Diversification of cardiac insulin signaling involves the p85alpha /beta subunits of phosphatidylinositol 3-kinase

Alexandra Kessler, Ingo Uphues, D. Margriet Ouwens, Martin Till, and Jürgen Eckel

Laboratory of Molecular Cardiology, Diabetes Research Institute, D-40225 Düsseldorf, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ventricular cardiomyocytes and cardiac tissue of lean and genetically obese (fa/fa) Zucker rats were used 1) to study the role of the p85 regulatory subunit isoforms p85alpha and p85beta for insulin signaling through the phosphatidylinositol (PI) 3-kinase pathway, and 2) to elucidate the implications of these mechanisms for cardiac insulin resistance. Western blot analysis of cardiomyocyte lysates revealed expression of p85alpha and p85beta but no detectable amounts of the splice variants of p85alpha . Essentially no p85alpha subunit of PI 3-kinase was found to be associated with insulin receptor substrate (IRS)-1 or IRS-2 in basal and insulin-stimulated (5 min) cardiomyocytes. Instead, insulin produced a twofold increase in p85beta associated with IRS-1, leading to a three- to fourfold increase in p85beta -associated PI 3-kinase activity. This response was significantly reduced in obese animals. Comparable results were obtained in the intact heart after in vivo stimulation. In GLUT-4-containing vesicles, an increased abundance (3.7 ± 0.7-fold over basal) of p85alpha was observed after insulin stimulation of lean animals, with no significant effect in the obese group. No p85beta could be detected in GLUT-4-containing vesicles. Recruitment of the p110 catalytic subunit of PI 3-kinase and a twofold increase in enzyme activity in GLUT-4-containing vesicles by insulin was observed only in lean rats. We conclude that, in the heart, p85alpha recruits PI 3-kinase activity to GLUT-4 vesicles, whereas p85beta represents the main regulator of IRS-1- and IRS-2-mediated PI 3-kinase activation. Furthermore, multiple defects of PI 3-kinase activation, involving both the p85alpha and the p85beta adaptor subunits, may contribute to cardiac insulin resistance.

GLUT-4-containing vesicles; obesity; insulin resistance; cardiac muscle


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS NOW WELL RECOGNIZED that phosphatidylinositol (PI) 3-kinase operates as an essential signal transduction element in most, if not all, of the cellular processes regulated by insulin in its target tissues (for review, see Refs. 1, 7, 40). In addition, PI 3-kinase is activated by a variety of growth factor tyrosine kinase receptors and thus regulates multiple and diverse cellular functions, including mitogenesis (10), membrane ruffling (25), cytoskeletal organization and cell motility (45), membrane trafficking (39), and insulin-stimulated glucose transport and glycogen synthesis (4, 20, 42). Class 1 PI 3-kinase is a heterodimeric enzyme consisting of a p110 catalytic subunit and a p85 regulatory subunit (1). The latter exerts adaptor functions by interacting with tyrosine-phosphorylated cognate motifs via two src homology 2 (SH2) domains, finally leading to an increased catalytic activity of the PI 3-kinase complex (32). The diversity of cellular functions controlled by PI 3-kinase requires a high level of specificity for the activation of this enzyme. This process, however, remains poorly understood.

One level of specificity relates to the tissue-specific expression of a considerable array of different adaptor subunits encoded by at least three different genes, termed p85alpha , p85beta , and p55gamma (40). The p85alpha gene generates two truncated splice variants, p55alpha and p50alpha , that lack the SH3 and several other NH3-terminal domains (2, 19). Isoform p50alpha was found to be highly expressed in human skeletal muscle (38) and exhibited the most pronounced effect of insulin on recruitment into antiphosphotyrosine immunoprecipitates (38). Furthermore, targeted disruption of the p85alpha gene resulted in increased insulin sensitivity, most probably resulting from the isoform switch to the p50alpha variant (43). It is therefore generally thought that the p85alpha regulatory subunit and its splice variants play a pivotal role in insulin signaling. The function of the p85beta isoform, which has a 62% amino acid homology with p85alpha (34), has remained controversial. In an earlier study, Baltensperger et al. (3) reported that p85beta does not mediate marked activation of PI 3-kinase, whereas in skeletal muscle this isoform contributes significantly to the activation process (38). An even higher association of p85beta with insulin receptor substrate (IRS)-1 compared with p85alpha was found in L6 myoblasts (13), and similar but distinct insulin receptor signaling complexes of the two isoforms were observed in rat hepatoma cells (41). These data suggest that the different adaptor subunit variants might play a prominent role in controlling downstream insulin signaling in a highly tissue-specific manner.

A second level of specificity relates to the compartmentalization of the IRS/PI 3-kinase signaling complexes (18). It is well known that insulin induces the recruitment of PI 3-kinase activity to intracellular membranes, whereas growth factors like platelet-derived growth factor (PDGF) shift this enzyme to the plasma membrane (33, 49). This difference is thought to explain the inability of PDGF to activate glucose transport despite increasing PI 3-kinase activity (33). A compelling hypothesis is that insulin recruits PI 3-kinase activity to the intracellular GLUT-4-containing vesicles, thus being directly linked to the process of GLUT-4 translocation (16). Three groups have confirmed the presence of PI 3-kinase activity in GLUT-4-containing vesicles after insulin stimulation of 3T3-L1 adipocytes (12, 16, 48); however, others (5) have failed to detect this colocalization. Furthermore, in skeletal muscle, PI 3-kinase activity was shown to be absent from GLUT-4-containing vesicles (26, 27). So far, no study has addressed the question of whether adaptor subunits may differentially recruit PI 3-kinase activity to different cellular compartments, certainly representing an additional, yet unexplored, level of specificity of PI 3-kinase signaling.

