Insulin Receptor Kinase Activity Research Articles

Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance.

Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.

Inhibitors such as staurosporine, H-7 or polymyxin B cannot be used in skeletal muscle to prove the role of protein kinase C on insulin action.

The precise role of protein kinase C in insulin action in skeletal muscle is not well defined. Based on the fact that inhibitors of protein kinase C block some insulin effects, it has been concluded that some of the biological actions of insulin are mediated via protein kinase C. In this study, we present evidence that inhibitors of protein kinase C such as staurosporine, H-7 or polymyxin B cannot be used to ascertain the role of protein kinase C in skeletal muscle. This is based on the following experimental evidences: a) staurosporine, H-7 and polymyxin B markedly block in muscle the effect of insulin on System A transport activity; however, this effect of insulin is not mimicked in muscle by TPA-induced stimulation of protein kinase C, b) H-7 and polymyxin B block insulin action on System A transport activity in an additive manner to the inhibitory effect of phorbol esters, c) staurosporine, H-7 and polymyxin B block the effect of insulin on lactate production, a process that is activated by insulin and TPA in an additive fashion, and d) staurosporine completely blocks the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle.

Alterations in insulin receptor and substrate phosphorylation in hypertensive rats.

Insulin stimulates tyrosine phosphorylation of the insulin receptor and of an endogenous substrate of approximately 185 kd (insulin receptor substrate 1 or IRS-1) in most cell types. Tyrosine phosphorylation of insulin receptor and of IRS-1 have been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the study presented here, the levels and phosphorylation state of the insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo have been examined in spontaneously hypertensive rats (SHR) by immunoblotting with antipeptide antibodies to insulin receptor and IRS-1 and antiphosphotyrosine antibodies. It was found that the levels of insulin receptor and IRS-1 protein in liver and muscle are similar in controls (Wistar-Kyoto rats) and SHR. By contrast, there is a decrease in autophosphorylation in the liver and muscle of SHR and a parallel decrease in phosphorylation of IRS-1. These data indicate that reduced insulin receptor kinase activity and reduced substrate phosphorylation may play an important role in the impaired insulin action in the hypertensive rat.

Pharmacological doses of insulin equalize insulin receptor phosphotyrosine content but not tyrosine kinase activity in plasmalemmal and endosomal membranes.

Following insulin administration to intact rats, the insulin receptor kinase activity of subsequently isolated cell fractions was significantly augmented. Of interest was the observation that the endosomal insulin receptor tyrosine kinase displayed four- to six-fold greater autophosphorylation activity than that of plasma membrane. Surprisingly, the endosomal insulin receptor tyrosine kinase displayed a decrease in beta-subunit phosphotyrosine content compared with that seen in the plasma membrane. These observations prompted the suggestion that insulin receptor tyrosine kinase phosphotyrosine dephosphorylation mediated by an endosome-specific phosphotyrosine phosphatase(s) yields activation of the endosomal insulin receptor tyrosine kinase. In a previous study we examined the effect of subsaturating doses of injected insulin. In this work we evaluated insulin receptor tyrosine kinase activity and phosphotyrosine content in plasma membrane and endosomes after a receptor-saturating pharmacological dose of insulin (150 micrograms/100 g body weight). At this dose the phosphotyrosine content per receptor was reduced compared with that seen earlier at insulin doses of 1.5 and 15 micrograms/100 g body weight. Endosomal insulin receptor tyrosine kinase was greater than that seen at the lower nonsaturating insulin doses. Furthermore, endosomal insulin receptor tyrosine kinase activity exceeded that of the plasma membrane, despite retaining about the same phosphotyrosine content per receptor. These data are consistent with the view that insulin receptor tyrosine kinase activity may be regulated by a particular pattern of phosphotyrosine content on the beta-subunit wherein both activating and inhibitory phosphotyrosine residues play a role.

Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain.

