Insulin-like effect of trypsin on the phosphorylation of rat adipocyte insulin receptor

Trypsin treatment of a partially purified insulin receptor preparation from rat adipocytes stimulated the phosphorylation of 90,000- and 72,000-Da polypeptides immunoprecipitated by anti-insulin receptor antibody. The phosphorylation of tyrosine residues alone was observed in both polypeptides. Trypsin concentrations which stimulated insulin receptor phosphorylation were the same as those previously shown to activate rat adipocyte glycogen synthase. Trypsin treatment of the insulin receptor fraction also stimulated the phosphorylation of an exogenous substrate of tyrosine kinase similarly to insulin treatment. Trypsin treatment of a highly purified insulin receptor from human placenta also activated the phosphorylation of the receptor-derived peptides. These results suggest that the insulin-stimulated protein kinase, a component of the insulin receptor, was activated by tryptic digestion to phosphorylate polypeptides derived from the insulin receptor itself. Thus, it is suggested that stimulation by trypsin of phosphorylation of the insulin receptor may be related to the insulin-like metabolic actions of trypsin observed in rat adipocytes.     

Tryptic activation of the insulin receptor. Proteolytic truncation of the alpha-subunit releases the beta-subunit from inhibitory control

Trypsin exerts insulin-like effects in intact cells and on partially purified preparations of insulin receptors. To elucidate the mechanism of these insulinomimetic effects, we compared the structures of insulin- and trypsin-activated receptor species with their functions, including insulin binding, autophosphorylation, and tyrosine kinase activity. In vitro treatment of wheat germ agglutinin-purified receptor preparations with trypsin resulted in proteolysis of both alpha- and beta-subunits. The activated form of the receptor had an apparent molecular mass of 110 kDa under nonreducing conditions, compared to the 400-kDa intact receptor, and was separated following reduction into an 85-kDa beta-subunit related fragment and a 25-kDa alpha-subunit related fragment. Treatment of whole cells with trypsin prior to isolation of the insulin receptor resulted in proteolytic modification of the alpha-subunit only. In this case, the total molecular mass of the activated species was 116 kDa, comprised of an intact 92-kDa beta-subunit and again a 25-kDa alpha-subunit related fragment. Values of Km for peptide substrate phosphorylation and Ki for inhibition of receptor autophosphorylation, and sites of autophosphorylation within the beta-subunits were similar for receptors activated either by insulin or trypsin. Insulin had no additional effect on the rate of autophosphorylation of the truncated receptor, and no binding of insulin by the truncated receptor was detected either by direct assay or cross-linking with bifunctional reagents. Based on the deduced amino acid sequence of the insulin receptor and the structural studies presented here we concluded that this activated form of the receptor resulted from tryptic cleavage at the dibasic site Arg576-Arg577. This was accompanied by loss of the insulin binding site and separation of alpha-beta heterodimers. As truncation of the alpha-subunit results in beta-subunit activation, it appears that the beta-subunit is a constitutively activated kinase and that the function of the alpha-subunit in the intact receptor is to inhibit the beta-subunit.     

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.     

The relationship of rat liver overt carnitine palmitoyltransferase to the mitochondrial malonyl-CoA binding entity and to the latent palmitoyltransferase.

Concanavalin A (ConA) stimulated the phosphorylation of the beta-subunit of the insulin receptor and an Mr-185,000 protein on serine and tyrosine residues in intact H-35 rat hepatoma cells. This Mr-185,000 protein whose phosphorylation was stimulated by ConA was identical to pp185, a protein reported previously to be a putative endogenous substrate for the insulin receptor tyrosine kinase in rat hepatoma cells. In Chinese hamster ovary (CHO) cells transfected with cDNA of the human insulin receptor, tyrosine-phosphorylation of pp185 was strongly enhanced by ConA compared with the controls, suggesting that the induction of tyrosine-phosphorylation of pp185 was due to stimulation of the insulin receptor kinase by ConA. Moreover, monovalent ConA only slightly induced the tyrosine-phosphorylation of pp185, which was enhanced by the addition of anti-ConA IgG, suggesting that ConA stimulated the insulin receptor kinase mainly by the receptor cross-linking or aggregation in intact cells. These data suggest that the insulin-mimetic action of ConA is related to the autophosphorylation and activation of the insulin receptor tyrosine kinase, as well as the subsequent phosphorylation of pp185 in intact cells.     

