The Effects of Intracellular Calcium Depletion on Insulin Signaling in 3T3-L1 Adipocytes

Dorothy Sears Worrall and Jerrold M. Olefsky

Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093; and San Diego Veterans Affairs Medical Center, San Diego, California 92161

Address all correspondence and requests for reprints to: Jerrold M. Olefsky or Dorothy Sears Worrall, Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: jolefsky@ ucsd.edu or dsears{at}ucsd.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 
We have examined the requirement for intracellular calcium (Ca2+) in insulin signal transduction in 3T3-L1 adipocytes. Using the Ca2+ chelator 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium (BAPTA-AM), we find both augmentation and inhibition of insulin signaling phenomena. Pretreatment of cells with 50 µM BAPTA-AM did not affect tyrosine phosphorylation of insulin receptor substrate (IRS)1/2 or insulin receptor (IR)ß. The decreased mobility of IRS1 normally observed after chronic stimulation with insulin, due to serine phosphorylation, was completely eliminated by Ca2+ chelation. Correlating with decreased insulin-induced serine phosphorylation of IRS1, phosphotyrosine-mediated protein-protein interactions involving p85, IRS1, IRß, and phosphotyrosine-specific antibody were greatly enhanced by pretreatment of cells with BAPTA-AM. As a result, insulin-mediated, phosphotyrosine-associated PI3K activity was also enhanced.

BAPTA-AM pretreatment inhibited other insulin-induced phosphorylation events including phosphorylation of Akt, MAPK (ERK1 and 2) and p70 S6K. Phosphorylation of Akt on threonine-308 was more sensitive to Ca2+ depletion than phosphorylation of Akt on serine-473 at the same insulin dose (10 nM). In vitro 3'-phosphatidylinositol-dependent kinase 1 activity was unaffected by BAPTA-AM. Insulin-stimulated insulin-responsive glucose transporter isoform translocation and glucose uptake were both inhibited by calcium depletion. In summary, these data demonstrate a positive role for intracellular Ca2+ in distal insulin signaling events, including initiation/maintenance of Akt phosphorylation, insulin-responsive glucose transporter isoform translocation, and glucose transport. A negative role for Ca2+ is also indicated in proximal insulin signaling steps, in that, depletion of intracellular Ca2+ blocks IRS1 serine/threonine phosphorylation and enhances insulin-stimulated protein-protein interaction and PI3K activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 
INTRACELLULAR CALCIUM (Ca2+) is an intracellular second messenger in many signal transduction pathways. Several reports indicate that intracellular calcium is involved in insulin signaling (1–3); however, its involvement in insulin action has been disputed (4). The role of Ca2+ in insulin signaling is likely to be subtle, relying on small, localized changes in intracellular concentration rather than large intracellular Ca2+ changes mediated by influx from extra- and intracellular compartments (5). Insulin is able to alter the affinity of Ca2+ for membranes isolated from insulin-target tissues, and it has been suggested that insulin induces fluxes in Ca2+ within small regions of the cell interior (Ref. 6; reviewed in Ref. 1). Although Ca2+ is required for proper insulin signal transduction, high concentrations of intracellular Ca2+ diminish insulin sensitivity (7). Obese and type 2 diabetic subjects exhibit elevated intracellular Ca2+ levels in many cell types, including insulin target tissues (8). High levels of intracellular Ca2+ are associated with insulin resistance in vivo (9–11) and in vitro (2, 12), which can be normalized by Ca2+ antagonists (9). Other studies (summarized in Ref. 9) show that pharmacological treatment of adipocytes with agents that elevate intracellular calcium can inhibit insulin-stimulated glucose uptake.

The present studies were conducted to investigate the importance of intracellular Ca2+ in a variety of insulin actions including insulin-responsive glucose transporter isoform (GLUT4) translocation and glucose transport. Using the intracellular Ca2+ chelator 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium (BAPTA-AM), our results show that Ca2+ has both negative and positive effects on the transduction of insulin signals in 3T3-L1 adipocytes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 
Role of Ca2+ in Insulin-Induced Protein Phosphorylation
BAPTA-AM is a cell-permeable molecule that is enzymatically converted to the noncell-permeable, calcium chelator BAPTA once it is within the cell. Thus, only intracellular calcium is chelated by treatment with this agent. Using this compound, we assessed the role of Ca2+ in basal and insulin-stimulated tyrosine and serine/threonine phosphorylation levels in whole-cell lysates as shown in Fig. 1Go. Tyrosine phosphorylation of insulin receptor substrate (IRS)1/2 and insulin receptor (IR)ß were unaffected (or slightly enhanced) by BAPTA-AM pretreatment (Fig. 1AGo, lanes 4–6), whereas the phosphotyrosine-containing band at 120–130 kDa is almost completely eliminated. Figure 1Go, A and B, shows that the mobility of IRS1 is significantly reduced after 90 min of insulin stimulation (left panel, lane 3), and preincubation of cells in 50 µM BAPTA-AM eliminates this mobility shift.



