Ana C.P. Thirone

Tissue-specific roles of IRS proteins in insulin signaling and glucose transport

In type 2-diabetes and impaired glucose tolerance, the muscle, fat and liver become resistant to insulin, and recent developments place dysregulation of insulin receptor substrate (IRS) expression and activation at the center of such defects. IRS1 and IRS2 are the major insulin receptor substrates leading to glucose homeostasis, and have distinct and overlapping roles in diverse organs. The majority of the published literature in this field suggests that IRS1 is the major substrate leading to stimulation of glucose transport in muscle and adipose tissues, whereas in liver, IRS1 and IRS2 have complementary roles in insulin signaling and metabolism.

Effect of Captopril, Losartan, and Bradykinin on Early Steps of Insulin Action

Insulin initiates its metabolic and growth-promoting effects by binding to the α subunit of its receptor, thereby activating the kinase in the β 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 insulinstimulated IRS-1 phosphorylation in both tissues. The acute administration of bradykinin increased insulinstimulated 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.

Topical insulin accelerates wound healing in diabetes by enhancing the AKT and ERK pathways: a double-blind placebo-controlled clinical trial.

Background Wound healing is impaired in diabetes mellitus, but the mechanisms involved in this process are virtually unknown. Proteins belonging to the insulin signaling pathway respond to insulin in the skin of rats. Objective The purpose of this study was to investigate the regulation of the insulin signaling pathway in wound healing and skin repair of normal and diabetic rats, and, in parallel, the effect of a topical insulin cream on wound healing and on the activation of this pathway. Research Design and Methods We investigated insulin signaling by immunoblotting during wound healing of control and diabetic animals with or without topical insulin. Diabetic patients with ulcers were randomized to receive topical insulin or placebo in a prospective, double-blind and placebo-controlled, randomized clinical trial (NCT 01295177) of wound healing. Results and Conclusions Expression of IR, IRS-1, IRS-2, SHC, ERK, and AKT are increased in the tissue of healing wounds compared to intact skin, suggesting that the insulin signaling pathway may have an important role in this process. These pathways were attenuated in the wounded skin of diabetic rats, in parallel with an increase in the time of complete wound healing. Upon topical application of insulin cream, the wound healing time of diabetic animals was normalized, followed by a reversal of defective insulin signal transduction. In addition, the treatment also increased expression of other proteins, such as eNOS (also in bone marrow), VEGF, and SDF-1α in wounded skin. In diabetic patients, topical insulin cream markedly improved wound healing, representing an attractive and cost-free method for treating this devastating complication of diabetes. Trial Registration ClinicalTrials.gov NCT01295177

Effect of chronic growth hormone treatment on insulin signal transduction in rat tissues

Growth hormone (GH) is known to produce insulin resistance, but the exact molecular mechanism remains unclear. We have chronically treated rats with GH and observed that the levels of insulin receptor in the liver or muscle were similar in both the GH-treated and non-treated rats. Insulin-stimulated receptor autophosphorylation was unaltered in the liver, but was reduced in the muscle of rats treated with GH. Insulin receptor substrate-1 (IRS-1) and phosphatidylinositol (PI) 3-kinase protein levels decreased in the liver but not muscle of GH-treated rats. There was no change in hepatic and muscle IRS-2 concentrations. A common finding in liver and muscle was the decrease in IRS-1 and IRS-2 tyrosine phosphorylation associated with a reduction in the interaction between these substrates and PI 3-kinase. These data suggest that changes in the early steps of insulin signal transduction may have a role in the insulin resistance observed in rats exposed to an excess of GH.

Opposite Effect of JAK2 on Insulin-Dependent Activation of Mitogen-Activated Protein Kinases and Akt in Muscle Cells Possible Target to Ameliorate Insulin Resistance

Many cytokines increase their receptor affinity for Janus kinases (JAKs). Activated JAK binds to signal transducers and activators of transcription, insulin receptor substrates (IRSs), and Shc. Intriguingly, insulin acting through its own receptor kinase also activates JAK2. However, the impact of such activation on insulin action remains unknown. To determine the contribution of JAK2 to insulin signaling, we transfected L6 myotubes with siRNA against JAK2 (siJAK2), reducing JAK2 protein expression by 75%. Insulin-dependent phosphorylation of IRS1/2 and Shc was not affected by siJAK2, but insulin-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-related kinase, p38, and Jun NH2-terminal kinase and their respective upstream kinases MKK1/2, MKK3/6, and MKK4/7 was significantly lowered when JAK2 was depleted, correlating with a significant drop in insulin-mediated cell proliferation. These effects were reproduced by the JAK2 inhibitor AG490. Conversely, insulin-stimulated Akt phosphorylation, glucose uptake, and GLUT4 translocation were not affected by siJAK2. Interestingly, in two insulin-resistant states, siJAK2 led to partial restoration of Akt phosphorylation and glucose uptake stimulation but not of the MAPK pathway. These results suggest that JAK2 may depress the Akt to glucose uptake signaling axis selectively in insulin-resistant states. Inhibition of JAK2 may be a useful strategy to relieve insulin resistance of metabolic outcomes.

