Luigi Mattiello

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

The insulin-induced platelet anti-aggregating effect is attributed to a nitric oxide (NO)-mediated increase of cyclic guanosine monophosphate (cGMP). The aim of this work, carried out in human platelets, is to show whether insulin increases NO synthesis in platelets and whether it enhances not only cGMP but also cyclic adenosine monophosphate (cAMP) in these cells. We observed that 1) insulin dose-dependently increases NO production, evaluated as citrulline synthesis from Larginine (n = 4, P = 0.015); 2) insulin dose-dependently increases not only cGMP but also cAMP: for instance, after 8 min of insulin incubation at 1,920 pmol/l, cAMP increased from 39.8 ± 1.4 to 121.3 ± 12.6 pmol/l/109platelets (in = 16, P = 0.0001);3) when insulin is incubated for 120 min, the increase of cGMP and cAMP shows a plateau between 2 and 20 min, and while the effect on cGMP is significant until 120 min, the effect on cAMP is no more significant at 60 and 120 min; 4) insulin increases the effects on cAMP of the adenylate cyclase agonists Iloprost and forskolin (n = 5, P = 0.0001) and enhances their platelet anti-aggregating effects (n = 6 and 8, respectively; P = 0.0001); and 5) the inhibition of NO synthase by NG-monomethyl-L-arginine blunts both the insulin effects on basal cGMP and cAMP (n = 4) and those on the Iloprost- and forskolin-induced cAMP increase (n = 5). Thus, insulin increases NO synthesis in human platelets, and, through NO, enhances both cGMP and cAMP. The platelet antiaggregating effect exerted by insulin is, therefore, a NO-mediated phenomenon involving both cGMP and cAMP.

Platelet Resistance to Nitrates in Obesity and Obese NIDDM, and Normal Platelet Sensitivity to Both Insulin and Nitrates in Lean NIDDM

OBJECTIVE Previous studies in our laboratory showed that the platelet anti-aggregating effect exerted by insulin, mediated by a nitric oxide (NO)-induced increase of guanosine-3′,5′-cyclic monophosphate (cGMP), is lost in the insulin-resistant of obesity and obese NIDDM. It is not clear 1) whether the alterations observed in obese NIDDM patients are attributable to the obesity-related insulin resistance or to diabetes per se and 2) whether insulin-resistant states present a normal or a blunted response to NO. This study has been conducted to investigate 1) the platelet sensitivity to insulin in lean NIDDM and 2) the platelet sensitivity to an NO donor, glyceryl trinitrate (GTN), in obesity and in both lean and obese NIDDM. RESEARCH DESIGN AND METHODS We determined 1) ADP-induced platelet aggregation and platelet cGMP content in platelet-rich plasma (PRP) obtained from 11 lean NIDDM patients, after a 3-min incubation with insulin (0, 240, 480, 960, 1,920 pmol/l) and 2) ADP-induced platelet aggregation and platelet cGMP content in PRP obtained from 9 obese subjects, 11 lean and 8 obese NIDDM patients, and 18 control subjects, after a 3-min incubation with 0, 20, 40, and 100 μmol/l GTN. RESULTS Insulin dose-dependently decreased platelet aggregation in lean NIDDM patients (P = 0.0001): with 1,920 pmol/l of insulin, ADP ED50 was 141.5 ± 6.4% of basal values (P = 0.0001). Furthermore, insulin increased platelet cGMP (P = 0.0001) from 7.5 ± 0.2 to 21.1 ± 3.7 pmol/109 platelets. These results were similar to those previously described in healthy subjects. GTN reduced platelet aggregation in all the groups (P = 0.0001) at all the concentrations tested (P = 0.0001), but GTN IC50 values were much higher in insulin-resistant patients: 36.3 ± 5.0 μmol/l in healthy control subjects, 26.0 ± 6.0 μmol/l in lean NIDDM patients (NS vs. control subjects), 123.6 ± 24.0 μmol/l in obese subjects (P = 0.0001 vs. control subjects), and 110.1 ± 19.2 μmol/l in obese NIDDM patients (P = 0.0001 vs. control subjects). GTN dose-dependently increased platelet cGMP in all the groups (P = 0.0001 in control subjects, lean NIDDM patients, and obese subjects; P = 0.04 in obese NIDDM patients). Values reached by obese subjects and obese NIDDM patients, however, were lower than those reached by control subjects (with 100 μmol/l of GTN, P = 0.001 and P = 0.0001, respectively). In healthy control subjects and in obese subjects, the insulin:glucose ratio, used as an indirect measure of insulin sensitivity, was positively correlated to GTN IC50 (r = 0.530, P = 0.008), further suggesting that the sensitivity to NO is reduced in the presence of insulin resistance. CONCLUSIONS The insulin anti-aggregating effect is preserved in lean NIDDM; platelet sensitivity to GTN in preserved in lean NIDDM but is reduced in the insulin-resistant states of obesity and obese NIDDM. Resistance to nitrates, therefore, could be considered another feature of the insulin-resistance syndrome.