We studied the inhibitory effect of non-phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143-1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine-phosphorylated peptides were synthesized using the N-(9-fluorenylmethoxycarbonyl)-O-dibenzylphosphono-L- tyrosine. The triphosphorylated peptide (1151-P3) and the non-phosphorylated peptide (1151-NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose-dependent and additive manner. When the receptor was pre-phosphorylated for 1 min with [gamma-32P]ATP, 1151-P3 decreased autophosphorylation to 60% of maximum, whereas 1151-NP had no further effect. In both non-activated and preactivated receptors, 1151-P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu4, Tyr) was dose-dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 1151-P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151-NP and 1151-P3 competition for the phosphorylation of a resin-immobilized 1151 peptide. While 1151-NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 1151-P3 caused a four-fold increase in the phosphorylation of 1151-NP--resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1314-1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non-phosphorylated receptor forms. Nevertheless, the 1322Ab immunoprecipitated a fully autophosphorylated receptor in the presence of 1151-NP, but not of 1151-P3, thus suggesting a conformational change induced by the non-phosphorylated peptide. In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 1151-peptide tyrosines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151-NP--resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.

Catalysis of serine and tyrosine autophosphorylation by the human insulin receptor.

The protein kinase activity of human insulin receptors purified from Sf9 insect cells after infection with a recombinant baculovirus was evaluated. The following experimental observations led to the unexpected conclusion that this receptor protein catalyzes both serine and tyrosine autophosphorylation at significant stoichiometries. (i) Phosphorylation of lectin-purified insulin receptors with [gamma-32P]ATP resulted in rapid receptor tyrosine phosphorylation (7 mol of P per high-affinity binding site) and the delayed onset of insulin-stimulated receptor serine phosphorylation (about 7% of total phosphorylation). The tyrosine kinase inhibitor (hydroxy-2-naphthalenylmethyl)phosphonic acid (HNMPA), which has no effect on protein kinase C or cyclic AMP-dependent protein kinase activities, inhibited both the receptor serine and tyrosine phosphorylation. (ii) Phosphorylation of a synthetic peptide substrate composed of insulin receptor residues 1290-1319 on serines-1305/1306 by partially purified insulin receptors was also inhibited by HNMPA. (iii) Insulin receptors sequentially affinity-purified on immobilized wheat germ agglutinin and immobilized insulin showed no apparent contaminant proteins on silver-stained SDS/polyacrylamide gels yet catalyzed autophosphorylation on receptor serine and tyrosine residues when incubated with [gamma-32P]ATP. These results suggest that the catalytic site of the insulin receptor tyrosine kinase also recognizes receptor serine residues as substrates for the phosphotransfer reaction. Furthermore, insulin-stimulated receptor serine phosphorylation in intact cells may occur in part by an autophosphorylation mechanism subsequent to tyrosine phosphorylation of the insulin receptor.

Normal insulin receptor tyrosine kinase activity and glucose transporter (GLUT 4) levels in the skeletal muscle of hyperinsulinaemic hypertensive rats.

The spontaneous hypertensive rat is an animal model characterized by a syndrome of hypertension, insulin resistance and hyperinsulinaemia. To elucidate whether in analogy to other insulin resistant animal models an inactivity of the insulin receptor kinase or an alteration of the glucose transporter (GLUT 4) level in the skeletal muscle might contribute to the pathogenesis of insulin resistance we determined insulin receptor kinase activity and GLUT 4 level in the hindlimbs of spontaneous hypertensive rats and normotensive control rats. Normotensive normoinsulinaemic Lewis and Wistar rats were used as insulin sensitive controls, obese Zucker rats were used as an insulin resistant control with known reduced skeletal muscle insulin receptor kinase activity. Binding of 125I-insulin, crosslinking of 125I-B26-insulin, autophosphorylation in vitro with 32P-ATP and phosphorylation of the synthetic substrate Poly (Glu 4: Tyr 1) were performed after partial purification of solubilized receptors on wheat germ agglutinin columns. GLUT 4 levels were determined by Western blotting of subcellular muscle membranes. Insulin receptors from spontaneous hypertensive rats compared to those from Lewis and Wistar rats showed no difference of the binding characteristics or the in vitro auto- and substrate phosphorylation activity of the receptor, while in the Zucker rats the earlier described insulin receptor kinase defect was clearly evident. Western blots of subcellular muscle membrane fractions with antibodies against GLUT 4 revealed no difference in transporter levels. These data suggest that insulin resistance in spontaneous hypertensive rats is caused neither by an insulin receptor inactivity nor by a decreased number of glucose transporters in the skeletal muscle.

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.

