Mutations in the juxtamembrane region of the insulin receptor impair activation of phosphatidylinositol 3-kinase by insulin.

CHO/IRF960/T962 cells express a mutant human insulin receptor in which Tyr960 and Ser962 in the juxtamembrane region of the receptor's beta-subunit are replaced by Phe and Thr, respectively. The mutant insulin receptor undergoes autophosphorylation normally in response to insulin; however, insulin fails to stimulate thymidine incorporation into DNA, glycogen synthesis, and tyrosyl phosphorylation of an endogenous substrate pp185 in these cells. Another putative substrate of the insulin receptor tyrosine kinase is phosphatidylinositol 3-kinase (Ptdlns 3-kinase). We have previously shown that Ptdlns 3-kinase activity in Chinese hamster ovary cells expressing the wild-type human insulin receptor (CHO/IR) increases in both antiphosphotyrosine [anti-Tyr(P)] immunoprecipitates and intact cells in response to insulin. In the present study a new technique (detection of the 85-kDa subunit of Ptdlns 3-kinase using [32P]phosphorylated polyoma virus middle T-antigen as probe) is used to monitor the Ptdlns 3-kinase protein. The 85-kDa subunit of Ptdlns 3-kinase is precipitated by anti-Tyr(P) antibodies from insulin-stimulated CHO/IR cells, but markedly less protein is precipitated from CHO/IRF960/T962 cells. The amount of Ptdlns 3-kinase activity in the immunoprecipitates was also reduced in the CHO/IRF960/T962 cells compared to CHO/IR cells. In intact CHO/IRF960/T962 cells, insulin failed to stimulate phosphate incorporation into one of the products of activated Ptdlns 3-kinase, phosphatidylinositol-3,4-bisphosphate [Ptdlns(3,4)P2], whereas it caused a 12-fold increase in CHO/IR cells. In contrast, phosphate incorporation into another product, phosphatidylinositol trisphosphate [PtdlnsP3], was only partially depressed in the CHO/IRF960/T962 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidylinositol-3-kinase is a non-tyrosine phosphorylated member of the insulin receptor signalling complex.

In rat HTC cells expressing a large number of human insulin receptors, insulin stimulated phosphatidylinositol-3-kinase (PI-3-kinase) activity. This activity was more effectively immunoprecipitated with anti-phosphotyrosine antibody (alpha-PY) than with anti-insulin receptor antibody (alpha-IR), suggesting that PI-3-kinase was not directly associated with the insulin receptor. alpha-PY immunoprecipitable PI-3 kinase activity, which was regulated by insulin, corresponded to a small pool of the total cellular PI-3-kinase activity. PI-3-kinase was not directly tyrosine phosphorylated by insulin treatment. A comparison of both catalytic activity and content of PI-3-kinase in alpha-PY immunoprecipitates indicated that after insulin treatment PI-3-kinase activity was enhanced by its association with tyrosine phosphorylated proteins. These studies suggest therefore that PI-3-kinase is a non-tyrosine phosphorylated member of the insulin receptor signalling complex.     

Insulin-dependent alterations of phorbol ester binding to adipocyte subcellular constituents. Evidence for the involvement of protein kinase C in insulin action

The binding of tritiated phorbol-12,13-dibutyrate (3H-PBu2) was employed to estimate the mass of protein kinase C associated with plasma membranes and cytosol isolated from untreated and insulin-treated adipocytes. Binding of 3H-PBu2 to both plasma membranes and cytosol was rapid, achieving a steady state within minutes. Treatment of cells with physiological concentration of insulin (0.67 nM) caused a 42% increase (from 0.92 +/- 0.08 to 1.30 +/- 0.12 pmol 3H-PBu2/mg protein, p less than 0.0001) and a 27% decrease (from 0.41 +/- 0.07 to 0.30 +/- 0.05 pmol 3H-PBu2/mg protein, p less than 0.020) in phorbol ester bound to cytosol and plasma membranes, respectively. The half-maximal concentrations of unlabelled PBu2 needed to displace 3H-PBu2 bound to cytosol from control and insulin-treated cells were 54 and 13 pM, respectively. These data indicate that insulin modifies protein kinase C in adipocytes.     