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Figure 1. Effect of Intracellular Calcium Depletion on Insulin-Induced Protein Phosphorylation

3T3-L1 adipocytes were pretreated with DMSO (C, control), 15 µM (B15), 25 µM (B25), or 50 µM (B50) BAPTA-AM for 30 min before treatment with insulin (1 nM, panels A and B, or 10 nM, panels C–E) for the indicated number of minutes. Representative Western blots from whole lysates immunoblotted (IB) with the indicated antibodies are shown. Number of repetitions (n) for each experiment in panels A–E are n = 10, n = 10, n = 10, n = 4, and n = 3, respectively. pY, Phosphotyrosine.

 
Insulin causes activation of Akt, MAPK (ERK1 and 2), and p70 S6K, and Fig. 1Go, C–E, shows that insulin- induced phosphorylation of these proteins is inhibited by BAPTA-AM. Protein levels of IRS1, IRß (data not shown), Akt, MAPK, and p70 S6K (Fig. 1Go, B–E) were unchanged by incubation with BAPTA-AM. Insulin’s calcium dependency does not rely on the influx of free calcium because chelation of extracellular calcium by 5 mM EGTA did not inhibit these events (data not shown).

Chelation of Ca2+ Enhances Insulin-Activated Protein-Protein Interaction and PI3K Activity
Figure 1Go, A and B, shows that chelation of intracellular calcium does not impair overall tyrosine phosphorylation of IRS1 and IRß. Antiphosphotyrosine immunoprecipitation studies were conducted to further assess tyrosine phosphorylation of these proteins with or without BAPTA-AM. Figure 2AGo shows that the amount of IRS1 immunoprecipitated with PY20 antibody after 5 min of submaximal insulin stimulation (1 nM) is enhanced by pretreatment with BAPTA-AM. PY20 immunoprecipitation of IRß was also slightly enhanced (Fig. 2BGo). Additionally, an antibody to the p85 regulatory subunit of PI3K was able to pull down 60% (±10%, SE) more IRS1 from the BAPTA-AM-pretreated, insulin-stimulated lysates than the non-pretreated, insulin-stimulated controls (Fig. 2CGo). Immunoprecipitation studies using an antibody against the p110{alpha} subunit of PI3K generated similar results with respect to IRS1-PI3K interaction (data not shown).



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Figure 2. Chelation of Intracellular Calcium Enhances Phosphotyrosine Dependent Protein-Protein Interactions

3T3-L1 adipocytes were pretreated with DMSO (C, control) or 50 µM (B50) BAPTA-AM for 30 min before treatment with 1 nM insulin for the indicated number of minutes. Whole lysates were immunoprecipitated (IP) and analyzed by immunoblotting (IB) with the indicated antibodies, as described in Materials and Methods. Representative blots are shown. Number of repetitions (n) for each experiment in panels A–C are n = 4, n = 3, and n = 2, respectively.

 
We also assessed the insulin-induced activity of PI3K under the same conditions. Thus, PI3K activity was measured in PY20 immunoprecipitates from whole-cell lysates and, as seen in Fig. 3Go, BAPTA-AM pretreatment enhanced 5-min insulin-stimulated PI3K activity by 57% (±2%, SE).



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Figure 3. Phosphotyrosine-Associated Insulin-Stimulated PI3K Activity Is Enhanced by Depletion of Intracellular Ca2+

Immunoprecipitated in vitro PI3K activity in insulin-stimulated cells (1 nM insulin for the indicated number of minutes) was measured as described in Materials and Methods. Graphical representation of phosphotyrosine-associated PI3K immunoprecipitated from cells stimulated with insulin. Cells were preincubated with DMSO control (C) or 50 µM BAPTA-AM for 30 min. Data are the average of two independent experiments ± SE, expressed as a percent of time-matched DMSO control values. A representative autoradiogram is shown with the in vitro reaction product (PI-3P) indicated with an arrow.

 
Ca2+ Requirements for Insulin-Stimulated Serine-473 and Threonine-308 Phosphorylation of Akt
To investigate the sensitivity of serine-473 and threonine-308 phosphorylation to Ca2+ depletion, we stimulated cells with a constant level of insulin (10 nM) for 5 min in the presence of increasing BAPTA-AM concentrations. BAPTA-AM preincubation more effectively inhibited insulin-stimulated threonine-308 phosphorylation than serine-473 phosphorylation (see Fig. 4AGo). At the lowest BAPTA-AM concentration used (5 µM), inhibition of phosphothreonine-308 was 2.9-fold greater than inhibition of phosphoserine-473 (Fig. 4AGo). Pretreatment of cells with BAPTA-AM before heat shock (44 C for 20 min) inhibited Akt phosphorylation at both sites, and again, threonine-308 phosphorylation was more sensitive than serine-473 phosphorylation (Fig. 4BGo).



