IRS-1 Mediates Inhibition of Ca2+ Mobilization by Insulin via the Inhibitory G-protein Gi

Abstract

Patients with diabetes mellitus have a 2-4-fold increased risk for coronary artery disease. They suffer from both microvascular (nephropathy and retinopathy) and macrovascular (peripheral artery disease) complications (1). Apart from increased concentrations of certain coagulation factors (2), patients with diabetes mellitus type I and II have platelets that show increased adhesion, aggregation, thromboxane production, and P-selectin expression (3). The hyperactivity might be caused by the absence of insulin inhibition, since intensive insulin treatment in diabetic patients reduced platelet aggregation (4).The insulin receptor is a heterotetrameric transmembrane glycoprotein composed of two extracellular ␣ subunits (135 kDa each) and two transmembrane ␤ subunits (95 kDa each) that function as allosteric enzymes in which the ␣ subunit inhibits the tyrosine kinase activity of the ␤ subunit. Insulin binding to the ␣ subunit relieves the inhibition of the kinase activity in the ␤ subunit leading to autophosphorylation of the ␤ subunits and a conformational change that further increases the kinase activity. The insulin receptor tyrosine kinase phosphorylates proteins such as Shc and the insulin receptor substrates IRS-1 (165-185 kDa) and . IRS-1 and IRS-2 have a highly conserved amino terminus, which contains a pleckstrin homology domain, a phosphotyrosine binding domain, and a carboxyl terminus with several tyrosine phosphorylation sites. IRS-1 and IRS-2 are complementary and act as "docking sites" to several Src homology 2 domains containing proteins, such as the regulatory subunits of phosphatidylinositol 3-kinase (PI3K) 1 (5). GTP-binding proteins (G-proteins) can also act as signal transducers for the insulin receptor. G-proteins are guanine nucleotide-binding regulatory proteins that function as molecular switches between a GTP-bound "on state" and a GDPbound "off state." These proteins amplify, transmit, and integrate signals. The major G-proteins involved in platelet aggregation and secretion are G q ␣, which mediates increases in cytosolic Ca 2ϩ concentration, [Ca 2ϩ ] i , and G i ␣, which inhibits adenylyl cyclase thereby suppressing cAMP that is an inhibitor of platelets (6). Receptors that couple to G-proteins are generally seven-transmembrane proteins, but there are important exceptions. The insulin-like growth factor II receptor has a single transmembrane domain and couples directly to G i2 in a manner similar to that of conventional G-protein-coupled receptors (7). Studies have been reported suggesting that the insulin receptor binds G i ␣ 2 (8, 9).The insulin receptor is present on muscle, liver, and adipose tissue but also on endothelial cells, lymphocytes, erythrocytes, and platelets. A human platelet contains ϳ570 insulin receptors (10). Insulin binding induces phosphorylation of the ␤ subunits (11,12), demonstrating that the receptor is func-

Greater Clinical Benefit of More Intensive Oral Antiplatelet Therapy With Prasugrel in Patients With Diabetes Mellitus in the Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition With Prasugrel–Thrombolysis in Myocardial Infarction 38

“…6 Physiologically, insulin reduces platelet aggregation by inhibiting the P2Y 12 receptor, although this inhibition is absent in subjects with DM and insulin resistance. 32,33 In previous mechanistic studies, insulin-treated subjects had higher levels of platelet aggregation after dual antiplatelet therapy than subjects with DM not treated with insulin, indicating that treatment with insulin may identify a group of subjects at particular risk for poor response to clopidogrel. 22 We did not collect age of onset (juvenile versus adult) or duration of diabetes, both factors that may influence insulin use.…”

Induction of insulin resistance by the adipokines resistin, leptin, plasminogen activator inhibitor-1 and retinol binding protein 4 in human megakaryocytes

“…9,29 Tyr phosphorylation of insulin receptor substrate 1 is under the control of insulin receptor substrate 1 Ser 307 but whether phospho-Ser 307 stimulates or inhibits insulin receptor substrate 1 function differs among cell types. In insulin-treated CHRF-288-11 cells, resistin induced a fall in phospho-Ser 307 , while cantharidin neutralized this inhibition ( Figure 2C).…”

“…This action interferes with the drop in cAMP induced by P2Y12 ligation, making insulin an inhibitor of Ca 2+ rises and platelet functions. 9 To clarify whether a similar mechanism was operational in CHRF-288-11 cells, thrombin-induced Ca 2+ mobilization was measured in the presence of AR-C69931MX, an inhibitor of the ADP-P2Y12 association. This treatment induced a dose-dependent decrease in Ca 2+ rises, confirming the presence of P2Y12 signaling in CHRF-288-11 cells (Online Supplementary Figure S2A).…”

“…9 To investigate whether adipokines could make platelets resistant to insulin, rises in Ca 2+ were measured in the presence of resistin, leptin and IL-6 at concentrations 10-fold those found under physiological conditions. These treatments did not change the decrease in Ca 2+ mobilization induced by insulin ( Figure 1A,B).…”

“…In healthy individuals, insulin interferes with the suppression of cAMP and accumulation of this inhibitor attenuates platelet functions and monocyte responses. 9,10 The insulin resistance observed in type 2 diabetes rescues the fall in cAMP, which promotes aggregation, secretion and procoagulant activity in platelets and tissue factor synthesis and interleukin-1β secretion in monocytes. Insulin also inhibits splicing of tissue factor pre-mRNA in platelets adhering to prothrombotic proteins and the loss of insulin responsiveness in type 2 diabetes might well contribute to the thrombogenicity of the platelet plug that forms upon plaque rupture.…”