Insulin rapidly increases glycerol-3-phosphate-acyltransferase activity in rat adipocytes.

Glycerol-3-phosphate acyltransferase (G3PAT) was activated by insulin in intact rat adipocytes within 1 min: this activation persisted for 10 min, and was due to a decrease in the Km of the enzyme. The addition of insulin to control adipocyte membranes also increased G3PAT activity, and this effect was mimicked by phosphatidylinositol-specific phospholipase C. Cytosol fractions from insulin-treated adipocytes stimulated G3PAT activity of control membranes, suggesting that a soluble mediator is released during insulin action, possibly through activation of a PI-specific PLC.     

Phosphatidylinositol 4-kinase is a component of glucose transporter (GLUT 4)-containing vesicles.

We recently developed a procedure for immunoisolating insulin-responsive membrane vesicles that contain the muscle/fat glucose transporter isoform, GLUT 4, from rat adipocytes. Utilizing this methodology, we are analyzing the components of these vesicles to gain an understanding of how they are regulated by insulin. In this report we identify a phosphatidylinositol (PtdIns) 4-kinase as a constituent of glucose transporter vesicles (GTVs). This kinase has the biochemical and immunological properties of a type II PtdIns 4-kinase as classified by Endeman et al. (Endemann, G., Dunn, S. N., and Cantley, L. C. (1987) Biochemistry 26, 6845-6852). A monoclonal antibody, 4C5G, which specifically inhibits the type II PtdIns 4-kinase, suppresses 80% of the GTV-PtdIns 4-kinase activity. In addition, the GTVs-PtdIns 4-kinase is maximally activated by the nonionic detergent Triton X-100, at a concentration of 0.2% and is inhibited by adenosine with a Ki of approximately 20-30 microM. We find that the GTVs do not contain any PtdIns4P 5-kinase or diacylglycerol kinase activities, whereas these activities were detected in the plasma membrane. An analysis of the subcellular distribution of PtdIns 4-kinase activity in the rat adipocyte shows that there are similar levels of activity in GTVs, plasma membranes, and the high and low density microsomal fractions, whereas the mitochondria- and nuclei-containing fractions have less than 5% of the activity seen in other fractions. Low density microsomes were subfractionated by sucrose density gradient centrifugation and PtdIns 4-kinase activity was found to correlate closely with the distribution of membrane protein, indicating that the activity is equally distributed throughout this heterogenous population of membranes. PtdIns 4-kinase activity measured in GTVs, plasma membranes, and low density microsomes, was not affected by prior treatment of the intact adipocytes with 35 nM insulin. We postulate that while the GTV-PtdIns 4-kinase is not regulated by insulin, it may play a role in defining the fusogenic properties necessary to mediate membrane movement between the GTVs, plasma membranes, and microsomes.     

Intracellular Localization of Phosphatidylinositide 3-kinase and Insulin Receptor Substrate-1 in Adipocytes: Potential Involvement of a Membrane Skeleton

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1–PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.     

Overexpression of Fer increases the association of tyrosine-phosphorylated IRS-1 with P85 phosphatidylinositol kinase via SH2 domain of Fer in transfected cells

We have reported that the protein-tyrosine kinase Fer is associated with signaling complexes containing insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI-3 kinase) in insulin-stimulated 3T3-L1 adipocytes [J. Biol. Chem. 275 (50) (2000) 38995]. We examined the subcellular localization of this complex in 3T3-L1 adipocytes and performed transfection study to know how this complex is formed. Interestingly we have detected that this complex is formed in LDM of insulin-stimulated 3T3-L1 adipocytes, which may be important for specific biological insulin effect. Based on transfection study, we have demonstrated that overexpression of both Fer and IRS-1 can induce Fer/IRS-1/P85 complexes without insulin stimulation and SH2 domain of Fer is essential for this complex. We have also demonstrated that Fer was an efficient substrate for insulin receptor kinase. Taken together, these data suggested that Fer may play a critically important role to form Fer/IRS-1/P85 complex in LDM of insulin-stimulated adipocytes and elicit biological effect through PI-3 kinase activity in LDM.     