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Figure 4. Insulin- and Heat Shock-Mediated Akt Serine-473 and Threonine-308 Phosphorylation Are Differentially Calcium Dependent

A, BAPTA-AM inhibition of Akt serine-473 and threonine-308 phosphorylation induced by 10 nM insulin for 5 min was determined by immunoblotting with the indicated phospho-specific antibodies. Data were calculated from four or five independent experiments and are expressed as percent inhibition of the DMSO control phosphorylation signal (C) ± SE. A representative immunoblot is shown. B, Phospho-specific immunoblots of lysates from cells treated with heat shock (44 C, 20 min). S473, Akt serine-473; T308, Akt threonine-308.

 
Ca2+ Chelation Does Not Affect in Vitro 3'-Phosphatidylinositol-Dependent Kinase 1 (PDK1) Activity
Insulin-mediated activation of Akt involves PI3K activity and subsequent phosphorylation of Akt by PDK1 and the as yet uncharacterized PDK2. Because phosphotyrosine-associated PI3K activity in insulin-stimulated whole-cell lysates is enhanced by intracellular calcium chelation (Fig. 3Go), it is possible that BAPTA-AM inhibition of Akt phosphothreonine-308 (Figs. 1Go and 4Go) is caused by inactivation of the Akt threonine-308 kinase, PDK1. To assess this possibility, immunoprecipitated PDK1 activity was assayed (see Materials and Methods). As seen in Fig. 5Go, BAPTA-AM treatment before insulin stimulation did not affect the constitutive activity of PDK1.



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Figure 5. Depletion of Intracellular Ca2+ Does Not Affect in Vitro PDK1 Activity

Immunoprecipitated PDK1 in vitro activity was measured as described in Materials and Methods. Cells were pretreated with DMSO or 100 µM BAPTA-AM for 30 min prior in stimulation with 10 nM insulin for 5 min. Data are the average of three independent experiments ± SE, expressed as a percent of maximal signal for each individual experiment. Data from negative controls (including control IgG IPs and kinase reactions without exogenous substrate) were all less than 1% of the maximal signal in each experiment.

 
Calcium Requirements for Insulin-Stimulated Glucose Uptake
We next assessed the effects of intracellular calcium depletion on insulin-mediated glucose uptake into 3T3-L1 adipocytes. Both basal and insulin-stimulated glucose uptake were inhibited by BAPTA-AM in a dose-dependent manner (Fig. 6AGo).



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Figure 6. Dose Response of Ca2+ Depletion and Inhibition of Insulin-Induced Glucose Transport

Basal and stimulated glucose uptake were measured after treatment with the indicated concentrations of BAPTA-AM for 10–30 min. Glucose uptake was measured as in Materials and Methods. A, Glucose uptake stimulated by insulin (16.7 nM for 20 min). Data are the average of four independent experiments ± SEM and are expressed as a percent of total counts per mg of protein. B, Glucose uptake stimulated by hydrogen peroxide (H2O2, 3 mM for 20 min). C, Glucose uptake stimulated by osmotic shock (600 mM sorbitol for 20 min). H2O2 and osmotic shock data are each from a single experiment done in triplicate, ± SE.

 
Glucose uptake can be stimulated by a variety of insulinomimetic stimuli including hyperosmotic shock and hydrogen peroxide (13, 14). BAPTA-AM pretreatment of 3T3-L1 adipocytes inhibited glucose transport stimulated by either of these two agents (600 mM Sorbitol or 3 mM hydrogen peroxide) (Fig. 6Go, B and C).

GLUT4 Translocation Requires Intracellular Calcium
Insulin stimulation of 3T3-L1 adipocytes leads to translocation of GLUT4 from an intracellular low density microsomal compartment (LDM) to the plasma membrane (PM). Cell fractionation and fluorescence microscopy studies were employed to observe the effects of intracellular calcium chelation on the location of GLUT4 in basal and insulin-stimulated cells. Cells were fractionated into their subcellular compartments as described in Materials and Methods, and the levels of GLUT4 protein in the LDM and PM fractions are shown in Fig. 7Go. Insulin stimulation causes a decrease in GLUT4 levels in the LDM fraction (Fig. 7AGo, lane 3, and 7B, left graph) and an increase in GLUT4 levels in the PM fraction (Fig. 7AGo, lane 7, and 7B, right graph). Pretreatment of cells with BAPTA-AM inhibits both the GLUT4 decrease in the LDMs and the increase in the PMs.



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Figure 7. BAPTA-AM Blocks Insulin-Stimulated GLUT4 Translocation As Measured in Subcellular Fractions

A, Cells were preincubated with DMSO (C) or 50 µM BAPTA-AM (B) for 30 min ± 20 min stimulation with 16.7 nM insulin. Subcellular fractionation and immunoblotting was done as described in Materials and Methods. Representative autoradiograms of the levels of GLUT4 in the LDMs and the PMs are shown. B, Graphical data representation of independent experiments similar to that shown in panel A, ± SE. Left graph shows the average LDM level of GLUT4 as a fraction of basal DMSO control (n = 4). Right graph shows the average PM level of GLUT4 as fold over basal DMSO control (n = 5).

