Insulin Receptor Kinase Activity Research Articles

Polyunsaturated fatty acid regulation of lipogenic enzyme gene expression in liver of genetically obese rat.

The polyunsaturated fatty acid regulation of lipogenic enzyme gene expression in genetically obese rats (Wistar fatty, non-insulin-dependent diabetes mellitus) has been investigated. The hepatic mRNA concentrations and activities of lipogenic enzymes in the fatty and lean rat were greatly increased by feeding a hydrogenated fat diet to fasted rats, and also reached similar maximum levels with similar time courses. By feeding a corn oil diet, however, the increases were markedly reduced in the lean rats, but were not significantly reduced in the fatty rats. Consequently, when the animals were fed corn oil, the mRNA concentrations and activities in the fatty rats were higher than those in the lean. Thus, it appeared that the higher gene expression in the fatty rats can be ascribed to the defects of polyunsaturated fatty acid suppression. On the other hand, insulin binding to receptors in the liver was reduced by the corn oil diet in the lean rats but was not reduced in the fatty rats (although the insulin binding level was lower in the Wistar fatty rats than in the lean). Changes in the insulin receptor autophosphorylation and kinase activity toward exogenous substrate were similar to the insulin binding. It is suggested that the polyunsaturated fatty acids may not suppress insulin binding activity to receptors in the livers of the fatty rats, probably due to down regulation by hyperinsulinemia. The defects of polyunsaturated fatty acid suppression of lipogenic enzyme gene expression may be one of the factors of obesity.

Effect of in vivo thyroid hormone status on insulin signalling and GLUT1 and GLUT4 glucose transport systems in rat adipocytes.

To examine the effect of thyroid hormone status on insulin action in isolated rat adipocytes, age- and weight-matched Sprague-Dawley rats were rendered hypothyroid (h) by i.p. injection of 2 mCi [131I]/kg. Another group of rats was made hyperthyroid (H) by i.p. injection of 500 micrograms L-thyroxine/kg/day for 7 days. The T4 levels in experimental groups were: controls, 33.5 +/- 0.95; h, 12.3 +/- 1.59; H, 133.2 +/- 8.8 micrograms/l. Adipocytes were isolated and 3-O-methylglucose transport (GT), insulin binding (IB) and insulin receptor kinase activity (IRKA) were determined. Subcellular membrane fractions (low-density microsomes, plasma membranes) were prepared and GLUT1 and GLUT4 glucose transporter immunodetected. Hyperthyroidism caused no significant effect on either IB or IRKA but increased insulin-stimulated GT by 43.6%. This increase of GT was associated with an increase of primarily GLUT4 glucose transporters. Hypothyroidism was associated with both increased insulin receptor affinity and enhanced IRKA. Despite a marked reduction of primarily GLUT4 glucose transporters, basal and insulin-stimulated GT was not reduced when compared with control. These results suggest that (1) in hyperthyroidism, increased insulin-stimulated glucose transport is associated with an increase of primarily GLUT4 glucose transporters, which may be responsible for the increment of peripheral glucose utilization in hyperthyroidism, and (2) the effect of hypothyroidism on insulin action in adipocytes is characterized by a state of increased insulin sensitivity, as indicated by the increase in insulin receptor affinity and tyrosine kinase activity. Despite the marked reduction of primarily GLUT4 glucose transporters, insulin-stimulated glucose transport is not diminished, which may suggest that functional activity of plasma membrane glucose transporters is enhanced in hypothyroidism.

Membrane glycoprotein PC-1 and insulin resistance in non-insulin-dependent diabetes mellitus.

Most patients with non-insulin-dependent diabetes mellitus are resistant to both endogenous and exogenous insulin. Insulin resistance precedes the onset of this disease, suggesting that it may be an initial abnormality. Insulin-receptor kinase activity is impaired in muscle, fibroblasts and other tissues of many patients with non-insulin-dependent diabetes mellitus, but abnormalities in the insulin-receptor gene do not appear to be the cause of this decreased kinase activity. Skin fibroblasts from certain insulin-resistant patients contain an inhibitor of insulin-receptor tyrosine kinase. Here we show that this inhibitor is a membrane glycoprotein, termed PC-1 (refs 10, 11). We find that PC-1 activity is increased in fibroblasts from seven of nine patients with typical non-insulin-dependent diabetes mellitus. In addition, overexpression of PC-1 in transfected cultured cells reduces insulin-stimulated tyrosine kinase activity. These studies raise the possibility that PC-1 has a role in the insulin resistance of non-insulin-dependent diabetes mellitus.