Functional interactions of phosphatidylinositol 3-kinase with GTPase-activating protein in 3T3-L1 adipocytes.

The role of phosphatidylinositol (PI) 3-kinase in specific aspects of insulin signaling was explored in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity by LY294002 or wortmannin significantly enhanced basal and insulin-stimulated GTPase-activating protein (GAP) activity in 3T3-L1 adipocytes. Furthermore, removal of the inhibitory influence of PI 3-kinase on GAP resulted in dose-dependent decreases in the ability of insulin to stimulate p21ras. This effect was specific to adipocytes, as inhibition of PI 3-kinase did not influence GAP in either 3T3-L1 fibroblasts, Rat-1 fibroblasts, or CHO cells. Immunodepletion of either of the two subunits of the PI 3-kinase (p85 or p110) yielded similar activation of GAP, suggesting that catalytic activity of p110 plays an important role in controlling GAP activity in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity in 3T3-L1 adipocytes resulted in abrogation of insulin-stimulated glucose uptake and thymidine incorporation. In contrast, effects of insulin on glycogen synthase and mitogen-activated protein kinase activity were inhibited only at higher concentrations of LY294002. It appears that in adipocytes, P1 3-kinase prevents activation of GAP. Inhibition of PI 3-kinase activity or immunodepletion of either one of its subunits results in activation of GAP and decreases in GTP loading of p21ras.     

Dissociation of insulin-stimulated glucose transport from the translocation of glucose carriers in rat adipose cells

Cycloheximide, a potent inhibitor of protein synthesis, has been used to examine the relationship between recruitment of hexose carriers and the activation of glucose transport by insulin in rat adipocytes. Adipocytes were preincubated +/- cycloheximide for 90 min then +/- insulin for a further 30 min. We measured 3-O-methylglucose uptake in intact cells and in isolated plasma membrane vesicles. The concentration of glucose transporters in plasma membranes and low density microsomes was measured using a cytochalasin B binding assay. Cycloheximide had no affect on basal or insulin-stimulated 3-O-methylglucose uptake in intact cells or in plasma membrane vesicles. However, the number of glucose carriers in plasma membranes prepared from cells incubated with cycloheximide and insulin was markedly reduced compared to that from cells incubated with insulin alone (14 and 34 pmol/mg protein, respectively). Incubation of cells with cycloheximide alone did not change the concentration of glucose carriers in either plasma membranes or in low density microsomes compared to control cells. When isolated membranes were analyzed with an antiserum prepared against human erythrocyte glucose transporter, decreased cross-reactivity was observed in plasma membranes prepared from cycloheximide/insulin-treated cells compared to those from insulin cells. The present findings indicate that incubation of adipocytes with cycloheximide greatly reduces the number of hexose carriers in the plasma membrane of insulin-stimulated cells. Despite this reduction, insulin is still able to maximally stimulate glucose uptake. Thus, these data suggest an apparent dissociation between insulin stimulation of glucose transport activity and the recruitment of glucose carriers by the hormone.     

Insulin stimulates phosphatidylinositol-3-kinase activity in rat adipocytes.

