Anti-insulin Receptor Research Articles

Development of vanadate sensitive human erythrocytes insulin receptor tyrosine phosphatase assay

The aim of this study was to investigate the effect of sodium orthovanadate on the alterations of human erythrocytes insulin receptor autophosphorylation. Human erythrocytes were incubated with insulin in a cell system and then lysed. The autophosphorylated insulin receptors were measured with the aid of a two-site immunofluorometric assay and using a monoclonal anti-insulin receptor antibody to label the insulin receptors and a monoclonal anti-phosphotyrosine antibody to assess tyrosine phosphorylation. When the erythrocytes were treated with insulin and then reincubated in insulin-free medium, vanadate completely inhibited insulin receptor dephosphorylation, although it had no effect on in vitro receptor autophosphorylation. Thus insulin receptor tyrosine phosphatase activity is postulated to be [% (autophosphorylated insulin receptors with vanadate−autophosphorylated insulin receptors without vanadate)/total insulin receptors] under overall steady conditions in a cell system. Using this assay, the insulin receptor tyrosine phosphatase activities of 25 control and 32 diabetic subjects were studied. There was no significant difference in insulin receptor tyrosine phosphatase activity between control subjects and diabetic subjects (0.173±0.062 vs 0.209−±0.057 autophosphroylated insulin receptors units/insulin receptors units). The assay used in this study requires only 0.6 ml of whole blood, and so should be a useful tool for detecting patients who are insulin-resistant due to abnormal insulin receptor tyrosine phosphatase activity.

Development of an Assay for Bioactive Insulin

A method to detect the biological activity of serum insulin has been developed. This method, called a bioactive insulin assay, determines the ability of serum insulin to stimulate the autophosphorylation of insulin receptors in an intact cell system. For this, intact Chinese hamster ovary cells which overexpress the human insulin receptor are treated with serum and then lysed. Autophosphorylation of the insulin receptors is then measured by a two-site immunofluorometric assay using monoclonal anti-insulin receptor antibodies and europium-labeled anti-phosphotyrosine antibodies. The detection limit of this assay is 1 μU/ml of insulin. Dilution and recovery test inter- and intraassay coefficient variations are permissible. The amount of insulin determined by this assay correlated well with the amount of insulin detected by a traditional immunological assay for insulin (r= 0.94,P< 0.001). In the case of a mutant insulin, the insulin from a Wakayama subject, the biologically active insulin was found to constitute 9% of the immunologically reactive insulin. Since this assay specifically measures the amount of biologically active insulin present in serum, it should be particularly useful in monitoring active insulin in patients with various mutant insulins.

Impaired activation of phosphoinositide 3-kinase by insulin in fibroblasts from patients with severe insulin resistance and pseudoacromegaly. A disorder characterized by selective postreceptor insulin resistance.

Some patients with severe insulin resistance develop pathological tissue growth reminiscent of acromegaly. Previous studies of such patients have suggested the presence of a selective postreceptor defect of insulin signaling, resulting in the impairment of metabolic but preservation of mitogenic signaling. As the activation of phosphoinositide 3-kinase (PI 3-kinase) is considered essential for insulin's metabolic signaling, we have examined insulin-stimulated PI 3-kinase activity in anti-insulin receptor substrate (IRS)-1 immunoprecipitates from cultured dermal fibroblasts obtained from pseudoacromegalic (PA) patients and controls. At a concentration of insulin (1 nM) similar to that seen in vivo in PA patients, the activation of IRS-1-associated PI 3-kinase was reduced markedly in fibroblasts from the PA patients (32+/-7% of the activity of normal controls, P < 0.01). Genetic and biochemical studies indicated that this impairment was not secondary to a defect in the structure, expression, or activation of the insulin receptor, IRS-1, or p85alpha. Insulin stimulation of mitogenesis in PA fibroblasts, as determined by thymidine incorporation, was indistinguishable from controls, as was mitogen-activated protein kinase phosphorylation, confirming the integrity of insulin's mitogenic signaling pathways in this condition. These findings support the existence of an intrinsic defect of postreceptor insulin signaling in the PA subtype of insulin resistance, which involves impairment of the activation of PI 3-kinase. The PA tissue growth seen in such patients is likely to result from severe in vivo hyperinsulinemia activating intact mitogenic signaling pathways emanating from the insulin receptor.