In the present investigation, we have analyzed and compared the involvement of different adaptor subunit isoforms in cardiac insulin signaling 1) at early steps by measuring the interaction with IRS proteins, and 2) at downstream reactions by monitoring the recruitment of PI 3-kinase to GLUT-4-containing vesicles. Furthermore, we have extended this analysis to insulin-resistant, genetically obese (fa/fa) Zucker rats. In our recent studies on this animal model, we have shown a failure of insulin-regulated recruitment of cardiac GLUT-4 (46) and an altered response of IRS-1-associated PI 3-kinase activity (24); however, the relative contribution of the different adaptor subunits to these defects is unknown. It is well established that cardiac insulin resistance plays an important role in a variety of disease states (8) and that the perpetual contractile activity of the myocardium may require highly specialized signaling pathways within the dynamic context of regulators of cardiac function (8). The present study shows that, in cardiac muscle, the p85alpha subunit recruits PI 3-kinase activity to GLUT-4-containing vesicles independent of IRS proteins, whereas p85beta mediates substantial activation of PI 3-kinase at the level of IRS-1. Both reactions are impaired in obesity, suggesting that multiple alterations of PI 3-kinase activation contribute to insulin resistance.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. 125I-labeled protein A (30 mCi/mg) and [gamma -32P]ATP (6,000 Ci/mmol) were purchased from Amersham (Braunschweig, Germany). Reagents for SDS-PAGE were supplied by Pharmacia and Sigma (Deisenhofen, Germany). Insulin (Actrapid HM, 100 units/ml) was supplied by Novo (Mainz, Germany). Protein G agarose and protein A-trisacryl were purchased from Pierce (Rockford, IL). The polyclonal GLUT-4 antiserum was obtained from a rabbit injected with a peptide corresponding to the COOH-terminal 12 amino acids of GLUT-4 (TELEYLGPDEND) coupled to keyhole limpet hemocyanin (Eurogentec, Belgium). The anti-IRS-1 antibody was kindly provided by Dr. J. A. Maassen (Leiden, The Netherlands). Rabbit polyclonal antiserum, raised against a glutathione S-transferase fusion protein corresponding to the amino-terminal SH2-domain of human p85alpha (p85alpha NSH2), was kindly supplied by Dr. P. R. Shepherd (London, UK). Rabbit polyclonal antiserum raised against a glutathione S-transferase (GST) fusion protein corresponding to the SH3-domain of p85beta was generously provided by Dr. T. Asano (Tokyo, Japan). Monoclonal anti-p85alpha PI 3-kinase antibody and the monoclonal horseradish peroxidase-conjugated anti-phosphotyrosine antibody (RC20) were purchased from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgG antibodies were from Promega (Mannheim, Germany). Polyclonal anti-p110alpha PI 3-kinase antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-p85 PI 3-kinase antibody (p85PAN), Jurkat cell lysate, and EGF-stimulated A431 cell lysate were obtained from UBI (Lake Placid, NY). All other chemicals were of the highest grade commercially available.

Isolation of cardiomyocytes and membrane preparation from whole heart. Genetically obese (fa/fa) male Zucker rats (12-16 wk old, 480-520 g) were kindly provided by Dr. L. Herberg (Düsseldorf, Germany). Blood samples were collected for determinations of glucose and insulin as outlined previously (9). Ca2+-tolerant cardiac myocytes were isolated by perfusion of adult rat heart with collagenase as detailed previously (24). The final cell suspension was incubated for 60 min until further use in HEPES buffer [130 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 25 mM HEPES, 5 mM glucose, 2% (wt/vol) BSA, pH 7.4, equilibrated with oxygen] containing MgSO4 and CaCl2 (final concentrations, 1 mM) at 37°C in a rotating water bath shaker. Cell viability was judged by determination of the percentage of rod-shaped cells and averaged 90-97% under all incubation conditions. Plasma and microsomal membranes from cardiac tissue were prepared as recently described by us (37). Briefly, cardiac tissue was removed and homogenized at 4°C in a buffer containing 10 mM Tris · HCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, and 2.6 mM dithiothreitol (DTT) by application of an Ultraturrax for 60 s. Homogenization was continued by 10 strokes in a glass-Teflon homogenizer followed by 3 × 3 strokes in a tight-fitting Potter-Elvehjem homogenizer. After centrifugation at 3,000 g, the supernatant was centrifuged at 200,000 g for 90 min to pellet the crude membrane fraction and obtain the cytosol (supernatant). Further purification was achieved by applying the pellet to a discontinuous gradient consisting of 0.57, 0.72, 1.07, and 1.43 M sucrose buffer and centrifugation at 40,000 g for 16 h. Membranes were harvested from each sucrose layer and stored at -70°C. Protein was determined using a modification of the Bio-Rad protein assay with BSA as a standard. Ouabain-sensitive Na+/K+-ATPase and Ca2+/K+-ATPase were used as marker enzymes for sarcolemma and microsomal membranes, respectively, and were determined as described (37). Membranes recovered from the 0.72 M sucrose layer were enriched five- to sevenfold in the activity of the Na+/K+-ATPase and were considered as a plasma membrane fraction (PM), whereas membranes obtained in the 1.07 M sucrose layer showed a 0.5-fold activity of this marker enzyme when compared with the homogenate. Furthermore, Ca2+/K+-ATPase increased fourfold in this fraction. The 1.07 M membrane fraction was henceforth termed the microsomal membrane fraction (MM). For the insulin-stimulated studies, rats received a tail vein injection of regular insulin (4 units/100 g), and hearts were removed 20 min later.