An extracellular domain of the β subunit is essential for processing, transport and kinase activity of insulin receptor

The extracellular portion of the insulin receptor (IR) beta-subunit has four cysteine and four asparagine residues which are potentially involved in disulphide bond formation between the alpha- and beta-subunits and N-linked glycosylation respectively. However, the function of this portion is not fully understood. In order to investigate the role of the extracellular domain of beta-subunit, we created a deletion mutant of IR cDNA which lacked 47 amino acid residues encoded by 141 bp corresponding to exon 13 of the IR gene. Insulin binding and surface labelling of COS 7 cells transiently expressing the mutant insulin receptors (IR delta Ex13) showed that the mutated receptors were not expressed on the cell surface. However, immunoblot analysis showed that uncleaved form (190 kDa) of the mutant receptors were intracellularly expressed. Deglycosylation with endoglycosidase H showed that the mutant receptors had mainly high-mannose oligosaccharide chains. The mutant IRs bound with high affinity to lentil lectin but with low affinity to wheat germ agglutinin. Therefore, it is suggested that misfolding of the mutant receptors inhibits transport to the Golgi apparatus where processing of oligosaccharide chains, as well as proteolytic cleavage into subunits, takes place. The binding affinity of the mutant receptors for insulin was 50% of normal. Furthermore, insulin-stimulated autophosphorylation of IR delta Ex13 was markedly impaired. These data provide the evidence for a critical role of the extracellular domain of IR beta-subunit for processing and transport as well as the intramolecular signal transduction to activate IR tyrosine kinase.

Insulin resistance in rats harboring growth hormone—secreting tumors: Decreased receptor number but increased kinase activity in liver

Growth hormone (GH) is a potent antagonist of insulin action, and this resistance occurs primarily at a post-binding step(s). To elucidate the underlying mechanisms, the effects of chronic GH excess on the structure and function of insulin receptors partially purified from the liver were examined in rats harboring GH-secreting tumors. Insulin resistance was established in this animal model of GH hypersecretion by a hyperinsulinemic euglycemic clamp. Specific binding of 125I-insulin and receptor number were reduced in tumor animals by 40% and 62%, respectively, reflecting downregulation of the insulin receptor by hyperinsulinemia in these animals. Receptors from tumor animals showed a 50% increase in β-subunit phosphorylation and in the kinase activity toward the synthetic polypeptide Glu4: Tyr1 when measured in vitro in the absence of insulin; however, the incremental stimulation by insulin (170 nmol/L) of the phosphorylation of either the β-subunit or Glu4:Tyr1 was not different between control and experimental animals. There was no difference between the two groups in Glu4:Tyr1 phosphorylation measured after immunodepletion of receptors by antibodies to the insulin receptor, indicating that the observed alteration in the kinase activity of tumor animals was intrinsic to the insulin receptor. Exposure to chronic GH excess did not alter insulin receptor structure, as evidenced by electrophoretic mobility under reducing and nonreducing conditions. The enhanced basal kinase activity of the receptor from tumor animals may reflect a more highly phosphorylated state of the receptor (and hence elevated enzyme activity) in these animals due to elevated serum insulin levels. These results demonstrate that the hepatic insulin resistance in rats chronically exposed to GH excess is not due to impaired insulin receptor kinase activity.

Corticosterone-Induced Insulin Resistance Is Not Associated with Alterations of Insulin Receptor Number and Kinase Activity in Chicken Kidney

Chicken renal insulin receptors have been recently characterized; their number and kinase activities vary in response to altered nutritional status. In the present study, the effect of chronic corticosterone treatment was examined in 5-week-old chickens. The development of an insulin resistance following corticosterone was suggested after 1 and 2 weeks of treatment by a significant decrease in the hypoglycemic effect of exogenous insulin and after 2 weeks by significant increases in plasma insulin levels (1.63 ± 0.13 vs 0.56 ± 0.14 ng insulin/ml in controls) and in renal cytosolic phosphoenolpyruvate carboxykinase activity (17.2 ± 0.8 vs 13.7 ± 0.7 nm/mn/mg tissue in controls). No significant changes were present at the level of insulin receptor number and kinase activity. Therefore, in kidney and, as previously observed, in muscles, corticosterone can induce insulin resistance at postreceptor steps in the cascade of events leading to insulin action.

