Insulin Receptor Kinase Activity Research Articles

ATP-dependent Desensitization of Insulin Binding and Tyrosine Kinase Activity of the Insulin Receptor Kinase: THE ROLE OF ENDOSOMAL ACIDIFICATION

Incubating endosomes with ATP decreased binding of 125I-insulin but not 125I-labeled human growth hormone. Increasing ATP concentrations from 0.1 to 1 mM increased beta-subunit tyrosine phosphorylation and insulin receptor kinase (IRK) activity assayed after partial purification. At higher (5 mM) ATP concentrations beta-subunit tyrosine phosphorylation and IRK activity were markedly decreased. This was not observed with nonhydrolyzable analogs of ATP, nor with plasma membrane IRK, nor with endosomal epidermal growth factor receptor kinase autophosphorylation. The inhibition of endosomal IRK tyrosine phosphorylation and activity was completely reversed by bafilomycin A1, indicating a role for endosomal proton pump(s). The inhibition of IRK was not due to serine/threonine phosphorylation nor was it influenced by the inhibition of phosphotyrosyl phosphatase using bisperoxo(1,10-phenanthroline)oxovanadate anion. Prior phosphorylation of the beta-subunit with 1 mM ATP did not prevent the inhibition of IRK activity on incubating with 5 mM ATP. To evaluate conformational change we incubated endosomes with dithiothreitol (DTT) followed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Without DTT the predominant species of IRK observed was alpha2 beta2. With DTT the alpha beta dimer predominated but on co-incubation with 5 mM ATP the alpha2 beta2 form predominated. Thus, ATP-dependent endosomal acidification contributes to the termination of transmembrane signaling by, among other processes, effecting a deactivating conformational change of the IRK.

An assay method for evaluating chemical selectivity of agonists for insulin signaling pathways based on agonist-induced phosphorylation of a target peptide.

An optical method for evaluating the physiologically relevant agonist and antagonist selectivity of an insulin signaling pathway based on an insulin-dependent on/off switching of phosphorylation of a target peptide via insulin receptor is described. Insulin receptor serves as a binding for insulin and a given insulin receptor-binding peptide as a target for an insulin-receptor complex. Upon binding of insulin to its receptor, the insulin receptor undergoes autophosphorylation which enables the receptor to have a kinase activity and phosphorylate various substrates. The phosphorylated tyrosine in the substrate was measured with a monoclonal anti-phosphotyrosine antibody. As the target substrate for insulin receptor, a Y939 peptide consisting of 12 amino acid residues derived from insulin receptor substrate 1 (IRS-1) was used. The present assay method involves different sequential steps: (1) immobilization of a biotin-coupled Y939 peptide on an avidin coated 96-well plate via biotin-avidin complexation; (2) insulin-dependent phosphorylation of the Y939 peptide by the insulin receptor; (3) enzymatic reaction and absorptiometric assay of the phosphorylated Y939 peptide using the anti-phosphotyrosine antibody labeled with horseradish peroxidase. An insulin-dependent absorbance was observed for insulin concentrations from 1.0 x 10(-10) to 1.0 x 10(-7) M, and it leveled off. The observed absorbance was explained to be due to an increase in the phosphorylated Y939 peptide caused by insulin and its receptor complexation. No signal was, however, induced by both vanadyl and vanadate ions at concentrations up to 1.0 x 10(-4) M; these results and previous intact cell level data taken together led to the conclusion that these ions did not induce phosphorylation of the Y939 peptide. Upon addition of tyrphostin 25, a specific inhibitor for insulin receptor kinase activity, phosphorylation of the Y939 peptide in the presence of 1.0 microM insulin was competitively inhibited over 1.0 x 10(-4) M tyrphostin 25. The present system thus exhibited "physiologically more relevant" agonist and antagonist selectivity, the principle of which is based in part on the insulin signal transduction rather than simply relying on the binding assay. The potential use of the present method for evaluating the selectivity of a wide range of agonists and antagonists toward the insulin signaling pathways is discussed.