Molecular cloning and characterization of Xenopus insulin-like growth factor-1 receptor: its role in mediating insulin-induced Xenopus oocyte maturation and expression during embryogenesis.

We have cloned a complementary DNA encoding the putative Xenopus insulin-like growth factor-1 (xIGF-1) receptor. Injection of messenger RNA derived from the cloned complementary DNA into Xenopus oocytes resulted in the expression and correct processing of the receptor's alpha- and beta-subunits. Using antibodies generated against protein expressed against the cloned sequence, we demonstrated that the endogenous xIGF-1 receptor in Xenopus oocytes was activated by nanomolar concentrations of mammalian IGF-1 and by insulin approximately 100-fold higher in concentration. This receptor activation profile correlated with hormone-induced Xenopus oocyte maturation. Furthermore, injection of a neutralizing antiinsulin receptor antibody into Xenopus oocytes inhibited hormone-induced xIGF-1 receptor activation. These results provide molecular and biochemical evidence supporting a role for xIGF-1 receptor in mediating insulin/IGF-1-induced Xenopus oocyte maturation. We also report here that embryonic transcription of xIGF-1 receptor is activated during the formation of the central nervous system in early Xenopus embryos.

Oral insulin is biologically active on rat immature enterocytes.

Oral insulin is biologically active on rat immature enterocytes.

Insulin-induced Activation of NADPH-dependent H2O2 Generation in Human Adipocyte Plasma Membranes Is Mediated by Gαi2

Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.

Defects in insulin signal transduction in liver and muscle of pregnant rats.

Pregnancy is known to induce resistance, but the exact molecular mechanism involved is unknown. In the present study, we have examined the levels and phosphorylation state of the insulin receptor and of insulin receptor substrate 1 (IRS-1), as well as the association between IRS-1 and phosphatidylinositol 3-kinase (PI 3-kinase) in the liver and muscle of pregnant rats (day 20 of gestation) by immunoprecipitation and immunoblotting with anti-insulin receptor, anti-IRS-1, anti-PI 3-kinase and anti-phosphotyrosine antibodies. There were no changes in the insulin receptor concentration in the liver and muscle of pregnant rats. However, insulin stimulation of receptor autophosphorylation, as determined by immunoblotting with antiphosphotyrosine antibody, was reduced by 30 +/- 6% (p < 0.02) in muscle and 36 +/- 5% (p < 0.01) in liver at day 20 of gestation. IRS-1 protein levels decreased by 45 +/- 6% (p < 0.002) in liver and by 56 +/- 9% (p < 0.002) in muscle of pregnant rats. In samples previously immunoprecipitated with anti-IRS-1 antibody and blotted with antiphosphotyrosine antibody, the insulin-stimulated IRS-1 phosphorylation levels in the muscle and liver of pregnant rats decreased by 70 +/- 9% (p < 0.01) and 75 +/- 8% (p < 0.01), respectively. The insulin-stimulated IRS-1 association with PI 3-kinase decreased by 81 +/- 6% in muscle (p < 0.01) and 79 +/- 11% (p < 0.01) in the liver during pregnancy. These data suggest that changes in the early steps of insulin signal transduction may have a role in the insulin resistance observed in pregnancy.

Protein tyrosine phosphatase 1B interacts with the activated insulin receptor.