Immunoadsorption of GLUT-4-containing vesicles. GLUT-4-enriched membrane vesicles were prepared essentially as previously described (47). Briefly, microsomal membranes were incubated for 14 h with protein G agarose at 4°C in PBS, pH 7.4, containing PMSF (0.1 mM), DTT (2.6 mM), EDTA (1 mM), and BSA (0.4%). After centrifugation, GLUT-4 antiserum was added to the supernatant, and the membranes were sonicated for 20 s. Control membranes were treated identically, except that the preimmune rabbit serum was added. After a 5-h incubation at 4°C, the membranes were pelleted, resuspended, and incubated for additional 16 h with protein-G agarose. After centrifugation, the beads were washed four times with PBS, and the vesicle proteins were eluted with Laemmli sample buffer (29) for SDS-PAGE or solubilized in a PBS-buffer (pH 7.4) containing PMSF (0.1 mM), DTT (2.6 mM), EDTA (1 mM), Na3VO4 (1 mM), and 2% Triton X-100 for immunoprecipitation.

Immunoprecipitation. Isolated cardiomyocytes or cardiac tissue of basal and insulin-stimulated lean and obese Zucker rats was homogenized at 4°C in a buffer containing 50 mM Tris, 150 mM NaCl, 20 mM NaF, 10 mM EDTA, 1 mM Na3VO4, 0.3 mM PMSF, 10 mM benzamidine, 15 µM pepstatin, and Triton X-100 (1% vol/vol), pH 7.4, by application of an Ultraturrax for 30 s. Homogenization was continued by 10 strokes in a glass-Teflon homogenizer followed by 9 strokes in a tight-fitting Potter-Elvehjem homogenizer. After incubation for 2 h, the suspension was centrifuged at 16,000 g for 2 min, and the supernatant was further incubated with a mixture of protein G agarose and protein A-trisacryl beads for 2 h at 4°C. After centrifugation, the supernatant was subjected to immunoprecipitation.

For immunoprecipitation of IRS-1, the IRS-1 antibody was preadsorbed to a mixture of protein G agarose and protein A-trisacryl beads for 2 h at 4°C. The adsorbed antibody was then added to the precleared lysate and incubated for 16 h at 4°C with gentle rotation. After centrifugation, the immunopellet was washed three times with complete lysis buffer and twice with PBS.

For immunoprecipitation of PI 3-kinase from GLUT-4 vesicles with either anti-p85 or anti-p85alpha antibody, the antibodies were preadsorbed to a mixture of protein G agarose and protein A-trisacryl beads for 2 h at 4°C. The adsorbed antibodies were then added to the solubilized GLUT-4 vesicles and incubated for 16 h at 4°C with gentle rotation. After centrifugation, the immunopellet was washed three times with complete solubilization buffer and twice with PBS.

Immunoblotting. Protein samples were separated by SDS/PAGE by use of 8-18% gradient gels and were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). Membranes were blocked for 90 min in PBS, pH 7.4, containing 0.05% Tween 20 and 10% milk powder, or, for phosphotyrosine detection, for 60 min in 10 mM Tris, pH 7.5, 50 mM NaCl, 0.1% Tween 20, and 1% BSA. Thereafter, membranes were incubated for 16 h at 4°C with dilutions from 1:250 to 1:1,000 of the different antibodies. After extensive washing with blocking buffer without milk powder or BSA, membranes were incubated for 2 h with 125I-protein A (0.3 µCi/ml) or with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG. Membranes were again extensively washed and either visualized by enhanced chemiluminescence on a LumiImager work station or visualized and quantified on a FUJIX BAS 1000 bioimaging analyzer (Fuji, Japan). Significance of reported differences was evaluated using the null hypothesis and t-statistics for unpaired data.