A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis

The cDNAs encoding the normal human insulin receptor (HIRc) and a receptor that had lysine residue 1018 replaced by alanine (A/K1018) were used to transfect Rat 1 fibroblasts. Lysine 1018 is a critical residue in the ATP binding site of the tyrosine kinase domain in the receptor beta-subunit. Untransfected Rat 1 cells express 1700 endogenous insulin receptors. Expressed HIRc receptors had levels of insulin-stimulable autophosphorylation in vitro comparable to normal receptors, whereas A/K1018 receptors had less than 1% of that activity. Stimulation by insulin of HIRc receptors in situ in intact cells led to phosphorylation of beta-subunit tyrosine residues and activation of tyrosine kinase activity that could be preserved and assayed in vitro after receptor purification. In contrast, A/K1018 receptors showed no such activation, either of autophosphorylation or of kinase activity toward histone. Cells expressing HIRc receptors display enhanced sensitivity to insulin of 2-deoxyglucose transport and glycogen synthase activity. This increased sensitivity was proportional to insulin receptor number at low but not at high levels of receptor expression. A/K1018 receptors were unable to mediate these biologic effects and actually inhibited insulin's ability to stimulate glucose transport and glycogen synthase through the endogenous Rat 1 receptors. Expressed HIRc receptors mediated insulin internalization and degradation, whereas A/K1018 receptors mediated little, if any. Endocytotic uptake of the expressed A/K1018 insulin receptors was also markedly depressed compared to normal receptors. Unlike HIRc receptors, A/K1018 receptors also fail to undergo down-regulation after long (24 h) exposures to high (170 nM) concentrations of insulin. We conclude the following. 1) Normal human insulin receptors expressed in Rat 1 fibroblasts display active tyrosine-specific kinase, normal intracellular itinerary after endocytosis, and normal coupling to insulin's biologic effects. 2) A receptor mutated to alter the ATP binding site in the tyrosine kinase domain had little if any tyrosine kinase activity. 3) This loss of kinase activity was accompanied by a nearly complete lack of both endocytosis and biologic activity.     

Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase

Both vanadate and hydrogen peroxide (H2O2) are known to have insulin-mimetic effects. We previously reported that the mixture of vanadate plus H2O2 results in the generation of a peroxide(s) of vanadate, which strongly enhances IGF-II binding to rat adipocytes (Kadota et al., 1987b). We now report that pervanadate mimics insulin in isolated rat adipocytes to (1) stimulate lipogenesis, (2) inhibit epinephrine-stimulated lipolysis, and (3) stimulate protein synthesis. The efficacy of pervanadate is comparable to that of insulin. However, it is 10(2)-10(3) times more potent than vanadate alone. Exposure of intact rat adipocytes to pervanadate was found to activate the WGA-purified insulin receptor tyrosine kinase assayed with the exogenous substrate poly(Glu80/Tyr20) in a dose-dependent manner to a maximum of 1464% of control at 10(-3) M compared with a maximum insulin effect of 1046% at 10(-6) M. In contrast, in vitro assayed autophosphorylation of the WGA-purified extract was increased 3-fold after exposure of intact cells to insulin but not significantly increased after pervanadate. Furthermore, high concentrations of pervanadate (10(-5) M) inhibited subsequent in vitro added insulin-stimulated autophosphorylation. In vitro addition of pervanadate to WGA-purified receptors could not stimulate autophosphorylation or exogenous tyrosine kinase activity and did not inhibit insulin-stimulated autophosphorylation. Labeling of intact adipocytes with [32P]orthophosphate followed by exposure to 10(-4) M pervanadate increased insulin receptor beta-subunit phosphorylation (7.9 +/- 3.0)-fold, while 10(-7) M insulin and 10(-4) vanadate increased labeling (5.3 +/- 1.8)- and (1.1 +/- 0.2)-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel mechanism for the insulin-like effect of vanadate on glycogen synthase in rat adipocytes