Phosphatidylinositol (PtdIns) 3-kinase is thought to participate in the signal transduction pathways initiated by the activation of receptor tyrosine kinases including the insulin receptor. To approach the physiological relevance of this enzyme in insulin signaling, we studied the activation of PtdIns-3-kinase in adipocytes, a major insulin target tissue for glucose transport and utilisation. To analyze possible interactions of the enzyme with cellular proteins, immunoprecipitations with the following antibodies were performed: (a) anti-phosphotyrosine antibodies, (b) two antibodies to the 85-kDa subunit of PtdIns-3-kinase (p85) and (c) an antibody to the 185-kDa major insulin receptor substrate (p185). We show that in cell extracts from adipocytes exposed to insulin, and after immunoprecipitation with an anti-phosphotyrosine antibody and an antibody to p85, we are able to detect a PtdIns-3-kinase activity stimulated by the hormone. Similarly, after immunoprecipitation with an antibody to p185, an increase in the PtdIns-3-kinase activity could be demonstrated. Taken together these results suggest that, upon insulin stimulation of fat cells, PtdIns-3-kinase itself is tyrosine phosphorylated and/or associated with an insulin receptor substrate, such as p185, which could function as a link between the insulin receptor and PtdIns-3-kinase. The PtdIns-3-kinase was activated within 1 min of exposure to insulin, and the half-maximal effect was reached at the same concentration, i.e. 3 nM, as for stimulation of the insulin receptor kinase. Subcellular fractionation showed that PtdIns-3-kinase activity was found both in the membranes and in the cytosol. Further, immunoprecipitation with an antibody to p85, which possesses the capacity to activate PtdIns-3-kinase, suggests that the presence of the enzyme in the membrane may be due to an insulin-induced recruitment of the PtdIns-3-kinase from the cytosol to the membrane. Finally, we used isoproterenol, which exerts antagonistic effects on insulin action. This drug was found to inhibit both the PtdIns-3-kinase and the insulin receptor activation by insulin, suggesting that the activation of the PtdIns-3-kinase was closely regulated by the insulin receptor tyrosine kinase. The occurrence of an insulin-stimulated PtdIns-3-kinase in adipocytes leads us to propose that this enzyme might be implicated in the generation of metabolic responses induced by insulin.     

Characterization of the adipocyte ghost (Na+,K+) pump. Insights into the insulin regulation of the adipocyte (Na+,K+) pump.

The (Na+,K+) ATPase in plasma membranes isolated from rat adipocytes is insensitive to insulin (Lytton J., Lin, J.C., and Guidotti, G. (1985) J. Biol. Chem. 260, 1177-1184). For this reason, the characteristics of the (Na+,K+) pump in adipocyte ghosts, prepared by hypotonic lysis of adipocytes (Rodbell, M. (1967) J. Biol. Chem. 242, 5744-5750), were studied. Herein it is demonstrated that the (Na+,K+) pump in ghosts is identical to that described in isolated plasma membranes, sharing the following characteristics: 1) the Ki values for ouabain are 1.3 x 10(-7) M and 4.5 x 10(-5) M for the alpha 2 and alpha 1 isozymes, respectively; 2) the K0.5 values for sodium are 11.4 +/- 1.6 and 7.2 +/- 3.8 mM for the alpha 2 and alpha 1 isozymes, respectively; 3) both forms of the (Na+,K+) pump are insensitive to insulin stimulation, presumably because the activities are already maximal. The ghosts are not in an insulin-stimulated state because the activity of the glucose transporter is not increased as it is in ghosts prepared from insulin-treated cells. In addition, presented evidence demonstrates that ghost internal sodium concentration, [Na+]i, is very sensitive to changes in the activity of the (Na+,K+) pump. If the [Na+]i, of adipocytes is also very sensitive to the activity of the (Na+,K+) pump, the mechanism of insulin stimulation of the adipocyte (Na+,K+) pump requires reexamination.     