Assay of PI 3-kinase activity. PI 3-kinase activity was measured as described previously (24). Briefly, 50 µl of a reaction mixture containing 0.2 mg/ml PI, 20 mM HEPES, pH 7.2, 0.4 mM EGTA, 0.4 mM Na2HPO4, and 10 mM MgCl2 with or without wortmannin (1 µM) were added to the immunoprecipitates. The kinase buffer was incubated with the immunoprecipitates for 5 min at room temperature, and the reaction was started by addition of [gamma -32P]ATP (40 µM and 0.2 µCi/µl). After 20 min, the reaction was stopped by the addition of 30 µl of 4 N HCl and 130 µl of chloroform-methanol (1/1). The organic phase was extracted and spotted on a silica gel thin-layer chromatography plate (Merck, Darmstadt, Germany) and was developed in chloroform-methanol-25% NH4OH-water (43:38:5:7, vol/vol). Plates were dried and subsequently visualized.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of p85 subunit variants in cardiac muscle and interaction with IRS proteins. Isoforms p85alpha and p85beta represent two widely expressed subunits of PI 3-kinase, whereas the splice variants of p85alpha exhibit a more tissue-specific expression (40). To determine the pattern of cardiac p85 expression, we used 1) a polyclonal anti-p85alpha NSH2 antiserum raised against a GST-fusion protein corresponding to the amino-terminal SH2-domain of human p85alpha , which antiserum readily detects the p50 and p55 truncated splice variants of p85alpha but does not crossreact with the beta -isoform (38); 2) an antiserum raised against the SH3-domain of p85beta with exclusive specificity for this isoform (19); and 3) a broad specific polyclonal p85-antibody (p85PAN). As shown in Fig. 1A, the p85alpha signal is clearly visible in cardiomyocyte lysates, whereas essentially no splice variants with the expected molecular mass of 48-55 kDa were observed (Fig. 1A, left lane). This observation is additionally supported by detection with the p85PAN antiserum. The p85beta isoform was detected with a slightly higher molecular mass compared with p85alpha . We therefore conclude that p85alpha and p85beta must be considered the major regulatory subunits of PI 3-kinase in the heart.


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Fig. 1.   Expression of p85 variants and effect of insulin on the association of p85alpha subunit of phosphatidylinositol (PI) 3-kinase to insulin receptor substrate (IRS)-1 and IRS-2 in cardiomyocytes of lean Zucker rats. A: cardiomyocytes were lysed, and cellular proteins (20 µg/lane) were separated by SDS-PAGE and immunoblotted with different p85 antisera using enhanced chemiluminescence (ECL) detection. B: cardiomyocytes (1 × 106 cells) were incubated in the absence or presence of insulin (350 nM) for 5 min. After cell lysis, p85, IRS-1, or IRS-2 immunoprecipitates (IP) were subjected to SDS-PAGE and transferred to nitrocellulose. Twenty micrograms of a Jurkat cell lysate was used as a control. Filters were then cut apart, and the upper part was analyzed (ID) for IRS-1 or IRS-2. The lower part of the filters was immunoblotted with anti-p85alpha NSH2 antiserum and 125I-labeled protein A. Representative blots of 4 independent experiments are presented. C: cell lysates were prepared as outlined above. Aliquots (20 µl) were then removed before and after immunoprecipitation with IRS-1 or IRS-2 antisera, respectively, and subjected to immunoblotting with the corresponding antiserum.

We then analyzed the insulin-regulated interaction of the two p85 isoforms with IRS proteins and the potential role of this interaction for PI 3-kinase activation. IRS-1 and IRS-2 were immunoprecipitated from basal and insulin-stimulated cells, and the association of p85alpha was determined by Western blotting of the immunoprecipitates (Fig. 1B). Surprisingly, insulin was completely unable to induce the recruitment of p85alpha to IRS-1 or IRS-2. Efficient immunoprecipitation of the IRS proteins was confirmed by Western blotting with IRS-1 and IRS-2 antibodies, respectively, whereas immunodetection of the p85alpha was verified on total lysates of Jurkat cells as well as on p85 immunoprecipitates from cardiac muscle (Fig. 1B). It may be argued that only a small percentage of the total IRS proteins was analyzed in the immunoprecipitates, limiting the sensitivity for p85alpha detection. However, as demonstrated in Fig. 1C, both the IRS-1 and the IRS-2 antisera were found to immunoprecipitate the IRS-1 proteins at nearly 100% efficiency. It is also worth noting that essentially the same results were obtained using shorter incubation times, ruling out a rapid dissociation of p85alpha from the IRS proteins.

To confirm the absence of IRS-1/-2 interaction with p85alpha , we immunoprecipitated p85alpha from basal and insulin-stimulated cells and analyzed the immunoprecipitates for IRS-1/-2 by Western blotting. As observed before, essentially no association of p85alpha with the two IRS proteins could be detected (Fig. 2A). Very recently (44), we reported that, in cardiomyocytes, the p85alpha subunit associates to a 200-kDa phosphoprotein in response to insulin, making it likely that, in this tissue, additional phosphoproteins different from IRS-1 and IRS-2 may serve to dock the p85alpha adaptor subunit. The question remains whether this interaction is able to enhance the intrinsic PI 3-kinase activity. We therefore determined the formation of PI-3-phosphate in p85alpha immunoprecipitates from basal and insulin-stimulated cardiomyocytes (Fig. 2B). In three separate experiments, a significant stimulation of PI 3-kinase activity to 151 ± 12% of control could be detected.


