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.     

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)     

Pertussis toxin inhibits autophosphorylation and activation of the insulin receptor kinase.

Pertussis toxin is an ADP-ribosyltransferase which alters the function of some of the GTP-binding proteins and inhibits some actions of insulin. In vivo, pertussis toxin (2 micrograms/ml/2h) inhibited insulin-stimulated tyrosyl autophosphorylation of the insulin receptor by 50% in FaO cells, and nearly completely inhibited phosphorylation of the cellular insulin receptor substrate pp185. Similarly, insulin-stimulated autophosphorylation and kinase activity of the insulin receptor purified on wheat germ agglutinin-agarose from pertussis toxin-treated FaO cells was diminished 50%; however, treatment of cells with the catalytically inactive B-oligomer of the toxin had no effect on receptor tyrosine kinase activity in vitro. Pertussis toxin did not alter insulin binding or the cellular levels of ATP, cAMP, and cGMP. Furthermore, immunoprecipitation of the insulin receptor from intact cells with anti-insulin receptor antibodies showed that pertussis toxin did not increase the phosphorylation of serine or threonine residues in the insulin receptor. These results suggest that pertussis toxin can modulate signal transduction of insulin at the level of the insulin receptor kinase.     

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.     

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.     

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)     

Shikonin stimulates glucose uptake in 3T3-L1 adipocytes via an insulin-independent tyrosine kinase pathway

Type 2 diabetes is due to defects in both insulin action and secretion. In an attempt to discover small molecules that stimulate glucose uptake, similar to insulin, a cell-based glucose uptake screening assay was performed using 3T3-L1 adipocytes. Shikonin, a substance originally isolated from the root of the Chinese plant that has been used as an ointment for wound healing, was thus identified. Shikonin stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. Stimulation of glucose uptake was also observed in rat primary adipocytes and cardiomyocytes. Like insulin, shikonin-stimulated glucose uptake was inhibited by genistein, a tyrosine kinase inhibitor, and enhanced by vanadate, a tyrosine phosphatase inhibitor. However, in contrast to insulin, shikonin-stimulated glucose uptake was not strongly inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). In vitro phosphorylation analyses revealed that shikonin did not induce tyrosine phosphorylation of the insulin receptor, but significantly induced both Thr-308 and Ser-473 phosphorylation of Akt. Our results suggest that in 3T3-L1 adipocytes, shikonin action is not mediated primarily via the insulin receptor/PI3K pathway, but rather via another distinct tyrosine kinase-dependent pathway leading to glucose uptake involving Akt phosphorylation.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes

To study molecular mechanisms for glucosamine-induced insulin resistance, we induced complete and reversible insulin resistance in 3T3-L1 adipocytes with glucosamine in a dose- and time-dependent manner (maximal effects at 50 mM glucosamine after 6 h). In these cells, glucosamine impaired insulin-stimulated GLUT-4 translocation. Glucosamine (6 h) did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 and -2 and weakly, if at all, impaired insulin stimulation of phosphatidylinositol 3-kinase. Glucosamine, however, severely impaired insulin stimulation of Akt. Inhibition of insulin-stimulated glucose transport was correlated with that of Akt activity. In these cells, glucosamine also inhibited insulin stimulation of p70 S6 kinase. Glucosamine did not alter basal glucose transport and insulin stimulation of GLUT-1 translocation and mitogen-activated protein kinase. In summary, glucosamine induced complete and reversible insulin resistance in 3T3-L1 adipocytes. This insulin resistance was accompanied by impaired insulin stimulation of GLUT-4 translocation and Akt activity, without significant impairment of upstream molecules in insulin-signaling pathway.     