Changes in the Kinase Activity of the Insulin Receptor Account for an Increased Insulin Sensitivity of Mammary Gland in Late Pregnancy

Mammary gland is an organ that undergoes cycles of growth, differentiation, and function during pregnancy and lactation. Although it is known that the gland enhances its sensitivity to insulin during lactation, it remains to be investigated whether this increased sensitivity develops during pregnancy and which are the molecular mechanisms underlying such a change. To address this issue, virgin and late-pregnant rats were subjected to a continuous infusion with 50% glucose for 72 h to produce a prolonged hyperinsulinemic-euglycemic condition. Insulin sensitivity in mammary gland was determined as the glucose utilization index by using 2-[3H]-deoxyglucose. Furthermore, binding characteristics and kinase activity were studied by means of both[ 125I]insulin binding and in vitro phosphorylation studies with insulin receptors partially purified from mammary gland. Whereas the glucose utilization index in mammary gland from nonpregnant rats remained unaffected by hyperinsulinemia, glands from pregnant rats displayed a high insulin-dependent glucose uptake. This effect was not paralleled by changes in the binding characteristics of insulin to the high-affinity receptor, suggesting that the high insulin sensitivity of mammary gland in pregnancy is not accounted for by changes at the level of hormone-receptor interaction. Autophosphorylation studies showed that insulin-stimulated kinase activity of insulin receptors from mammary gland was 6- and 20-fold higher in pregnant than in virgin animals under normo- and hyperinsulinemic conditions, respectively. Moreover, insulin dose-response curves revealed that the efficacy of insulin to stimulate kinase activity of the insulin receptor was markedly higher in pregnant than in virgin rats, whereas its potency (ED50 ∼ 15 nm) was not changed. These data, therefore, show that mammary glands develop increased insulin sensitivity during late pregnancy, caused by an augmented kinase activity of the insulin receptor.

Changes in the kinase activity of the insulin receptor account for an increased insulin sensitivity of mammary gland in late pregnancy.

Mammary gland is an organ that undergoes cycles of growth, differentiation, and function during pregnancy and lactation. Although it is known that the gland enhances its sensitivity to insulin during lactation, it remains to be investigated whether this increased sensitivity develops during pregnancy and which are the molecular mechanisms underlying such a change. To address this issue, virgin and late-pregnant rats were subjected to a continuous infusion with 50% glucose for 72 h to produce a prolonged hyperinsulinemic-euglycemic condition. Insulin sensitivity in mammary gland was determined as the glucose utilization index by using 2-[3H]-deoxyglucose. Furthermore, binding characteristics and kinase activity were studied by means of both [125I]insulin binding and in vitro phosphorylation studies with insulin receptors partially purified from mammary gland. Whereas the glucose utilization index in mammary gland from nonpregnant rats remained unaffected by hyperinsulinemia, glands from pregnant rats displayed a high insulin-dependent glucose uptake. This effect was not paralleled by changes in the binding characteristics of insulin to the high-affinity receptor, suggesting that the high insulin sensitivity of mammary gland in pregnancy is not accounted for by changes at the level of hormone-receptor interaction. Autophosphorylation studies showed that insulin-stimulated kinase activity of insulin receptors from mammary gland was 6- and 20-fold higher in pregnant than in virgin animals under normo- and hyperinsulinemic conditions, respectively. Moreover, insulin dose-response curves revealed that the efficacy of insulin to stimulate kinase activity of the insulin receptor was markedly higher in pregnant than in virgin rats, whereas its potency (ED50 approximately 15 nM) was not changed. These data, therefore, show that mammary glands develop increased insulin sensitivity during late pregnancy, caused by an augmented kinase activity of the insulin receptor.

Early signaling events triggered by peroxovanadium [bpV(phen)] are insulin receptor kinase (IRK)-dependent: specificity of inhibition of IRK-associated protein tyrosine phosphatase(s) by bpV(phen).

