Agostino Consoli

In Vivo Formation of 8-Iso-Prostaglandin F2α and Platelet Activation in Diabetes Mellitus Effects of Improved Metabolic Control and Vitamin E Supplementation

Background—Diabetes mellitus (DM) is associated with enhanced lipid peroxidation and persistent platelet activation. We tested the hypothesis that the in vivo formation of the F2-isoprostane 8-iso-prostaglandin (PG)F2α, a bioactive product of arachidonic acid peroxidation, is enhanced in DM and contributes to platelet activation. Methods and Results—Urine samples were obtained from 85 diabetic patients and 85 age- and sex-matched healthy subjects for measurement of immunoreactive 8-iso-PGF2α and 11-dehydro-thromboxane B2 (TXM), an in vivo index of platelet activation. Sixty-two had non–insulin-dependent (NID)DM, and 23 had insulin-dependent (ID) DM. Vitamin E supplementation, metabolic control, and cyclooxygenase inhibitors were used to investigate the mechanisms of formation of 8-iso-PGF2α in this setting. Urinary 8-iso-PGF2α excretion was significantly higher (P=0.0001) in NIDDM patients (419±208 pg/mg creatinine; range 160 to 1014) than in age-matched control subjects (208±92; 41 to 433). Urinary 8-iso...

Predominant Role of Gluconeogenesis in Increased Hepatic Glucose Production in NIDDM

Excessive hepatic glucose output is an important factor in the fasting hyperglycemia of non-insulindependent diabetes mellitus (NIDDM). To determine the relative contributions of gluconeogenesis and glycogenolysis in a quantitative manner, we applied a new isotopic approach, using infusions of [6-3H]glucose and [2-14C]acetate to trace overall hepatic glucose output and phosphoenolpyruvate gluconeogenesis in 14 postabsorptive NIDDM subjects and in 9 nondiabetic volunteers of similar age and weight. Overall hepatic glucose output was increased nearly twofold in the NIDDM subjects (22.7 ± 1.0 vs. 12.0 ± 0.6 μmol · kg−1 · min−1 in the nondiabetic volunteers, P < .001); phosphoenolpyruvate gluconeogenesis was increased more than threefold in the NIDDM subjects (12.7 ± 1.4 vs. 3.6 ± 0.4 μmol kg−1 min−1 in the nondiabetic subjects, P < .001) and was accompanied by increased plasma lactate, alanine, and glucagon concentrations (all P < .05). The increased phosphoenolpyruvate gluconeogenesis accounted for 89 ± 6% of the increase in overall hepatic glucose output in the NIDDM subjects and was significantly correlated with the fasting plasma glucose concentrations (r = .67, P < .01). Glycogenolysis, calculated as the difference between overall hepatic glucose output and phosphoenolpyruvate gluconeogenesis, was not significantly different in the NIDDM subjects (9.9 ± 0.06 μmol · kg−1 · min−1) and the nondiabetic volunteers (8.4 ± 0.3 μmol kg−1 · min−1). We conclude that increased gluconeogenesis is the predominant mechanism responsible for increased hepatic glucose output in NIDDM.

Mechanisms of uremic erythrocyte-induced adhesion of human monocytes to cultured endothelial cells†

In end‐stage renal disease (ESRD) endothelium may represent a key target for the action of circulating elements, such as modified erythrocytes (RBC) and/or plasmatic factors, that may facilitate inflammation and the vasculopathy associated with uremia. We have previously demonstrated that phosphatidylserine (PS) exposure on the surface of RBC from ESRD patients increases RBC‐human umbilical vein endothelial cell (HUVEC) interactions and causes decreased nitric oxide (NO) production. We postulated that, besides the pro‐inflammatory effects due to decreased NO bio‐availability, enhanced ESRD‐RBC‐HUVEC interactions might directly stimulate pro‐inflammatory pathways leading to increased vascular adhesion molecule expression. ESRD‐RBC‐endothelial cell interactions induced a time‐dependent up‐regulation of VCAM‐1 and ICAM‐1 (measured by Western blot (WB) and real‐time PCR), associated with mitogen‐activated protein kinase (MAPK) activation and impairment of the Akt/endothelial nitric oxide synthase (eNOS) signaling cascade, measured by WB. In reconstitution experiments, normal RBC incubated with uremic plasma showed increased PS exposure and significantly increased VCAM‐1 and ICAM‐1 mRNA levels when incubated on HUVEC. Interestingly, ESRD‐RBC induced increased expression of adhesion molecules was prevented by Annexin‐V (AnV, able to mask PS on RBC surface), anti‐integrin‐αvβ3, anti‐thrombospondin‐1 (TSP‐1), and PD98059 (a selective inhibitor of MAPK phosphorylation). Moreover, AnV reversed the ESRD‐RBC effects on MAPK and Akt/eNOS signaling pathways. Our data demonstrate that, possibly via a direct interaction with the endothelial thrombospondin‐(αvβ3) integrin complex, ESRD‐RBC‐HUVEC adhesion induces a vascular inflammatory phenotype. Thus, intervention targeting ESRD‐RBC increased adhesion to endothelium and/or MAPK and Akt/eNOS pathways may have the potential to prevent vascular lesions under uremic conditions. J. Cell. Physiol. 213:699–709.