Effect of aging on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipocytes

The effect of aging on the insulin binding parameters and on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipose tissue has been investigated. Using solubilized receptors from adipocyte plasma membranes, no significant differences were identified in either affinity or receptor number in adult vs old rats. Time courses for in vitro receptor phosphorylation revealed that both the initial rate of autophosphorylation and the maximal 32P incorporation were decreased by 40% in old (24-month) animals as compared to adult (3-month) control rats. The tyrosine phosphatase activity associated with the adipocyte plasma membranes does not account for the decreased kinase activity found in old rats. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in both groups of rats. However, the kinase activity showed a decreased responsiveness to the hormone in the old rats. Double reciprocal plot analysis of receptor phosphorylation revealed that the K m for ATP was not modified. In contrast, the insulinstimulated V max value was decreased by two-fold in 24-month-old rats. The decrease in V max does not appear to be related to an increased basal phosphorylation level on Ser/Thr residues of the C terminus of the receptor β-subunit. Thus, we conclude that the reduced insulin receptor kinase activity in adipose tissue from old rats is due, at least in part, to a defect of the intrinsic kinase activity of the insulin receptor.

Modulation of rat brain insulin receptor kinase activity in diabetes

Insulin receptors from rat brain regions were studied for insulin binding and receptor associated kinase activity, in alloxan induced short-term and long-term diabetes, and insulin induced hypoglycemia. Insulin receptor activity was assessed by [ 123I]insulin binding, and basal as well as insulin stimulated kinase activity of the receptor, expressed as phosphorylation of the synthetic peptide poly (Glu-Tyr (4:1)). Regional distribution pattern elicited the highest binding and kinase activity in the olfactory bulb. Diabetes caused a significant increase in the kinase activity. The data suggests that brain insulin receptor kinase is regulated differently compared to peripheral tissues and supports the concept of an active brain insulin receptor in vivo.

Decrease in beta-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase.

In a previous study, we showed that the rat hepatic insulin receptor (IR) kinase of endosomes (ENs) was transiently activated to levels exceeding those of plasma membrane (PM) receptors following insulin injection. Phosphatase treatment of EN receptors abolished IR kinase activation implicating beta-subunit autophosphorylation as a mediator of the activation process (Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Foster, B., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931-12940). In the present study, the phosphotyrosine (PY) content of the IR beta-subunit in PM and ENs was estimated by two different methods. In one method, direct in vivo labeling with 32Pi followed by receptor immunoprecipitation was carried out. In the second method, immunoblotting with antibodies against the submembrane domain of the IR beta-subunit, encompassing residue 960 (alpha 960), and with antibodies against PY (alpha PY) was used to determine the content of PY/beta-subunit in PM and ENs following injection of insulin. By both methods, it was found that the PY content of PM IR was significantly greater than that of IR in ENs. With doses of 1.5 micrograms of insulin/100 g body weight (50% receptor occupancy) or 15 micrograms/100 g body weight (receptor saturation), the PY/beta-subunit of PM IR attained a level 2.0 to 2.5-fold of that observed for the IR of ENs. Surprisingly, the IR of ENs incorporated 3 to 5 times more PY/beta-subunit than those of PM consequent to autophosphorylation. Exogenous IR kinase activity (poly(Glu:Tyr)) in PM changed only slightly with insulin dose. In contrast, EN receptors exhibited a dose-dependent increase in kinase activity coincident with the decrease in PY/beta-subunit levels. A comparison of the proportion of receptor and kinase activity immunoprecipitated by alpha PY both before and after autophosphorylation indicated that ENs but not PM contained a small population of lightly phosphorylated but highly activated receptors. Since Thr12-Lys (IR kinase residues 1142-1153) efficiently inhibited IR autophosphorylation of both PM and EN receptors, Tris phosphorylation of beta-subunit regulatory tyrosines was unlikely. These results may be explicable by a dephosphorylation-dependent activation of IR kinase, as seen with the src family of tyrosine kinases.