Peroxovanadiums (pVs) are potent protein tyrosine phosphatase (PTP) inhibitors with insulin-mimetic properties in vivo and in vitro. We have established the existence of an insulin receptor kinase (IRK)-associated PTP whose inhibition by pVs correlates closely with IRK tyrosine phosphorylation, activation, and downstream signaling. pVs have also been shown to activate various tyrosine kinases (TKs) that could participate in activation of the insulin-signaling pathway. In the present study we have sought to determine whether pV-induced IRK tyrosine phosphorylation requires the intrinsic kinase activity of the IRK, and whether IRK activation is necessary to realize the early steps in the insulin-signaling cascade. To address this we evaluated the effect of a pure pV compound, bis peroxovanadium 1,10-phenanthroline [bpV(phen)], in HTC rat hepatoma cells overexpressing normal (HTC-IR) or kinase-deficient (HTC-M1030) mutant IRKs. We showed that at a dose of 0.1 mM, but not 1 mM, bpV(phen) induced IRK-dependent events. Thus, 0.1 mM bpV(phen) increased tyrosine phosphorylation and IRK activity in HTC-IR but not HTC-M1030 cells. Tyrosine phosphorylation of insulin signal-transducing molecules was promoted in HTC-IR but not HTC-M1030 cells by bpV(phen). The association of p185 and p60 with the src homology-2 (SH2) domains of Syp and the p85-regulatory subunit of phosphatidylinositol 3'-kinase was induced by bpV(phen) in HTC-IR, but not in HTC-M1030 cells, as was insulin receptor substrate-1-associated phosphatidylinositol 3'-kinase activity. Thus autophosphorylation and activation of the IRK by bpV(phen) is effected by the IRK itself, and the early events in the insulin- signaling cascade follow from this activation event. This establishes a critical role for PTP(s) in the regulation of IRK activity. bpV(phen) could be distinguished from insulin only in its ability to activate ERK1 in HTC-M1030 cells, thus indicating that this event is IRK independent, consistent with our previous hypothesis that bpV(phen) inhibits a PTP involved in the negative regulation of mitogen-activated protein kinases.

Protein kinase C (PKC) ϵ enhances the inhibitory effect of TNFα on insulin signaling in HEK293 cells

Recently we have shown that PKC β1 and β2 are able to inhibit the tyrosine kinase activity of the human insulin receptor (HIR). Now we have investigated whether a distinct PKC isoform might be involved in the inhibitory effect of TNFα on insulin signaling in HEK293 cells. TNFα induces a rapid translocation of the PKC isoform ϵ (TNFα 10 −9 M, maximal effect within 5–10 min) in rat-1 fibroblasts, while no effect occurred on other isoforms. Cotransfection of HIR with PKC ϵ did not significantly reduce the insulin stimulated receptor kinase activity; however, when cells were incubated with TNFα for 10 min (10 −9 M) a 62±17% ( n=5) inhibition of the insulin receptor kinase activity was observed which was significantly ( P<0.01) higher than that observed in cells which were not transfected with PKC (32±11.5%, n=5). The data suggest that translocation of PKC ϵ induced by TNFα enables this PKC isoform to interact with insulin signaling and to inhibit the insulin receptor kinase activity.

Synthetic Multinuclear Chromium Assembly Activates Insulin Receptor Kinase Activity: Functional Model for Low-Molecular-Weight Chromium-Binding Substance

The biologically-active form of chromium, low-molecular-weight chromium-binding substance (LMWCr), activates the insulin-dependent tyrosine protein kinase activity of insulin receptor (IR). The site of activation was shown to be on the active site fragment of the β subunit of IR. As LMWCr previously has been proposed to contain a multinuclear chromic assembly, the ability of multinuclear chromium assemblies to activate IR kinase activity has been probed. The trinuclear cation [Cr3O(O2CCH2CH3)6(H2O)3]+ (1) has been found to activate IR activity in a fashion almost identical to that of LMWCr using rat adipocytic membrane fragments and an active site fragment of IR, while a variety of other chromic complexes have in contrast been found to be ineffective or to inhibit kinase activity. The activation of IR kinase activity by complex 1, its stability in aqueous and strongly acidic solution, and its low molecular weight suggest that it potentially could be used in a treatment for adult-onset diabetes.

Mechanisms by which insulin and muscle contraction stimulate glucose transport.