1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells.

Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits. This interaction takes place via the phosphorylated YMXM motifs of IRS-1 and the Src homology region 2 (SH2) domains of p85. In this study, the stable overexpression in a Chinese hamster ovary (CHO) cell line of a mutant p85 alpha (delta p85) protein, which lacks a binding site for p110, disrupted the complex formation between IRS-1 and the catalytic subunit of PI 3-kinase in intact cells during insulin stimulation. Activation of insulin receptor kinase and the tyrosine phosphorylation of IRS-1 remained unaffected. In this cell line, both insulin-stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate and the insulin-stimulated glucose uptake due to the translocation of GLUT1 glucose transporters were markedly impaired, whereas neither phorbol 12-myristate 13-acetate-stimulated glucose uptake nor the insulin-stimulated activation of RAS was impaired. These results suggest that PI 3-kinase is required for glucose transport in insulin signaling in CHO cells.

A peptide that stimulates phosphorylation of the plant insulin-binding protein. Isolation, primary structure and cDNA cloning.

The soybean seed basic 7S globulin (Bg) is capable of binding bovine insulin and insulin-like growth factors, and has protein kinase activity which corresponds to about two thirds of the tyrosine kinase activity of the rat insulin receptor. A 4-kDa peptide named leginsulin, which can bind to Bg and compete with insulin for binding to Bg, was isolated from radicles of germinated soybean seeds. The leginsulin had a stimulatory effect on the phosphorylation activity of Bg, suggesting that it is involved in cellular signal transduction. The leginsulin was sequenced by automated Edman degradation and electrospray ionization mass spectrometry. It consisted of 37 amino acid residues with six half-cystines in three disulfide bridges. The mass spectrometric analysis revealed that a portion of the peptide is processed to delete the C-terminal glycine like a number of animal peptide hormones, but not C-terminally amidated. The cDNA encoding the leginsulin was cloned, sequenced and considered to code for a precursor polypeptide consisting of a putative signal peptide, the leginsulin, a linker peptide, a 6-kDa peptide and a C-terminal peptide. Although there is no sequence similarity between the leginsulin and insulin or insulin-like growth factors, the leginsulin is a possible candidate for plant peptide hormones.

Phenylarsine oxide inhibits insulin-stimulated protein phosphatase 1 activity and GLUT-4 translocation.

Phenylarsine oxide (PAO) has previously been shown to inhibit insulin-stimulated glucose transport without affecting insulin binding and tyrosine kinase activity of insulin receptor (S. C. Frost and M. D. Lane. J. Biol. Chem. 260: 2646-2652, 1985). This study examines the effect of PAO on insulin's ability to activate adipocyte protein phosphatase 1 (PP-1) and dephosphorylate GLUT-4, the insulin-sensitive glucose transporter. In particulate fractions, insulin stimulated PP-1 activity (40% increase over basal with phosphorylase a) in a time- and dose-dependent manner (half-maximal effect of 0.89 nM in 1 min). Insulin did not alter cytosolic PP-1 activity. With GLUT-4 as a substrate, insulin caused more than twofold stimulation of particulate PP-1 activity. Addition of PAO (5 microM) before or after insulin treatment abolished insulin's effect on PP-1 activation. The presence of 2,3-dimercaptopropanol (200 microM) prevented the effect of PAO on PP-1 activation and glucose uptake. In addition, PAO significantly increased GLUT-4 phosphorylation, blocked insulin-stimulated dephosphorylation, and partially diminished insulin-stimulated translocation of GLUT-4. We conclude that PAO may interfere with the components of insulin signal transduction pathways that lead to the activation of PP-1 and this may be responsible for the observed inhibition in insulin action.

Characterization of insulin receptor from the muscle of the shrimp Penaeus japonicus (Crustacea: Decapoda)

The beta-subunit of the insulin receptor from the muscle of the shrimp Penaeus japonicus exists as multiple subtypes with M(r) of 79,000, 77,000 and 75,000. Only the subunit of M(r) 79,000 is autophosphorylated after the addition of insulin. The autophosphorylation occurred specifically at Tyr residues, as demonstrated by the specific subsequent dephosphorylation by the phosphotyrosyl protein phosphatase from the human placenta. The detergent, Triton X-100, and the metal ion, Mn2+, caused a noticeable enhancement of the autophosphorylation of shrimp insulin receptors from the muscle. Okadaic acid activated the kinase activity of the insulin-stimulated insulin receptor, but not the basal activity of the insulin receptor without the addition of insulin. Further studies comparing the insulin binding of the shrimp insulin receptor in the regulation of kinase activity of the multiple beta-subunit subtypes from the shrimp muscle are under way.