Protein tyrosine phosphatase 1B (PTP1B) is a protein tyrosine phosphatase of unknown function, although increasing evidence supports a role for this phosphatase in insulin action. We have investigated the interaction of PTP1B with the insulin receptor using a PTP1B glutathione S-transferase (GST) fusion protein with a point mutation in the enzyme's catalytic domain. This fusion protein is catalytically inactive, but the phosphatase's phosphotyrosine binding site is maintained. The activated insulin receptor was precipitated from purified receptor preparations and whole-cell lysates by the inactive PTP1B-GST, demonstrating a direct association between the insulin receptor and PTP1B. A p120 of unknown identity was also precipitated from whole-cell lysates by the PTP1B fusion protein, but IRS-1 (pp185) was not. A catalytically inactive [35S]PTP1B-fusion protein bound directly to immobilized insulin receptor kinase domains and was displaced in a concentration-dependent manner. Finally, tyrosine-phosphorylated PTP1B was precipitated from whole-cell lysates by an anti-insulin receptor antibody after insulin stimulation. The site of interaction between PTP1B and the insulin receptor was studied using phosphopeptides modeled after the receptor's kinase domain, the NPXY domain, and the COOH-terminal. Each phosphopeptide inhibited the PTP1B-GST:insulin receptor interaction. Study of mutant insulin receptors demonstrated that activation of the kinase domain is necessary for the PTP1B:insulin receptor interaction, but receptors with deletion of the NPXY domain or of the COOH-terminal can still bind to the PTP1B-GST. We conclude that PTP1B can associate directly with the activated insulin receptor at multiple different phosphotyrosine sites and that dephosphorylation by PTP1B may play a significant role in insulin receptor signal transduction.

Insulin Induces Tyrosine Phosphorylation of JAK2 in Insulin-sensitive Tissues of the Intact Rat

The Janus kinase family of protein tyrosine kinases constitutes a novel type of signal transduction pathway activated in response to a wide variety of polypeptide ligands and has four known members: JAK1, JAK2, JAK3, and Tyk2. In this study, we examined the ability of insulin to stimulate JAK2 tyrosine phosphorylation in insulin-sensitive tissues of the intact rat using immunoprecipitation and immunoblotting. The results demonstrate that after an infusion of insulin, JAK2 is rapidly tyrosine phosphorylated (and the kinase is activated) in the liver, adipose tissue, skeletal muscle, heart, and isolated adipocytes. The presence of phosphorylated JAK2 was detectable after an infusion of insulin that increased serum insulin to physiological postprandial levels (40-70 microunits/ml). Co-immunoprecipitation with anti-insulin receptor antibody, anti-JAK2 antibody, and anti-IRS-1 antibody showed that JAK2 interacts with the insulin receptor and IRS-1 to form stable complexes following stimulation by insulin. In two animal models of insulin resistance the regulation of JAK2 tyrosine phosphorylation after insulin infusion paralleled the phosphorylation of the insulin receptor and of IRS-1. In conclusion, our data indicate that after physiological stimulation by insulin in the intact animal, JAK2 associates with the insulin receptor and is tyrosine phosphorylated in insulin-sensitive tissues in a time- and dose-dependent fashion.

Isolation of a nuclease-resistant decoy RNA that selectively blocks autoantibody binding to insulin receptors on human lymphocytes.

An RNA containing 2'-amino pyrimidines has been isolated using in vitro selection techniques that specifically and avidly (apparent Kd approximately 30 nM) binds a mouse monoclonal antibody called MA20. This 2'-amino-derivatized RNA is at least 10,000-fold more stable than unmodified RNA in serum, and can act as a decoy and block MA20 binding to its natural antigen, the human insulin receptor, on lymphocytes. Furthermore, this RNA decoy can inhibit MA20-mediated downmodulation of insulin receptor expression on human lymphocytes in culture by up to 90%. Surprisingly, the decoy RNA cross-reacts with autoantibodies from patients with extreme insulin resistance and can inhibit these antiinsulin receptor antibodies from downmodulating insulin receptor expression by up to 80% without impeding insulin binding to its receptor. These results suggest that in vitro-selected decoy RNAs may be able to specifically and selectively block oligoclonal autoimmune responses to self-antigens in patients with autoimmune diseases.

Insulin-like growth factor I and insulin down-regulate growth hormone (GH) receptors in rat osteoblasts: evidence for a peripheral feedback loop regulating GH action.