Insulin binding to its receptor activates a tyrosine kinase that initiates a cascade of signaling events, the initial step being the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1). Subsequent IRS-1 association and activation of phosphatidylinositiol 3-kinase (PI 3-kinase) is believed to be involved in the events leading to the translocation of glucose transporters (GLUT4) to the plasma membrane resulting in uptake of glucose into the cell. Muscle contractions increase insulin sensitivity, but also stimulate muscle glucose uptake independent of insulin. The contraction signaling pathway is distinct from the insulin pathway because the effect of insulin and contractions on glucose uptake are additive, and contractions do not increase insulin receptor kinase or PI 3-kinase activity. In contrast, studies indicating that contractions cause the translocation of GLUT4 and that both contractions and insulin-stimulated glucose transport can be blocked by calcium channel blockers suggest that the two pathways may converge. However, the possibility that two distinct GLUT4 pools may be targeted, one by insulin the other by contractions, indicates that additional research is needed to better define the mechanisms by which glucose transport is stimulated in muscle.

Analysis of Thermal Injury-induced Insulin Resistance in Rodents

Burn injury is associated with insulin resistance. The molecular basis of this resistance was investigated by examining insulin receptor signaling in rats after thermal injury. The impaired insulin-stimulated transport of [3H]2-deoxyglucose into soleus muscle strips confirmed the insulin resistance following burns. In vivo insulin-stimulated phosphoinositide 3-kinase activity, pivotal in translocation of GLUT4, was decreased in burns when assessed by its insulin receptor substrate-1 (IRS-1)-associated activity. Insulin-induced tyrosine kinase activity of insulin receptor (IR) and tyrosine phosphorylation of IRS-1 were also attenuated. Immunoprecipitated IR, however, appeared to have normal insulin-responsive kinase activity. Finally, immunoprecipitated IRS-1 was tested for its effect on partially purified recombinant IR and was found to inhibit its kinase activity. This inhibitory effect of IRS-1 was abolished by prior treatment of IRS-1 with alkaline phosphatase, indicating that burn injury-related hyperphosphorylation of IRS-1 is similar to that observed in TNFalpha-induced inhibition of IR signaling. All of these changes were observed in the absence of quantitative changes in IR, IRS-1, and phosphoinositide 3-kinase. Alterations in postreceptor insulin signaling, therefore, may be responsible for the insulin resistance after thermal injury.

Interaction between cAMP-dependent and insulin-dependent signal pathways in tyrosine phosphorylation in primary cultures of rat hepatocytes.

The present studies were undertaken to determine whether the interaction between cAMP-dependent and insulin-dependent pathways in primary cultures of rat hepatocytes affects biological functions and tyrosine phosphorylation. Quiescent hepatocytes were pretreated with dibutyryl cAMP or cAMP-generating agents such as glucagon, and then treated or not with insulin. Preincubation for 6 h with dibutyryl cAMP or glucagon enhanced the effect of insulin on DNA synthesis, but not the effect of insulin on amino acid transport or glycogen and protein synthesis. Tyrosine phosphorylation of intracellular proteins was determined by immunoblot analysis using an anti-phosphotyrosine antibody. Maximum tyrosine phosphorylation of a 195 kDa protein, which may be a substrate of insulin receptor kinase, of 175-180 kDa proteins, including insulin receptor substrate (IRS)-1, and of 90-95 kDa proteins, including the insulin receptor beta-subunit, was reached within 30 s of incubation with insulin. Pretreatment for about 3 h with dibutyryl cAMP or cAMP-generating agents clearly increased insulin-dependent tyrosine phosphorylation of the 195 kDa protein, but not IRS-1, IRS-2 or the insulin receptor beta-subunit. Because dibutyryl cAMP and cAMP-generating agents did not increase insulin receptor number or its kinase activity, the effect of cAMP on this potentiation of tyrosine phosphorylation is assumed to be exerted at a step distal to insulin receptor kinase activation. The potentiation by cAMP pretreatment of insulin-stimulated tyrosine phosphorylation may in part be secondary to inhibition of phosphotyrosine phosphatase activity, because cAMP pretreatment blunted the effect of Na3VO4 on the net tyrosine phosphorylation of the 195 kDa protein as compared with cells pretreated with no additive. In summary, the interactions between cAMP-dependent and insulin-dependent pathways that lead to augmentation of DNA synthesis appear to parallel the changes in tyrosine phosphorylation. Further studies will be required to determine whether there is a causal relationship between these phenomena.