Expression of a dominant-negative mutant human insulin receptor in the muscle of transgenic mice.

To examine the in vivo effects of a kinase-deficient mutant human insulin receptor, we used the muscle creatine kinase promoter to express a putative dominant-negative receptor: Ala1134-->Thr (Moller, D. E., Yokota, A., White, M. F., Pazianos, A. G., and Flier, J. S. (1990) J. Biol. Chem. 265, 14979-14985) in transgenic mice. Two lines were generated, where receptor expression was restricted to striated muscle and was increased by 5-12-fold in skeletal muscle. Transgenic gluteal muscle insulin receptor kinase activity was reduced by approximately 80% after maximal in vitro insulin stimulation. Glycogen content in this muscle was reduced by 45% in transgenic mice. Insulin levels were approximately 2-fold higher, and glucose concentrations were 12% higher in transgenics fed ad libitum. Transgenic mice exhibited reduced in vivo sensitivity to low dose (0.1 milliunits/g) intravenous insulin. In isolated soleus muscles from transgenics, where mutant receptors were expressed at lower levels, insulin-stimulated receptor kinase activity was reduced by 42%, but insulin-stimulated 2-deoxyglucose uptake was unaffected. These results indicate that (i) overexpression of a kinase-deficient human insulin receptor in muscle causes dominant-negative effects at the level of receptor kinase activation, (ii) impairment of insulin-stimulated muscle receptor tyrosine kinase activity can cause decreased insulin sensitivity in vivo, (iii) kinase-defective receptor mutants may be used to create novel animal models of tissue-specific insulin resistance.

High Glucose Condition Activates Protein Tyrosine Phosphatases and Deactivates Insulin Receptor Function in Insulin-Sensitive Rat 1 Fibroblasts

To investigate the mechanism for the impairment of insulin receptor kinase activity induced by high glucose (HG) in Rat 1 fibroblasts that expressed human insulin receptors (HIRc), we measured protein tyrosine phosphatase (PTPase) activity in HG cells. Incubating HIRc cells for 4 days in 27 mM D-glucose (HG) stimulated cytosolic PTPase activities, but not particulate PTPase activity as determined by two methods using the dephosphorylation of insulin receptors. Furthermore, PTP1B, a major non-transmembrane PTPase in the cytosolic fraction,was increased in HG cells according to Western blots. These results indicate that desensitization of insulin receptor function by a high glucose condition is associated with the activation of PTPase activity.

Elevated protein tyrosine phosphatase activity and increased membrane viscosity are associated with impaired activation of the insulin receptor kinase in old rats

Insulin resistance is very common in the elderly, and may be associated with glucose intolerance or frank diabetes. In previous studies we demonstrated that insulin resistance in old Wistar rats is associated with decreased autophosphorylation and activation of the hepatic insulin receptor kinase (IRK) in vivo. We now show that this defect can be reproduced in vitro, where the extent of insulin-induced activation of IRK in liver membranes of old rats was decreased by approximately 50% compared with young controls. The defect could be largely abolished after solubilization of the membranes with Triton X-100. We also show that: (a) the viscosity of membranes from the old rats was significantly (P < 0.001, n = 4) higher (by 15%) compared with young controls; (b) incubation of plasma membranes from old animals with lecithin liposomes, which lowered their cholesterol levels, partially abolished the defect in IRK activation; and (c) Triton extracts of liver membranes prepared from old rats did not interfere with the activation of IRK derived from young controls. Additionally, non-membrane components did contribute to the development of this defect. We observed a significant (approximately 30%) (P < 0.001, n = 18) elevation of cytosolic protein tyrosine phosphatase (PTP) activity directed against the beta subunit of the insulin receptor in livers of old rats. No such elevation of PTP activity could be demonstrated with synthetic substrates. Our findings are consistent with a model in which increased membrane viscosity as well as enhancement of a cytosolic PTP activity both markedly inhibit the activation in vivo of the hepatic IRK in old animals.

Troglitazone prevents glucose-induced insulin resistance of insulin receptor in rat-1 fibroblasts.

Troglitazone (CS045), a compound belonging to the thiazolidine diones, is being tested as a new oral antidiabetic agent. Evidence exists from animal studies and clinical trials with non-insulin-dependent diabetes mellitus patients that Troglitazone might reduce insulin resistance. The molecular mechanism of this effect is not understood. In this study, we investigated whether Troglitazone might interfere with the mechanism of glucose-induced insulin resistance. Several studies indicate that hyperglycemia reduces the kinase activity of the insulin receptor in different cell types. This effect is paralleled by translocation of several protein kinase C (PKC) isoforms, and it can be prevented by PKC inhibitors, which suggests that glucose-induced receptor desensitization is mediated by activation of PKC. We studied the effect of hyperglycemia on the insulin receptor kinase activity and its modulation by Troglitazone in rat-1 fibroblasts that stably overexpress the human insulin receptor. Before stimulation with insulin (10(-7) M), cells were acutely exposed to hyperglycemic conditions in the absence or presence of Troglitazone (0.01-2 micrograms/ml). The insulin receptor was solubilized from a plasma membrane fraction or whole cell lysates, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted against antiphosphotyrosine and anti-insulin receptor beta-subunit (CT 104) antibodies. Acute hyperglycemia (25 mM glucose) induced a significant inhibition of the insulin receptor kinase (IRK) activity within 30 min (inhibition to 30 +/- 12.5% of maximal insulin-stimulated beta-subunit phosphorylation, n = 9, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose Transporter (GLUT 4) Content and Insulin Receptor Kinase Activity in Muscles of the LA/N-cp Rat

In this study we compared insulin binding activity, insulin receptor tyrosine kinase activity, and GLUT 4 protein content in six muscles from LA/N-cp rats and their lean controls. LA/N-cp rats had an approximate 20-fold increase in insulin concentration (837 +/- 113 vs 40 +/- 1), associated with significant (p < 0.01) decreases in both insulin binding activity per mg muscle and in muscle GLUT 4 content. Maximum insulin tyrosine kinase activity was also lower in muscle from LA/N-cp rats, but no difference was noted when tyrosine kinase activity was expressed per receptor. These data indicate that there are at least two defects in the insulin action cascade in muscle from LA/N-cp rats that contribute to the insulin resistance in these animals.

Vanadate augments insulin-stimulated insulin receptor kinase activity and prolongs insulin action in rat adipocytes. Evidence for transduction of amplitude of signaling into duration of response.

Vanadate, a protein tyrosine phosphatase inhibitor, preserves insulin-stimulated lipogenesis after removal of insulin. To investigate the mechanism of this action of vanadate, lipogenesis was studied in isolated rat adipocytes exposed to vanadate for 60 min followed by insulin for 15 min at 37 degrees C. Vanadate (10-50 microM) prolonged insulin-stimulated lipogenesis. The half-time (t1/2) of the decay in insulin (0.34 nM)-stimulated lipogenesis after removal of insulin by washing in pH 7.0 followed by pH 7.6 buffer was 21 min in the absence and 59 min in the presence of vanadate. During these conditions, vanadate did not alter insulin binding nor the removal of insulin by the series of washes. In contrast to lipogenesis, the t1/2 of the decay in insulin receptor tyrosine kinase (IRK) activity, assayed with the artificial substrate Poly[Glu:Tyr] (4:1), was not significantly prolonged by vanadate (6 vs. 6.8 min). However, insulin-stimulated IRK activity was markedly augmented by vanadate to 319 +/- 19% of insulin alone, associated with a similar augmentation of phosphotyrosine incorporation into the insulin receptor beta-subunit determined by Western blotting with antiphosphotyrosine antibodies. To determine the relationship between prolongation of lipogenesis and the increase in IRK, adipocytes were exposed to 17.2 nM insulin to activate the IRK to the same extent as insulin (0.34 nM) plus vanadate (maximum activation). During these two conditions, the decay of lipogenesis was similar and after stimulation with 17.2 nM insulin was not prolonged by vanadate. We conclude that vanadate prolongs insulin action at insulin concentrations that do not maximally activate the IRK by augmenting IRK activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Vanadate augments insulin-stimulated insulin receptor kinase activity and prolongs insulin action in rat adipocytes. Evidence for transduction of amplitude of signaling into duration of response

Vanadate augments insulin-stimulated insulin receptor kinase activity and prolongs insulin action in rat adipocytes. Evidence for transduction of amplitude of signaling into duration of response

Mechanisms of cellular insulin resistance in human pregnancy

OBJECTIVE: The cellular mechanism(s) of insulin resistance developed during pregnancy were studied by investigating the functionality of insulin receptors and glucose transport. STUDY DESIGN: Abdominal adipose tissue was obtained from eight lean pregnant and nine control subjects, matched for insulin resistance by intravenous glucose tolerance testing. Insulin receptor binding and glucose transport were measured in freshly isolated adipocytes. Receptor kinase activity was measured on partially purified receptors. Data were analyzed by Student t test. RESULTS: High-affinity insulin receptors were reduced in cells from pregnant compared with normal controls (2.0 ± 0.4 vs 5.8 ± 1.3 × 104 sites per cell, p < 0.05). Kinase activity of insulin receptors was unaltered in pregnancy. Adipocytes from pregnant subjects displayed a threefold decrease in insulin sensitivity for glucose transport (median effective concentration 324 ± 93 vs 93 ± 14 pmol/L, p < 0.025) and a reduction in maximal insulin-stimulated glucose transport (1.58 ± 0.15 vs 2.33 ± 0.24 pmol/105 cells/10 seconds, p < 0.025). CONCLUSION:These results show that adipocytes from pregnant subjects exhibit decreased insulin receptor number and an impaired insulin sensitivity in the absence of functional alterations of receptor kinase activity. (AM J OBSTET GYNECOL 1994;170:635-41.)

Stimulation of insulin receptor tyrosine kinase activity by an amino terminal sequence of human growth hormone

The in vitro effect of a hypoglycaemic fragment of human growth hormone containing the sequence H 2N-LeuSerArgLeuPheAsu 11AsnAlaCOOH (Asu 11hGH 613) on tyrosine kinase of rat hepatic insulin receptors was examined. Insulin receptor kinase activity was evaluated using the synthetic polypeptide poly(GluTyr) (4:1) as substrate. The hypoglycaemic Asu 11hGH 613 appeared to enhance the phosphorylation of the exogenous substrate by the stimulation of insulin receptor kinase activity. The levels of poly(GluTyr)(4:1) phosphorylation were significantly higher in the insulin receptor preparations incubated in the presence of the Asu 11hGH 6 613 peptide. A dose dependent stimulation of receptor kinase activity was observed and this stimulatory effect was found to be further enhanced by the addition of increasing concentrations of insulin. In hepatic extracts depleted of insulin receptor, no stimulation of kinase activity by the Asu 11hGH 613 was observed. From these data, it is concluded that the increase of poly (GluTyr)(4:1) phosphorylation is the result of the interaction between the Asu 11hGH 613 and the hepatic insulin receptor.

Delayed onset of insulin activation of the insulin receptor kinase in vivo in human skeletal muscle.

During the infusion of insulin in vivo, the rate of activation of glucose disposal lags significantly behind the rate of increase in serum insulin levels. To determine whether this delay was related to transcapillary transport of insulin, we determined increments in serum insulin levels, glucose disposal rates (GDR), and insulin receptor (IR) kinase activity measured during continuous infusions of insulin (40 and 120 mU.m-2.min-1) administered to 8 nondiabetic males; similar studies were done at 1,200.m-2.min-1 in 2 of the subjects. Half-maximal insulin levels were achieved at a mean of 4.9 and 7.2 min during the 40 and 120 mU.m-2.min-1 clamps, respectively, with corresponding half-maximal GDR stimulation at a mean of 59 and 47 min. Unlike the rise in insulin levels, IR kinase activation was much slower with half-maximal activity occurring at approximately 40-60 min in the 2 clamps. Thus, the rise in serum insulin levels in each clamp was much faster than the increment in either kinase activity or glucose disposal. Insulin infusion increased both IR kinase and GDR maximally approximately 10-fold, with half-maximal stimulation at approximately 3,600 and approximately 700 pM, indicating spare kinase for glucose disposal. These results demonstrate that the delay in stimulation of glucose disposal by insulin is related to a rate-limiting step between the intravascular space and the cell-surface of skeletal muscle. This may involve delayed transendothelial transport of insulin.