Insulin activates vascular endothelial growth factor in vascular smooth muscle cells: influence of nitric oxide and of insulin resistance

Background  We aimed to evaluate whether insulin influences vascular endothelial growth factor (VEGF) synthesis and secretion in cultured vascular smooth muscle cells (VSMCs) via nitric oxide (NO) and whether these putative effects are lost in insulin‐resistant states.

Insulin increases cyclic nucleotide content in human vascular smooth muscle cells: a mechanism potentially involved in insulin-induced modulation of vascular tone

SummaryIt has been suggested that insulin exerts a vasodilating effect, but the mechanisms involved are not completely understood. Since cyclic nucleotides mediate the vasodilation induced by endogenous substances, such as prostacyclin and nitric oxide, we aimed to investigate the influence of insulin (concentration range 240–960 pmol/l) on both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) content in human vascular smooth muscle cells. Insulin dose-dependently increased both nucleotides (cAMP: from 0.7±0.1 to 2.6±0.4 pmol/106 cells, p=0.0001; cGMP: from 1.3±0.2 to 3.4±0.7 pmol/106 cells, p=0.033). This increase is receptor-mediated, since it was blunted when cells were preincubated with the tyrosine kinase inhibitor genistein. The effect of insulin remained significant (p=0.0001) when preincubation with the phosphodiesterase inhibitor theophylline prevented cyclic nucleotide catabolism. The increase of cGMP was blunted when the cells were preincubated with the guanylate cyclase inhibitor methylene blue, and with the nitric oxide-synthase inhibitor NG-monomethyl-l-arginine. At all the concentrations tested, insulin potentiated the increase of cAMP induced by the stable prostacyclin analogue Iloprost (p=0.0001), whereas only at 1920 pmol/l did it potentiate the cGMP increase induced by glyceryltrinitrate (p=0.05). This study demonstrates that the vasodilating effects exerted by insulin may at least in part be attributable to an increase of both cGMP and cAMP via a receptor-mediated activation of adenylate and guanylate cyclases in human vascular smooth muscle cells and that the insulin effect on cGMP is mediated by nitric oxide.

Insulin Increases Guanosine-3′ ,5′-Cyclic Monophosphate in Human Platelets: A Mechanism Involved in the Insulin Anti-Aggregating Effect

To investigate whether insulin reduces platelet aggregability through a modulation of the guanosine-3′,5′-cyclic monophosphate (cGMP) concentrations, we determined by a radioimmunoassay the cGMP values in the platelet-rich plasma (PRP) obtained from 17 healthy volunteers and incubated for 3 min with different concentrations of human recombinant insulin (0, 240, 480, 720, 960, and 1,920 pM). Insulin induced a dose-dependent cGMP increase, from 18.5 ± 3.3 to 42.0 ± 6.4 pmol/109 platelets (P = 0.0001). This increase was completely blunted when PRP was preincubated for 20 min with the tyrosine kinase inhibitor genistein (10 μM) or with the guanylate cyclase inhibitor methylene blue (10 μM), but the increase remained highly significant (P = 0.003 and 0.009) when PRP was preincubated for 20 min with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 500 μM) or with the nitric oxide synthase inhibitor NG-mono-methyl-L-arginine (L-NMMA, 30 μM). Finally, the insulin-induced decrease of platelet aggregability to collagen and ADP was completely blunted when PRP was preincubated with 10 μM of the guanylate cyclase inhibitor methylene blue. This study demonstrates that the platelet anti-aggregatory effect exerted by insulin is attributable to the insulin-induced increase of cGMP that is due to a direct receptor-mediated platelet guanylate cyclase activation.

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

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 Ca2+-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 Ca2+ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as l-(3H)-citrulline production from l-(3H)-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/106 cells at 30 min (p = 0.0001); cAMP rose from 0.9 ± 0.09 to 11.65 ± 0.74 pmol/106 cells at 15 min (p = 0.0001). ii) Sodium nitroprusside (100 μmol/l) and glyceryltrinitrate (100 μmol/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 μmol/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]

In central obesity, weight loss restores platelet sensitivity to nitric oxide and prostacyclin.