Insulin stimulation of human adipocytes activates the kinase of only a fraction of the insulin receptors.

The degree of insulin receptor kinase activation by in situ stimulation was studied in isolated human adipocytes. Although maximal in situ stimulation increased the kinase activity approximately 10-fold, this activity could again be doubled by subsequent activation in a cell-free system. To investigate how in situ stimulation resulted in incomplete activation, receptors binding or not binding to anti-phosphotyrosine antibody (alpha-PY) were studied separately. Even after maximal insulin stimulation of the cells, approximately 50% of the receptors did not bind to alpha-PY and had low kinase activity. In the cell-free system, however, these receptors reached activity levels similar to the other receptors, suggesting that they were intact and that factors in their cellular environment had prevented their activation. The activity of the alpha-PY-binding receptors could only be slightly increased in the cell-free system, suggesting that almost complete activation had been attained in situ. In situ stimulation with increasing insulin concentrations increased the number of activated receptors rather than their individual activity. We conclude that factors in the in situ environment prevent insulin activation of approximately 50% of the insulin receptors in human adipocytes and might therefore be important regulators of insulin signaling.

Abnormal glucose transport and GLUT1 cell-surface content in fibroblasts and skeletal muscle from NIDDM and obese subjects.

Glucose transport and GLUT1 expression were studied in fibroblasts from 7 lean and 5 obese non-insulin-dependent diabetic (NIDDM) subjects with at least 2 NIDDM first-degree relatives and from 12 lean and 5 obese non-diabetic subjects with no family history of diabetes. The obese individuals also had a strong family history of obesity. Fibroblasts from all of the subjects exhibited no difference in insulin receptor binding, autophosphorylation, and kinase and hexokinase activity. At variance, basal 2-deoxyglucose (2-DG) uptake and 3H-cytochalasin B binding were 50% increased in cells from individuals with NIDDM (p < 0.001) and/or obesity (p < 0.01) as compared to the lean non-diabetic subjects. Insulin-dependent (maximally stimulated-basal) 2-DG uptake and cytochalasin B binding were decreased three-fold in cells from the diabetic and/or obese subjects (p < 0.01). GLUT1 mRNA and total protein levels were comparable in fibroblasts from all the groups. However, basal GLUT1 cell-surface content was 50% greater in fibroblasts from the NIDDM and/or obese subjects as compared to the lean non-diabetic individuals while insulin-dependent GLUT1 recruitment at the cell surface was diminished three-fold. Increased basal GLUT1 content in the plasma membrane was also observed in skeletal muscle of 4 NIDDM and 3 non-diabetic obese individuals (p < 0.05 vs the lean non diabetic subjects). Basal 2-DG uptake in fibroblasts from diabetic/obese individuals and lean control subjects strongly correlated with the in vivo fasting plasma insulin concentration of the donor. A negative correlation was demonstrated between the magnitude of insulin-dependent glucose uptake by the fibroblasts and plasma insulin levels in vivo. We conclude that a primary abnormality in glucose transport and GLUT1 cell-surface content is present in fibroblasts from NIDDM and obese individuals. The abnormal GLUT1 content is also present in skeletal muscle plasma membranes from NIDDM and obese individuals.

Effect of cations on the tyrosine kinase activity of the insulin receptor: inhibition by fluoride is magnesium dependent.