 
Insulin-stimulated GLUT4 translocation was also evaluated by fluorescence microscopy (described in Materials and Methods). Fixed, nonpermeabilized cells were stained with fluorescently labeled concanavalin A (red color in Fig. 8AGo), which binds to glycosylated residues on cell-surface proteins and defines the location of the PM. GLUT4 protein (green color in Fig. 8A) and nuclei stained with DAPI (blue color in Fig. 8A) are also shown. Five-section, volume-view, overlay images ("ALL") are shown for each condition. Colocalized red and green signals appear yellow in these images; thus, a yellow PM ring is indicative of GLUT4 translocation (see Fig. 8AGo, InsulinDMSO/ALL image; DMSO, dimethylsulfoxide).



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Figure 8. BAPTA-AM Blocks Insulin-Stimulated GLUT4 Translocation As Measured by Quantitative Digital Fluorescence Microscopy

Cells were preincubated with DMSO or 50 µM BAPTA-AM (B) for 30 min ± 20 min stimulation with 16.7 nM insulin. Cell staining and microscopic imaging were performed as described in Materials and Methods. A, Green staining represents GLUT4 localization, red staining represents concanavalin A (Con A) binding of glycosylated residues on the surface of intact cells, and blue staining represents DAPI binding within nuclei. Five-section, volume-view, overlay images are shown ("ALL" column). Yellow color indicates the colocalization of green and red staining (GLUT4 and Con A). B, Digital fluorescence images overlaid in panel A, which are comprised of five serial sections in volume view, are represented quantitatively as described in Materials and Methods. Quantitative images of voxel intensity for red staining ("Red" column, Con A signal) and green staining ("Green" column, GLUT4 signal) are shown and correspond to the fluorescent images (panel A, "ALL" column). Computer-generated, quantitative values are indicated by a rainbow color scale (example is shown, bottom right) where violet is the least fluorescence intensity value and red is the greatest fluorescence intensity value. C, Graphical representation of numerical data generated from image values visually represented in panel B (see detailed description in Materials and Methods). B50; 50 µM BAPTA-AM, B100; 100 µM BAPTA-AM. Data are compiled from five-section, deconvoluted volume views and are the average of five PM measurements for each of five cells ± SEM, expressed as a ratio of green (GLUT4) signal intensity to red (Con A) signal intensity located within the PM region as defined by red fluorescence.

 
Figure 8AGo shows that the yellow ring that appears at the PM after insulin stimulation (InsulinDMSO/ALL) fails to appear in insulin-stimulated cells that were pretreated with BAPTA-AM (InsulinBAPTA/ALL). This effect of BAPTA-AM is clearly and quantitatively seen in Fig. 8BGo. Figure 8CGo is a graphical representation of GLUT4 translocation and shows that pretreatment of cells with 50 or 100 µM BAPTA-AM blocks insulin-induced translocation of GLUT4 to the PM without affecting basal GLUT4 levels (Fig. 8CGo).

Subcellular Localization of Akt
We next analyzed the subcellular distribution of Akt, hypothesizing that the BAPTA-AM-induced decrease in Akt phosphorylation may be a consequence of Akt inaccessibility to PDK1 and 2 at the PM. The distribution of Akt and phosphoserine-473 Akt was analyzed using protein- and phospho-specific antibodies that recognize all three Akt isoforms (Fig. 9Go). The amount of Akt protein in the basal and insulin-stimulated PM fractions was greater in BAPTA-AM-treated cells compared with control cells. Insulin stimulation did not cause an increase in Akt localization at the PM, compared with basal levels, in control or BAPTA-pretreated cells. BAPTA-AM strongly inhibited insulin-stimulated, serine-473 phosphorylation of Akt in the cytosol and PM (Fig. 9AGo, compare lanes 3 with lanes 4 in pS-Akt blot). The ratio of phosphoserine-473 Akt signal relative to total Akt protein signal in each fraction is shown in Fig. 9BGo. Interestingly, in control cells, the PM fraction contained 9.4 times more serine-phosphorylated Akt protein than the cytosol fraction, although the majority of total Akt protein was in the cytosol (Fig. 9BGo).



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Figure 9. Effects of Ca2+ Depletion On the Subcellular Localization of Total Akt Protein and Insulin-Induced Phosphoserine-473 Akt. Cells Were Stimulated with 16.7 nM Insulin for 20 Min

Subcellular fractionation and Western blotting were done as described in Materials and Methods. Localization and phosphorylation of Akt within whole lysate (WL), cytosol, and PM fractions in basal and insulin-stimulated cells ± 100 µM (B100) BAPTA-AM preincubation. A, Representative Western blots are shown using antibodies specific for total Akt and phosphoserine-473 Akt (pS-Akt), as indicated. Lanes 1, Basal, DMSO control. Lanes 2, Basal, 100 µM BAPTA-AM. Lanes 3, Insulin, DMSO control. Lanes 4, Insulin, 100 µM BAPTA-AM. B, Graph represents the ratio of phosphoserine-473 Akt signal (pS-Akt) to Akt protein signal in different fractions.