The anabolic actions of GH are mediated by the production of insulin-like growth factor I (IGF-I) from the liver and by local production of IGF-I in extrahepatic tissues. Insulin facilitates the hepatic production of IGF-I by up-regulating GH receptors (GHRs) in the liver and augmenting the IGF-I response to GH. Although GHRs have also been identified in extrahepatic tissues that produce IGF-I, the possibility that IGF-I and insulin might partake in GHR regulation, thereby modulating the effects of GH locally has not received detailed study. The aim of this study was to investigate whether IGF-I and insulin are involved in the local regulation of GHRs, using osteoblasts as a model of GH-responsive extrahepatic tissues. We have used UMR106.06, a well differentiated rat osteoblast-like cell line that expresses GHRs and exhibits a mitogenic response to GH. IGF-I and insulin (0-10 nM) increased cell number and reduced [125I]GH binding in a concentration-dependent manner, with ED50 values of 0.8 and 0.3 nM, respectively. Although IGF-I increased cell number maximally by 36.9 +/- 1.2% (mean +/- SE) above the control value and insulin by 104.8 +/- 5.7% (P < 0.001), they decreased GH binding to 47.0 +/- 9.3% (P < 0.01) and 29.8 +/- 8.7% of the control value (P < 0.001), respectively. Scatchard analysis revealed that the down-regulation of GH binding was attributed to reduced receptor numbers and not binding affinity. The effects of IGF-I and insulin at submaximal concentrations were additive, although the combined effects did not exceed the maximal effect of either growth factor alone. Addition of an anti-IGF-I receptor antibody (alpha IR3) reversed the inhibition of GH binding induced by IGF-I, but not that caused by insulin; similarly, an antiinsulin receptor antibody (29B4) attenuated the inhibitory effect of insulin only. Addition of alpha IR3 alone or an ant-IGF-I antibody (Sm1.2) decreased cell number and increased GH binding in a concentration-dependent mode. GH at 1.5 nM significant increased cell number by 19.3 +/- 2.4% above the control level (P < 0.01), an increase that was reversed by alpha IR3. GH increased GH binding by 32.4 +/- 7.2% (P < 0.05) in cells treated with alpha IR3 to remove the secondary effect of IGF-I. In summary, IGF-I and insulin acted via specific receptors to stimulate cell proliferation and down-regulate GHRs in osteoblasts. GH stimulated cell proliferation, an action mediated by local production of IGF-I, and GH enhanced its own binding. The collective data suggest the presence of a peripheral negative feedback loop that allows IGF-I to limit locally the response of extrahepatic tissues to circulating GH.

A Peroxovanadium Compound InducesXenopusOocyte Maturation: Inhibition by a Neutralizing Anti-insulin Receptor Antibody

Synthetic peroxovanadium compounds are a new class of potent inhibitors of protein phosphotyrosine phosphatases. These compounds exhibit insulin-like activity bothin vitroand in experimental animals. However, the molecular mechanism by which these compounds exert their biological effect is not well defined. We demonstrate here that several of these compounds induceXenopusoocyte maturationin vitro,as indicated by germinal vesicle breakdown. Using one of these compounds for further studies, we show that the induction is dose-dependent and is accompanied by activation of maturation promoting factor as well as activation ofXenopusMAP kinase. Like insulin, bpV(pic) causes an acute accumulation of PI(3,4,5)P3(phosphotidylinositol-3,4,5-trisphosphate), a product of PI 3-kinase. More importantly, bpV(pic)-induced oocyte maturation was abolished by microinjection of a neutralizing monoclonal anti-insulin receptor antibody (17A3) into oocytes or preincubation of oocytes with a PI 3-kinase inhibitor (wortmannin). These results suggest that bpV(pic) acts upstream of theXenopusIGF-1 receptor in the induction of meiotic maturation, presumably by neutralizing an inhibitory protein tyrosine phosphatase(s) that may regulate the receptor. Finally, using an oocyte–follicle cell complex that responded to human chorionic gonadotropin (hCG) to undergo GVBD, we showed that injection of 17A3 anti-insulin receptor antibody into oocytes did not affect hCG-induced oocyte maturation.

Insulin-induced activation of phosphoinositide 3-kinase in Fao cells.