Role of Liver in Pathophysiology of NIDDM

Glucose homeostasis is physiologically maintained by the balance between glucose production by the liver and glucose utilization by the peripheral tissues. Insulin controls hepatic glucose production and promotes glucose utilization by the skeletal muscle. In non-insulin-dependent diabetes mellitus (NIDDM), postabsorptive hepatic glucose production is increased and exhibits a positive correlation with fasting plasma glucose concentration. This increase in hepatic glucose production is the main cause of fasting hyperglycemia in NIDDM. Between the two processes by which the liver produces glucose (gluconeogenesis and glycogenolysis), gluconeogenesis appears to be drastically increased in NIDDM. The increase in gluconeogenesis accounts for most of the increased hepatic glucose production in this condition, and a positive correlation has been found in NIDDM subjects between the rates of gluconeogenesis and fasting plasma glucose concentration. Increased production of gluconeogenic precursors (lactate, alanine, glycerol) fuels this increased gluconeogenesis, but some type of intrahepatic mechanism is also present in NIDDM that increases the hepatic conversion of these substrates into glucose. Hyperglucagonemia and increased hepatic free fatty acid oxidation might be responsible for this increase hepatic gluconeogenic efficiency in NIDDM. Reduced suppression of hepatic glucose production after carbohydrate ingestion also plays an important role in the impairment in postprandial glucose homeostasis in NIDDM. In NIDDM subjects splanchnic extraction of an oral glucose load is not decreased, but hepatic glucose production is suppressed less than in nondiabetic subjects after the load. Residual hepatic glucose production after glucose ingestion is also correlated with fasting plasma glucose in NIDDM. Preliminary data suggest that in the postprandial state increased gluconeogenesis represents the primary mechanism responsible for impaired suppression of hepatic glucose production. Given the primary role of increase hepatic gluconeogenesis in the pathogenesis of hyperglycemia in NIDDM, development of new drugs aimed at correcting the factors that might cause increased gluconeogenesis (e.g., increased free fatty acid oxidation and hyperglucagonemia) might open the way for new form of treatment of this disorder.

G972R IRS-1 Variant Impairs Insulin Regulation of Endothelial Nitric Oxide Synthase in Cultured Human Endothelial Cells

Background—Impaired insulin-mediated vasodilation might contribute to vascular damage in insulin-resistant states. Little is known about insulin regulation of nitric oxide (NO) synthesis in insulin-resistant cells. The aim of this work was to investigate insulin regulation of NO synthesis in human umbilical vein endothelial cells (HUVECs) carrying the IRS-1 gene G972R variant, known to be associated with impaired insulin activation of the PI3-kinase (PI3-K) pathway in transfected cells. Methods and Results—HUVECs were screened for the presence of the G972R-IRS-1 (HUVEC-G972R) variant by restriction fragment length polymorphisms. After 24-hour exposure to 10−7 mol/L insulin, endothelial NO synthase (eNOS) mRNA (reverse transcription–polymerase chain reaction), eNOS protein levels (Western blotting), and NOS activity (conversion of [3H]arginine into [3H]citrulline) were increased in wild-type HUVECs (HUVEC-WT), whereas they did not change from baseline in HUVEC-G972R. Compared with HUVEC-WT, in HUVEC-G972R after 2 and 10 minutes of insulin stimulation, IRS-1–associated PI3-K activity was reduced by 47% and 32%, respectively; Akt phosphorylation was decreased by 40% at both time points; and eNOS-Ser1177 phosphorylation was reduced by 38% and 51%, respectively. In HUVEC-WT, eNOS-Thr495 phosphorylation decreased after insulin stimulation. In contrast, in HUVEC-G972R, eNOS-Thr495 phosphorylation increased after insulin stimulation and was 40% greater than in HUVEC-WT. Conclusions—Our data demonstrate that genetic impairment of the (IRS)-1/PI3-K/PDK-1/Akt insulin signaling cascade determines impaired insulin-stimulated NO release and suggest that the G972R-IRS-1 polymorphism, through a direct impairment of Akt/eNOS activation in endothelial cells, may contribute to the genetic predisposition to develop endothelial dysfunction and cardiovascular disease.