Vanadate activated rat adipocyte glycogen synthase similarly to insulin in a dose- and time-dependent manner. No additional effect was observed when insulin and vanadate were added together. Vanadate also partially counteracted the effect of epinephrine to activate rat adipocyte glycogen phosphorylase similarly to insulin. Inhibition of Na+K+ATPase or stimulation of hydrogen peroxide generation were shown not to be the mechanisms of the insulin-like action of vanadate on glycogen synthase. Vanadate stimulated the phosphorylation of the 95,000-dalton subunit of the insulin receptor on tyrosine residues both in intact adipocytes and in a solubilized insulin receptor fraction. Vanadate also stimulated the phosphorylation of the 95,000-dalton subunit of a highly purified insulin receptor from human placenta. Neither the insulin receptor fraction from rat adipocyte nor the highly purified insulin receptor from human placenta contained any detectable phosphotyrosine phosphatase activity. Potassium fluoride had no stimulatory effect on the phosphorylation of the insulin receptor. Vanadate caused a 10-fold decrease in the Km for ATP, for tyrosine kinase, and enhanced the phosphorylation of histone H2B. These results demonstrate that vanadate enhances the phosphorylation of the insulin receptor by stimulating the kinase reaction in a similar but not identical manner to insulin.     

Insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function.

We have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (Tyr1158, Tyr1162, and Tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. A single-Phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. The double-Phe replacements showed a 60-70% reduction, and substitution of all 3 tyrosine residues with Phe or Ser reduced insulin-stimulated tyrosine autophosphorylation by greater than 80%. Phosphopeptide mapping each mutant revealed that all remaining tyrosine autophosphorylation sites were phosphorylated normally following insulin stimulation, and no new sites appeared. The single-Phe mutants showed insulin-stimulated kinase activity toward a synthetic peptide substrate of 50-75% when compared with wild-type receptor kinase activity. Insulin-stimulated kinase activity was further reduced in the double-Phe mutants and barely detectable in the triple-Phe mutants. In contrast to the wild-type receptor, all of the mutant receptor kinases showed a significant reduction in activation following in vitro insulin-stimulated autophosphorylation. When studied in intact Chinese hamster ovary cells, insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of the cellular substrate pp185 in the single-Phe and double-Phe mutants was progressively lower with increased tyrosine replacement and did not exceed the basal levels in the triple-Phe mutants. However, all the mutant receptors, including the triple-Phe mutant, retained the ability to undergo insulin-stimulated Ser and Thr phosphorylation. Thus, full activation of the insulin receptor tyrosine kinase is dependent on insulin-stimulated Tris phosphorylation of the kinase domain, and the level of autophosphorylation in the kinase domain provides a mechanism for modulating insulin receptor kinase activity following insulin stimulation. By contrast, insulin stimulation of receptor phosphorylation on Ser and Thr residues by cellular serine/threonine kinases can occur despite markedly reduced tyrosine autophosphorylation.     

Contrasting interactions between phorbol ester and insulin on the regulation of glycogen synthase activity and p33 mRNA accumulation in rat hepatoma cells

We have compared the effect of phorbol 12-myristate 13-acetate (PMA) with that of insulin on three targets of insulin action in H4IIEC3 (H4) rat hepatoma cells. These parameters are the phosphorylation state and tyrosine kinase activity of the insulin receptor, the activation state of glycogen synthase, and the accumulation of p33 mRNA. Under conditions where insulin treatment of H4 cells clearly activated receptor serine and tyrosine phosphorylation on the insulin receptor beta-subunit in situ, activated receptor tyrosine kinase activity in vitro, and activated glycogen synthase and p33 mRNA accumulation in situ, PMA alone did not influence the insulin receptor phosphorylation state or tyrosine kinase activity and did not affect glycogen synthase activity, but markedly increased p33 mRNA accumulation. When PMA was added in the presence of insulin, particularly if PMA was preincubated, the receptor phosphorylation state and the tyrosine kinase activity again were not affected, but insulin-activated glycogen synthase was significantly diminished or abolished. In contrast, increased p33 mRNA accumulation by PMA was additive with that of insulin. Thus, under conditions where no effect was observed on the insulin receptor phosphorylation state or the tyrosine kinase activity, PMA acted in an insulin-antagonistic manner on glycogen synthase and in an insulin-like manner on p33 mRNA accumulation, indicating that these actions of PMA are unrelated to early events in the pathway of the insulin action. Effects on glycogen synthase are most readily explained by an effect of protein kinase C-activated phosphorylation of glycogen synthase.     