Insulin receptor binding and kinase activity in liver and skeletal muscles of lactating goats

Lactation in goats has been shown to modify in vivo insulin action. [Debras, E., J. Grizard, E. Aina, S. Tesseraud, C. Champredon, and M. Arnal. Am. J. Physiol. 256 (Endocrinol. Metab. 19): E295-E302, 1989]. To further elucidate the mechanism of insulin action, we studied insulin binding and insulin receptor tyrosine kinase activity in solubilized and partially purified receptor preparations from liver and skeletal muscles (longissimus dorsi, tensor fascia lata, diaphragm, and masseter) from lactating and nonlactating goats. Lactation did not alter insulin receptors in the various skeletal muscles and had a minor influence on liver receptors (where only a 20% increase in receptor number was visible, P less than 0.05). Insulin-stimulated autophosphorylation and the kinase activity against polyglutamyltyrosine (4:1) were not significantly modified in skeletal muscle receptor preparations from lactating goats when compared with nonlactating animals. They tended to decrease in liver preparations, but not significantly. Thus the changes in insulin action in vivo during lactation in goats were not related to modifications in insulin kinase activity but were probably localized at a postreceptor level.

Pioglitazone increases insulin sensitivity by activating insulin receptor kinase.

A new oral agent, 5-[4-(2-(5-ethyl 12-pyridyl)ethoxy]- benzoyl]-2,4-thiazolidinedione (pioglitazone), has been developed for treatment of non-insulin-dependent diabetes mellitus (NIDDM). This agent increases insulin sensitivity in vivo in genetically obese Wistar fatty rats. Administration of the agent (3 mg/kg/day) for 10 days to the rats ameliorated hyperglycemia and hyperinsulinemia, indicating that it decreased insulin resistance. To clarify the mechanism of the drug to increase insulin sensitivity, we examined insulin binding and kinase activity of insulin receptors from muscles of both untreated and treated rats. Pioglitazone treatment did not change insulin binding in Wistar fatty rats but increased insulin-stimulated autophosphorylation of insulin receptors to 78% over the level in the control but not the basal state. Kinase activity toward exogenous substrate, poly Glu4Tyr1, was also increased to 87% over the level of untreated control obese rats. In contrast, in lean rats, pioglitazone treatment did not increase autophosphorylation and kinase activity toward exogenous substrates. To further elucidate the mechanism, we incubated insulin receptors with the agent and measured kinase activity. Incubation of solubilized receptors with the agent did not increase kinase activity. However, the receptors from IM-9 cells, which were incubated with 10(-8) M pioglitazone for 7 days, showed a 46% increase over the control in insulin-stimulated autophosphorylation and kinase activity. These results suggested that pioglitazone increased insulin sensitivity in part by activating kinase of the receptors through indirect effect on insulin receptors and that the drug may have useful benefits in insulin resistance of NIDDM.

Insulin receptor kinase activity in muscles and white adipose tissue during course of VMH obesity.

Early after lesion of the ventromedial hypothalamus nuclei (VMH), insulin-induced glucose utilization is increased in white adipose tissue (WAT), whereas oxidative and glycolytic muscles are, respectively, normoresponsive or resistant to insulin. Five weeks later, all of the muscles are resistant, whereas WAT returns to normal responsiveness. The aim of this study was to characterize the insulin receptor kinase activity in WAT and muscles 1 and 6 wk after lesion. The number and affinity of insulin receptors were not modified in any of the tissues studied. Autophosphorylation and phosphorylation of an exogenous substrate were similar in oxidative and glycolytic muscles of VMH and control rats both 1 and 6 wk after the lesion. Insulin receptors from WAT of 1-wk VMH rats exhibited a 2.5-fold increase in insulin-stimulated autophosphorylation and phosphorylation. Six weeks after the lesion, both autophosphorylation and phosphorylation returned to normal values. This suggests that insulin receptor tyrosine kinase activity does not play a significant role in the insulin resistance of skeletal muscles but has a crucial role in mediating the variations of insulin action on WAT observed during the development of VMH obesity.

Anti-insulin receptor antibody and insulin resistance

Type B insulin resistance syndrome is characterized by the presence of a circulating anti-insulin receptor antibody which inhibits insulin action. The syndrome is usually associated with autoimmune diseases, acanthosis nigricans and hyperinsulinemia. The antibody causes insulin resistance by inhibiting insulin binding to insulin receptors, decreasing insulin receptor number, i.e. down regulation of insulin receptors, and desensitizing insulin receptors which leads to decreased insulin signal transduction through insulin receptors. It also has the ability to elicit insulin action associated with or without increased autophosphorylation of insulin receptors. The antibody usually binds to the insulin receptor with decreased association rate and decreased dissociation rate. The antibody which elicits insulin action without activation of kinase activity of insulin receptors may induce conformational change of insulin receptors. These characteristic features of the anti-insulin receptor antibody may be informative in solving the mechanism of insulin action and may be a useful tool to study insulin receptor functions.

