beta(3)-adrenergic stimulation differentially inhibits insulin signaling and decreases insulin-induced glucose uptake in brown adipocytes

Activity of the sympathetic nervous system is an important factor involved in the pathogenesis of insulin resistance and associated metabolic and vascular abnormalities. In this study, we investigate the molecular basis of cross-talk between beta(3)-adrenergic and insulin signaling systems in mouse brown adipocytes immortalized by SV40 T infection. Insulin-induced tyrosine phosphorylation of the insulin receptor, insulin receptor substrate 1 (IRS-1), and IRS-2 was reduced by prestimulation of beta(3)-adrenergic receptors (CL316243). Similarly, insulin-induced IRS-1-associated and phosphotyrosine-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity, but not IRS-2-associated PI 3-kinase activity, was reduced by beta(3)-adrenergic prestimulation. Furthermore, insulin-stimulated activation of Akt, but not mitogen-activated protein kinase, was diminished. Insulin-induced glucose uptake was completely inhibited by beta(3)-adrenergic prestimulation. These effects appear to be protein kinase A-dependent. Furthermore inhibition of protein kinase C restored the beta(3)-receptor-mediated reductions in insulin-induced IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity. Together, these findings indicate cross-talk between adrenergic and insulin signaling pathways. This interaction is protein kinase A-dependent and, at least in part, protein kinase C-dependent, and could play an important role in the pathogenesis of insulin resistance associated with sympathetic overactivity and regulation of brown fat metabolism.     

Membrane-targeted phosphatidylinositol 3-kinase mimics insulin actions and induces a state of cellular insulin resistance.

Phosphatidylinositol (PI) 3-kinase plays an important role in various insulin-stimulated biological responses including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between PI 3-kinase and these biological responses is still unclear. We have investigated whether targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes is sufficient and necessary to induce PI 3-kinase dependent signaling responses, characteristic of insulin action. We overexpressed Myc-tagged, membrane-targeted p110 (p110(CAAX)), and wild-type p110 (p110(WT)) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer. Overexpressed p110(CAAX) exhibited approximately 2-fold increase in basal kinase activity in p110 immunoprecipitates, that further increased to approximately 4-fold with insulin. Even at this submaximal PI 3-kinase activity, p110(CAAX) fully stimulated p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas, p110(WT) had little or no effect on these downstream effects. Interestingly p110(CAAX) did not activate MAP kinase, despite its stimulation of p21(ras). Surprisingly, p110(CAAX) did not increase basal glycogen synthase activity, and inhibited insulin stimulated activity, indicative of cellular resistance to this action of insulin. p110(CAAX) also inhibited insulin stimulated, but not platelet-derived growth factor-stimulated mitogen-activated protein kinase phosphorylation, demonstrating that the p110(CAAX) induced inhibition of mitogen-activated protein kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells. Moreover, p110(CAAX) stimulated IRS-1 Ser/Thr phosphorylation, and inhibited IRS-1 associated PI 3-kinase activity, without affecting insulin receptor tyrosine phosphorylation, suggesting that it may play an important role as a negative regulator for insulin signaling. In conclusion, our studies show that membrane-targeted PI 3-kinase can mimic a number of biologic effects normally induced by insulin. In addition, the persistent activation of PI 3-kinase induced by p110(CAAX) expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact.     

Akt mediates sequestration of the beta(2)-adrenergic receptor in response to insulin.

The counterregulation of catecholamine action by insulin includes insulin-stimulated sequestration of the beta(2)-adrenergic receptor. Herein we examined the signaling downstream of insulin receptor activation, focusing upon the role of 1-phosphatidylinositol 3-kinase and the serine-threonine protein kinase Akt (also known as protein kinase B) in the internalization of beta(2)-adrenergic receptors. Inhibition of 1-phosphatidylinositol 3-kinase by LY294002 blocks insulin-induced sequestration of the beta(2)-adrenergic receptor, implicating Akt in downstream signaling to the beta(2)-adrenergic receptor. Phosphorylation studies of the C-terminal cytoplasmic domain of the beta(2)-adrenergic receptor by Akt in vitro identified Ser(345) and Ser(346) within a consensus motif for Akt phosphorylation. Double mutation (i.e. S345A/S346A) within this motif abolishes insulin counterregulation of beta-adrenergic stimulation of cyclic AMP accumulation as well as insulin-stimulated sequestration. Furthermore, expression of constitutively activated Akt (T308D/S473D) mimics insulin action on cyclic AMP responses and beta(2)-adrenergic receptor internalization. Expression of the dominant-negative version of Akt (K179A/T308A/S473A), in contrast, abolishes both insulin counterregulation of the cyclic AMP response as well as insulin-stimulated sequestration of the beta(2)-adrenergic receptor. The action of the serine-threonine protein kinase Akt in insulin counterregulation mirrors the central role of protein kinase A in beta-agonist-induced desensitization.     

