiochem (La Jolla, CA), and the rabbit polyclonal antibody to GLUT1 was a generous gift from Dr. Michael Mueckler (Washington University, St. Louis, MO.). The GLUT4 antibody (IA02) was isolated as described previously (50). The phosphotyrosine (PY20H) and IRS1 antibodies were purchased from Transduction Laboratories, Inc. (Lexington, KY) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. The phosphoserine- and threonine-specific PKB antibodies were from New England Biolabs, Inc. (Beverly, MA). Lissamine rhodamine-conjugated donkey antirabbit IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), and Vectashield was obtained from Vector Laboratories, Inc. (Burlingame, CA). The EGFP-PH/Grp1 plasmid was generated by cloning the PH domain of Grp1 (kindly provided by Dr. Michael Czech, University of Massachusetts Medical Center) into the HindIII and BamHI sites of pEGFP-C2 vector (CLONTECH Laboratories, Inc., Palo Alto, CA).

Cell Culture
3T3L1 adipocytes (American Type Culture Collection, Manassas, VA) were cultured in DMEM containing 25 mM glucose and 10% calf serum at 37 C in an 8% CO2 atmosphere. At confluence, cells were differentiated by incubation in medium containing 25 mM glucose, 10% FBS, 1 µg/ml insulin, 1 mM dexamethasone, and 0.5 mM isobutyl-1-methylxanthine. After 4 days the medium was changed to DMEM, 25 mM glucose, and 10% FBS. Cells were routinely used at 10–12 days post differentiation. Before use, cells were washed two times with PBS and serum-starved in DMEM with 0.1% BSA for at least 2 h.

Plasma Membrane Sheet Assay and Confocal Microscopy
Preparation of plasma membrane sheets from 3T3L1 adipocytes was performed by the method of Robinson et al. (51). Briefly, after growth factor treatment, cells were washed in ice-cold PBS and incubated for 30 sec in ice-cold 0.5 mg/ml poly-L-lysine in PBS. The cells were then swollen in 1/3x KHMgE buffer (1x concentration, 70 mM KCl, 30 mM HEPES, pH 7.5, 5 mM MgCl2, 3 mM EGTA) by three rinses. The swollen cells were placed in 1x KHMgE buffer with 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride and sonicated for 2 sec using a microtip at setting 4.8 on a 550 Sonic Dismembrator (Fisher Scientific, Hampton, NH). The bound membrane sheets were fixed for 20 min in 2% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA), quenched in 100 mM glycine/PBS for 15 min at room temperature, and washed three times in PBS. The sheets were then blocked for 30 min at room temperature in 5% donkey serum/PBS and incubated for 1 h with either a 1:100 dilution of GLUT4 antibody or 1:500 dilution of GLUT1 antibody. The sheets were then washed three times with PBS and incubated for 1 h with a 1:100 dilution of the lissamine rhodamine-conjugated donkey antirabbit antibody. After incubation with the secondary antibody, the sheets were washed three more times in PBS, coverslipped with Vectashield, and viewed on a Bio-Rad Laboratories, Inc. (Richmond, CA) laser confocal microscope.

Immunoblotting
3T3L1 cell lysates were prepared from six-well dishes of adipocytes that had been treated with calmodulin inhibitors and growth factors or left untreated. Cells were washed twice with ice-cold PBS, scraped into lysis buffer (25 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EGTA, 1% NP40, 50 mM NaF, 2 mM Na4P2O7, 1 mM Na3VO4, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride), and incubated with rotation for 20 min at 4 C. Insoluble material was removed by microcentrifugation for 10 min at 4 C. The lysates were then subjected to reducing SDS-PAGE (8% acrylamide) and transferred to polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA). The membranes were immunoblotted with either the phosphotyrosine antibody or the PKB antibodies.

Immunoprecipitation and PI 3-Kinase Assay
Whole-cell detergent lysates were immunoprecipitated for 2 h at 4 C with either a phosphotyrosine antibody conjugated to agarose (PT-66) or the IRS1 antibody followed by 1 h incubation with protein A+-agarose. The immunoprecipitated lipid kinase activity was determined as described by Turinsky et al. (52). Briefly, the immunoprecipitates were incubated with 40 µCi of [{gamma}-32P]ATP plus 20 µg of phosphatidylinositol (Avanti Polar Lipids, Birmingham, AL) for 15 min at room temperature. The radiolabeled phospholipid product was spotted onto silica plates (Analtech, Newark, DE), subjected to TLC, and visualized by autoradiography.

Transient Transfection
Differentiated 3T3L1 adipocytes were transiently transfected by a modification of the electroporation method described previously (53). Briefly, fully differentiated adipocytes were electroporated (0.16 kV and 950 microfarads) with 50 µg of the EGFP-PH/Grp1 plasmid DNA per cuvette. After electroporation, the adipocytes were replated on collagen-coated tissue culture plates and allowed to recover for 24 h before use.


    ACKNOWLEDGMENTS
 
We wish to thank Dr. Kenneth Coker and Diana Boeglin for their assistance in this study.


    FOOTNOTES
 
Address requests for reprints to: Jeffrey E. Pessin, Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242-1109.

This work was supported by research grants DK-33823, DK-49871, and DK-25295 from the NIH (J.E.P.) and a grant from the American Diabetes Association (D.B.S.). J.S.E. was the recipient of Postdoctoral Fellowship Training Grant 398234 from the Juvenile Diabetes Foundation.

Received for publication October 19, 1999. Revision received November 23, 1999. Accepted for publication December 1, 1999.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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