Phosphoinositide 3-kinase (PI3-kinase) plays a crucial role in insulin signal transduction. We studied the molecular mechanism of the insulin-induced activation of PI3-kinase in rat hepatoma Fao cells using an antibody against the 110-kDa catalytic subunit (p110) and two against the 85-kDa regulatory subunit (p85 alpha). PI3-kinase activity increased 1.6-fold in anti-p85 immunoprecipitates after insulin stimulation, whereas it did not increase when cell lysates were first immunoprecipitated with anti-phosphotyrosine or anti-insulin receptor substrate-1 (IRS-1), then with anti-p85, suggesting that the PI3-kinase which associates with tyrosyl phosphoproteins including IRS-1 is responsible for the increase in kinase activity. The activated PI3-kinase molecules constituted 4-6% of the total PI3-kinase, and their specific activity was 11-14 times higher than that of the basal state. Anti-p110 recognized the catalytically active form of p110, and immunoprecipitated p110 only after exposure to insulin. Hence, the epitope of anti-p110, P200-C215, seems to be included in the portion of p110, the conformation of which is changed by insulin stimulation. We conclude that, in response to insulin stimulation, only a small fraction of p85 in the PI3-kinase pool associates with tyrosyl phosphoproteins including IRS-1, and that the specific activity of p110 is increased presumably through a conformational change including the P200-C215 region.

Two Naturally Occurring Mutant Insulin Receptors Phosphorylate Insulin Receptor Substrate-1 (IRS-1) but Fail to Mediate the Biological Effects of Insulin: EVIDENCE THAT IRS-1 PHOSPHORYLATION IS NOT SUFFICIENT FOR NORMAL INSULIN ACTION

Two naturally occurring mutant insulin receptors, Arg-1174 --> Gln and Leu-1178 --> Pro, found in patients with dominantly inherited Type A insulin resistance, showed unusual signaling properties when stably expressed in Chinese hamster ovary (CHO) cells. Both mutant receptors were expressed on the cell surface and bound insulin normally, but showed markedly impaired autophosphorylation in response to insulin. In addition, the in vitro tyrosine kinase activity of both mutant receptors toward an artificial substrate was also severely impaired. Despite these defects of kinase activity, anti-phosphotyrosine immunoblotting of whole cell lysates and anti-phosphotyrosine immunoprecipitation of 32P-labeled cells showed insulin-stimulated tyrosine phosphorylation of a protein of approximately 185 kDa to an extent comparable to that seen in CHO cells expressing wild-type human insulin receptors. Anti-insulin receptor substrate-1 (IRS-1) immunoprecipitation followed by anti-phosphotyrosine immunoblotting confirmed that this tyrosine-phosphorylated protein was IRS-1. In contrast, CHO cells expressing an insulin receptor mutated at the ATP binding site (Lys-1030 --> Arg) showed no insulin-stimulated autophosphorylation or phosphorylation of IRS-1. Despite exhibiting apparently normal insulin stimulation of IRS-1 tyrosine-phosphorylation, cells expressing the Arg-1174 --> Gln or Pro-1178 --> Leu receptors showed marked impairment in insulin stimulation of glycogen synthesis, thymidine incorporation, and activation of MAP kinase. The inability of these mutant receptors to signal normally to metabolic and mitogenic responses suggests that insulin-stimulated tyrosine phosphorylation of IRS-1 alone is insufficient to fully mediate insulin action.

Insulin receptor mediates inhibitory effect of insulin, but not of insulin-like growth factor (IGF)-I, on IGF binding protein 1 (IGFBP-1) production in human granulosa cells.