Insulin-mediated translocation of glucose transporters from intracellular membranes to plasma membranes: sole mechanism of stimulation of glucose transport in L6 muscle cells

Plasma membranes and light microsomes were isolated from fused L6 muscle cells. Pre-treatment of cells with insulin did not affect marker enzyme or protein distribution in isolated membranes. The number of glucose transporters in the isolated membranes was calculated from the D-glucose-protectable binding of [3H]cytochalasin B. Glucose transporter number was higher in plasma membranes and lower in intracellular membranes derived from insulin-treated cells than in the corresponding fractions from untreated cells. The net increase in glucose transporters in plasma membranes was identical to the net decrease in glucose transporters in light microsomes (2 pmol/1.23 x 10(8) cells). The fold increase in glucose transporter number/mg protein in plasma membranes (2-fold) was similar to the fold increase in glucose transport caused by insulin. This suggests that recruitment of glucose transporters from intracellular membranes to the plasma membrane is the major mechanism of stimulation of hexose transport in L6 muscle cells. This is the first report of isolation of the two insulin-sensitive membrane elements from a cell line, and the results indicate that, in contrast to rat adipocytes, there is not change in the intrinsic activity of the transporters in response to insulin.     

Naturally occurring ether-linked phosphatidylcholine activates phosphatidylinositol 3-kinase and stimulates cell growth

Phosphatidylcholine (PC) from marine invertebrates is enriched in ether-linked forms. PCs from ray fish, Dasyatis sp., and bivalve, Macoma birmanica, used in the present study, contain 65% and 75% (w/w of total PC) of ether-linked forms, respectively. Ether-linked PCs also occur in mammalian membranes. Agonist-mediated hydrolysis of PC generates second messengers which participate in cellular responses. In this study, we tested whether PCs from marine invertebrates directly affect mammalian cell growth and activity of phosphatidylinositol (PI-3-kinase). PI-3-kinase participates in mitogenesis initiated by a variety of growth factors. PI-3-kinase converts polyphosphoinositides to 3′ phosphorylated isomers and these products accumulate in response to mitogenic stimuli. Whether cell membrane lipids regulate PI-3-kinase activity is not known. The marine animal–derived PCs and dioleoyl DAG (dioleoylglycerol) stimulated growth of murine pre-B lymphocytes, whereas chicken PC (egg lecithin) inhibited growth of these cells. Egg lecithin is also a potent inhibitor of PI-3-kinase activity in vitro. We studied the effect of PCs and DAG on PI-3-kinase activity. Unlike egg lecithin, marine animal PCs enhanced PI-3-kinase activity. We investigated the effect of lipids on PI-3-kinase substrate utilization. PCs enriched in ether-linked species increased utilization of substrates by PI-3-kinase. PCs purified from marine organisms also contain a substantially higher percentage of the cis-unsaturated fatty acids, especially of the ? ω3 series (25% and 30% of total fatty acids for Dasyatis sp. and Macoma birmanica, respectively), as compared to vertebrate sources. In spite of differences in fatty acid composition, marine PCs and dioleoyl DAG showed similar effects on cell growth and PI-3-kinase activity. These findings indicate that ether-linked phospholipids activate PI-3-kinase and may participate in mitogenic responses. © 1994 Wiley-Liss, Inc.     

Convergence and divergence of the signaling pathways for insulin and phosphoinositolglycans.