 

    Discussion
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 
In this study, we have examined the effects of intracellular calcium depletion on various aspects of insulin action and have found that intracellular calcium can play both a positive and negative role at different steps in the insulin signaling cascade. For example, we have found that depletion of intracellular calcium inhibits insulin-stimulated glucose transport as well as translocation, suggesting an important role for calcium in vesicular trafficking and fusion of GLUT4 vesicles. Intracellular calcium depletion also inhibits activation of a variety of serine/threonine kinases, including Akt, p70 S6K, and MAPK. Interestingly, the decrease in insulin-stimulated Akt phosphorylation was not due to impaired PI3K activation because, if anything, calcium depletion enhanced insulin stimulation of PI3K. Taken together, these studies demonstrate a critical role for intracellular calcium in the more distal components of the insulin action pathway, particularly related to GLUT4 translocation.

Treatment of cells with BAPTA-AM to deplete intracellular calcium had no inhibitory effect on early steps of insulin action, such as insulin receptor autophosphorylation, IRS-1 tyrosine phosphorylation, or activation of PI3K. In fact, we found some evidence for increased activity of these initial events. For example, there was an increase in the amount of PI3K activity associated with IRS-1 in the insulin-stimulated state. Most likely, this was due to the ability of calcium depletion to inhibit IRS-1 serine/threonine phosphorylation and thus, enhance the efficiency of PI3K binding to phosphorylated IRS-1. In agreement with these results, it has been shown that serine/threonine phosphorylation of IRS1 is associated with insulin resistance (15–19) and that serine phosphorylation causes a reduction in IRS1’s ability to act as a docking site for PI3K (20, 21).

Akt phosphorylation is downstream of PI3K activity, and we found that in the BAPTA-AM-treated cells, there was a marked impairment in insulin-stimulated and heat shock-stimulated Akt phosphorylation. Clearly, in insulin-stimulated cells, this was not due to decreased PI3K activity because, if anything, PI3K activity was increased. Therefore, the data show that intracellular calcium depletion interferes with insulin-stimulated Akt activation at a step downstream of PI3K, assuming that in vitro PI3K activity reflects in vivo activity.

The level of insulin-induced serine-473 phosphorylation per Akt protein is inhibited in the cytosol and absent in the PM fractions from BAPTA-AM- pretreated cells. In contrast, generation of serine-473 phosphorylation per Akt protein in insulin-stimulated control cells was significantly greater in the PM fraction compared with the cytosol. This distinct subcellular distribution of phosphorylated Akt protein in control cells is not surprising given that activation of Akt by insulin requires its translocation to the PM and subsequent phosphorylation by PDK1 and the as yet uncharacterized PDK2 (22). These data suggest that Ca2+ is not required for Akt to fractionate with the PM and that the inhibition/lack of phospho-Akt signal in these fractions results from some Akt-localization-independent effect of Ca2+ chelation. Interestingly, insulin-mediated phosphorylation of Akt on threonine-308 and serine-473 exhibit different sensitivities to intracellular Ca2+ depletion that may be mediated by site-specific kinases and/or phosphatases. We have shown that the in vitro intrinsic activity of the threonine-308 kinase PDK1 is unaffected by BAPTA-AM pretreatment. Although the serine-473 candidate kinases are not known to be dependent on Ca2+ (22–25), conflicting data exists regarding the role of Ca2+ and Akt serine-473 phosphorylation (26, 27). Ca2+ depletion may also activate Akt phosphatase(s), similar to the work of Draznin et al. (2) showing that elevated levels of intracellular Ca2+ inhibit phosphatases specific for glycogen synthase and GLUT4.

Phospholipid binding by certain phospholipid-binding domains, including the Akt PH (pleckstrin homology) domain, can be affected by relatively low levels of Ca2+ (28, 29) and by phosphorylation levels of phosphatidylinositol. Chelation of intracellular calcium did not inhibit the localization of Akt at the PM in our study but may have perturbed the PH domain-mediated localization of PDK1 and/or the hypothetical PDK2. This would also explain the BAPTA-AM sensitivity of insulin-induced p70 S6K phosphorylation, the activation of which is dependent upon PDK1 (30). Alternatively, phosphatidylinositol phosphatase (e.g. SHIP, SH2-containing inositol polyphosphate 5-phosphatase) activity may be affected by intracellular Ca2+ depletion, resulting in PH domain-mediated effects on Akt phosphorylation. The mode of Ca2+’s site-specific effects on Akt phosphorylation are unclear; however, we demonstrate that the minimum level of Ca2+ required for Akt threonine-308 phosphorylation is higher than that required for serine-473 phosphorylation.