Insulin-like growth factor binding proteins (IGFBPs) may participate in regulating ovarian function by modifying effects of insulin-like growth factors (IGFs) or by directly affecting ovarian steroidogenesis in both normal and pathological circumstances. The latter include hyperinsulinemic insulin resistant states, such as polycystic ovary syndrome. We examined regulation of IGFBP-1 production in human granulosa cells by insulin and IGF-I. The cells were obtained during in vitro fertilization, plated in McCoy-5A tissue culture medium supplemented with 10% fetal calf serum (10(5) cells/0.5 mL), and incubated at 37 C, 90% humidity, 5% CO2 for 48 h. After additional 24 h incubation without fetal calf serum, 1, 10, or 100 ng/mL of insulin or IGF-I were added with or without 2 h preincubation with 10 micrograms/mL monoclonal anti insulin receptor antibody IR-47-9. After 48 h incubation with insulin or IGF-I, the medium was collected and IGFBP-1 and progesterone concentrations were measured, using kits from Diagnostic Systems Laboratories, Webster, TX. Progesterone concentration ranged between 50-100 ng/mL/10(5) cells, without consistent stimulatory effect of either insulin or IGF-I. Control cells produced 7.0 +/- 1.7 ng/mL of IGFBP-1. Incubation with 1 or 10 ng/mL of insulin resulted in culture medium IGFBP-1 concentrations of 7.1 +/- 1.3 ng/mL and 5.4 +/- 0.7 ng/mL, respectively (P = NS). Incubation with 100 ng/mL of insulin reduced IGFBP-1 culture medium concentration to 1.6 +/- 0.3 ng/mL (P < 0.01, compared with controls). 1, 10, and 100 ng/mL of IGF-I inhibited IGFBP-1 concentrations in the conditioned culture medium to 1.3 +/- 0.3 ng/mL, 0.4 +/- 0.1 ng/mL and 0.3 +/- 0.1 ng/mL, respectively (P < 0.01, compared with controls). Preincubation with antiinsulin receptor antibody IR-47-9 alleviated inhibitory effect of insulin, but not of IGF-I on IGFBP-1 production. After preincubation with IR-47-9, IGFBP-1 culture medium concentrations were 5.9 +/- 0.8 ng/mL, 4.9 +/- 1.2 ng/mL, and 4.8 +/- 1.3 ng/mL for 1, 10, and 100 ng/mL of insulin, respectively. The latter number was significantly higher than IGFBP-1 concentration in the medium collected from cells incubated with 100 ng/mL of insulin without IR-47-9 (1.6 +/- 0.3 ng/mL, P < 0.01) and not significantly different from the control cells. For cells preincubated with IR-47-9 and then incubated with 1, 10, or 100 ng/mL of IGF-I, the IGFBP-1 conditioned culture medium concentrations were 1.7 +/- 0.1 ng/mL, 0.5 +/- 0.2 ng/mL, and 0.3 +/- 0.1 ng/mL, respectively. None of these were significantly different from the IGFBP-1 concentrations in the medium collected from cells incubated with the respective concentrations of IGF-I without preincubation with IR-47-9. We conclude that 1) both insulin and IGF-I inhibit IGFBP-1 production by cultured human granulosa cells; 2) IGF-I is a more potent inhibitor of IGFBP-1 production than insulin; 3) in the range of hormone concentrations tested, insulin exerts its inhibitory effect on IGFBP-1 production via insulin receptor, while IGF-I appears to exert its effect via another receptor.

A Case of Chronic Thyroiditis that Subsequently Developed Diabetes Mellitus Due to Anti-insulin Receptor Antibodies

A Case of Chronic Thyroiditis that Subsequently Developed Diabetes Mellitus Due to Anti-insulin Receptor Antibodies

Insulin action in human granulosa cells from normal and polycystic ovaries is mediated by the insulin receptor and not the type-I insulin-like growth factor receptor.

In order to account for the effects of insulin on the polycystic ovary (PCO), despite peripheral insulin resistance in women with polycystic ovary syndrome (PCOS), it has been suggested that insulin may act through the type-I insulin-like growth factor (IGF) receptor and not the insulin receptor. We have tested this hypothesis by investigating the effect of anti-insulin receptor and anti-type-I IGF receptor antibodies on insulin-stimulated steroidogenesis in human granulosa cells in vitro from normal (N) and PCO. Insulin-stimulated estradiol and progesterone production was inhibited by an anti-insulin receptor antibody. In contrast, anti-type-I IGF receptor antibodies had no effect on insulin-stimulated steroidogenesis in granulosa cell cultures from N or PCO. Hence insulin acts via its own receptor in human granulosa cells from both N and PCO.