Regulation of IRS-1/SHP2 interaction and AKT phosphorylation in animal models of insulin resistance.

Insulin stimulates tyrosine kinase activity of its receptor, resulting in phosphorylation of its cytosolic substrate, insulin receptor substrate-1, which, in turn, associates with proteins containing SH2 domains, including phosphatidylinositol 3-kinase (Pl 3-kinase) and the phosphotyrosine phosphatase SHP2. The regulation of these associations in situations of altered insulin receptor substrate-1 (IRS-1) phosphorylation was not yet investigated. In the present study, we investigated insulin-induced IRS-1/SHP2 and IRS-1/PI 3-kinase associations and the regulation of a downstream serine-kinase AKT/PKB in liver and muscle of three animal models of insulin resistance: STZ diabetes, epinephrine-treated rats, and aging, which have alterations in IRS-1 tyrosine phosphorylation in common. The results demonstrated that insulin-induced IRS-1/PI 3-kinase association has a close correlation with IRS-1 tyrosine phosphorylation levels, but insulin-induced IRS-1/SHP2 association showed a modulation that did not parallel IRS-1 phosphorylation, with a tissue-specific regulation in aging. The integration of the behavior of IRS-1/PI 3-kinase and with IRS-1/SHP2 associations may be important for insulin signaling downstream as AKT phosphorylation. In conclusion, the results of the present study demonstrated that insulin-induced IRS-1/SHP2 association can be regulated in insulin-sensitive tissues of animal models of insulin resistance and may have a role in the control of AKT phosphorylation, which may be implicated in the control of glucose metabolism.

Regulation of insulin-stimulated tyrosine phosphorylation of Shc and IRS-1 in the muscle of rats: effect of growth hormone and epinephrine

Insulin receptor substrate‐1 (IRS‐1) and Shc protein have the same binding site at the insulin receptor and compete in their association with the phosphorylated receptor. The present study demonstrates that a decrease in the level of muscle insulin receptor phosphorylation induced by chronic growth hormone (GH) treatment or acute epinephrine infusion is accompanied by a reduction in the level of IRS‐1 phosphorylation and in the association with phosphatidylinositol 3‐kinase. In contrast, no change is observed in insulin‐stimulated Shc tyrosine phosphorylation, or in the association of this substrate with Grb2. These data suggest that a reduction in insulin receptor phosphorylation may affect post‐receptor processes differentially by preserving the phosphorylation of some substrates and pathways, but not of others.

G120K-PEG, a human GH antagonist, decreases GH signal transduction in the liver of mice.

After receptor binding, growth hormone (GH) induces GH receptors (GHR) dimerization and JAK2 is activated after its association with a dimerized GHR, stimulating the tyrosyl phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2 and Shc proteins. G120K-PEG, a GH antagonist is produced by a mutation that blocks GH action by preventing the GHR dimerization. This study shows that the inhibitory effect of G120K-PEG was maximal with a GH:G120K-PEG ratio of 1:100, as no increase in JAK2 tyrosyl phosphorylation was observed with this dose of GH. When the dose of GH was increased and with a GH:G120K-PEG ratio of 1:10 some tyrosyl phosphorylation of JAK2 could be observed. Additionally, GH-induced IRS-1, IRS-2 and SHC tyrosyl phosphorylation was inhibited approximately 50% at equimolar concentrations of the antagonist of GH and almost abolished with a GH:G120K-PEG ratio of 1:100. The results clearly show that G120K-PEG inhibits GH signal transduction in mouse liver.

Statement of Retraction. Effect of Captopril, Losartan, and Bradykinin on Early Steps of Insulin Action. Diabetes 1997;46:1950–1957. DOI: 10.2337/diab.46.12.1950

The above-cited article has been retracted by the American Diabetes Association, the publisher of Diabetes . This article was previously the subject of an expression of concern in the March 2015 issue of the journal (Diabetes 2015;64:1068–1070. DOI: 10.2337/db15-ec03). As noted in the March 2015 expression of concern, the American Diabetes Association asked the corresponding author’s institution, the University of Campinas, to review the following issue with the …