We, in the present study, and others have shown that intracellular calcium is required for glucose uptake stimulated by insulin and other stimuli (2, 31, 32). We also find that BAPTA-AM preincubation completely blocked insulin-stimulated GLUT4 translocation as assayed using subcellular fractionation and fluorescence imaging. The role of Akt in mediating these effects is unclear from our studies. Whether Akt is upstream of insulin-activated glucose transport is controversial, BAPTA-AM’s effect on glucose transport and Akt phosphorylation may be correlative rather than causal. The requirement of Ca2+ in synaptic vesicle fusion and in vesicle fusion/exocytosis in nonneuronal cells is well documented. Specific levels of Ca2+ regulate interactions between vesicle associated proteins (synaptotagmins and syntaxins) required for vesicle fusion in an isoform-specific manner (33). Studies of vesicle fusion in nonneuronal cell types suggest that exocytosis is mediated by small, localized changes in Ca2+ flux that occur at the site of fusion (34, 35). Such small changes would not be detectable by traditional means of measuring intracellular Ca2+ levels (e.g. the fluorescent Ca2+ indicator Fura-2) but are inhibitable with BAPTA-AM (34). Ca2+/calmodulin has been shown to be required for vacuolar fusion (36) and also for insulin-stimulated GLUT4 translocation (3) and glucose transport (Refs. 37 and 38, and our unpublished observations). Thus, calmodulin and some vesicle-associated proteins (SNAREs, soluble N-ethyl maleimide sensitive factor attachment protein receptor) are Ca2+-regulated proteins required for vesicle fusion, possibly including fusion of GLUT4 vesicles with the PM after insulin-mediated translocation.

There are many studies in the literature regarding the involvement of Ca2+ in insulin signaling, and although large changes in intracellular levels of Ca2+ do not occur in response to insulin (5), it is clear that an optimal Ca2+ concentration within cells is essential for insulin mediated events (2). It has been suggested that small changes in Ca2+ concentration or Ca2+ fluxes may be mechanistically important for insulin signaling (1, 2). Also, elevated levels of intracellular calcium are associated with in vitro and in vivo insulin-resistant states (7–11, 39), and it is possible that high intracellular levels of Ca2+ would prevent an insulin target cell from sensing acute insulin-induced small changes in Ca2+ flux. This may provide one reason why elevated intracellular Ca2+ levels are associated with insulin resistant states.

In summary, we have found that Ca2+ depletion results in enhanced proximal insulin action including PI3K activation and IRS1/PI3K association. In contrast, distal insulin signaling events are inhibited by Ca2+ depletion. We find that Ca2+ is required for insulin-induced Akt phosphorylation and that this Ca2+-dependent activation of Akt is downstream of PI3K. Lastly, we have demonstrated that Ca2+ depletion blocks insulin-induced GLUT4 translocation and glucose transport. Thus, intracellular Ca2+ is an important component of insulin action at multiple steps in the insulin signaling cascade.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 
Materials
DMEM and Glutamax were obtained from Life Technologies, Inc. (Rockville, MD). Penicillin-streptomycin and FCS were obtained from Omega Scientific (Tarzana, CA). Insulin was a gift from Eli Lilly & Co. (Indianapolis, IN). Silica-coated thin-layer chromatography plates and all chemicals, unless otherwise noted, were obtained from Sigma (St. Louis, MO). Recombinant protein A-agarose and antibodies to IRS1, p85, p110{alpha}, PDK1 kinase assay kit, ETC were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Akt (pan) antibody and phospho-specific antibodies for Akt, p70 S6K, and MAPK were from New England Biolabs, Inc. (Beverly, MA). The GLUT4 antibody was obtained from Chemicon (Temecula, CA). Mouse monoclonal antiphosphotyrosine (PY20) was from Transduction Laboratories (San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated antirabbit antibody was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Anti-IRß antibody and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All radioisotopes were obtained from NEN Life Science Products (Boston, MA). Enhanced chemiluminescence reagent was obtained from Pierce Chemical Co. (Rockford, IL). "Complete" protease tablets were from Roche (Indianapolis, IN). DAPI and tetramethyl-rodamine isothiocyanate (TRITC)-conjugated concanavalin A were from Molecular Probes, Inc. (Eugene, OR).

Cell Culture
3T3-L1 fibroblasts were maintained in DMEM-high glucose (25 mM glucose, 1.8 mM CaCl2) medium containing 10% calf serum. Postconfluent fibroblasts were differentiated into adipocytes by adding 1 µg/ml insulin, 0.1 µg/ml dexamethasone, and 112 µg/ml isobutylmethylxanthine to the medium. The differentiation medium was removed after 3 d and replaced with DMEM-low glucose (5 mM glucose, 1.8 mM CaCl2) medium containing 10% FCS, Glutamax, and 1% penicillin-streptomycin. Seven days after the addition of the differentiation mix, the cells were plated in culture dishes for each given experiment. The medium was changed every third day until use, 10–16 d post differentiation. Approximately 90% of the cells exhibited large lipid droplets indicative of adipocytes. Before each experiment, cells were serum-starved for 2–4 h in DMEM-low glucose containing 0.1% BSA. This study protocol was used in all our experiments. During experiments, DMSO concentration in the media/incubation buffer was equal in control samples to that used as vehicle for inhibitors in all experiments and never exceeded 0.1%. BAPTA-AM was diluted in media or buffer and cells were pretreated for 10–30 min before initiating stimulation with insulin, etc. The drug was present during stimulation only unless otherwise noted.