Central obesity shows impaired platelet responses to the antiaggregating effects of nitric oxide (NO), prostacyclin, and their effectors—guanosine 3′,5′‐cyclic monophosphate (cGMP) and adenosine 3′,5′‐cyclic monophosphate (cAMP). The influence of weight loss on these alterations is not known. To evaluate whether a diet‐induced body‐weight reduction restores platelet sensitivity to the physiological antiaggregating agents and reduces platelet activation in subjects affected by central obesity, we studied 20 centrally obese subjects before and after a 6‐month diet intervention aiming at reducing body weight by 10%, by measuring (i) insulin sensitivity (homeostasis model assessment of insulin resistance (HOMAIR)); (ii) plasma lipids; (iii) circulating markers of inflammation of adipose tissue and endothelial dysfunction, and of platelet activation (i.e., soluble CD‐40 ligand (sCD‐40L) and soluble P‐selectin (sP‐selectin)); (iv) ability of the NO donor sodium nitroprusside (SNP), the prostacyclin analog Iloprost and the cyclic nucleotide analogs 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) and 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) to reduce platelet aggregation in response to adenosine‐5‐diphosphate (ADP); and (v) ability of SNP and Iloprost to increase cGMP and cAMP. The 10 subjects who reached the body‐weight target showed significant reductions of insulin resistance, adipose tissue, endothelial dysfunction, and platelet activation, and a significant increase of the ability of SNP, Iloprost, 8‐Br‐cGMP, and 8‐Br‐cAMP to reduce ADP‐induced platelet aggregation and of the ability of SNP and Iloprost to increase cyclic nucleotide concentrations. No change was observed in the 10 subjects who did not reach the body‐weight target. Changes of platelet function correlated with changes of HOMAIR. Thus, in central obesity, diet‐induced weight loss reduces platelet activation and restores the sensitivity to the physiological antiaggregating agents, with a correlation with improvements in insulin sensitivity.

Insulin influences the renin-angiotensin-aldosterone system in humans

This study investigates whether insulin influences the renin-angiotensin-aldosterone system in humans. Six healthy male volunteers were placed on a 30-minute euglycemic insulin clamp at 160 microU/mL; euglycemia was maintained also in the following 60 minutes by means of appropriate dextrose infusion. Throughout the study, plasma renin activity, angiotensin II, aldosterone, and factors involved in the regulation of the renin-angiotensin-aldosterone system were measured: catecholamines, angiotensin-converting enzyme, sodium, and potassium. A significant increase of plasma renin activity and angiotensin II was observed, and a decrease of aldosterone was also detected. These changes can be ascribed to the effects of the rapid insulin-induced plasma potassium decrease on plasma renin activity and aldosterone secretion because they did not occur in a control clamp study with a potassium infusion.

Impaired synthesis and action of antiaggregating cyclic nucleotides in platelets from obese subjects: possible role in platelet hyperactivation in obesity.

Background  Subjects with central obesity exhibit platelet hyperactivity, which is involved in the atherosclerotic process and therefore can account for the increased risk of cardiovascular morbidity and mortality. The aim of the study was to evaluate whether alterations of platelet function in obesity involve synthesis and/or action of the two antiaggregating cyclic nucleotides adenosine 3′,5′‐cyclic monophosphate (cAMP) and guanosine 3′,5′‐cyclic monophosphate (cGMP).

The Insulin-Induced Increase of Guanosine-3′,5′-Cyclic Monophosphate in Human Platelets Is Mediated by Nitric Oxide

To investigate whether the insulin-induced increase of guanosine-3ʹ,5ʹ-cyclic monophosphate (cGMP) in human platelets is mediated by nitric oxide or is influenced by the nitric oxide precursor L-arginine, we measured cGMP in platelet-rich plasma obtained from healthy volunteers incubated for 3 min with human recombinant insulin (0, 240, 480, 960, and 1,920 pmol/l) both with and without 1) a 20-min incubation with the nitric oxide–synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) (50, 70, 100, and 1,000 μmol/l; n = 5 for each dose) and 2) a 20-min incubation with the nitric oxide precursor L-arginine (300 μmol/l; n = 6). In a first set of experiments, insulin induced a dose-dependent cGMP increase, from 9.8 ± 0.8 to 45.6 ± 5.5 pmol/l09 platelets (P = 0.0001); in the presence of 1 mmol/l L-NMMA, this increase was blunted, cGMP being 8.9 ± 1.4 and 11.1 ± 2.2 pmol/l09 platelets at 0 and 1,920 pmol/l insulin, respectively (NS). In the experiments with 70 and 100 μmol/l L-NMMA, the insulin effect on cGMP was inhibited, whereas 50 μmol/l L-NMMA did not blunt this insulin effect. In another set of experiments carried out to investigate the effects of L-arginine, insulin induced a dose-dependent cGMP increase, from 23.6 ± 6.9 to 59.0 ± 12.0 pmol/l09 platelets (P = 0.0001); with L-arginine, basal cGMP values increased to 35.5 ± 6.6 pmol/l09 platelets (P = 0.05), and insulin maintained its ability to enhance dose-dependently cGMP values, which rose to 76.8 ± 19.4 pmol/l09 platelets (P = 0.003). This study carried out in human platelets demonstrates that the cGMP increase induced by insulin, which accounts for the antiaggregating effect of the hormone, is mediated by nitric oxide.