Elevation of Serum Insulin Concentration During Euglycemic Hyperinsulinemic Clamp Studies Leads to Similar Activation of Insulin Receptor Kinase in Skeletal Muscle of Subjects With and Without NIDDM

The role of skeletal muscle insulin receptor kinase in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM) was investigated. Muscle biopsies from 13 patients with NIDDM and 10 control subjects at fasting serum insulin concentrations and approximately 1,000 pmol/l steady-state serum insulin during euglycemic hyperinsulinemic clamps were immediately frozen. The biopsies were then solubilized, and the receptors were immobilized to anti-insulin receptor antibody-coated microwells. Receptor kinase and binding activities were consecutively measured in these wells. The increase in serum insulin concentration (73 +/- 14 to 1,004 +/- 83 and 45 +/- 7 to 1,07 +/- 77 pmol/l in the NIDDM and control groups, respectively) had similar effects on receptor kinase activity in both study groups (12 +/- 1 to 42 +/- 5 and 12 +/- 2 to 47 +/- 5 amol P.fmol binding activity-1. min-1 in the NIDDM and control groups, respectively). Moreover, by selecting only the receptors that bound to anti-phosphotyrosine antibody, we found similar hyperinsulinemia-induced increases of this receptor fraction and its kinase activity in both study groups. In vitro activation of the immobilized receptors with 2 mmol/l ATP and insulin further increased their kinase activity to almost similar levels, independently of whether they had been previously stimulated in vivo or were from diabetic or nondiabetic subjects. Compared with this activity reached in vitro, the kinase activity obtained by in vivo stimulation at the clamp insulin concentration was only approximately 12%, because most receptors remained inactive and only a few reached almost the in vitro activation level.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of human, porcine and bovine insulin to insulin receptors from human brain, muscle and adipocytes and to expressed recombinant alternatively spliced insulin receptor isoforms

Previous studies have suggested that human and porcine insulin exert identical effects on blood glucose and counter-regulatory hormones but elicit different neurophysiological reactions. A major goal of the present study was to investigate whether this could be caused by different relative affinities of the insulins from different species to insulin receptors from the brain compared to other tissues. Insulin receptors isolated from human brain, muscle or adipocytes as well as from cultured cells over-expressing either of the human insulin receptor isoforms (exon 11- or exon 11 +) were immobilized to microwells coated with monoclonal anti-insulin receptor antibody. Subsequently the binding of human, porcine and bovine insulin was measured. While the receptors derived from the different tissues had different affinities for insulin, there were no tissue-specific differences in the relative binding of the insulins of the three species. The insulins of the three species were also not different with regard to their binding to the receptor isoforms. Finally, in human brain homogenates no differences in the degradation rates for human, porcine and bovine insulin were detected. Thus, our data do not support the hypothesis that different neurophysiological reactions during hypoglycaemia due to human or porcine insulin are caused by differences of the binding of the insulins to human brain insulin receptors or their degradation in the human brain.

Mechanisms of Enhanced Transmembrane Signaling by an Insulin Receptor Lacking a Cytoplasmic β-Subunit Domain

We have recently characterized a mutant insulin receptor (HIR delta 978) in which the insulin receptor beta-subunit was truncated at amino acid residue 978. Compared with parental Rat1 cells, the cells expressing the truncated receptor exhibited enhanced sensitivity to insulin's biologic actions. All of these effects are now extended to transcriptional events, since we now show enhanced sensitivity to insulin stimulation of c-fos mRNA expression. These effects were insulin-specific, since insulin-like growth factor-1 stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, and thymidine incorporation into DNA were normal. In addition, the truncated receptor exhibited enhanced sensitivity only in vivo, but not in vitro, since the kinase activity of wheat germ agglutinin-purified receptor preparations was comparable between HIR delta 978 and parental Rat1 insulin receptors. Parental rat endogenous insulin-like growth factor-1 receptors and transfected human insulin receptors form hybrid receptors as well as homologous tetrameric receptors. The normal heterotetrameric receptors possess kinase activity in vivo leading to tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with the p85 regulatory subunit of phosphatidyl inositol 3-kinase. Interestingly, preincubation with human-specific anti-insulin receptor antibody abolished the increased insulin sensitivity in glucose incorporation into glycogen in HIR delta 978 cells. Furthermore, microinjection of anti-IRS-1 antibody into HIR delta 978 cells inhibited insulin stimulation of DNA synthesis. In summary: 1) truncated receptors on the cell surface confer enhanced insulin sensitivity in vivo; 2) the normal heterotetrameric receptors are functionally active and couple to IRS-1 efficiently; and 3) IRS-1 is an important molecule transmitting insulin's biological signals in HIR delta 978 cells.