In situ inhibition of tyrosine protein kinase by erbstatin

Erbstatin inhibited the kinase activity of the receptor for epidermal growth factor in cultured A431 cells, while it did not alter turnover of the receptor protein. It also inhibited autophosphorylation of the src gene product p60src in Rous sarcoma virus-infected normal rat kidney cells. Erbstatin did not inhibit the binding of epidermal growth factor to its receptor but did inhibit internalization of epidermal growth factor-receptor complexes. It did not inhibit the epidermal growth factor-stimulated phosphatidylinositol turnover in A431 cells. Thus, erbstatin inhibited two oncogene product-related tyrosine protein kinases in situ and thus is a useful tool to study the role of tyrosine protein kinase.     

Characterization of phosphatidylinositol kinase activity associated with the insulin receptor

Various lipids were tested as substrates for the insulin receptor kinase using either receptor partially purified from rat hepatoma cells by wheat-germ-agglutinin-Sepharose chromatography or receptor purified from human placenta by insulin-Sepharose affinity chromatography. Phosphatidylinositol was phosphorylated to phosphatidylinositol 4-phosphate by the partially purified insulin receptor. In contrast, phosphatidylinositol 4-phosphate and diacylglycerol were not phosphorylated. In some, but not all preparations of partially purified insulin receptor, the phosphatidylinositol kinase activity was stimulated by insulin (mean effect 33%). Phosphatidylinositol kinase activity was retained in insulin receptor purified to homogeneity. Insulin regulation of the phosphatidylinositol kinase was lost in the purified receptor; however, dithiothreitol stimulated both autophosphorylation of the purified receptor and phosphatidylinositol kinase activity in parallel about threefold. (Glu80Tyr20)n, a polymeric substrate specific to tyrosine kinases, inhibited the phosphatidylinositol kinase activity of the purified receptor by greater than 90% and inhibited receptor autophosphorylation by 67%. Immunoprecipitation by specific anti-receptor antibodies depleted by greater than 90% the phosphatidylinositol kinase activity in the supernatant of the purified receptor and the phosphatidylinositol kinase activity was recovered in the precipitate in parallel with receptor autophosphorylation activity. These characteristics of the phosphatidylinositol kinase activity of the purified insulin receptor and its metal ion preference paralleled those of the receptor tyrosine kinase activity and differed from bulk phosphatidylinositol kinase activity in cell extracts, which was not significantly inhibited by (Glu80Tyr20)n, stimulated by dithiothreitol or depleted by immunoprecipitation with anti-(insulin receptor) antibody. These results suggest that the insulin receptor is associated with a phosphatidylinositol kinase activity; however, this activity is not well regulated by insulin. This kinase appears to be distinct from the major phosphatidylinositol kinase(s) of cells. Its relationship to insulin action needs further study.     

Effect of phosphotyrosyl-IRS-1 level and insulin receptor tyrosine kinase activity on insulin-stimulated phosphatidylinositol 3, MAP,and S6 kinase activities

The role of tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) was studied utilizing parental CHO cells or CHO cells that overexpress IRS-1, the insulin receptor, or both IRS-1 and the insulin receptor. Insulin stimulation of these four cell lines led to progressive levels of IRS-1 tyrosine phosphorylation of one, two, four, and tenfold. Maximal insulin-stimulated IRS-1 associated Ptdlns 3′-kinase activit in these cells was 1-, 1.5-, 3-, and 3-fold, while insulin sensitivity, as determined by ED₅₀, was 1-, 2.5-, 10-, and 10-fold. Both sensitivity and maximal response paralleled the increased level of phosphotyrosyl-IRS-1; however, the increased level of phosphotyrosyl-IRS-1 seen in CHO/IR/IRS-1 cells did not further increase these responses. Likewise, maximal insulin-stimulated MAP kinase activity in these cell lines increased in parallel with IRS-1 tyrosine phosphorylation except in the CHO/IR/IRS-1 cell lines with activity levels of one-, five-, nine-, and ninefold. However, insulin sensitivity of the MAP and S6 kinases and maximal insulin-stimulated S6 kinase activity was not changed by a twofold increase in phosphotyrosyl-IRS-1, but an increase was observed with insulin-stimulated receptor autophosphorylation and kinase activity in CHO/IR cells which led to a tenfold increase in insulin receptor autophosphorylation and a fourfold increase in IRS-1 tyrosine phosphorylation. Thus, these three kinase activities may be differentially coupled to the activation of the insulin receptor kinase activity via IRS-1 and other possible cellular substrates. © 1995 Wiley-Liss, Inc.     