Alteration of insulin receptor kinase in obese, insulin-resistant mice

We have studied the properties of muscle insulin receptors obtained from genetically or experimentally-induced obese mice that are both insulin-resistant. Insulin receptors, partially purified by wheat germ agglutinin--agarose chromatography, were studied in a cell-free system for autophosphorylation, for their ability to phosphorylate a synthetic glutamate--tyrosine copolymer and for their binding characteristics. Insulin receptor number was decreased by 25% in muscles from obese mice without any change in their binding affinity. The insulin stimulatory action on its beta-subunit receptor phosphorylation was diminished in preparations from genetically- or experimentally-induced obese mice to a higher degree than the decrease in insulin receptor number. HPLC analysis of the phosphopeptides generated by trypsin treatment of the labeled receptor beta-subunit was identical in lean and obese mice. Similar alteration of the kinase activity was found in obese mice when the phosphorylation of casein or polyglutamate--tyrosine was measured. Trypsin treatment of the receptor preparations was less effective in stimulating the kinase activity in obese mice than in lean mice. These results suggest that the defect in insulin receptor kinase activity reflects an alteration in the transmission of the message from the alpha- to the beta-subunit or an impairment of the enzyme functioning by environmental conditions.     

Stimulation of glycogen synthesis by insulin requires S6 kinase and phosphatidylinositol-3-kinase in HTC-IR cells

In order to study the role of phosphatidylinositol-3-kinase (PI3K), PKB, FRAP, S6 kinase, and MAP kinase in insulin-stimulated glycogen synthesis, we used a specific inhibitor of PI3K, LY294002, the immunosuppressant inhibitor of FRAP, rapamycin, and the inhibitor of MAPK kinase (MEK)/MAPK, PD98059, in rat HTC hepatoma cells overexpressing human insulin receptors. The PI3K inhibitor LY294002 completely blocks insulin-stimulated glycogen synthesis by inhibiting glycogen synthase, PKB (Akt-1), and FRAP (RAFT) autophosphorylation, as well as p70 S6 kinase activation, whereas insulin receptor substrates tyrosine phosphorylation and MEK activity were not affected. However, rapamycin only partially blocks insulin-stimulated glycogen synthesis by partial inhibition of glycogen synthase, whereas it completely blocks S6 kinase activation and FRAP autophosphorylation, but does not affect either PKB autophosphorylation, MEK activity, or insulin receptor tyrosine phosphorylation. Insulin-stimulated glycogen synthesis and glycogen synthase were not affected by the MEK/MAPK inhibitor PD98059. These data suggest that the PI3K, and not the MAPK pathway plays an important role in the insulin-stimulated glycogen synthesis in the hepatocyte, partly mediated by FRAP and S6 kinase activation. However, the inhibition of FRAP and S6 kinase activation is not sufficient to block insulin-stimulated glycogen synthesis, suggesting an important role of a branching pathway upstream of S6 kinase and downstream of PI3K, which is probably mediated by PKB in the signaling of the insulin receptor in hepatoma HTC cells.     

The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes.

Competitive hormone binding studies with membrane and partially purified receptors from Xenopus laevis oocytes revealed that the oocyte possesses high affinity (KD = 1-3 nM) binding sites for both insulin growth factors 1 and 2 (IGF-1 and IGF-2), but not for insulin. Consistent with these findings, IGF-1 activates hexose uptake by Xenopus oocytes with a KA (3 nM) identical with its KD, while IGF-2 and insulin activate hexose uptake with KA values of 50 nM and 200-250 nM, respectively, suggesting activation mediated through an IGF-1 receptor. Both IGF-1 and insulin activate receptor beta-subunit autophosphorylation and, thereby, protein substrate (reduced and carboxyamidomethylated lysozyme, i.e. RCAM-lysozyme) phosphorylation with KA values comparable to their respective KD values for ligand binding and KA values for activation of hexose uptake. The autophosphorylated beta-subunit(s) of the receptor were resolved into two discrete components, beta 1 and beta 2 (108 kDa and 94 kDa, respectively), which were phosphorylated exclusively on tyrosine and which exhibited similar extents of IGF-1-activated autophosphorylation. When added prior to autophosphorylation, RCAM-lysozyme blocks IGF-1-activated autophosphorylation and, thereby, IGF-1-activated protein substrate (RCAM-lysozyme) phosphorylation. Based on these findings, we conclude that IGF-1-stimulated autophosphorylation of its receptor is a prerequisite for catalysis of protein substrate phosphorylation by the receptor's tyrosine-specific protein kinase. The IGF-1 receptor kinase is implicated in signal transmission from the receptor, since anti-tyrosine kinase domain antibody blocks IGF-1-stimulated kinase activity in vitro and, when microinjected into intact oocytes, prevents IGF-1-stimulated hexose uptake.     