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Fig. 2.   Effect of insulin on the association of IRS-1 and IRS-2 to p85alpha and p85alpha -associated PI 3-kinase activity in cardiomyocytes of lean Zucker rats. A: cardiomyocytes were treated with insulin as outlined in Fig. 1. After cell lysis, p85alpha IP were subjected to immunoblotting with IRS-1 or IRS-2 antisera, respectively. B: cells were stimulated with insulin, and lysates were incubated with anti-p85alpha antibody. Immune complexes were subsequently collected on protein A beads, washed, and assayed for PI 3-kinase activity in the absence or presence of wortmannin. Lipid products were then separated by thin-layer chromatography followed by autoradiography, as described in MATERIALS AND METHODS. PI 3-P, PI 3-phosphatase. A representative experiment out of 3 is shown.

In our earlier studies with the use of ventricular cardiomyocytes, we reported that insulin increases the association of p85 to IRS-1 and the IRS-1-associated PI 3-kinase activity about four- to sixfold (31). To confirm that this effect is mediated by the p85beta subunit, we determined 1) the association of p85beta with IRS-1 in response to insulin and 2) the PI 3-kinase activity in p85beta and IRS-1 immunoprecipitates from basal and insulin-stimulated cells. In cardiomyocytes from lean rats, insulin was clearly able to recruit p85beta to IRS-1 (1.8 ± 0.3-fold increase over basal, n = 3), although an appreciable amount was already present in the basal state (Fig. 3A). In obese rats, no significant effect of insulin on the recruitment of p85beta to IRS-1 could be detected. It is worth noting that the insulin-stimulated tyrosine phosphorylation of IRS-1 in cardiomyocytes from obese rats is not different from that seen in the lean controls (24). The question remains whether the recruitment of PI 3-kinase to IRS-1 by the p85beta subunit will also result in changes in the intrinsic activity of the enzyme. Isoform p85beta was therefore immunoprecipitated from basal and insulin-stimulated cells, and PI 3-kinase activity was determined in the immunoprecipitates. As depicted in Fig. 3B, a three- to fourfold increase was observed in lean control cells stimulated with insulin for 5 min, with a 50% lower response in obese animals. Taking into account the actual amount of p85beta being associated to IRS-1 (Fig. 3A), it can be estimated that the increase in intrinsic activity of PI 3-kinase in response to insulin is the same in both lean and obese rats, suggesting that defective recruitment of p85beta to IRS-1 underlies the reduced responsiveness of cardiac PI 3-kinase in obese Zucker rats. A similar result was obtained for IRS-1-associated PI 3-kinase activity (Fig. 3, B and C), with a sixfold increase in lean and a threefold response in obese animals.


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Fig. 3.   Insulin-stimulated association of p85beta subunit of PI 3-kinase to IRS-1 and p85beta - and IRS-1-associated PI 3-kinase activity in cardiomyocytes from lean and obese Zucker rats. A: cardiomyocytes of lean and obese Zucker rats were incubated as outlined in Fig. 1, and IRS-1 was immunoprecipitated. Immunopellet was separated by SDS-PAGE and immunoblotted with a polyclonal anti-p85beta antiserum with ECL detection. Equal loading was ensured by probing the upper part of the filter with the IRS-1 antiserum. Representative blots are shown. B: cells were stimulated with insulin, and lysates (1.5 mg protein) were incubated with anti-p85beta or anti-IRS-1 antibody. Immune complexes were then collected on protein A beads, washed, and assayed for PI 3-kinase activity. Lipid products were separated by thin-layer chromatography followed by autoradiography, as detailed in MATERIALS AND METHODS. C: quantification of IRS-1-associated PI 3-kinase activity. Data are mean values ± SE of 4 separate experiments. *Significantly different from basal (P > 0.05).

Additional proof for a differential interaction of p85alpha and p85beta with the IRS proteins was obtained from in vivo experiments in which the animals received a tail vein injection of insulin followed by removal of the heart after 20 min. This protocol was established in our earlier investigations and was found to result in a prominent translocation of GLUT-4 to the plasma membrane (46, 47). Figure 4A confirms the results obtained with cardiomyocytes (see Fig. 1) and shows that no p85alpha adaptor subunit was detected in IRS-1 immunoprecipitates from cardiac muscle lysates either from basal or insulin-treated rats, whereas this antibody clearly revealed an immunoreactive protein in A-431 cell lysates and a p85 immunoprecipitate from cardiac muscle lysate. However, the p85beta antibody revealed an insulin-dependent association of this adaptor subunit to both IRS-1 and IRS-2 (1.9 ± 0.2-fold increase over basal, n = 3) with a blunted response in obese rats (Fig. 4, B and C). Insulin-dependent tyrosine phosphorylation of IRS-1 was monitored in IRS-1 immunoprecipitates from cardiac muscle lysates by immunodetection of phosphotyrosine proteins. In agreement with the data obtained in cardiomyocytes, no difference was observed in the efficacy of insulin-stimulated tyrosine phosphorylation of IRS-1 between lean and obese rats (data not shown).