Phosphoinositolglycan molecules isolated from insulin-sensitive mammalian tissues have been demonstrated in numerous in vitro studies to exert partial insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive cells. However, their ill-defined structures, heterogeneous nature, and limited availability have prohibited the analysis of the underlying molecular mechanism. Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure prepared in large scale from the core glycan of a glycosyl-phosphatidylinositol-anchored membrane protein from Saccharomyces cerevisiae has recently been shown to stimulate glucose transport as well as a number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10 microns) of the maximal insulin effect in isolated rat adipocytes, cardiomyocytes, and diaphragms (G. Müller et al., 1997, Endocrinology 138: 3459-3476). Consequently, we used this PIG-P for the present study in which we compare its intracellular signaling with that of insulin. The activation of glucose transport by both PIG-P and insulin in isolated rat adipocytes and diaphragms was found to require stimulation of phosphatidylinositol (PI) 3-kinase but to be independent of functional p70S6kinase and mitogen-activated protein kinase. The increase in glycerol-3-phosphate acyltransferase activity in rat adipocytes in response to PIG-P and insulin was dependent on both PI 3-kinase and p70S6kinase. This suggest that the signaling pathways for PIG-P and insulin to glucose transport and metabolism converage at the level of PI 3-kinase. A component of the PIG-P signaling pathway located up-stream of PI 3-kinase was identified by desensitization of isolated rat adipocytes for PIG-P action by combined treatment with trypsin and NaCl under conditions that preserved cell viability and the insulin-mimetic activity of sodium vanadate but completely blunted the insulin response. Incubation of the cells with either trypsin or NaCl alone was ineffective. The desensitized adipocytes were reconstituted for stimulation of lipogenesis by PIG-P by addition of the concentrated trypsin/salt extract. The reconstituted adipocytes exhibited 65-75% of the maximal PIG-P response and similar EC50 values for PIG-P (2 to 5 microns) compared with control cells. A proteinaceous N-ethylmaleimide (NEM)-sensitive component contained in the trypsin/salt extract was demonstrated to bind in a functional manner to the adipocyte plasma membrane of desensitized adipocytes via bipolar interactions. An excess of trypsin/salt extract inhibited PIG-P action in untreated adipocytes in a competitive fashion compatible with a receptor function for PIG-P of this protein. The presence of the putative PIG-P receptor protein in detergent-insoluble complexes prepared from isolated rat adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma membrane may play a role in insulin-mimetic signaling by PIG-P. Furthermore, treatment of isolated rat diaphragms and adipocytes with PIG-P as well as with other agents exerting partially insulin-mimetic activity, such as PI-specific phospholipase C (PLC) and the sulfonylurea glimepiride, triggered tyrosine phosphorylation of the caveolar marker protein caveolin, which was apparently correlated with stimulation of lipogenesis. Strikingly, in adipocytes subjected to combined trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride failed completely to provoke insulin-mimetic effects. A working model is presented for a signaling pathway in insulin-sensitive cells used by PIG(-P) molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive receptor protein for PIG-P, and additional proteins located in caveolae/detergent-insoluble complexes.     

The association of insulin-elicited phosphotyrosine proteins with src homology 2 domains.

The interactions of the phosphotyrosine (Tyr(P))-containing proteins in basal and insulin-stimulated 3T3-L1 adipocytes with src homology 2 (SH2) domains from phosphatidylinositol 3-kinase (PI3K), ras GTPase-activating protein (GAP), and phospholipase C gamma have been examined. The Tyr(P) forms of the insulin receptor and its 160-kDa substrate protein (pp160) associated with fusion proteins containing either or both the SH2 domains of PI3K, but not with fusion proteins containing the two SH2 domains of GAP or phospholipase C gamma. These results demonstrate a specificity for the association of the Tyr(P) form of the insulin receptor and pp160 with SH2 domains that parallels the reported effects of insulin on PI3K, GAP, and phospholipase C gamma in vivo. Immunoprecipitates of pp160 from the cytosol of insulin-treated, but not basal, 3T3-L1 adipocytes contained PI3K activity. Moreover, the Tyr(P) form of pp160 with associated PI3K activity migrated at 10 S on a sucrose velocity gradient, whereas the Tyr(P) form without associated activity migrated at 6 S. These findings indicate that the Tyr(P) form of pp160 associates directly with PI3K in vivo.     

Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes.