A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.     

Insulin-induced up-regulated uncoupling protein-1 expression is mediated by insulin receptor substrate 1 through the phosphatidylinositol 3-kinase/Akt signaling pathway in fetal brown adipocytes

To investigate the role of insulin receptor substrate-1 (IRS-1) and its downstream signaling in insulin-induced thermogenic differentiation of brown adipocytes, we have reconstituted IRS-1-deficient fetal brown adipocytes (IRS-1(-/-)) with wild-type IRS-1 (IRS-1(wt)). The lack of IRS-1 resulted in the inability of insulin to induce IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity and Akt phosphorylation in IRS-1(-/-) brown adipocytes. In addition, these cells showed an impairment in activating alpha-Akt, beta-Akt, and gamma-Akt isoforms upon insulin stimulation. Reconstitution of IRS-1(-/-) brown adipocytes with IRS-1(wt) restored the IRS-1/PI 3-kinase/Akt signaling pathway. Treatment of wild-type brown adipocytes with insulin for 24 h up-regulated uncoupling protein-1 (UCP-1) expression and transactivated the UCP-1 promoter; this effect was abolished in the absence of IRS-1 or in the presence of an Akt inhibitor and further recovered after IRS-1(wt) reconstitution. Neither UCP-2 nor UCP-3 was up-regulated by insulin in wild-type and IRS-1-deficient brown adipocytes. Insulin stimulated the expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) and its DNA binding activity in wild-type brown adipocytes but not in IRS-1(-/-) cells. However, insulin stimulation of both C/EBPalpha expression and binding activity was restored after IRS-1(wt) reconstitution of deficient cells. Retrovirus-mediated expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma in IRS-1(-/-) brown adipocytes up-regulated UCP-1 protein content and transactivated UCP-1 promoter regardless of insulin stimulation. Both C/EBPalpha and peroxisome proliferator-activated receptor gamma reconstituted FAS mRNA expression, but only C/EBPalpha restored insulin sensitivity in the absence of IRS-1. Finally, reconstitution of IRS-1(-/-) brown adipocytes with the IRS-1 mutants IRS-1(Phe-895), which lacks IRS-1/growth factor receptor binding protein 2 binding but not IRS-1/p85-PI 3-kinase binding, or with IRS-1(Tyr-608/Tyr-628/Tyr-658), which only binds p85-PI 3-kinase, induced UCP-1 expression and transactivated the UCP-1 promoter. These data provide strong evidence for an essential role of IRS-1 through the PI 3-kinase/Akt signaling pathway inducing UCP-1 gene expression by insulin.     

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.     

Diversification of cardiac insulin signaling involves the p85 alpha/beta subunits of phosphatidylinositol 3-kinase

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 p85 alpha and p85 beta 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 p85 alpha and p85 beta but no detectable amounts of the splice variants of p85 alpha. Essentially no p85 alpha 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 p85 beta associated with IRS-1, leading to a three- to fourfold increase in p85 beta-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 p85 alpha was observed after insulin stimulation of lean animals, with no significant effect in the obese group. No p85 beta 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, p85 alpha recruits PI 3-kinase activity to GLUT-4 vesicles, whereas p85 beta 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 p85 alpha and the p85 beta adaptor subunits, may contribute to cardiac insulin resistance.     

Association of phosphatidylinositol 3-kinase composed of p110beta-catalytic and p85-regulatory subunits with the small GTPase Rab5.

A family of phosphatidylinositol 3-kinases (PI 3-kinase), comprising three major classes (I-III) in terms of substrate specificity and regulation, play important roles in a variety of cell functions. We previously reported that the class-I heterodimeric PI 3-kinase consisting of p110beta-catalytic and p85-regulatory subunits is synergistically activated by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating trimeric G proteins. Here we report an additional unique feature of the p110beta/p85 PI 3-kinase. The small GTPase Rab5 was identified as a binding protein for the p110beta-catalytic subunit in a yeast two-hybrid screening system. The interaction appears to require at least two separated amino-acid sequences present specifically in the beta isoform of p110 and the GTP-bound form of Rab5. The expressions of constitutively active and dominant negative mutants of Rab5 in THP-1 cells induce the stimulation and inhibition, respectively, of protein kinase B activity, which is dependent on the PI 3-kinase product phosphatidylinositol 3,4,5-triphosphate. These results suggest that there is a specific interaction between GTP-bound Rab5 and the p110beta/p85 PI 3-kinase, leading to efficient coupling of the lipid kinase product to its downstream target, protein kinase B.     

Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signaling

Class IA phosphoinositide (PI) 3-kinase is composed of a p110 catalytic subunit and a p85 regulatory subunit and plays a pivotal role in insulin signaling. To explore the physiological roles of two major regulatory isoforms, p85 alpha and p85 beta, we have established brown adipose cell lines with disruption of the Pik3r1 or Pik3r2 gene. Pik3r1-/- (p85 alpha-/-) cells show a 70% reduction of p85 protein and a parallel reduction of p110. These cells have a 50% decrease in PI 3-kinase activity and a 30% decrease in Akt activity, leading to decreased insulin-induced glucose uptake and anti-apoptosis. Pik3r2-/- (p85 beta-/-) cells show a 25% reduction of p85 protein but normal levels of p85-p110 and PI 3-kinase activity, supporting the fact that p85 is more abundant than p110 in wild type. p85 beta-/- cells, however, exhibit significantly increased insulin-induced Akt activation, leading to increased anti-apoptosis. Reconstitution experiments suggest that the discrepancy between PI 3-kinase activity and Akt activity is at least in part due to the p85-dependent negative regulation of downstream signaling of PI 3-kinase. Indeed, both p85 alpha-/- cells and p85 beta-/- cells exhibit significantly increased insulin-induced glycogen synthase activation. p85 alpha-/- cells show decreased insulin-stimulated Jun N-terminal kinase activity, which is restored by expression of p85 alpha, p85 beta, or a p85 mutant that does not bind to p110, indicating the existence of p85-dependent, but PI 3-kinase-independent, signaling pathway. Furthermore, a reduction of p85 beta specifically increases insulin receptor substrate-2 phosphorylation. Thus, p85 alpha and p85 beta modulate PI 3-kinase-dependent signaling by multiple mechanisms and transmit signals independent of PI 3-kinase activation.     

Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes.

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for many insulin-induced metabolic and mitogenic responses. However, it is unclear whether PI3K activation is sufficient for any of these effects. To address this question we increased PI3K activity in differentiated 3T3-L1 adipocytes by adenovirus-mediated expression of both the inter-SH2 region of the regulatory p85 subunit of PI3K (iSH2) and the catalytic p110 alpha subunit (p110). Coexpression resulted in PI3K activity that exceeded insulin-stimulated activity by two- to fivefold in cytosol, total membranes, and the low density microsome (LDM) fraction, the site of greatest insulin stimulation. While insulin increased glucose transport 15-fold, coexpression of iSH2-p110 increased transport (5.2-) +/- 0.7-fold with a parallel increase in GLUT4 translocation to the plasma membrane. Constitutive activation of PI3K had no effect on maximally insulin-stimulated glucose transport. Neither basal nor insulin-stimulated activity of glycogen synthase or mitogen-activated protein kinase was altered by iSH2-p110 coexpression. DNA synthesis was increased twofold by insulin in control 3T3-L1 adipocytes transduced with beta-galactosidase-encoding recombinant adenovirus, while iSH2-p110 coexpression increased DNA synthesis fivefold. These data indicate that (i) increased PI3K activity is sufficient to activate some but not all metabolic responses to insulin, (ii) activation of PI3K to levels exceeding the effect of insulin in adipocyte LDM results in only a partial stimulation of glucose transport, and (iii) increased PI3K activity in the absence of growth factor or oncoprotein stimulation is a potent stimulus of DNA synthesis.     

DHEA administration increases brown fat uncoupling protein 1 levels in obese OLETF rats

We found that UCP-1 and UCP-3 mRNA expression levels and the UCP-1 protein content in brown adipose tissue (BAT) were reduced in prediabetic OLETF rats than the lean LETO rats. Administration of dehydroepiandrosterone (DHEA) for 17 days induced remarkable weight loss, which was in part attributed to an enhanced utilization of ingested energy. DHEA administration significantly increased the levels of BAT UCP-1 and UCP-3 mRNA expression. Among the upstream signals for UCP-1 regulation, expression levels of the beta 3 adrenergic receptor (beta(3)AR) and PPAR gamma coactivator-1 (PGC-1) were significantly decreased in the OLETF rats and increased by DHEA administration. The decreased expression levels of UCP-1 and its upstream regulators, beta(3)AR and PGC-1, in BAT may contribute to inefficient energy utilization and obesity in OLETF rats, which was corrected by DHEA treatment.     