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Fig. 4.   Association of p85beta subunit of PI 3-kinase to IRS-1 and IRS-2 in cardiac tissue under in vivo conditions. A: cardiac tissue was removed from control or insulin-treated lean Zucker rats, and immunoprecipitations were performed with either anti-IRS-1 or polyclonal anti-p85 (p85PAN) antiserum, as described in MATERIALS AND METHODS. The cell-lysate (A-431) and the immunopellets (p85 IP, IRS-1 IP) were then analyzed by SDS-PAGE and immunoblotted with a monoclonal antibody directed against p85alpha . The polyvinylidene difluoride (PVDF) sheets were incubated with horseradish peroxidase-conjugated anti-mouse IgG and processed for ECL detection. B and C: lean and obese Zucker rats were treated with insulin, and PI 3-kinase was coimmunoprecipitated with anti-IRS-1 or anti-IRS-2 antiserum, as described. The immunopellet was separated by SDS-PAGE and immunoblotted with p85beta antiserum. The PVDF sheets were incubated with [125I]protein A and submitted to autoradiography. Representative blots out of 4 independent experiments are shown.

Recruitment of p85 isoforms and PI 3-kinase activity to GLUT-4-containing vesicles in response to insulin. We then addressed the question of whether the two p85 adaptor isoforms might be involved in the recruitment of PI 3-kinase activity to intracellular compartments, potentially in an isoform-specific fashion. This was of particular interest, because only p85beta associates to IRS-1 in the heart (see above) and because IRS-1 is assumed to mediate the downstream targeting of PI 3-kinase to GLUT-4-containing vesicles (16). To examine the colocalization of PI 3-kinase and GLUT-4 and to evaluate possible alterations at this level of insulin signaling in the insulin-resistant state, lean and obese Zucker rats were treated with insulin, and GLUT-4-containing vesicles from cardiac muscle were obtained as outlined in MATERIALS AND METHODS.

Western blotting analysis of GLUT-4-containing vesicles with either the anti-p85alpha NSH2 or the p85beta antiserum demonstrated only marginal differences between nonspecific control vesicles prepared by using preimmune serum and GLUT-4-containing vesicles in the basal state of both lean and obese Zucker rats, respectively. Thus PI 3-kinase is absent from the GLUT-4 compartment in the basal state. However, in vivo insulin stimulation clearly increased the abundance of p85alpha in GLUT-4-containing vesicles of lean control rats (Fig. 5A). This effect was completely blunted in GLUT-4-containing vesicles prepared form cardiac tissue of obese animals. Quantification of p85alpha signals showed that the abundance of p85alpha in GLUT-4-containing vesicles was increased to 365 ± 65% of basal in response to insulin in lean control animals, with essentially no response in the obese group (Fig. 5B). It should be noted that we calculated the actual effect of insulin without normalization to the abundance of GLUT-4 in the vesicles, which decreased by 20-30% in lean rats (46). Despite the significant interaction with IRS-1 after insulin stimulation, the p85beta subunit remained undetectable in the GLUT-4-containing vesicles (Fig. 5).


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Fig. 5.   Colocalization of p85 isoforms and GLUT-4 in cardiac muscle of lean and obese Zucker rats. A: microsomal membranes (MM) from control or insulin-treated Zucker rats were incubated with GLUT-4-antiserum or with a nonspecific preimmune serum of the same rabbit. Immunoadsorption was then performed by addition of protein-G agarose as described in MATERIALS AND METHODS. The immunopellets were separated by SDS-PAGE and immunoblotted with antisera against p85alpha , p85beta , or GLUT-4. The PVDF sheets were incubated with horseradish peroxidase-conjugated anti-rabbit IgG and submitted to ECL detection in the case of p85alpha and beta  or incubated with [125I]protein A for GLUT-4 detection. B: quantification of 3-6 independent p85alpha immunoblot experiments. Data represent means ± SE; *significantly different from basal (P < 0.01).

As shown in Fig. 6A, insulin treatment of lean Zucker rats was also found to increase the abundance of the catalytic subunit p110 of PI 3-kinase in the GLUT-4-containing vesicles. To determine whether GLUT-4-associated PI3-kinase is activated, anti-GLUT-4-immunoadsorbed vesicles were solubilized in detergent-containing buffer, immunoprecipitated with anti-p85 IgG, and preadsorbed to a mixture of protein A/G agarose, and the p85 immunoprecipitates were subjected to a PI 3-kinase assay, as outlined in MATERIALS AND METHODS. Representative thin-layer chromatography bioimages of PI 3-kinase activity obtained from GLUT-4-containing vesicles of lean and obese rats are shown in Fig. 6B. In vivo insulin stimulation increased the enzyme activity present in the GLUT-4 vesicles by about twofold in the lean group, whereas the hormone was unable to produce any significant stimulation of PI 3-kinase activity in GLUT-4 vesicles of obese rats (Fig. 6C), in agreement with the absence of regulatory subunits under these conditions.