Cross-linking of the B cell AgR results in activation of mature B cells and tolerization of immature B cells. The initial signaling events stimulated by membrane immunoglobulin (mIg) cross-linking are tyrosine phosphorylation of a number of proteins. Among the targets of mIg-induced tyrosine phosphorylation are the tyrosine kinases encoded by the lyn, blk, fyn, and syk genes, the mIg-associated proteins MB-1 and Ig-beta, phospholipase C-gamma 1 and -gamma 2, as well as many unidentified proteins. In this report we show that mIg cross-linking also regulates phosphatidylinositol 3-kinase (PtdIns 3-kinase), an enzyme that phosphorylates inositol phospholipids and plays a key role in mediating the effects of tyrosine kinases on growth control in fibroblasts. Cross-linking mIg on B lymphocytes greatly increased the amount of PtdIns 3-kinase activity which could be immunoprecipitated with anti-phosphotyrosine (anti-tyr(P) antibodies. This response was observed after mIg cross-linking in mIgM- and mIgG-bearing B cell lines and after cross-linking either mIgM or mIgD in murine splenic B cells. Thus, regulation of PtdIns 3-kinase is a common feature of signaling by several different isotypes of mIg. This response was rapid and peaked 2 to 3 min after the addition of anti-Ig antibodies. The anti-Ig-stimulated increase in PtdIns 3-kinase activity associated with anti-Tyr(P) immunoprecipitates could reflect increased tyrosine phosphorylation of PtdIns 3-kinase, increased activity of the enzyme, or both. In favor of the first possibility, the tyrosine kinase inhibitor herbimycin A blocked the increase in ant-Tyr(P)-immunoprecipitated PtdIns 3-kinase activity as well as the anti-Ig-induced tyrosine phosphorylation. Moreover, this response was not secondary to phospholipase C activation but rather seemed to be a direct consequence of mIg-induced tyrosine phosphorylation. Activation of the phosphoinositide pathway by a transfected M1 muscarinic acetylcholine receptor expressed in WEHI-231 B lymphoma cells did not increase the amount of PtdIns 3-kinase activity which could be precipitated with anti-Tyr(P) antibodies. Similarly, inhibition of the phosphoinositide pathway did not abrogate the ability of mIg cross-linking to stimulate this response. Thus, mIg-induced tyrosine phosphorylation regulates PtdIns 3-kinase, an important mediator of growth control in fibroblasts and potentially an important regulatory component in B cells as well.     

Biological properties of antibodies against rat adipocyte intrinsic membrane proteins. Dependence on multivalency for insulin-like activity.

Antisera from rabbits injected with rat adipocyte plasma membranes or intrinsic proteins from such membranes, obtained by a dimethylmaleic anhydride extraction step, mimicked the action of insulin on both glucose transport and lipolysis in intact adipocytes. Biological activity in both types of antisera was mediated by immunoglobulin binding to one or more intrinsic proteins of the adipocyte plasma membrane since fat cells were unresponsive to all antisera absorbed with dimethylmaleic anhydride-extracted membranes. Acid treatment of immunoprecipitates released antibodies which activated glucose uptake and reacted with solubilized adipocyte membranes on immunodiffusion plates. The biologically active immunoglobulin preparations failed to form immunoprecipitin lines when tested against membranes from brain, liver, lung, muscle, kidney, and spleen. Insulin-sensitive glucose uptake in rat soleus muscle did not respond to the antisera. The antibodies activated hexose uptake into fat cells and reacted with solubilized adipocyte membranes on immunodiffusion plates when rat or mouse adipocytes were studied, but not when monkey fat cells were used. The anti-membrane antibody preparations readily activated hexose uptake in trypsinized fat cells which had lost the capacity to bind or respond to insulin. These data are consistent with the concept previously proposed (Pillion, D.J., and Czech, M.P. (1978) J. Biol. Chem. 253, 3761-3764) that the anti-membrane immunoglobulins do not interact with the insulin binding site of the insulin receptor. Monovalent Fab fragments of the biologically active antisera, prepared by papain digestion of the native anti-membrane immunoglobulins, were ineffective in enhancing glucose uptake in adipocytes. However, biological activity of the anti-membrane Fab fragments was restored by the addition of goat anti-rabbit Fab antisera to cells treated with the Fab fraction. Anti-rabbit Fab antisera alone or in combination with Fab fragments prepared from control rabbit sera exhibited no biological activity. These results demonstrate that the ability of anti-membrane antisera to mimic the biological activity of insulin on isolated fat cells is critically dependent on immunoglobulin binding to one or more intrinsic plasma membrane proteins and the multivalent nature of immunoglobulin structure.     

