Franco Cavalot

Postprandial blood glucose predicts cardiovascular events and all-cause mortality in type 2 diabetes in a 14-year follow-up: lessons from the San Luigi Gonzaga Diabetes Study.

OBJECTIVE To evaluate whether postprandial blood glucose predicts cardiovascular events and all-cause mortality in type 2 diabetes in a long-term follow-up taking into account A1C and the main cardiovascular risk factors. RESEARCH DESIGN AND METHODS Consecutive type 2 diabetic patients (n = 505) followed up at our diabetes clinic were evaluated at baseline (1995) for the main cardiovascular risk factors and for five glycemic control parameters (fasting blood glucose, blood glucose 2 h after breakfast, blood glucose 2 h after lunch, blood glucose before dinner, and A1C); all-cause mortality and the first cardiovascular events occurring during the 14-year follow-up were measured. RESULTS We observed 172 cardiovascular events (34.1% of the population) and 147 deaths (29.1% of the population). Using the Cox analysis with the backward method, we categorized the variables according to the therapeutic targets of the American Diabetes Association. Our observations were as follows. When the five glycemic control parameters were considered together, the predictors were 1) for cardiovascular events, blood glucose 2 h after lunch (hazard ratio 1.507, P = 0.010) and A1C (1.792, P = 0.002); and 2) for mortality, blood glucose 2 h after lunch (1.885, P < 0.0001) and A1C (1.907, P = 0.002). When blood glucose 2 h after lunch and A1C were considered together with the main cardiovascular risk factors, the following glycemic control parameters were predictors: 1) for cardiovascular events, blood glucose 2 h after lunch (1.452, P = 0.021) and A1C (1.732, P = 0.004); and 2) for mortality, blood glucose 2 h after lunch (1.846, P = 0.001) and A1C (1.896, P = 0.004). CONCLUSIONS In type 2 diabetes, both postprandial blood glucose and A1C predict cardiovascular events and all-cause mortality in a long-term follow-up.

Influence of Physical Training on Blood Glucose Control, Glucose Tolerance, Insulin Secretion, and Insulin Action in Non-insulin-dependent Diabetic Patients

This study has been designed to investigate, in five non-insulin-dependent diabetic patients, the influence of physical training (1 h a day, 7 days a wk for 6 wk, at 50–60% maximum oxygen uptake) on blood glucose control, glucose tolerance, insulin secretion, and insulin action. Physical training resulted in a significant improvement in blood glucose control, glucose tolerance, and insulin action. These results suggest that short-term intense physical training ameliorates the main metabolic derangements of non-insulin-dependent diabetes mellitus.

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.

Clinical significance of nonalbuminuric renal impairment in type 2 diabetes.

Objective In type 2 diabetes, prevalence of nonalbuminuric renal impairment is increasing worldwide, though its clinical significance remains unclear. This large-cohort study aimed at evaluating the association of this phenotype with cardiovascular risk factors and other complications. Methods Type 2 diabetic patients from the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study (n = 15 773), visiting consecutively 19 hospital-based Diabetes Clinics in years 2007–2008, were examined. Serum creatinine was assessed by the Jaffe method; albuminuria was measured by immunonephelometry or immunoturbidimetry. Results Of patients with renal impairment, as identified by an estimated glomerular filtration rate (eGFR) less than 60 ml/min per 1.73 m2, 56.6% were normoalbuminuric, 30.8% were microalbuminuric, and 12.6% were macroalbuminuric. Percentages were similar when GFR was estimated using the more accurate Chronic Kidney Disease Epidemiology Collaboration equation instead of the simplified Modification of Diet in Renal Disease formula, and were independent of age, thus indicating that the increasing prevalence of this phenotype does not reflects misclassification of elderly patients. Nonalbuminuric renal impairment was not associated with HbA1c and correlated less strongly with retinopathy and hypertension than albuminuria, either alone or associated with reduced eGFR. It was associated with a higher prevalence of cardiovascular disease (CVD) than albuminuria alone, but lower than albuminuric renal impairment. Female sex correlated with nonalbuminuric renal impairment and male sex with the albuminuric forms. Conclusions These data show that type 2 diabetic patients with nonalbuminuric renal impairment exhibit distinct clinical features, suggesting predominance of macroangiopathy as underlying renal pathology, and that this phenotype is associated with significant CVD burden.

Insulin Directly Reduces Platelet Sensitivity to Aggregating Agents: Studies In Vitro and In Vivo