Diabetic state-induced modification of insulin-stimulated, glucose uptake into and kinase activity in, the diaphragm muscle of genetically diabetic KK-CAy mice.

The effect of insulin on glucose and 2-deoxyglucose uptake into isolated diaphragm was investigated in genetically diabetic KK-CAy mice, and in part in alloxan-treated mice. Concentration-response curves of insulin for glucose uptake (8.3 mM in vitro) for 1 h were shifted to the left in two kinds of diabetic model mice. Insulin-stimulated glucose uptake was biphasic; it was high 1 week, and returned to the normal level 4 weeks, after alloxan injection despite high blood glucose levels. Insulin-stimulated glucose uptake was the same at higher glucose levels (25 mM) as at 8.3 mM glucose for 1 h, despite an increase in basal glucose uptake (without insulin) in KK-CAy mice. Insulin-receptor kinase activity in diabetic KK-CAy mouse diaphragm also changed biphasically as the glucose concentration increased: an increase at 8.3 mM, but no increase at 16.7 mM or 25.0 mM glucose for 3 h-pretreatment including 1 h-insulin treatment. These results suggest that although the initial state of diabetes enhances insulin action, the prolonged hyperglycemia rather suppressed the insulin action in vivo.

Prevention by protein kinase C inhibitors of glucose-induced insulin-receptor tyrosine kinase resistance in rat fat cells.

Hyperglycemia causes insulin-receptor kinase (IRK) resistance in fat cells. We characterized the mechanism of IRK inhibition and studied whether it is the consequence of a glucose-induced stimulation of protein kinase C (PKC). Fat cells were incubated for 1 or 12 h in culture medium containing either a low-(5-mM) or high- (25-mM) glucose concentration. IRK was isolated, insulin binding was determined, and autophosphorylation was studied in vitro with [gamma-32P]ATP or was determined by Western blotting with anti-phosphotyrosine antibodies. Substrate phosphorylation was investigated with the artificial substrate poly(Glu80-Tyr20). Partially purified insulin receptor from rat fat cells, which were cultured under high-glucose conditions for 1 or 12 h, showed no alteration of insulin binding but a reduced insulin effect on autophosphorylation (30 +/- 7% of control) and poly(Glu80-Tyr20) phosphorylation (55.5 +/- 9% of control). Lineweaver-Burk plots of the enzyme kinetics revealed, beside a reduced Vmax, and increased KM (from 30 microM to 80 microM) for ATP of IRK from high-glucose-treated cells. Because a similar inhibition pattern was earlier found for IRK from fat cells after acute phorbol ester stimulation, we investigated whether activation of PKC might be the cause of the reduced IRK activity. We isolated PKC from the cytosol and the membrane fraction of high- and low-glucose fat cells and determined the diacylglycerol- and phospholipid-stimulated PKC activity toward the substrate histone. There was no significant change of cytosolic PKC; however, membrane-associated PKC activity was increased in high-glucose-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hyperinsulinemia and hyperglycemia on insulin receptor function and glycogen synthase activation in skeletal muscle of normal man

Insulin receptor function, glycogen synthase activity, and activation by phosphatases were studied in biopsies of human skeletal muscle under conditions of hyperglycemia and/or hyperinsulinemia for 150 minutes. Twenty-one healthy volunteers underwent either (A) a hyperinsulinemic, euglycemic clamp (serum insulin, 160.0 ± 7.7 mU/L; plasma glucose, 4.9 ± 0.1 mmol/L; n = 9), (B) a hyperglycemic clamp during normoinsulinemia (serum insulin, 18.1 ± 3.3 mU/L; plasma glucose, 12.9 ± 0.2 mmol/L; n = 6), or (C) a combined hyperinsulinemic, hyperglycemic clamp (serum insulin, 158.3 ± 15.0 mU/L; plasma glucose, 11.4 ± 0.8 mmol/L; n = 6). During all studies, the endogenous insulin secretion was inhibited with somatostatin. Insulin binding and kinase activity of insulin receptors solubilized from vastus lateralis muscle biopsies were unaffected by hyperglycemia and/or hyperinsulinemia. Hyperinsulinemia activated the muscle glycogen synthase with a decrease in the half-maximal activation constant (A 0.5) for glucose-6-phosphate (G6P) from 0.53 ± 0.04 to 0.21 ± 0.02 mmol/L (study A, P < .02) and from 0.53 ± 0.06 to 0.19 ± 0.05 mmol/L (study C, P < .03). In addition, the rate of glycogen synthase activation by phosphatases increased from 0.078 ± 0.017 to 0.134 ± 0.029 U/min/mg protein (study A, P < .03) and from 0.082 ± 0.013 to 0.145 ± 0.033 U/min/mg protein (study C, P = .05). Hyperglycemia during normoinsulinemia did not affect A 0.5 or phosphatase activity. In conclusion, (1) hyperinsulinemia for 2 1 2 hours increases glycogen synthase activity and activation by phosphatases independently on the glycemia; and (2) insulin receptor binding and basal and insulin-stimulated receptor kinase activity are not modified during short-term hyperinsulinemia and/or hyperglycemia.