Dissociation of PTPase levels from their modulation of insulin receptor signal transduction

Protein tyrosine phosphatases have been implicated in the regulation of receptor tyrosine kinase signalling pathways, including that of the insulin receptor. Here, cell density-dependent changes in PTPase expression have been exploited to investigate the relationship between cellular PTPase levels and the insulin receptor signal transduction pathway. Increasing cell density (20%, 50%, and >90%) in the rat McA-RH7777 hepatoma cell line resulted in increased protein expression of the receptor-like PTPase LAR (14-fold), and the nonreceptor PTPases PTP1B (11-fold) and SHP2 (10-fold). Each of these PTPases has previously been implicated in regulating insulin receptor signal transduction. Despite these marked increases, maximum insulin receptor autophosphorylation as well as receptor expression actually increased 2-fold. MAP kinase also increased approximately 2-fold as a function of cell density and paralleled increases in expression levels. Neither sensitivity nor maximum responsiveness to insulin were decreased at increasing cell densities as assessed by activation of PI 3-kinase. Duration of response was also unimpaired. These results suggest that expression levels of relevant PTPases are not the primary determinant in their modulation of insulin receptor kinase activity. Restricted accessibility at the molecular level or involvement of accessory proteins may be more critical parameters.     

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism.

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.     

Insulin stimulates spreading of skeletal muscle cells involving the activation of focal adhesion kinase,phosphatidylinositol 3-kinase and extracellular signal regulated kinases

Insulin plays an important role in muscle cell survival and proliferation. However, there is no report showing the role of insulin in spreading of muscle cells. In the present report, we showed that insulin enhances muscle cell spreading concomitant with enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Moreover, insulin can stimulate the cell spreading even in presence of integrin alpha5 blockers although to a lesser extent as compared to control. Cell adhesion was not dependent on insulin and serum, and decreased in presence of integrin blockers. We found direct association of FAK with affinity purified insulin receptors using in vitro kinase assay. The increase in FAK tyrosine phosphorylation was associated with increase in its kinase activity and further supported by increased phosphotyrosine accumulation on focal adhesions and increased membrane localization of FAK after stimulation by insulin. Moreover, insulin-mediated muscle cell spreading was dependent upon phosphatidylinositol 3-kinase (PI 3-kinase) activity. PI 3-kinase activity was found to be associated with FAK and the FAK associated PI 3-kinase activity enhanced when cells were plated in presence of insulin. We also observed activation of MAP kinases, i.e., ERK-1/-2 during insulin mediated muscle cell spreading. In conclusion, FAK, PI 3-kinase, and MAP kinase are important components of pathway(s) that regulate insulin stimulated muscle cell spreading.     

Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.     

Over-expression of the p110beta but not p110alpha isoform of PI 3-kinase inhibits motility in breast cancer cells

Phosphoinositide 3-kinase (PI 3-kinase) activity is required for growth factor-induced cytoskeletal regulation and cell migration. We previously found that in MTLn3 rat adenocarcinoma cells, EGF-stimulated induction of actin barbed ends and lamellipod extension specifically requires the p85/p110alpha isoform of PI 3-kinase. To further characterize signaling by distinct PI 3-kinase isoforms, we have developed MTLn3 cells that transiently or stably overexpress either p110alpha or p110beta. Transient overexpression of p110beta inhibited EGF-stimulated lamellipod extension, whereas p110alpha-transfected cells showed normal EGF-stimulated lamellipod extension. Similar results were obtained by overexpression of kinase-dead p110beta, suggesting that effects on cytoskeletal signaling were due to competition with p85/p110alpha complexes. Stable overexpression of p110alpha appeared to be toxic, based on the difficulty in obtaining stable overexpressing clones. In contrast, cells expressing a 2-fold increase in p110beta were readily obtainable. Interestingly, cells stably expressing p110beta showed a marked inhibition of EGF-stimulated lamellipod extension. Using computer-assisted analysis of time-lapse images, we found that overexpression of p110beta caused a nearly complete inhibition of motility. Cells overexpressing p110beta showed normal activation of Akt and Erk, suggesting that overall PI 3-kinase signaling was intact. A chimeric p110 molecule containing the p85-binding and Ras-binding domains of p110alpha and the C2, helical, and kinase domains of p110beta, was catalytically active yet also inhibited EGF-stimulated lamellipod extension. These data highlight the differential signaling by distinct p110 isoforms. Identification of effectors that are differently regulated by p110alpha versus p110beta will be important for understanding cell migration and its role in metastasis.