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Fig. 6.   Insulin-induced PI 3-kinase activity in GLUT-4-containing vesicles. GLUT-4-containing vesicles from cardiac tissue of insulin-stimulated lean and obese Zucker rats were obtained by immunoadsorption as described. A: GLUT-4-containing vesicles of lean animals were separated by SDS-PAGE and immunoblotted with a polyclonal anti-p110alpha antibody. The PVDF sheets were incubated with [125I]protein A and submitted to autoradiography. B: GLUT-4-containing vesicles were solubilized and subjected to immunoprecipitation with p85PAN antibodies, as discussed in MATERIALS AND METHODS. Immunoprecipitates were then subjected to the PI 3-kinase reaction, which was performed in the absence or presence of wortmannin (1 µmol/l). Radiolabeled lipid products were separated by thin-layer chromatography followed by autoradiography as described in MATERIALS AND METHODS. C: quantification of PI 3-kinase activity was performed with a bioimaging analyzer. Values are means ± SE (n = 4); *significantly different from basal (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Class 1 PI 3-kinase represents a key element of the insulin-signaling cascade; however, this enzyme is activated by many receptor and nonreceptor tyrosine kinases mediating a variety of effects different from insulin (1, 7, 10, 36, 40). Therefore, the question of specificity of insulin-regulated PI 3-kinase activation remains a key step to an improved understanding of insulin action. Two apparently independent characteristics determine the specificity of PI 3-kinase activation. First, a panel of regulatory adaptor subunit isoforms with a tissue-specific expression exists (40); second, the compartmentalization of the PI 3-kinase signaling complexes plays a crucial role in directing insulin action to the downstream effector sites (18, 33, 49). In the present study, we have analyzed these processes in isolated cardiomyocytes and cardiac tissue after in vivo stimulation with insulin. We now present for the first time conclusive evidence for a functionally different involvement of the p85alpha and the p85beta adaptor subunit in insulin signaling, pointing to an additional level of specificity in the insulin-PI 3-kinase pathway.

Two widely expressed isoforms of the PI 3-kinase adaptor subunit, p85alpha and p85beta , have been described (34), and these proteins were also detected in adult cardiomyocytes. However, the truncated splice variants of p85alpha , p50 and p55, were below the detection level in cardiac tissue. This differs substantially from skeletal muscle, in which the p50 variant exhibits an even higher expression than p85alpha (38). It is worth noting that we have used the p85alpha NSH2 antiserum of Ref. 38, and that this antiserum does not recognize any splice variants of p85alpha in 3T3-L1 adipocytes (33), a cell line not expressing p85beta (3). The tissue-specific expression of the adaptor subunit variants supports the notion (40) of individual functions of these proteins, most probably in a cell-specific context. The present study demonstrates such a functional difference between the p85alpha and the p85beta isoform in the heart, in that 1) only p85beta associates to IRS-1 and IRS-2 in response to insulin, 2) p85beta mediates a significant activation of PI 3-kinase, and 3) only p85alpha is recruited to GLUT-4-containing vesicles.

The two isoforms of p85 share 62% amino acid homology (34) and form stable complexes with the catalytic p110 subunit of PI 3-kinase (15). Several functional differences between p85alpha and p85beta have been reported. Thus triggering of the CD3 antigen complex in T-cells was found to induce a rapid serine phosphorylation of p85alpha , whereas p85beta remained unaffected (35). Furthermore, Giorgino et al. (13) reported a selective upregulation of p85alpha expression in L6 myoblasts, and similar but distinct insulin receptor signaling complexes of the two isoforms were observed in rat hepatoma cells (41). We show here that, in the heart, insulin-regulated activation of IRS-1-associated PI 3-kinase is mediated by p85beta , whereas p85alpha does not interact with IRS-1 or IRS-2. The latter finding may be explained by the interaction of p85alpha with a yet-unidentified phosphoprotein, pp200, which was observed in contraction- and insulin-stimulated cardiomyocytes (44). In their earlier study using transfected cell lines, Baltensperger et al. (3) hypothesized that PI 3-kinase is recruited to IRS-1 irrespective of the isoform of the regulatory subunit and that, in a second step, the intrinsic activity increases when associated with p85alpha , whereas p85beta was found to be insulin insensitive. We report here a completely different situation in a primary cell, showing that p85beta mediates a substantial activation of PI 3-kinase, in agreement with recent results in skeletal muscle (38). We therefore conclude that, in the heart, the p85beta adaptor subunit represents an essential element of insulin signaling with a function different from that of p85alpha .

A second key finding of the present investigation consists of the observation that insulin stimulation results in a significant recruitment of PI 3-kinase activity to GLUT-4-containing vesicles and that this activity is associated with the p85alpha regulatory subunit of the enzyme. The p85beta adaptor subunit could not be detected in the GLUT-4-containing vesicles despite its association with IRS-1 and IRS-2. We therefore conclude that the regulatory subunit serves to mediate the assembly of PI 3-kinase and GLUT-4-vesicles in this downstream-signaling complex of insulin action. Our data fit with a scenario in which the two isoforms of the p85 adaptor subunit of PI 3-kinase mediate diversification of cardiac insulin signaling in that 1) p85alpha recruits PI 3-kinase activity to the GLUT-4-vesicles and thus participates in GLUT-4 translocation, and 2) p85beta serves to activate PI 3-kinase upstream of the translocation process. This latter reaction will lead to the stimulation of protein kinase B (PKB)/Akt, which is known to regulate cardiac glycolysis (6), protein synthesis, and gene expression (1).