Acute hyperglycemia and acute hyperinsulinemia decrease plasma fibrinolytic activity and increase plasminogen activator inhibitor type 1 in the rat

Abstract Decreased plasma fibrinolysis may contribute to accelerated atherothrombosis in diabetes. To observe whether hyperglycemia and hyperinsulinemia, common findings in type 2 diabetes, acutely affect plasma fibrinolysis in vivo, we evaluated plasma fibrinolysis (lysis of fibrin plates, free PAI-1 activity and t-PA activity) in the rat after a hyperglycemic euinsulinemic clamp (n=8), an euglycemic hyperinsulinemic clamp (n=7) or a saline infusion (n=15). Plasma fibrinolytic activity was sharply reduced after both the hyperglycemic and hyperinsulinemic clamps as compared to the respective controls (mean lysis areas on the fibrin plate, 139±21 vs. 323±30 mm2, p<0.001; 78±27 vs. 312±27 mm2p<0.001, respectively). Plasma PAI-1 activity was greater after both hyperglycemic and hyperinsulinemic clamps as compared to saline infusion (6.6±2.6 vs. 1.6±0.6 IU/ml, p<0.001; 26±4 vs. 1.3±0.7 IU/ml, p<0.0001, respectively). Plasma t-PA activity was significantly reduced both after the hyperglycemic (0.36±0.15 vs. 2.17±0.18 IU/ml in controls, p<0.001) and the hyperinsulinemic (0.3±0.1 vs. 2.3±0.3 IU/ml in control, p<0.001) clamps. These data show that in vivo both acute hyperglycemia and acute hyperinsulinemia can decrease plasma fibrinolytic potential and that this is due to increased plasma PAI-1 and decreased free t-PA activities.

Plasma exosome microRNA profiling unravels a new potential modulator of adiponectin pathway in diabetes: effect of glycemic control.

CONTEXT Type 2 diabetes is a chronic disease characterized by inadequate β-cell response to the progressive insulin resistance. MicroRNAs (miRNAs) are short, endogenous, noncoding RNAs representing a class of powerful gene expression modulators. Previous population studies observed a modulation of circulating miRNAs in diabetic patients; however, few data are presently available on miRNA modulation in diabetic patients naïve to pharmacological treatment as well as the effect of glycemic control on this. OBJECTIVE We aimed at studying circulating miRNA expression in diabetic patients naïve to treatment and at investigating the influence on this of glycemic control. DESIGN This was a case-control study. PARTICIPANTS Eighteen treatment-naïve diabetic patients with poor metabolic control and 12 control patients participated in the study. MAIN OUTCOME MEASURES Wide miRNA expression profiling was performed, and the expression of miRNAs found to be dysregulated was then validated by quantitative RT-PCR. Finally, algorithm-identified putative miRNA targets were evaluated by quantitative RT-PCR and ELISA. RESULTS In diabetic patients, microarray analysis showed that four miRNAs are increased, whereas 21 miRNAs are decreased. Quantitative RT-PCR validation confirmed the significant up-regulation of miR-326 (P = .004) and down-regulation of let-7a (P < .001) and let-7f (P = .003). Notably, an inverse negative correlation was found between circulating miR-326 and its putative target adiponectin (p = -0.479, P = .009). After 12 months of antidiabetic treatment, quantitative RT-PCR data analysis showed that miR-326 levels were unaffected, whereas the levels of let-7a and let-7f were significantly increased. CONCLUSIONS Treatment-naïve, poorly controlled diabetic patients show a significant dysregulation of miRNAs involved in the regulation of the adiponectin pathway, a phenomenon that may be reversed, at least in part, by improved glycemic control.