Direct modulation of insulin receptor protein tyrosine kinase by vanadate and anti-insulin receptor monoclonal antibodies

Sodium vanadate activates "in vitro" insulin receptor autophosphorylation and protein tyrosine kinase in a dose-dependent manner. Insulin receptor protein tyrosine kinase is directly activated also by the anti-insulin receptor beta subunit monoclonal antibody 18-44. We previously demonstrated that the anti-insulin receptor monoclonal antibody MA-10 decreases insulin-stimulated receptor protein tyrosine kinase activity "in vitro", without inhibiting insulin receptor binding. In this report we show that insulin receptor protein tyrosine kinase, activated by sodium vanadate or by monoclonal antibody 18-44, is inhibited by MA-10 antibody. These data suggest that insulin receptor protein tyrosine kinase activity can be either activated and inhibited through mechanisms different from insulin binding.     

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.     

Insulin stimulates phosphorylation of the beta 2-adrenergic receptor by the insulin receptor, creating a potent feedback inhibitor of its tyrosine kinase.

Insulin counterregulates catecholamine action at several levels, primarily in liver, fat, and adipose tissue. Herein we observe that expression of increased levels of beta(2)-adrenergic receptor increasingly inhibits insulin-stimulated phosphorylation of its primary downstream substrates (IRS-1,2). In Chinese hamster ovary cells, the insulin receptor phosphorylates dominantly Tyr(364) in the C-terminal cytoplasmic domain of the beta-receptor. A Y364A mutant form of the beta(2)-adrenergic, in contrast, loses it ability to inhibit insulin-stimulated phosphorylation of IRS-1,2. Upon phosphorylation, the C-terminal cytoplasmic domain of the beta(2)-adrenergic receptor demonstrates a potent inhibitory feedback action that can block both insulin-stimulated autophosphorylation of the insulin receptor and phosphorylation of IRS-1,2 in NIH mouse 3T3-L1 adipocyte membranes. Studies in vitro with purified insulin receptor and the C-terminal cytoplasmic domain of the beta(2)-adrenergic receptor demonstrate that the tyrosine-phosphorylated beta-receptor domain is a potent counterregulatory inhibitor of the insulin receptor tyrosine kinase.     

Decreased tyrosine kinase activity of insulin receptor isolated from rat adipocytes rendered insulin-resistant by catecholamine treatment in vitro.

Catecholamine treatment of isolated rat adipocytes decreases insulin binding and inhibits insulin stimulation of the glucose-transport system. There is increasing evidence that the insulin signal is transmitted after insulin is bound to the receptor via a tyrosine kinase, which is an intrinsic part of the receptor. To find whether the receptor kinase is modified by catecholamines, we solubilized and partially purified the insulin receptor of isoprenaline-treated adipocytes and studied the effect of insulin on its kinase activity. (1) Insulin increased the tyrosine autophosphorylation of the insulin receptor kinase from catecholamine-treated cells only 4-fold, compared with a 12-fold stimulation in control cells. (2) The rate of insulin-stimulated 32P incorporation into the receptor of isoprenaline-treated cells at non-saturating [32P]ATP concentrations (5 muM) was decreased to 5-8% of the values for receptor from control cells. (3) 125I-insulin binding to the partially purified receptor from catecholamine-treated cells was also markedly decreased. The insulin receptor from catecholamine treated cells bound 25-50% of the amount of insulin bound by the receptor from control cells at insulin concentrations of 10 pM-0.1 muM. Part of the impaired insulin-responsiveness of the receptor kinase of catecholamine-treated cells is therefore explained by impaired binding properties; however, an additional inhibition of the kinase activity of the insulin receptor from catecholamine-treated cells is evident. (4) This inhibition of kinase activity decreased when the concentration of [gamma-32P]ATP in the phosphorylation assay was increased. A Lineweaver-Burk analysis revealed that the Km for ATP of the receptor kinase from isoprenaline-treated cells was increased to approx. 100 muM, compared with approx. 25 muM for receptor of control cells. (5) We conclude from the data that catecholamine treatment of rat adipocytes modulates the kinase activity of the insulin receptor by increasing its Km for ATP and that this is part of the mechanism leading to insulin-resistance in these cells.     

Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.     

Phorbol ester-mediated protein kinase C interaction with wild-type and COOH-terminal truncated insulin receptors.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and insulin were compared in wild-type human insulin receptors (HIRc cells) and human insulin receptors lacking 43 COOH-terminal amino acid residues (HIR delta CT cells). TPA increased total phosphorylation of the wild-type insulin receptor and inhibited insulin-stimulated autophosphorylation by 32 +/- 10% in HIRc cells. TPA inhibited insulin-stimulated autophosphorylation by 46 +/- 14% in HIR delta CT cells and also caused a 65% decrease in basal phosphorylation. Insulin-stimulated tyrosine kinase activity for poly(Glu4/Tyr1) was inhibited by TPA in HIRc and HIR delta CT cells by 50 and 40%, respectively. TPA decreased insulin-stimulated glucose incorporation into glycogen by 50% in HIRc cells and to near basal levels in HIR delta CT cells; this inhibitory effect of TPA was reversed in both cell lines by staurosporine. In conclusion, 1) TPA-induced inhibition of insulin receptor tyrosine autophosphorylation was linked to concomitant inhibition of the biological effects of insulin in cells expressing either wild-type or COOH-terminal truncated insulin receptors; and 2) the inhibitory effects of TPA were not dependent upon phosphorylation of COOH-terminal residues and furthermore appeared to be independent of phosphorylation of any insulin receptor serine/threonine residues. These findings suggest a novel protein kinase C mechanism that results in altered insulin receptor function without increasing phosphorylation of the receptor.     

The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies.

The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp 185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Anti-receptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.     

Cross talk of pp125(FAK) and pp59(Lyn) non-receptor tyrosine kinases to insulin-mimetic signaling in adipocytes

Signaling molecules downstream from the insulin receptor, such as the insulin receptor substrate protein 1 (IRS-1), are also activated by other receptor tyrosine kinases. Here we demonstrate that the non-receptor tyrosine kinases, focal adhesion kinase pp125(FAK) and Src-class kinase pp59(Lyn), after insulin-independent activation by phosphoinositolglycans (PIG), can cross talk to metabolic insulin signaling in rat and 3T3-L1 adipocytes. Introduction by electroporation of neutralizing antibodies against pp59(Lyn) and pp125(FAK) into isolated rat adipocytes blocked IRS-1 tyrosine phosphorylation in response to PIG but not insulin. Introduction of peptides encompassing either the major autophosphorylation site of pp125(FAK), tyrosine 397, or its regulatory loop with the twin tyrosines 576 and 577 inhibited PIG-induced IRS-1 tyrosine phosphorylation and glucose transport. PIG-induced pp59(Lyn) kinase activation and pp125(FAK) tyrosine phosphorylation were impaired by the former and latter peptide, respectively. Up-regulation of pp125(FAK) by integrin clustering diminished PIG-induced IRS-1 tyrosine phosphorylation and glucose transport in nonadherent but not adherent adipocytes. In conclusion, PIG induced IRS-1 tyrosine phosphorylation by causing (integrin antagonized) recruitment of IRS-1 and pp59(Lyn) to the common signaling platform molecule pp125(FAK), where cross talk of PIG-like structures and extracellular matrix proteins to metabolic insulin signaling may converge, possibly for the integration of the demands of glucose metabolism and cell architecture.     

Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.