During subsequent drug infusions, the mean of 10 measurements was taken at each study stage. Invasively determined MAP was recorded with every other LBF determination. Protocol. Vascular responses were measured as changes in LBF as above, Wortmannin PI3K inhibitor with vasoactive agents administered via the ipsilateral femoral artery. Continuous bedside monitoring of heart rate and intra-arterial blood pressure allowed full hemodynamic assessment. ET-1 blockade was achieved with BQ-123 (Clinalfa, Basel, Switzerland), a high-affinity competitive inhibitor of ET-1 type A receptors. We have previously demonstrated increased endogenous endothelin activity in obese and diabetic subjects compared with lean subjects using an intrafemoral arterial infusion rate of 0.6 mg/min (1 ��mol/min) (29), and this rate was used in the current study protocol.

NG-monomethyl-l-arginine (l-NMMA) (Clinalfa), a competitive antagonist of l-arginine, was infused at 16 mg/min in the femoral artery to block NO generation by nitric oxide synthase (NOS). This is the standard infusion rate used in our laboratory (4, 30), chosen on the basis that it provides near-maximal effect across all populations. Four-hour hyperinsulinemic-euglycemic glucose clamps were performed using a different insulin exposure for lean (10 mU?m?2?min?1) and obese (30 mU?m?2?min?1) subjects, by design imposing hyperinsulinemic conditions that would provide matched effects on skeletal muscle glucose metabolism and therefore on insulin-stimulated NO across the groups (28) but achieving different circulating insulin levels.

Insulin was administered systemically via an antecubital vein. The protocol is presented diagrammatically in Fig. 1. All studies were performed following an overnight fast. Female subjects with a regular menstrual cycle were studied in the menstrual phase (days 1�C7) of their cycle. Subjects were studied in random sequence on two occasions, receiving insulin alone on one occasion and insulin with concurrent BQ-123 on the other occasion. On each study day, basal vascular responses were measured before the initiation of the hyperinsulinemic-euglycemic clamp. Steady state was defined as the stage when glucose infusion rate changes of ��5% were required to maintain euglycemia, and was at least 210 min from the initiation of the clamp procedure. Further vascular measurements were made at this stage, followed by measurements during the coinfusion of l-NMMA.

Fig. 1. Study schema. Subjects were studied on 2 occasions in random sequence, receiving a systemic hyperinsulinemic-euglycemic clamp (300 mU?m?2?min?1) alone or in combination with intrafemoral arterial endothelin type A (ETA Entinostat … Laboratory. Blood for serum glucose determinations was put in untreated polypropylene tubes and centrifuged using an Eppendorf microcentrifuge (Brinkman, Westbury, NY).