Association of Metformin's effect to increase insulin-stimulated glucose transport with potentiation of insulin-induced translocation of glucose transporters from intracellular pool to plasma membrane in rat adipocytes.

To examine the cellular mechanism of the antihyperglycemic action of metformin, we studied its effect on various functional and molecular parameters involved in the pathogenesis of insulin resistance. Isolated rat adipocytes were incubated with or without metformin (1-100 micrograms/ml) for 2 h at 37 degrees C followed by an incubation with or without insulin (1.72 nM). Metformin treatment had no significant effect on basal 3-O-methylglucose uptake. In contrast, metformin increased insulin-stimulated glucose transport in a dose-dependent manner up to 43 +/- 7%. This effect was neither associated with a significant effect of metformin on trace insulin binding (1.74 +/- 0.20% without metformin vs. 1.89 +/- 0.30% with metformin; P greater than 0.05) nor with an effect of metformin on insulin-receptor kinase activity as measured by 32P incorporation into the 95,000-Mr beta-subunit of the insulin receptor and an exogenous substrate, histone 2B.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro phosphorylation of the adipocyte lipid-binding protein (p15) by the insulin receptor. Effects of fatty acid on receptor kinase and substrate phosphorylation

Phosphorylation of the adipocyte lipid-binding protein (ALBP) isolated from 3T3-L1 cells has been studied in vitro utilizing the wheat germ agglutinin-purified 3T3-L1 adipocyte insulin receptor and the soluble kinase domain of the human insulin receptor. Following insulin-stimulated, ATP-dependent autophosphorylation of the wheat germ agglutinin-purified receptor beta-subunit, ALBP was phosphorylated exclusively on tyrosine 19 in the sequence Glu-Asn-Phe-Asp-Asp-Tyr19, analogous to the substrate phosphorylation consensus sequence observed for several tyrosyl kinases. The concentration of insulin necessary for half-maximal receptor autophosphorylation (KIR0.5) was identical to that necessary for half-maximal ALBP phosphorylation (KALBP0.5), 10 nM. Kinetic analysis indicated that stimulation of ALBP phosphorylation by insulin was attributable to a 5-fold increase in the Vmax (to 0.33 fmol/min/fmol insulin-binding sites) while the Km for ALBP was largely unaffected. By utilizing the soluble kinase domain of the human receptor beta-subunit, the presence of oleate bound to ALBP increased the kcat/Km greater than 3-fold. Oleate dramatically inhibited autophosphorylation of the 38-kDa fragment of the soluble receptor kinase in a concentration dependent fashion (I0.5 approximately 4 microM). The 48-kDa kinase exhibited much less sensitivity to the effects of oleate (I0.5 approximately 190 microM). The inhibition of autophosphorylation of the 48-kDa soluble kinase by oleate was reversed by adding saturating levels of ALBP. These results demonstrate that in vitro the murine adipocyte lipid-binding protein is phosphorylated on tyrosine 19 in an insulin-stimulated fashion by the insulin receptor and that the presence of a bound fatty acid on ALBP increases the affinity of insulin receptor for ALBP. Inhibition of insulin receptor kinase activity by unbound fatty acids suggests that the end products of the lipogenic pathway may feedback inhibit the tyrosyl kinase and that fatty acid-binding proteins have the potential to modulate such interaction.