Glucose and insulin independently reduce the fibrinolytic potential of human vascular smooth muscle cells in culture

Summary Hyperglycaemia and hyperinsulinaemia have both been related to accelerated atherosclerosis in non-insulin-dependent diabetes mellitus (NIDDM). Plasma fibrinolytic potential is reduced in NIDDM and it is known that glucose and insulin can modulate plasminogen activator inhibitor (PAI-1) and tissue-plasminogen activator (t-PA) secretion and can therefore regulate local fibrinolysis. Vascular smooth muscle cells (vSMC) play an important role in the development of atherosclerotic lesions; however, the role of insulin and glucose in regulating PAI-1 and t-PA production in vSMC is presently not known. Therefore, we cultured arterial vSMC explanted from human umbilical cords and exposed them to increasing concentrations of glucose (5, 12, 20, 27, 35 mmol/l) or insulin (0.1, 0.5, 1, 10 nmol/l) in a serum free medium. After 24 h, PAI-1 and t-PA antigens and activity were evaluated in the culture medium; in cells exposed to 20 mmol/l glucose and to 0.5 nmol/l insulin PAI-1 gene expression was also evaluated. An increase in PAI-1 antigen was observed at each glucose concentration (by 138, 169, 251 and 357 % as compared to 5 mmol/l glucose) which was paralleled by an increase in PAI-1 activity. t-PA concentration was also increased by glucose but its activity was sharply reduced. An increase in PAI-1 antigen was detected at each insulin level (by 121, 128, 156 and 300 % as compared to no insulin). PAI-1 activity was slightly increased at the lowest insulin concentrations but markedly increased by 10 nmol/l insulin. t-PA antigen was also increased by insulin; however, its activity was markedly reduced at each concentration. As compared to control cells, PAI-1 mRNA was increased by 2.5 and 2.0 fold by 20 mmol/l glucose and 0.5 nmol/l insulin, respectively. We conclude that in human vSMC both glucose and insulin can affect the fibrinolytic balance so as to reduce fibrinolytic potential. This might contribute to decreased local fibrinolysis and thereby might accelerate the atherothrombotic process in NIDDM subjects. [Diabetologia (1996) 39: 1425–1431]

Phenotype modulation in cultures of vascular smooth muscle cells from diabetic rats: Association with increased nitric oxide synthase expression and superoxide anion generation

Proliferative modification of vascular smooth muscle cell (vSMC) and impaired bioavailability of nitric oxide (NO) have both been proposed among the mechanisms linking diabetes and atherosclerosis. However, diabetes induced modifications in phenotype and nitric oxide synthase(s) (NOS) expression and activity in vSMC have not been fully characterized. In this study, cell morphology, proliferative response to serum, alpha‐SMactin levels, eNOS expression and activity, cGMP intracellular content, and superoxide anion release were measured in cultures of vSMC obtained from aorta medial layer of ten diabetic (90% pancreatectomy, DR) and ten control (sham surgery, CR) rats. Vascular SMC from DR showed a less evident “hill and valley” culture morphology, increased growth response to serum, greater saturation density, and lower levels of α‐SMactin. In the same cells, as compared to CR cells, eNOS mRNA levels and NOS activity were increased, while intracellular cGMP level was lower and superoxide anion production was significantly greater. These data indicate that chronic hyperglycemia might induce, in the vascular wall, an increased number of vSMC proliferative clones which persist in culture and are associated with increased eNOS expression and activity. However, upregulation of eNOS and increased NO synthesis occur in the presence of a marked concomitant increase of O2− production. Since NO bioavailabilty, as reflected by cGMP levels, was not increased in DR cells, it is tempting to hypothesize that the proliferative phenotype observed in DR cells is associated with a redox imbalance responsible quenching and/or trapping of NO, with the consequent loss of its biological activity. J. Cell. Physiol. 196: 378–385, 2003.

Decreased in vivo oxidative stress and decreased platelet activation following metformin treatment in newly diagnosed type 2 diabetic subjects.

In type 2 diabetes, metformin reduces cardiovascular risk beyond the effect of glycaemic control. Since oxidative stress and the consequent enhanced platelet activation contribute to accelerated atherosclerosis in diabetes, we hypothesized that metformin could reduce oxidative stress in this condition.