Insulin Stimulates Nitric Oxide Synthesis in Human Platelets and, Through Nitric Oxide, Increases Platelet Concentrations of Both Guanosine-3′, 5′-Cyclic Monophosphate and Adenosine-3′, 5′-Cyclic Monophosphate

Trovati, Mariella; Anfossi, Giovanni; Massucco, Paola; Mattiello, Luigi; Costamagna, Costanzo; Piretto, Valentina; Mularoni, E.; Cavalot, Franco; Bosìa, Amalia; Ghigo, Dario

doi:10.2337/diab.46.5.742

Cited by 104 publications

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“…Trovati et al (24) reported earlier that platelets suspended in plasma are inhibited by insulin through a rise in [cAMP]. Also cAMP production induced by the stable PGI 2 analogue iloprost and the adenylyl cyclase activator forskolin was enhanced by insulin.…”

Section: ؉mentioning

confidence: 93%

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

Ferreira

Eybrechts

Mocking

et al. 2004

Journal of Biological Chemistry

102

116

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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-

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Section: ؉mentioning

confidence: 93%

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

Ferreira

Eybrechts

Mocking

et al. 2004

Journal of Biological Chemistry

102

116

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“…Furthermore, not only was NO capable of activating tyrosine kinase or PI 3-kinase but also the "messenger" NO could activate NOS in the absence of either tyrosine kinase or PI 3-kinase (or both) in the NOS system (unpublished results), whereby NO would probably amplify its own generation by "feedback" activation of NOS and, consequently, would amplify the effect of insulin itself in the system. It has been reported previously that NO can stimulate glucose metabolism in tissues (25) and that insulin treatment of platelets, cells that do not require insulin for glucose metabolism, results in the increased production of NO (26). However, it has not been previously appreciated that the production of NO as a result of the activation of NOS by insulin is actually an essential step-beyond the activation of both PI 3-and tyrosine kinases by insulin-in the transduction of messages that affect glycemic control.…”

Section: Discussionmentioning

confidence: 99%

Nitric Oxide: The “Second Messenger” of Insulin

et al. 2000

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SummaryIncubation of various tissues, including heart, liver, kidney, muscle, and intestine from mice and erythrocytes or their membrane fractions from humans, with physiologic concentration of insulin resulted in the activation of a membrane-bound nitric oxide synthase (NOS). Activation of NOS and synthesis of NO were stimulated by the binding of insulin to speci c receptors on the cell surface. A Lineweaver -Burk plot of the enzymatic activity demonstrated that the stimulation of NOS by insulin was related to the decrease in the K m for L-arginine, the substrate for NOS, with a simultaneous increase of V max . Addition of N G -nitro-L-arginine methyl ester (LNAME), a competitive inhibitor of NOS, to the reaction mixture completely inhibited the hormone-stimulated NO synthesis in all tissues. Furthermore, NO had an insulin-like effect in stimulating glucose transport and glucose oxidation in muscle, a major site for insulin action. Addition of NAME to the reaction mixture completely blocked the stimulatory effect of insulin by inhibiting both NO production and glucose metabolism, without affecting the hormone-stimulated tyrosine or phosphatidylinositol 3-kinases of the membrane preparation. Injection of NO in alloxan-induced diabetic mice mimicked the effect of insulin in the control of hyperglycemia (i.e., lowered the glucose content in plasma). However, injection of NAME before the administration of insulin to diabetic-induced and nondiabetic mice inhibited not only the insulin-stimulated increase of NO in plasma but also the glucose-lowering effect of insulin.

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“…We measured the effects of insulin on cGMP and cAMP in platelets at close intervals ranging from a few seconds to 120 min to show that insulin action on both nucleotides is very rapid, reaching the maximum effect after only 2 min [13]. Furthermore, we observed that in these cells insulin increases NO production in a few minutes [13].…”

Section: : 831±839]mentioning

confidence: 99%

“…We recently showed in both human VSMC (hVSMC) and in platelets that insulin increases intracellular concentrations of cGMP [8±13], by a mechanism which is blocked both by the guanylate cyclase inhibitor methylene blue and by the NOS inhibitor l-NMMA [8,9,12,13]. We also showed that insulin can, in both hVSMC and platelets, increase the intracellular concentrations of adenosine 3':5'-cyclic monophosphate (cAMP), with a mechanism blocked by l-NMMA [9,13], that could be attributed to cGMP [14±17] or directly to NO [18] as we will explain more extensively in the Discussion.…”

Section: : 831±839]mentioning

confidence: 99%

Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3′ : 5′-cyclic monophosphate and adenosine 3′ : 5′-cyclic monophosphate concentrations

et al. 1999

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Aims/hypothesis. Insulin incubation of human vascular smooth muscle cells (hVSMC) for 120 min increases both guanosine 3¢ : 5¢-cyclic monophosphate (cGMP) and adenosine 3¢ : 5¢-cyclic monophosphate (cAMP) and these effects are blocked by inhibiting nitric oxide synthase (NOS). These data suggest that insulin activates a constitutive Ca 2+ -dependent NOS (cNOS), not described at yet in hVSMC. To test this hypothesis, we evaluated in hVSMC: i) the kinetics of the insulin-induced enhancement of the two cyclic nucleotides; ii) the ability of nitric oxide (NO) to increase both cyclic nucleotides; iii) NO involvement in the short-term influence of insulin on both cyclic nucleotides; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of a cNOS activity; vi) the expression of mRNA for cNOS. Methods. In hVSMC incubated with insulin, NO donors and the Ca 2+ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as l-( 3 H)-citrulline production from l-( 3 H)-arginine; by northern blot hybridization, we measured the expression of cNOS mRNA. Results. i) By incubating hVSMC with 2 nmol/l insulin for 0±240 min, we observed an increase of both cGMP and cAMP (ANOVA: p = 0.0001). Cyclic GMP rose from 0.74 ± 0.01 to 2.62 ± 0.10 pmol/10 6 cells at 30 min (p = 0.0001); cAMP rose from 0.9 ± 0.09 to 11.65 ± 0.74 pmol/10 6 cells at 15 min (p = 0.0001). ii) Sodium nitroprusside (100 mmol/l) and glyceryltrinitrate (100 mmol/l) increased both cGMP and cAMP (p = 0.0001). iii) The effects of insulin on cyclic nucleotides were blocked by NOS inhibition. iv) An increase of NO was observed by incubating hVSMC for 5 min with 2 nmol/l insulin (p = 0.0001). v) Ionomycin (1 mmol/l) enhanced NO production (p = 0.0001) and increased both cyclic nucleotides (p = 0.0001). vi) hVSMC expressed mRNA of cNOS. Conclusion/interpretation. Human VSMC express cNOS, which is rapidly activated by insulin with a consequent increase of both cGMP and cAMP, suggesting that insulin-induced vasodilation in vivo is not entirely endothelium-mediated. [Diabetologia (1999) 42: 831±839]

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Insulin Stimulates Nitric Oxide Synthesis in Human Platelets and, Through Nitric Oxide, Increases Platelet Concentrations of Both Guanosine-3′, 5′-Cyclic Monophosphate and Adenosine-3′, 5′-Cyclic Monophosphate

Cited by 104 publications

References 0 publications

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

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

Nitric Oxide: The “Second Messenger” of Insulin

Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3′ : 5′-cyclic monophosphate and adenosine 3′ : 5′-cyclic monophosphate concentrations

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