Lysates, Immunoprecipitations, and Immunoblottting
Cells were rinsed two times with ice-cold PBS and lysed at 4 C in lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 4 mM sodium orthovanadate, 20 mM sodium pyrophosphate, 200 mM sodium fluoride, 2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 10% glycerol, pH 7.4. Lysates were vortexed well and placed on ice for 10 min. Lysates were then centrifuged at 14,000 x g for 10 min at 4 C. Supernatants were separated from the resulting fat layer and protein concentrations were determined by Bradford assay. Lysates for immunoprecipitation or affinity chromatography were incubated with 2–4 µg of the indicated antibody or GST-fusion with protein A-sepharose or glutathione-agarose, respectively, at 4 C for 2–14 h with gentle agitation. Pellets were washed three times in lysis buffer. Laemmli’s buffer was added to the pellets and boiled for 5 min. Samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Immunoblotting with various antibodies was conducted as per company instructions. Immunoblots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody and subsequently analyzed by enhanced chemiluminescence and autoradiography. Autoradiographs were quantitated using densitometric scanning and NIH Image software.

2-Deoxyglucose Uptake
After pretreatment as indicated in figure legends, 3T3-L1 adipocytes were stimulated with 16.7 nM insulin, 600 mM sorbitol, or 3 mM H2O2 for 20 min at 37 C. Glucose transport was determined by the addition of 0.1 mM 2-deoxyglucose containing 0.2 µCi of 2-[3H] deoxyglucose as described previously (40) in HEPES/salts buffer with 0.1% BSA (10 mM HEPES, 2.5 mM NaH2PO4, 130 mM NaCl, 4.7 mM KCl, 1.24 mM MgSO4, 2.47 mM CaCl2, pH 7.4). Nonspecific uptake was assessed using 0.1 mM L-glucose containing 0.2 µCi of L-[3H]glucose. The reaction was stopped after 10 min by aspiration and extraneous glucose was removed by three washes with ice-cold PBS. Cells were lysed in 1 N NaOH, and glucose uptake was assessed by scintillation counting. Samples were normalized for protein content by Bradford protein assay.

Subcellular Fractionation
After pretreatment as indicated, cells from one 10-cm dish per condition were incubated with insulin as described and washed three times with ice-cold PBS. Cells were scraped into ice-cold HES buffer (255 mM sucrose, 20 mM HEPES, 1 mM EDTA, 4 mM Na3VO4, 200 mM sodium fluoride, 20 mM sodium pyrophosphate, pH 7.4) supplemented with protease inhibitors ("Complete" protease cocktail tablet). Cells were then homogenized using an Potter-Ejlerham homogenizer. Subcellular fractionation was carried out as described previously (41).

In Vitro PI3K and PDK1 Activity Assays
3T3-L1 adipocytes were pretreated and stimulated as indicated, then lysed and immunoprecipitated as described above with the following modifications. After incubation with the antibody, bead pellets were washed three times with Buffer A (Tris-buffered saline, 1% NP-40, and 100 µM Na3VO4, pH 7.4), three times with Buffer B (100 mM Tris, 500 mM LiCl2, and 100 µM Na3VO4, pH 7.4), and twice with Buffer C (10 mM Tris, 100 mM NaCl, 1 mM EDTA, and 100 µM Na3VO4, pH 7.4). Pellets were resuspended in Buffer C without Na3VO4. As described previously (42), PI3K activity was assessed by the phosphorylation of phosphatidylinositol in the presence of 20 µCi of [{gamma}-32P]ATP for 20 min. The reactions were stopped with 20 µl of 8 N HCl and 160 µl of CHCl3:methanol (1:1) and centrifuged. The lower organic phase was removed and applied to 1%-CDTA-coated silica gel TLC plates. After the separation of lipids by TLC using the borate-buffered system (43), phosphatidylinositol 3-phosphate was visualized by autoradiography. Autoradiographs were scanned and quantitated as described above.

In vitro PDK1 kinase assays were conducted as per manufacturer’s instructions using immunoprecipitated PDK1 from cells pretreated 30 min with DMSO or 100 µM BAPTA-AM before stimulation with 10 nM insulin for 5 min. One protocol exception was that rabbit antisheep antibody and protein A-agarose were used to precipitate the primary sheep anti-PDK1 antibody. Briefly, PDK1 was immunoprecipitated from cell lysates [Lysis buffer A: 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% (vol/vol) 2-mercaptoethanol, 1% (vol/vol) Triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 mM sodium ß-lycerophosphate, 0.1 mM pheynylmethylsulfonyl fluoride, 1 µM microcystin, 1 mg/ml of aprotinin, pepstatin, leupeptin] overnight at 4 C. Activity of PDK1 is measured by the PDK1-dependent activation of SGK (serum- and glucocorticoid-induced protein kinase) in a two-step reaction. In step 1, immunoprecipitated PDK1 was incubated with purified, inactivated SGK protein in PDK1 assay dilution buffer (supplied in kit) for 30 min at 30 C with shaking. In step 2, exogenous Akt/PKB specific substrate peptide was added with [{gamma}-32P] ATP for an additional 10 min. Labeled substrate peptide was spotted onto P81 paper and washed with 0.75% phosphoric acid. Incorporation of radioisotope was measured by scintillation counting. Results are the average of three independent experiments. Data from negative controls (including control IgG IPs and kinase reactions without exogenous substrate) were all less than 1% of the maximal signal in each experiment.