We have recently reported that fluoride interacts directly with the insulin receptor, which causes inhibition of its phosphotransferase activity. The inhibitory effect of fluoride on phosphotransferase activity is not due to the formation of complexes with aluminium and occurs in the absence of alterations to the binding of ATP or insulin. In this report we substantiate that the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle shows a strict requirement of Mg2+ ions (Ka near 11 mM). This effect of Mg2+ was inhibited in a competitive manner by Mn2+, which is compatible with competition of both divalent ions for binding sites. The inhibition of tyrosine kinase activity caused by fluoride was dependent on the concentration of Mg2+ in the medium and no inhibitory effect was detected at low concentrations of Mg2+. Moreover, the addition of increasing concentrations of Mn2+ in the presence of a constant high concentration of Mg2+, led to a gradual decrease in the inhibitory effect of fluoride. These results indicate that the Mg-insulin receptor complex is the major fluoride-susceptible form. Based on the characteristics of the inhibition of tyrosine kinase shown by fluoride it might be proposed that its action is exerted by the formation of multi-ionic MgF complexes analogous to Pi, which bind to the insulin receptor kinase.

Functional Association between the Insulin Receptor and the Transmembrane Protein-tyrosine Phosphatase LAR in Intact Cells

The receptor-type protein-tyrosine phosphatase LAR (for leukocyte common antigen-related) has been implicated as a physiological regulator of the insulin receptor. To demonstrate a functional interaction between LAR and the insulin receptor, we incubated CHO cells overexpressing the human insulin receptor with an antibody to the extracellular domain of LAR and found a 47% decrease in insulin receptor autophosphorylation and kinase activity. A physical association between LAR and the insulin receptor was then shown by immunoprecipitation of LAR from cell lysates and immunoblotting with antibody to the insulin receptor, or vice versa. Up to 11.8% of the LAR protein in the lysates of CHO cells overexpressing both the insulin receptor and LAR co-immunoprecipitated with the insulin receptor. The LAR/insulin receptor association was related to the level of LAR or insulin receptor overexpression and was increased 6.5-fold by chemical cross-linking and 3.9-fold by treatment with insulin, suggesting that receptor activation influences the affinity of LAR for the insulin receptor. In insulin-stimulated rat liver, LAR was temporally enriched in endosomes with the insulin receptor, and incubation of endosomes with neutralizing LAR antibodies decreased insulin receptor dephosphorylation in situ by 28% (p = 0.01 versus control). These data provide more direct evidence of a role for LAR in the physiological regulation of insulin action at the receptor level.

Severe resistance to insulin and insulin-like growth factor-I in cells from a patient with leprechaunism as a result of two mutations in the tyrosine kinase domain of the insulin receptor

We studied the biological properties of insulin receptors (IRs) and insulin-like growth factor-I (IGF-I) receptors in cultured fibroblasts from a patient with leprechaunism (leprechaun Par-1). Patient cells displayed normal insulin binding capacity and affinity. Basal in vivo auto phosphorylation and in vitro exogenous kinase activity of patient IRs were elevated twofold to threefold compared with control receptors, and insulin had no further effect on these processes. Moreover, patient IRs were unable to promote the stimulation of metabolic and mitogenic pathways. IR substrate-1 (IRS-1) and mitogen-activated protein (MAP) kinase tyrosine phosphorylation and glycogen and DNA synthesis were not increased in the basal state in patient fibroblasts and were also insensitive to the stimulatory effect of insulin. As for IGF-I, although binding and receptor kinase activity were normal, the ability to stimulate glycogen and DNA synthesis was altered in patient cells. Two mutant alleles of the IR gene were detected by denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The maternal allele contained a point mutation in exon 18 encoding the tryptophan-for-arginine substitution at position 1092, and the paternal allele had a point mutation in exon 20 substituting lysine for glutamic acid at codon 1179. Thereby, leprechaun Par-1 was a compound heterozygote for two missense mutations located in the IR β-subunit. The present investigation provides the first evidence that leprechaunism can be causally related to structural alterations in the tyrosine kinase domain of the IR. These alterations result in severe impairment of insulin and IGF-I action.

Role of the insulin receptor C-terminal acidic domain in the modulation of the receptor kinase by polybasic effectors.