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.     

Two different protein kinase activities are associated with the insulin receptor.

In intact rat hepatocytes insulin stimulates the phosphorylation of the beta-subunit of its receptor exclusively on serine residues, which are also phosphorylated in the absence of insulin. In contrast, in partially purified insulin receptors derived from these same cells and in highly purified insulin receptors obtained by immunoprecipitation with anti-receptor antibodies, the receptor beta-subunit is phosphorylated solely on tyrosine residues. For both cell-free systems, insulin's stimulatory action on receptor phosphorylation leads to an increase in phosphotyrosine. When partially purified receptors were used to phosphorylate two exogenous substrates, casein and histone, insulin was found to stimulate the phosphorylation of both tyrosine and serine. However, the basal and insulin-stimulated kinase activity of immunoprecipitated receptors was only tyrosine-specific. From these observations we propose that the insulin-receptor complex consists of two different insulin-stimulatable kinase activities: (1) a tyrosine-specific kinase, which is a constituent of the insulin-receptor structure and whose activation is likely to be the first post-binding event in insulin action; and (2) a serine-specific kinase, which is closely associated with the receptor in the cell membrane.     

Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.     

Regulation of insulin receptor kinase by multisite phosphorylation

The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its beta subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse-phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor beta subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100% higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor beta subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated beta subunit autophosphorylation activities when incubated with [gamma-32 P]ATP. Similarly, the ability of insulin to stimulate the receptor beta subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the beta-catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of 32P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Diacylglycerols modulate phosphorylation of the insulin receptor from human mononuclear cells

It has been found that 1,2- but not 1,3-diacylglycerols stimulated phosphorylation of the insulin receptor of cultured human monocyte-like (U-937) and lymphoblastoid (IM-9) cells both in the intact- and broken-cell systems. The stimulation of the receptor's beta-subunit phosphorylation was dose-dependent, with optimal effect at 100 micrograms/ml of diacylglycerol. The effects of insulin and 1,2-diacylglycerols on the phosphorylation of partially purified insulin receptors were additive. Phosphoamino acid analysis showed a major effect of diacylglycerols on phosphorylation of tyrosine residues. The diacylglycerols also stimulated tyrosine kinase activity of the partially purified U-937 and IM-9 insulin receptors 2.5-3.5-fold when measured by phosphorylation of an exogenous substrate, poly(Glu80Tyr20) in the absence of any added insulin, calcium or phospholipid. Since this diacylglycerol effect could not be reproduced under conditions optimal for protein kinase C activation and the purified protein kinase C did not stimulate phosphorylation of the beta-subunit of the insulin receptor in this system, it is unlikely that the diacylglycerol effect was mediated by protein kinase C. Since these exogenous 1,2-diacylglycerols at the same high concentration also inhibited 125I-insulin binding to the insulin receptor of the intact U-937 and IM-9 cells, diacylglycerols could modulate the function of the insulin receptor and insulin action in human mononuclear cells.     

Diacylglycerols modulate insulin action in rat adipocytes

Effect of 1,2-diacylglycerols on the insulin receptor function and insulin action in rat adipocytes was studied. 1,2-dioctanoylglycerol (100 micrograms/ml) did not alter insulin binding but it did stimulate phosphorylation of the beta-subunit of the insulin receptor as well as its tyrosine kinase activity. However, dioctanoylglycerol inhibited insulin-stimulated receptor autophosphorylation. This concentration of dioctanoylglycerol inhibited insulin-stimulated CO2 metabolism, lipogenesis and 3-O-methyl-glucose transport in a dose-dependent manner but did not alter any of these bioeffects in absence of insulin. While there was no direct link between diacylglycerol effect on tyrosine kinase activity of the insulin receptor and insulin action in rat adipocytes, the parallel inhibition of insulin-stimulated receptor autophosphorylation and insulin bioeffects by dioctanoylglycerol suggests its direct or indirect role in insulin signalling in rat fat cells.     

Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-naphthalenyl-methyl) phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa beta-subunit of the insulin receptor (IC50 = 200 microM). The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 microM, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa beta-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. Our data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases.