Early Steps Of Insulin Action Research Articles

Interaction between IGFBP7 and insulin: a theoretical and experimental study.

Insulin-like growth factor binding protein 7 (IGFBP7) can bind to insulin with high affinity which inhibits the early steps of insulin action. Lack of recognition mechanism impairs our understanding of insulin regulation before it binds to insulin receptor. Here we combine computational simulations with experimental methods to investigate the interaction between IGFBP7 and insulin. Molecular dynamics simulations indicated that His200 and Arg198 in IGFBP7 were key residues. Verified by experimental data, the interaction remained strong in single mutation systems R198E and H200F but became weak in double mutation system R198E-H200F relative to that in wild-type IGFBP7. The results and methods in present study could be adopted in future research of discovery of drugs by disrupting protein–protein interactions in insulin signaling. Nevertheless, the accuracy, reproducibility, and costs of free-energy calculation are still problems that need to be addressed before computational methods can become standard binding prediction tools in discovery pipelines.

Acute physical exercise reverses S‐nitrosation of the insulin receptor, insulin receptor substrate 1 and protein kinase B/Akt in diet‐induced obese Wistar rats

Early evidence demonstrates that exogenous nitric oxide (NO) and the NO produced by inducible nitric oxide synthase (iNOS) can induce insulin resistance. Here, we investigated whether this insulin resistance, mediated by S-nitrosation of proteins involved in early steps of the insulin signal transduction pathway, could be reversed by acute physical exercise. Rats on a high-fat diet were subjected to swimming for two 3 h-long bouts, separated by a 45 min rest period. Two or 16 h after the exercise protocol the rats were killed and proteins from the insulin signalling pathway were analysed by immunoprecipitation and immunoblotting. We demonstrated that a high-fat diet led to an increase in the iNOS protein level and S-nitrosation of insulin receptor beta (IR beta), insulin receptor substrate 1 (IRS1) and Akt. Interestingly, an acute bout of exercise reduced iNOS expression and S-nitrosation of proteins involved in the early steps of insulin action, and improved insulin sensitivity in diet-induced obesity rats. Furthermore, administration of GSNO (NO donor) prevents this improvement in insulin action and the use of an inhibitor of iNOS (L-N6-(1-iminoethyl)lysine; L-NIL) simulates the effects of exercise on insulin action, insulin signalling and S-nitrosation of IR beta, IRS1 and Akt. In summary, a single bout of exercise reverses insulin sensitivity in diet-induced obese rats by improving the insulin signalling pathway, in parallel with a decrease in iNOS expression and in the S-nitrosation of IR/IRS1/Akt. The decrease in iNOS protein expression in the muscle of diet-induced obese rats after an acute bout of exercise was accompanied by an increase in AMP-activated protein kinase (AMPK) activity. These results provide new insights into the mechanism by which exercise restores insulin sensitivity.

Effect of thiopental, pentobarbital and diethyl ether on early steps of insulin action in liver and muscle of the intact rat

A large number of experimental studies have investigated insulin signaling in rats. In these studies different anaesthetics have been used to anaesthetize rats. However, the direct effects of anaesthetics on the regulation of the early steps of insulin action are not known. In the present study, we investigated the effect of thiopental, pentobarbital and diethyl ether on the plasma glucose disappearance rate, IR, IRS-1 and IRS-2 tyrosine phosphorylation, IRSs association with PI 3-kinase, Akt and Erk phosphorylation, in liver and muscle of rats. Fasting plasma glucose levels were higher in animals anaesthetized with ether. No differences in plasma glucose disappearance rates were observed, however. Insulin-induced IR, IRS-1 and IRS-2 tyrosine phosphorylation, association of these substrates with PI 3-kinase and Akt and ERK phosphorylation were similar in the three groups of animals in both tissues. These data suggest that both thiopental and pentobarbital may be used in studies where changes in insulin signaling are being measured and where adequate general anaesthesia is required.

The phosphatidylinositol/AKT/atypical PKC pathway is involved in the improved insulin sensitivity by DHEA in muscle and liver of rats in vivo

DHEA improves insulin sensitivity and has anti-obesity effect in animal models and men. However, the molecular mechanisms by which DHEA improves insulin action have not been clearly understood. In the present study, we examined the protein levels and phosphorylation state of insulin receptor (IR), IRS-1 and IRS-2, the association between IRSs and PI3K and SHP2, the insulin-induced IRSs associated PI 3-kinase activities, and the phosphorylation status of AKT and atypical PKCζ/λ in the liver and the muscle of 6 month-old Wistar rats treated with DHEA. There was no change in IR, IRS-1 and IRS-2 protein levels in both tissues of treated rats analysed by immunoblotting. On the other hand, insulin-induced IRS-1 tyrosine phosphorylation was increased in both tissues while IRS-2 tyrosyl phosphorylation was increased in liver of DHEA treated group. The PI3-kinase/AKT pathway was increased in the liver and the PI3K/atypical PKCζ/λ pathway was increased in the muscle of DHEA treated rats. These data indicate that these regulations of early steps of insulin action may play a role in the intracellular mechanism for the improved insulin sensitivity observed in this animal model.

Evidence for a direct effect of captopril on early steps of insulin action in BC3H-1 myocytes

Captopril, an angiotensin-converting enzyme (ACE) inhibitor, has been reported to improve insulin sensitivity. However, despite extensive investigation, the mechanisms responsible for this effect are not fully understood. Reduction of plasma angiotensin II and inhibition of kininase II have been suggested to contribute to improve insulin sensitivity. Insulin binding was measured at tracer insulin concentration in intact cells with or without captopril treatment. Specific binding, expressed as percent of total insulin added, was not different in control and captopril-treated cells. However, captopril treatment caused an increase in insulin-induced insulin receptor substrate-1 (IRS-1) phosphorylation accompanied by an increased association of IRS-1 with phosphoinositide-3 kinase (PI-3 kinase), despite no change on insulin receptor (IR) autophosphorylation. There was also an increased threonine kinase B (AKT) phosphorylation in captopril-treated cells followed by enhanced basal and insulin-stimulated glucose uptake. These results indicate that captopril treatment has a direct effect on early phosphorylation events induced by insulin in BC3H-1 myocytes. Copyright 2003, Elsevier Science (USA). All rights reserved.

Chronic hyperinsulinism induced down-regulation of insulin post-receptor signaling transduction in Hep G2 cells.

To study the regulatory effect of acute and chronic insulin treatment on insulin post-receptor signaling transduction pathway in a human hepatoma cell line (Hep G2), Hep G2 cells were incubated in the presence or absence of insulin with different concentrations in serum free media for 16 h and then stimulated with 100 nmol/L insulin for 1 min. Protein levels of insulin receptor beta-subunit (IR beta), insulin receptor substrate-1 (IRS-1) and p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) were determined in total cell lysates by Western-immunoblot. Phosphorylated proteins IR beta, IRS-1 and interaction of PI 3-kinase with IRS-1 were determined by immunoprecipitation. Results showed that 1-min insulin stimulation rapidly induced tyrosine phosphorylation of IR beta and IRS-1, which in turn, resulting in association of PI 3-kinase with IRS-1. 1-100 nmol/L chronic insulin treatment induced a dose-dependent decrease in the protein level of IR beta and a slight decrease in the protein level of IRS-1. There was a more marked reduction in the phosphorylation of IR beta, IRS-1, reaching a nadir of 22% (P < 0.01) and 15% (P < 0.01) of control levels, respectively, after 16 h treatment with 100 nmol/L insulin. The association between IRS-1 and PI 3-kinase was decreased by 66% (P < 0.01). There was no significant change in PI 3-kinase protein levels. These data suggest that chronic insulin treatment can induce alterations of IR beta, IRS-1 and PI 3-kinase three early steps in insulin action, which contributes significantly to insulin resistance, and may account for desensitization of insulin action.

Role of 17β-estradiol administration on insulin sensitivity in the rat: implications for the insulin receptor

The role of 17β-estradiol in the early steps of insulin action is only partially known, although its effect on glucose homeostasis has been reported. In this paper, we attempt to prove the influence of 17β-estradiol on the insulin receptor of ovariectomized rats treated with different hormonal doses. Our results show that high doses of estradiol impair insulin sensitivity while low doses improve it. We think that these results are the consequence of changes at a molecular level, because high doses of estradiol produced lower expression of the insulin receptor gene, lower content of this receptor in target tissues, and lower phosphorylation of insulin receptor in these tissues. However, low doses of estradiol seem to produce just the opposite. The possible existence of consensus response elements in the insulin receptor gene promoter to estradiol could be controlling the expression of this gene, this control being dose and timing dependent. Moreover, we cannot discard a possible effect of estradiol on the activity of protein tyrosine phosphatases, and therefore, on the activity of the insulin receptor. These new findings improve knowledge about the possible risk for insulin resistance in women taking oral contraceptives or receiving hormonal replacement therapy around the menopause, but could also open the door towards the possible utilization of 17β-estradiol in some diabetes cases.

Endurance training improves responsiveness to insulin and modulates insulin signal transduction through the phosphatidylinositol 3-kinase/Akt-1 pathway.

Endurance training increases insulin-stimulated muscle glucose transport and leads to improved metabolic control in diabetic patients. To analyze the effects of endurance training on the early steps of insulin action in muscle of rats. Male rats submitted to daily swimming for 6 weeks were compared with sedentary controls. At the end of the training period, anesthetized animals received an intravenous (i.v.) injection of insulin and had a fragment of their gastrocnemius muscle excised for the experiments. Associations between insulin receptor, insulin receptor substrates (IRS)-1 and -2 and phosphatidylinositol 3-kinase (PI3-kinase) were analyzed by immunoprecipitation and immunoblotting. Akt-1 serine phosphorylation and specific protein quantification were detected by immunoblotting of total extracts, and IRS-1/IRS-2-associated PI3-kinase activity were determined by thin-layer chromatography. Insulin-induced phosphorylation of IRS-1 and IRS-2 increased respectively by 1.8-fold (P<0.05) and 1.5-fold (P<0.05), whereas their association with PI3-kinase increased by 2.3-fold (P<0.05) and 1.9-fold (P<0.05) in trained rats as compared with sedentary controls, respectively. The activity of PI3-kinase associated with IRS-1 and IRS-2 increased by 1.8-fold (P<0.05) and 1.7-fold (P<0.05) respectively, in trained rats as compared with their untrained counterparts. Serine phosphorylation of Akt-1/PKB increased 1.7-fold (P<0.05) in trained rats in response to insulin. These findings were accompanied by increased responsiveness to insulin as demonstrated by a reduced area under the curve for insulin during an i.v. glucose tolerance test, by increased glucose disappearance rate during an insulin tolerance test, and by increased expression of glucose transporter-4. The increased responsiveness to insulin induced by chronic exercise in rat skeletal muscle may result, at least in part, from the modulation of the insulin signaling pathway at different molecular levels.

Effect of treatment with different doses of 17–β–estradiol on the insulin receptor

The mechanism for the development of insulin resistance in normal pregnancy is complex and is associated with serum levels of sex hormones. However, the influence of these hormones on the early steps of insulin action has not been extensively studied, although the potentially beneficial effect of estradiol on glucose homeostasis has been reported. In this paper, we attempted to determine the effect of 17–β–estradiol on the insulin receptor of ovariectomized rats treated with different doses of hormones. Our results showed a tissue-dependent response to estradiol. We found that low doses of estradiol increased the amount of insulin receptors in liver and muscle on days 6 and 11 of treatment but not in adipose tissue, and after 16 days only the muscle responsed in this way. On the other hand, high doses of estradiol significantly decreased the amount of insulin receptors, at least in muscle and adipose tissue. We believe that the low concentrations of 17–β–estradiol (similar to early pregnancy) could be responsible for the increase in insulin sensitivity by increasing the amount of insulin receptors in peripheral tissues. When the hormone levels were high (similar to late pregnancy) the amount of insulin receptors decreased in peripheral tissues, and insulin sensitivity is diminished just as in late pregnancy. The specific molecular mechanism for this action is as yet unknown.

Early Steps of Insulin Action in the Skin of Intact Rats

Insulin is an important regulator of growth and initiates its action by binding to its receptor, which undergoes tyrosyl autophosphorylation and further enhances its tyrosine kinase activity towards other intermediate molecules, including insulin receptor substrate 1, insulin receptor substrate 2, and Shc. Insulin receptor substrate proteins can dock various src-homology-2-domain-containing signaling proteins, such as the 85 kDa subunit of phosphatidylinositol 3 kinase and growth-factor-receptor-bound protein 2. The serine-threonine kinase is activated downstream to phosphatidylinositol 3 kinase. Shc protein has been shown to directly induce the association with growth-factor-receptor-bound protein 2 and downstream the activation of the mitogen-activated protein kinase. In this study we investigated insulin signal transduction pathways in skin of intact rats by immunoprecipitation and immunoblotting with specific antibodies, and also by immunohistochemistry with anti-insulin-receptor antibody. Our results showed that skin fragments clearly demonstrated the presence of insulin receptor in cell bodies of the epidermis and hair follicles and some faint staining was also detected in fibroblasts of the dermis. It was also observed that acute stimulation with insulin can induce tyrosyl phosphorylation of insulin receptor, that the insulin receptor substrates and Shc proteins serve as signaling molecules for insulin in skin of rats, and that insulin is able to induce association of insulin receptor substrate 1/phosphatidylinositol 3 kinase and Shc/growth-factor-receptor-bound protein 2 in this tissue, as well as phosphorylation of mitogen-activated protein kinase and serine-threonine kinase, demonstrating that proteins involved in early steps of insulin action are expressed in skin of intact rats and are quickly activated after insulin stimulation.

Tissue-specific regulation of early steps in insulin action in septic rats

Sepsis is known to induce insulin 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 septic rats 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 and phosphorylation levels in the liver and muscle of septic rats. IRS-1 protein levels were decreased by 40 ± 3% (p < 0.01) in muscle but not in liver of septic 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 of septic rats decreased by 38 ± 5% (p < 0.01) and insulin-stimulated IRS-1 association with PI 3-kinase decreased by 44 ± 7% in muscle (p < 0.01) but no changes were seen in liver. These data suggest that there is a tissue-specific regulation of early steps of insulin signal transduction in septic rats, and the changes observed in muscle may have a role in the insulin resistance of these animals.

A high-fructose diet induces changes in pp185 phosphorylation in muscle and liver of rats.

Insulin stimulates the tyrosine kinase activity of its receptor resulting in the tyrosine phosphorylation of pp185, which contains insulin receptor substrates IRS-1 and IRS-2. These early steps in insulin action are essential for the metabolic effects of insulin. Feeding animals a high-fructose diet results in insulin resistance. However, the exact molecular mechanism underlying this effect is unknown. In the present study, we determined the levels and phosphorylation status of the insulin receptor and pp185 (IRS-(1/2)) in liver and muscle of rats submitted to a high-fructose diet evaluated by immunoblotting with specific antibodies. Feeding fructose (28 days) induced a discrete insulin resistance, as demonstrated by the insulin tolerance test. Plasma glucose and serum insulin and cholesterol levels of the two groups of rats, fructose-fed and control, were similar, whereas plasma triacylglycerol concentration was significantly increased in the rats submitted to the fructose diet (P<0.05). There were no changes in insulin receptor concentration in the liver or muscle of either group. However, insulin-stimulated receptor autophosphorylation was reduced to 72 +/- 4% (P<0.05) in the liver of high-fructose rats. The IRS-1 protein levels were similar in both liver and muscle of the two groups of rats. In contrast, there was a significant decrease in insulin-induced pp185 (IRS-(1/2)) phosphorylation, to 83 +/- 5% (P<0.05) in liver and to 77 +/- 4% (P<0.05) in muscle of the high-fructose rats. These data suggest that changes in the early steps of insulin signal transduction may have an important role in the insulin resistance induced by high-fructose feeding.

A High Fructose Diet Affects the Early Steps of Insulin Action in Muscle and Liver of Rats

A high fructose diet induces insulin resistance in rats, although the exact molecular mechanism involved is unknown. In this study, we used immunoprecipitation and immunoblotting to examine the levels and phosphorylation status of the insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), as well as the association of the IRS-1 with phosphatidylinositol 3-kinase (PI 3-kinase), and phosphotyrosine phosphatase (SHP2) in the liver and muscle of rats fed a control or high fructose diet for 28 d. There were no differences in IR and the IRS-1 protein levels in the liver and muscle of rats fed the control and high fructose diets. However, tyrosine-phosphorylation of the insulin receptor after insulin stimulation was reduced to 71 +/- 2% (P < 0.05) of control in the liver of the fructose-fed rats. In samples previously immunoprecipitated with anti-IRS-1 antibody and blotted with antiphosphotyrosine antibody, the insulin-stimulated IRS-1 phosphorylation levels in the liver and muscle of the fructose-fed group were only 70 +/- 6% (P < 0.05) and 76 +/- 5% (P < 0.05) of those of control rats, respectively. The insulin-stimulated IRS-1 association with PI 3-kinase was reduced to 84 +/- 3% (P < 0.05) in the liver and to 84 +/- 4% (P < 0.05) in the muscle of the fructose-fed group compared with control rats. Insulin-stimulated IRS-1 association with SHP2 was reduced to 79 +/- 5% (P < 0.05) in liver of the fructose-fed rats. These data suggest that changes in the early steps of insulin signal transduction may have an important role in the insulin resistance observed in these rats.

Magnesium Deficiency Modulates the Insulin Signaling Pathway in Liver but Not Muscle of Rats

Altered insulin secretion and sensitivity have been observed in Mg-deficient animals. However, the effects of Mg deficiency and supplementation on intracellular signaling events triggered by insulin are unknown. Therefore, we studied the early steps of insulin action in muscle and liver of rats fed Mg-deficient (DF-6, DF-11) or control (CO-6, CO-11) diets for 6 or 11 wk, respectively, and Mg-deficient or control diets for 6 wk, followed by Mg supplementation for 5 wk (SDF and SCO groups, respectively). There were no differences in the glucose disappearance rate (K(itt)) or insulin signaling between CO-6 and DF-6 rats. Between the two groups of rats fed for 11 wk, the DF-11 group had a significantly greater K(itt). SDF and SCO rats had K(itt) that did not differ from CO-11 rats, but that were significantly lower than in DF-11 rats. In the latter rats, insulin receptor and insulin receptor substrate-1 protein and phosphorylation levels were elevated in liver and there was a greater association between the insulin receptor substrate-1 and p85 subunit of phosphatidyl-inositol 3-kinase compared with CO-11 rats. There were no differences in the early steps of insulin action in SDF and control rats. These results suggest that the normal insulin sensitivity maintained by Mg supplementation and the increased insulin sensitivity produced by a long period of Mg deprivation may result, at least in part, from alterations in or maintenance of the early molecular steps of insulin action in hepatic tissue.

Loss of sensitivity to insulin at early events of the insulin signaling pathway in the liver of growth hormone-transgenic mice

Growth hormone (GH) excess is associated with secondary hyperinsulinemia, but the molecular mechanism and consequences of this alteration are poorly understood. To address this problem we have examined the levels and phosphorylation state of the insulin receptor (IR) and the insulin receptor substrate-1 (IRS-1), the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) as well as the PI 3-kinase activity in the livers of GH-transgenic mice. As expected, IR levels were reduced in the liver of GH-transgenic mice (55% of normal values) as determined by immunoblotting with an anti-IR beta-subunit antibody. IR and IRS-1 phosphorylation as determined by immunoblotting with antiphosphotyrosine antibody were increased in basal conditions by 315% and 560% respectively. After a bolus administration of insulin in vivo, IR phosphorylation increased by 40% while IRS-1 phosphorylation did not change. Insulin administration to control (normal) mice produced 670% and 300% increases in the IR and IRS-1 phosphorylation respectively. In the GH-transgenic animals, basal association of PI 3-kinase with IRS-1 as well as PI 3-kinase activity in liver was increased by 200% and 280% respectively, and did not increase further after administration of insulin in vivo, indicating a complete insensitivity to insulin at these levels. In conclusion, GH excess and the resulting secondary hyperinsulinemia were associated with alterations at the early steps of insulin action in liver. IR concentration was reduced, while IR and IRS-1 phosphorylation, IRS-1/PI 3-kinase association, and PI 3-kinase activity appeared to be maximally activated under basal conditions, thus making this tissue insensitive to further stimulation by exogenous insulin in vivo.

Angiotensin-converting enzyme inhibitor increases insulin-induced pp185 phosphorylation in liver and muscle of obese rats.

The clinical use of angiotensin-converting enzyme (ACE) inhibitors has been associated with increased insulin sensitivity. However, the molecular mechanism is unknown. The authors examined the early steps in insulin action, i.e., the phosphorylation status of the insulin receptor and of the pp185 in liver and muscle of obese rats treated acutely with captopril, using immunoblotting with antiphosphotyrosine antibodies. Following treatment with captopril there was an improvement in insulin-induced insulin receptor and pp185 phosphorylation in the liver and muscle of obese rats. This finding contribute to an explanation of the mechanism by which ACE inhibitors appear to improve insulin sensitivity.

Protein Deficiency and Nutritional Recovery Modulate Insulin Secretion and the Early Steps of Insulin Action in Rats

Maternal malnutrition was shown to affect early growth and leads to permanent alterations in insulin secretion and sensitivity of offspring. In addition, epidemiological studies showed an association between low birth weight and glucose intolerance in adult life. To understand these interactions better, we investigated the insulin secretion by isolated islets and the early events related to insulin action in the hind-limb muscle of adult rats fed a diet of 17% protein (control) or 6% protein [low (LP) protein] during fetal life, suckling and after weaning, and in rats receiving 6% protein during fetal life and suckling followed by a 17% protein diet after weaning (recovered). The basal and maximal insulin secretion by islets from rats fed LP diet and the basal release by islets from recovered rats were significantly lower than that of control rats. The dose-response curves to glucose of islets from LP and recovered groups were shifted to the right compared to control islets, with the half-maximal response (EC50) occurring at 16.9 ± 1.3, 12.4 ± 0.5 and 8.4 ± 0.1 mmol/L, respectively. The levels of insulin receptor, as well as insulin receptor substrate-1 and phosphorylation and the association between insulin receptor substrate-1 and phosphatidylinositol 3-kinase were greater in rats fed a LP diet than in control rats. In recovered rats, these variables were not significantly different from those of the other two groups. These results suggest that glucose homeostasis is maintained in LP and recovered rats by an increased sensitivity to insulin as a result of alterations in the early steps of the insulin signal transduction pathway.

Early Steps in Insulin Action

The biological effects of insulin are initiated by the binding of insulin to the insulin receptor. Insulin binds to the extracellular domain of the insulin receptor and induces conformational changes in the receptor, leading to autophosphorylation of the receptor on intracellular tyrosine residues. These phosphorylated tyrosine residues act as binding sites for proteins which subsequently may be phosphorylated by the insulin receptor. As a result, yet other proteins can be recruited to form larger complexes and, in the case of enzymes, changes in their activity may take place. By a combination of these processes, the activated insulin receptor initiates cascades of biochemical events which are regulated mainly by specific phosphorylation or dephosphorylation reactions. Intermediates which are involved in the normal insulin signalling pathway are subjects of expanding research.

Effect of captopril, losartan, and bradykinin on early steps of insulin action

Effect of captopril, losartan, and bradykinin on early steps of insulin action

Effect of captopril, losartan, and bradykinin on early steps of insulin action.

Insulin initiates its metabolic and growth-promoting effects by binding to the alpha subunit of its receptor, thereby activating the kinase in the beta subunit. This event leads to tyrosyl phosphorylation of its cytosolic substrate, insulin receptor substrate 1 (IRS-1), which in turn associates with and activates phosphatidylinositol (PI) 3-kinase. The clinical use of ACE inhibitors has been associated with increased insulin sensitivity. However, the exact molecular mechanism is unknown. In the present study, we examined the phosphorylation status of the insulin receptor and IRS-1, as well as the association between IRS-1 and PI 3-kinase in the liver and muscle of 20-month-old rats treated acutely with captopril, using immunoprecipitation with antipeptide antibodies to the insulin receptor and IRS-1, and immunoblotting with antiphosphotyrosine and anti-PI 3-kinase antibodies. Insulin stimulation increased receptor autophosphorylation to 462 +/- 253% (P < 0.05) in the liver and 697 +/- 78% (P < 0.001) in the muscle of ACE inhibitor-treated rats. There were also increases to 250 +/- 17% (P < 0.001) and 280 +/- 50% (P < 0.05) in the insulin-stimulated IRS-1 phosphorylation levels in the liver and muscle, respectively, of animals treated with captopril. The insulin-stimulated IRS-1 association with PI 3-kinase rose to 305 +/- 20% (P < 0.001) in liver and 267 +/- 48% (P < 0.05) in muscle. Losartan, an ANG receptor blocker, had no significant effect on insulin-stimulated IRS-1 phosphorylation in both tissues. The acute administration of bradykinin increased insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 in the liver and muscle. These data demonstrate that ACE inhibitors modulate the early steps of insulin signaling, and that this effect may be simulated by the administration of bradykinin.