Basic polymers such as polylysine have been found to activate insulin receptor autophosphorylation and kinase activity toward substrates. It was suggested that acidic receptor domains may be involved in the interaction of the receptor with these basic effectors. In a previous study, we have shown that the receptor acid-rich C-terminal sequence, including residues 1270-1280, is involved in the regulation of the receptor kinase activity. Moreover, this domain may be the site of interaction with histone, which is a modulator of the receptor kinase. In this study, we investigated whether the insulin receptor domain comprising amino acids 1270-1280 is involved in the interaction with polybasic effectors. We used anti-peptide serum directed to this sequence, and basic activators such as polylysine, polyarginine and protamine sulfate. Our antibodies inhibit polylysine-induced receptor autophosphorylation, whereas they have no effect on receptor phosphorylation stimulated by concanavalin A which is a non-basic activator of the insulin receptor. Polylysine-induced receptor aggregation was blocked by the antibodies (Fab fragments or whole Ig), indicating that competition occurs between the antibody and polylysine at the level of their binding site to the receptor. Finally, we observed a direct interaction of the 125I-peptide corresponding to receptor sequence 1270-1280 with the basic polymers in dot-blot experiments. Interestingly, the peptide did not bind spermine, a basic molecule which is not an activator of the insulin receptor kinase. Our data indicate that the insulin receptor C-terminal acidic domain including residues 1270-1280 is involved in the interaction of polylysine and other polybasic molecules with the receptor. Since this receptor region has been implicated in the regulation of the receptor kinase activity, we propose that interaction of basic effectors with this domain may be responsible for their activating properties.

Overexpression of bovine growth hormone in transgenic mice is associated with changes in hepatic insulin receptors and in their kinase activity.

Transgenic mice expressing a hybrid gene produced by linking the promoter regulatory region of phosphoenolpyruvate carboxykinase (PEPCK) gene to the bovine growth hormone (bGH) gene, were used to investigate the effects of GH on insulin binding and insulin dependent tyrosine kinase activity of hepatic insulin receptors. Transgenic mice had normal levels of blood glucose, despite hyperinsulinemia, indicating that these animals were insulin resistant. The number of insulin receptors in the liver of transgenic mice was significantly decreased in both the particulate fraction (25%) and the solubilized membranes (40%) indicating that expressed (functional) and non-expressed (cryptic) receptors were affected. Scatchard analysis of competitive binding curves for insulin indicated that the affinity of the receptor did not differ between transgenic and normal mice. Insulin dependent tyrosine kinase activity in insulin receptors partially purified by wheat germ agglutin (WGA) agarose chromatography from solubilized liver membranes, was measured. The stimulatory action of insulin on phosphorylation of the synthetic substrate (a copolymer Glu-Tyr, 4:1) was increased 100% in transgenic, as compared to normal mice, using the same binding activity. Since transgenic mice are hyperinsulinemic, it is likely that the decreased insulin binding in this group reflects down regulation of the expressed and non-expressed insulin receptors, and the increased kinase activity represents a compensatory mechanism. We conclude that alterations in the insulin receptor number and in the tyrosine kinase activity develop in response to changes in insulin levels. Thus, insulin resistance detected in the liver of transgenic mice overexpressing GH may be due to post receptor defects.

The inhibition of the insulin receptor by the receptor protein PC-1 is not specific and results from the hydrolysis of ATP.

The membrane protein plasma cell differentiation antigen 1 (PC-1) has been purified as an inhibitor of insulin receptor tyrosine kinase activity and has been implicated in the pathogenesis of NIDDM. However, we show here that PC-1 is a general protein kinase inhibitor in vitro and that this inhibition results from the hydrolysis of ATP by the intrinsic nucleotide pyrophosphatase activity of PC-1. Thus, the inhibition diminished with increasing ATP concentrations, and it was nullified when the ATP concentration was kept constant with a regenerating system or when ATP was added repetitively. When care was taken to avoid ATP depletion, PC-1 did not affect the insulin sensitivity of insulin receptor autophosphorylation. We conclude that the reported inhibition of insulin signaling by PC-1 does not result from a direct inhibition of the insulin receptor kinase activity.

Effect of plane of nutrition and growth hormone treatment on insulin receptor gene expression and kinase activity of sheep muscle and adipose tissue.