Although it is well accepted that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular membranes (33, 49), the issue of PI 3-kinase colocalizing to GLUT-4 has remained controversial (5, 12, 16, 26, 27, 48). Heller-Harrison et al. (16) reported that, in response to insulin, activated complexes of IRS-1 · PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT-4-containing vesicles prepared from 3T3-L1 adipocytes. They also showed that insulin treatment caused a two- to threefold increase in the amount of the regulatory subunit p85 and the enzymatic activity of PI 3-kinase in GLUT-4-containing vesicles prepared from these cells, in excellent agreement with our observations in the heart. Klip's group [Wang et al. (48)] confirmed the presence of PI 3-kinase activity in GLUT-4-containing vesicles obtained from 3T3-L1 adipocytes and suggested that actin filaments might be involved in the relocalization of PI 3-kinase to GLUT-4 vesicles. However, in skeletal muscle, PI 3-kinase was shown to be absent from GLUT-4-containing vesicles (26, 27), although it is generally believed that GLUT-4-containing vesicles from these two tissues are very similar (21). The present study on cardiac muscle clearly shows that, in this muscle tissue, the PI 3-kinase can be found in GLUT-4-containing vesicles in response to insulin. As outlined by Kristiansen et al. (26), this may be explained by the tissue-dependent copurification of cytoskeletal elements in the GLUT-4 vesicle preparation, since the cytoskeletal network may participate in binding and directing PI 3-kinase to the GLUT-4 vesicles. The precise function of PI 3-kinase activity in the GLUT-4-containing vesicles and its relationship to GLUT-4 translocation is yet unknown. One possibility relates to the recent report that Akt-2 associates to GLUT-4-containing vesicles and phosphorylates their component proteins in response to insulin (28). In this context, the production of phosphatidylinositol 3,4,5-trisphosphates and phosphatidylinositol 3,4-bisphosphates by PI 3-kinase may serve as binding sites for Akt (11). It is worth noting that we also observe the insulin-dependent recruitment of Akt-2 to our GLUT-4 vesicle preparation obtained from cardiac muscle (22), using the same in vivo protocol as described here for the recruitment of PI 3-kinase. Certainly, additional mechanisms, like binding of phosphoinositides to proteins that mediate intracellular vesicle trafficking, must also be considered. Glucose-regulated protein-1 and cytohesin-1, two proteins that bind phosphoinositides through a pleckstrin homology domain and display a region of high sequence similarity to the yeast Sec7 protein, represent recently identified members of this family (23, 30).

The data obtained in insulin-resistant Zucker rats lend further support to the notion that PI 3-kinase activity within or in association with the GLUT-4-containing vesicles may serve to trigger the trafficking process. The complete lack of PI 3-kinase activity in GLUT-4-containing vesicles of obese rats and the inability of insulin to recruit p85alpha to the vesicles correlate with our earlier observations of a complete block of insulin-stimulated GLUT-4 translocation in cardiac muscle of these animals (46) and makes it likely that the defective relocalization of PI 3-kinase to GLUT-4 vesicles represents an essential element of the insulin-resistant state. The molecular basis of this defect remains undefined; however, it must be different from defects at the level of IRS-1 and IRS-2, because cardiac p85alpha does not interact with these proteins. Earlier studies had shown a defect in PI 3-kinase activation by insulin in muscle tissue of obese rodents (17) and patients (14); however, these defects were attributed to a diminished tyrosine phosphorylation of IRS-1 and are located upstream of GLUT-4 translocation. As described here and in our earlier study (24), we do not see any alteration in the insulin-regulated tyrosine phosphorylation of IRS-1 in cardiac tissue of obese Zucker rats. Instead, we have reported the hyperphosphorylation of IRS-1 on serine/threonine leading to a disturbed PI 3-kinase activation in cardiomyocytes of obese Zucker rats (24). Consistently, we show here a reduced IRS-1/p85beta PI 3-kinase activity in this animal model, most probably resulting from a defective recruitment of PI 3-kinase to IRS-1.

In summary, the present study shows that, in cardiac muscle, the two regulatory subunit isoforms of PI 3-kinase, p85alpha and p85beta , have different functional implications for mediating downstream insulin signaling. Recruitment of p85alpha and PI 3-kinase activity to GLUT-4-containing vesicles in response to insulin and the complete loss of this effect in the insulin resistance of obesity make it likely that an adaptor isoform-specific compartmentalization of PI 3-kinase represents an additional level of specificity in insulin signaling.


    ACKNOWLEDGEMENTS

This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Bundesministerium für Gesundheit, the Deutsche Forschungsgemeinschaft (SFB 351 C2), EU COST Action B5, and BIOMED Concerted Action 3084. D. M. Ouwens is supported by the Royal Netherlands Academy of Sciences (Casimir-Ziegler Fellowship) and by the Netherlands Organization for Scientific Research (NWO GB-MW).


    FOOTNOTES

Address for reprint requests and other correspondence: J. Eckel, Diabetes Research Institute, Auf'm Hennekamp, D-40225 Düsseldorf, Germany (E-mail: eckel{at}uni-duesseldorf.de).

Current address of I. Uphues: Boehringer Ingelheim Pharma KG, 88397 Biberach, Germany; current address of D. M. Ouwens: Dept. of Molecular Cell Biology, Leiden University Medical Center, 2333 AL Leiden, The Netherlands.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 February 2000; accepted in final form 19 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 280(1):E65-E74
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