The aim of this study was to investigate the influence of insulin on platelet function, both in vitro and in vivo. For the in vitro investigation, we evaluated whether insulin affects platelet function at a physiological hormone concentration by incubating the platelet-rich plasma (PRP) of fasting subjects with human regular insulin at the final concentration of 40 μU/ml for 30 min; we observed a significant reduction of platelet sensitivity to all the aggregating agents employed, i.e., ADP, platelet-activating factor (PAF), epinephrine, collagen, and Na+ arachidonate. To investigate whether the insulin effect on platelets is dose dependent, we incubated the PRP of fasting subjects with different concentrations of human regular insulin (40, 80, 120, and 160 μU/ml) for 5 min, and we observed that the insulin-induced reduction of platelet sensitivity to aggregating agents is a dose-dependent phenomenon. Furthermore, the comparison between the platelet responses after 5 and 30 min of incubation with insulin showed that the insulin effect on platelet aggregation is time dependent. The lack of specificity of its inhibiting activity suggests that insulin does not interfere with the initial binding of each aggregating agent at specific sites but does influence a common step of platelet aggregation. Our study rules out the possibility that insulin reduces platelet-function–modifying intraplatelet cAMP levels or thromboxane A2 production, because this hormone decreases the platelet concentrations of cAMP–a phenomenon that, per se, promotes platelet aggregation–and does not modify collagen or Na+ arachidonate–induced platelet production of thromboxane A2, measured by radioimmunoassay of its stable-metabolite thromboxane B2. Insulin seems to help in modifying platelet membrane properties, as has already been shown for erythrocytes. The in vivo investigation comprised three studies of the influence of insulin on platelet function in male volunteers: 1) a euglycemic-hyperinsulinemic (40-μU/ml) clamp for 90 min followed by 60 min of euglycemia; 2) a euglycemic-hyperinsulinemic (160-μU/ml) clamp for 30 min followed by 60 min of euglycemia; and 3) an intravenous bolus of human regular insulin (3.84 U/m2). Throughout the three studies, we serially measured platelet sensitivity to ADP, PAF, epinephrine, collagen, and Na+ arachidonate. We observed that insulin in vivo and at the physiologic concentration of 40 μU/ml reduced platelet aggregation. For some aggregating agents, we demonstrated a dose and time dependence of the insulin effect. The latter was reversed after the insulin infusion. When insulin was administered as an intravenous bolus and platelet aggregation was studied before the appearance of hypoglycemia, we observed that insulin influence on platelets can be detected after only 10 min. In conclusion, this study suggests that insulin may have a role in the physiological modulation of platelet function and that the long-term insulin deficiency might account for the enhanced platelet aggregability frequently observed in diabetic patients.

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.

HbA1c Variability as an Independent Correlate of Nephropathy, but Not Retinopathy, in Patients With Type 2 Diabetes: The Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study

OBJECTIVE To examine the association of hemoglobin (Hb) A1c variability with microvascular complications in the large cohort of subjects with type 2 diabetes from the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study. RESEARCH DESIGN AND METHODS Serial (3–5) HbA1c values collected in a 2-year period before enrollment were available from 8,260 subjects from 9 centers (of 15,773 patients from 19 centers). HbA1c variability was measured as the intraindividual SD of 4.52 ± 0.76 values. Diabetic retinopathy (DR) was assessed by dilated funduscopy. Chronic kidney disease (CKD) was defined based on albuminuria, as measured by immunonephelometry or immunoturbidimetry, and estimated glomerular filtration rate (eGFR) was calculated from serum creatinine. RESULTS Median and interquartile range of average HbA1c (HbA1c-MEAN) and HbA1c-SD were 7.57% (6.86–8.38) and 0.46% (0.29–0.74), respectively. The highest prevalence of microalbuminuria, macroalbuminuria, reduced eGFR, albuminuric CKD phenotypes, and advanced DR was observed when both HbA1c parameters were above the median and the lowest when both were below the median. Logistic regression analyses showed that HbA1c-SD adds to HbA1c-MEAN as an independent correlate of microalbuminuria and stages 1–2 CKD and is an independent predictor of macroalbuminuria, reduced eGFR, and stages 3–5 albuminuric CKD, whereas HbA1c-MEAN is not. The opposite was found for DR, whereas neither HbA1c-MEAN nor HbA1c-SD affected nonalbuminuric CKD. CONCLUSIONS In patients with type 2 diabetes, HbA1c variability affects (albuminuric) CKD more than average HbA1c, whereas only the latter parameter affects DR, thus suggesting a variable effect of these measures on microvascular complications.

Studies on Mechanisms Involved in Hypoglycemia-Induced Platelet Activation

The aim of our study was to investigate the mechanisms involved in hypoglycemia-induced platelet activation. Sixteen healthy male subjects received a 60-min intravenous infusion of human regular insulin at the rate of 64 mU · m−2 · min−1: throughout 150 min, we serially measured plasma concentrations of glucose, insulin, and counterregulatory hormones; platelet sensitivity to ADP, thrombin and platelet-activating factor; plasma concentrations of platelet markers for specific proteins of in vivo release reaction (β-thromboglobulin and platelet factor 4). Our study showed that insulin-induced hypoglycemia causes a significant increase in platelet sensitivity to aggregating agents in vitro and a platelet release reaction in vivo. Hypoglycemia-induced platelet activation was not correlated with plasma glucose concentrations at nadir and occurred before the increase of plasma growth hormone and cortisol. To further elucidate the mechanisms of hypoglycemia-induced platelet activation, we incubated in vitro platelet-rich plasma (PRP) of seven fasting healthy subjects with the same concentrations of insulin, epineph-rine, glucagon, growth hormone, and cortisol measured in vivo during insulin-induced hypoglycemia. Only epinephrine was able to increase platelet sensitivity to aggregating agents. To investigate the role of α-adrenergic receptors in this phenomenon, we also studied four healthy subjects on another occasion, repeating the above-described insulin infusion together with intravenous infusion of phentolamine (–15 to + 150 min), 5 mg over 2 min followed by 500 μg/min. α-Blockade was able to suppress hypoglycemia-induced increase of platelet sensitivity to aggregating agents. A further study in vitro confirmed these results obtained in vivo, showing that incubation with phentolamine is able to inhibit the epinephrine-induced increase of platelet aggregation in response to ADP, thrombin, and platelet-activating factor. In conclusion, insulin-induced hypoglycemia deeply influences platelet function, causing an increase of platelet sensitivity to aggregating agents in vitro and a release reaction in vivo. Through α-adrenoreceptors, epinephrine is responsible for the hypoglycemia-induced increase of platelet aggregation in response to ADP, thrombin, and platelet-activating factor.

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.