Staining of GLUT4 Translocation
3T3-L1 adipocytes on coverslips were pretreated and stimulated as indicated, then rinsed two times with ice-cold PBS. Cells were fixed in 3.7% formaldehyde in PBS for 10 min. Coverslips were rinsed two times with PBS, and all further incubations were shielded from light. Cells were stained with TRITC-concanavalin A for 30 min then washed two times with PBS. Cells were permeabilized in 0.1% Triton X-100, 2% FCS in PBS for 10 min. Coverslips were incubated with GLUT4 antibody overnight at 4 C then washed in 2% FCS in PBS for 10 min. Coverslips were then incubated with FITC-conjugated antirabbit secondary antibody for 60 min then washed with 2% FCS in PBS for 10 min. Nuclei were stained with 50 ng/ml DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) in PBS for 5 min then washed with 2% FCS in PBS for 10 min. Coverslips were rinsed in water before mounting on Gelvatol on microscope slides.

Imaging of GLUT4 Translocation
Images were captured using a DeltaVision deconvolution microscope system (Applied Precision, Inc., Issaquah, WA). The system includes a Photometrics charge-coupled device mounted on a Nikon microscope. In general, 75 optical sections spaced by 0.2 µm were taken of cells mounted and stained on coverslips. Pixel intensities were kept in the linear response range of the digital camera. The lens used for these images was 40x (NA1.3); the resulting pixel size is 120 nm2. Data sets were deconvoluted and analyzed using SoftWorx software (Applied Precision, Inc.) on a Silicon Graphics (Mountain View, CA) Octane workstation. Images used for quantitation were volume views made from 5 consecutive sections of the 75 sections that include the entire volume of the cells. We used the 5 sections beginning at section no. 17, counting away from the cell bottom/coverslip for all measurements (Fig. 8AGo). These five-section, volume views were used for analysis because they best represented the cell edges and the staining distribution within the PM of each cell.

DataInspector application was used to quantitatively analyze the five-section volume views. The images shown in Fig. 8BGo are quantitative volume views define the voxel intensity of each wavelength in a unique false-color scale (very low voxel intensity as violet, very strong as red). Importantly, these quantitations are computer-derived using the original digital fluorescent image data and are not dependent upon the brightness of the cell images projected on the computer screen or prints. Voxel intensity values can be displayed in a color scale form (Fig. 8BGo) or can be displayed in numerical form (not shown, but graphically represented in Fig. 8CGo). Levels of FITC-labeled (green), anti-GLUT4 antibody at the PM were measured relative to staining of TRITC-concanavalin A (red) used as a plasma membrane control marker. Levels of TRITC-concanavalin A at the PM relative to TRITC-concanavalin A in the cytoplasm did not change between matched control and BAPTA-AM-pretreated samples. Thus, changes in the GLUT4/concanavalin A ratio reflect changes in GLUT4 levels only. Each measurement was determined using the summed voxel intensity within a circular cursor with a diameter of 480 nm. Values graphed in Fig. 8CGo are the average of five PM measurements for each of five cells ± SEM.


    ACKNOWLEDGMENTS
 
We thank Donna Reichart and Jay Nelson for excellent technical assistance, which contributed to this work. We thank Dr. James Feramisco and Brian Smith (of the UCSD Cancer Center Digital Imaging Shared Resource Facility for Deconvolution Microscopy) for their imaging expertise and advice on image analysis.


    FOOTNOTES
 
This work was supported by NIH Grant DK-33651 and the Veterans Administration Medical Research Service. D.S.W. was supported by NIH/NIDDK Individual NRSA Grant DK-09595.

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Abbreviations: BAPTA-AM, 1,2-bis(o-Aminophenoxy)ethane- N,N,N',N'-tetraacetic acid, sodium; CDTA, trans-1,2-diaminocyclo-hexane-N,N,N',N'-tetra-acetic acid; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; GLUT4, insulin- responsive glucose transporter isoform; IR, insulin receptor; IRS, insulin receptor substrate; LDM, low density microsomal compartment; PDK1 or PDK2, 3'-phosphatidylinositol-dependent kinase 1 or 2; PH, pleckstrin homology; PM, plasma membrane; TRITC, tetramethyl-rodamine isothiocyanate.

Received for publication January 16, 2001. Accepted for publication October 19, 2001.