In growing animals muscle growth initially takes precedence over adipose tissue accretion [I]. The mechanisms responsible for this are unknown, but tissue specific adaptation of the insulin signalling system could be involved. We have investigated this possibility by varying growth rate in young sheep by feedmg different levels of nutrition and treating chrol4cay. with growth h o n e (GH) and determining effects on insulin receptor kinase activity and receptor protein and mRNA concentration. Thirty-two castrated male lambs of approximately 6 months of age and weighmg between 30 and 40kg were randomly assigned to four treatment groups each of which was fed varying amounts of a lamb fattener diet (55% barley, 35% oats, 2.5% soya; metabolisable energy 12.4 MJ/kg DM and a crude protein content of 112gkg DM) to achieve growth rates of either 50, 100,200 or 350glday over a 3 week period. During the last week, half of the lambs of each group were injected daily for 7 days with GH (O.2mgkgI Liuey, Somidobove, lot 505ALO). Lambs were killed by electrostatic stunning followed by exsanguination. Samples of skeletal muscle (Vastus lateralis) and perirenal adipose tissue were taken and . d a t e l y frozen in liquid N2 prior to membrane preparation and RNA isolation. Muscle membranes were prepared by homogenisation of powdered tissue in 5OmM Tris-HCI, pH 7.4 containing 5mM EDTA, 2pg/mI leupeptin, 5pdmI soyabean trypsin inhibitor, 15pdmI h a m i d i n e , using a polytron. An equal volume of 1M KCI was added, s the sheep insulin -tor CD was 92.6% and 86.0% similar to the comsponding regions of the human [8] and rat [9] insulin receptor cDNAs. Mcasurancnt of insulin receptor mRNA was determined by an RNase protection assay using standard protocols [ 101. Briefly, an antiscnsc transaipt was generated from pSIR restricted with EcoRl using SP6 RNA polymerase and then hybridiscd with 40pg samples of RNA. The produds ofthe hyhdmhon wcrc digested with RNase A and RNase T, and then precipitated with 5% (w/v) hichloroacetic acid onto GFK filters and their radioactivity determined by liquid scintillation counting.

Mechanisms Behind Insulin Resistance in Rat Skeletal Muscle After Oophorectomy and Additional Testosterone Treatment

The absence of female sex hormones, as well as testosterone treatment of oophorectomized (OVX) female rats has been demonstrated to result in decreased whole-body insulin-mediated glucose uptake. The cellular mechanism behind this insulin resistance and the role of low levels of female sex hormones as a risk factor for development of peripheral insulin resistance are not yet fully clarified. We assessed the protein expression of GLUT4 and glycogen synthase, as well as insulin-induced translocation of GLUT4 to the plasma membrane, in soleus skeletal muscle from control rats, OVX rats, and OVX rats treated for 8 weeks with testosterone (OVX + T). Whole-body insulin-mediated glucose uptake assessed by the hyperinsulinemic-euglycemic clamp procedure was 25% lower in OVX rats (P < 0.001) and addition of testosterone treatment further decreased insulin-mediated glucose uptake in OVX + T rats by 48% (P < 0.001) compared with controls. GLUT4 protein expression in soleus muscles was unaltered in the OVX and OVX + T rats compared with controls. Insulin induced a 3.7-fold increase (P < 0.05) in the plasma membrane content of GLUT4 in soleus muscle from control rats, whereas plasma membrane content of GLUT4 in soleus muscle from OVX or OVX + T rats was unaltered in response to insulin. Glycogen synthase protein expression in muscle homogenates was decreased by 25% in the OVX group (P < 0.05) and by 37% in the OVX + T group (P < 0.05) when compared with the control group. Insulin receptor and tyrosine kinase activities in the basal and insulin-stimulated states did not differ between the OVX and OVX + T rats. In conclusion, the absence of female sex hormones appears to decrease insulin-mediated whole-body glucose uptake via an impaired insulin-stimulated translocation of GLUT4 to the plasma membrane and by decreased protein expression of glycogen synthase. Testosterone treatment further impairs whole-body insulin-mediated glucose uptake, presumably by additional impairment of glycogen synthase expression.