Retention of insulin-stimulated D-glucose transport activity by adipocyte plasma membranes following extraction of extrinsic proteins

Plasma membrane vesicles prepared from adipocytes incubated with insulin exhibited accelerated D-glucose transport activity characteristic of insulin action on intact fat cells. Both control and insulin-stimulated D-glucose transport activities were inhibited by cytochalasin B and thiol reagents. Extraction of plasma membranes with dimethylmaleic anhydride eluted 80% of the protein from plasma membrane vesicles. The two major glycoprotein bands (94,000 and 78,000 daltons) and small amounts of a 56,000-dalton band were retained in dodecyl sulfate gels of the extracted membranes. Both control and insulin-activated D-glucose transport activities were retained by plasma membrane vesicles extracted with dimethylmaleic anhydride. Cytochalasin B binding activity was also retained by extracted membrane vescles and D-glucose uptake into extracted vescles derived from untreated or insulin-treated fat cells was inhibited by cytochalasin B. These results suggest that the modification of the adipocyte hexose transport system elicited by insulin action is not altered by a major purification step which involves quantitative extraction of extrinsic membrane proteins.     

Oxidative stress disrupts insulin-induced cellular redistribution of insulin receptor substrate-1 and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. A putative cellular mechanism for impaired protein kinase B activation and GLUT4 translocation

In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H2O2 concentrations display impaired insulin stimulated GLUT4 translocation from internal membrane pools to the plasma membrane (Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kannety, H., and Bashan, N. (1998) Diabetes 47, 1562-1569). In this study we further characterize the cellular mechanisms responsible for this observation. Two-hour exposure to approximately 25 microM H2O2 (generated by adding glucose oxidase to the medium) resulted in disruption of the normal insulin stimulated insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI) 3-kinase cellular redistribution between the cytosol and an internal membrane pool (low density microsomal fraction (LDM)). This was associated with reduced insulin-stimulated IRS-1 and p85-associated PI 3-kinase activities in the LDM (84 and 96% inhibition, respectively). The effect of this finding on the downstream insulin signal was demonstrated by a 90% reduction in insulin stimulated protein kinase B (PKB) serine 473 phosphorylation and impaired activation of PKBalpha and PKBgamma. Both control and oxidized cells exposed to heat shock displayed a wortmannin insensitive PKB serine phosphorylation and activity. These data suggest that activation of PKB and GLUT4 translocation are insulin signaling events dependent upon a normal insulin induced cellular compartmentalization of PI 3-kinase and IRS-1, which is oxidative stress-sensitive. These findings represent a novel cellular mechanism for the induction of insulin resistance in response to changes in the extracellular environment.     

Enhancement by adenosine of insulin-induced activation of phosphoinositide 3-kinase and protein kinase B in rat adipocytes.

The role of adenosine receptor in regulation of insulin-induced activation of phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B was studied in isolated rat adipocytes. Rat adipocytes are known to spontaneously release adenosine, which in turn binds and stimulates the adenosine A1 receptors on the cells. In the present study, we observed that degradation of this adenosine by adenosine deaminase attenuated markedly the insulin-induced accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), a product of PI 3-kinase. p-Aminophenylacetyl xanthine amine congener (PAPA-XAC), an inhibitor of the adenosine A1 receptor, also inhibited the insulin-induced PtdIns(3,4,5)P3 accumulation. When extracellular adenosine was inactivated by adenosine deaminase, phenylisopropyladenosine, an adenosine A1 receptor agonist, potentiated the insulin-induced accumulation of PtdIns(3,4,5)P3. Insulin-induced activation of protein kinase B, the activity of which is controlled by the lipid products of PI 3-kinase, was also potentiated by adenosine. Prostaglandin E2, another activator of a pertussis toxin-sensitive GTP-binding protein in these cells, potentiated the insulin actions. Thus, the receptors coupling to the GTP-binding protein were found to positively regulate the production of PtdIns(3,4,5)P3, a putative second messenger for insulin actions, in physiological target cells of insulin.