The normal action of insulin to vasodilate and redistribute blood flow in support of skeletal muscle metabolism is impaired in insulin-resistant states. Increased endogenous endothelin contributes to endothelial dysfunction in obesity and diabetes. Here, we test the hypothesis that increased endogenous endothelin action also contributes to skeletal muscle insulin resistance via impairments in insulin-stimulated vasodilation. We studied nine lean and seven obese humans, measuring the metabolic and hemodynamic effects of insulin (300 mU · m−2 · min−1) alone and during femoral artery infusion of BQ123 (an antagonist of type A endothelin receptors, 1 μmol/min). Endothelin antagonism augmented skeletal muscle responses to insulin in obese subjects through changes in both leg blood flow (LBF) and glucose extraction. Insulin-stimulated LBF was significantly increased in obese subjects only. These changes, combined with differential effects on glucose extraction, resulted in augmented insulin-stimulated leg glucose uptake in obese subjects (54.7 ± 5.7 vs. 107.4 ± 18.9 mg/min with BQ123), with no change in lean subjects (103.7 ± 11.4 vs. 88.9 ± 16.3, P = 0.04 comparing BQ123 across groups). BQ123 allowed augmented leg glucose extraction in obese subjects even in the face of NOS antagonism. These findings suggest that increased endogenous endothelin action contributes to insulin resistance in skeletal muscle of obese humans, likely through both vascular and tissue effects.

Increased circulating levels of endothelin have been described in a number of insulin-resistant states, including type 2 diabetes (1,2), relatives of subjects with type 2 diabetes (1), obesity (35), and polycystic ovarian syndrome (6). Recent work from our laboratory and others has shown that increased endogenous endothelin activity in subjects with obesity and diabetes contributes to the impaired endothelium-dependent vasodilation that characterizes these states (7,8).

We know that a portion of the net metabolic actions of insulin depends on flow redistribution (911), which insulin regulates by stimulating production of nitric oxide (NO) in the vasculature (12,13). On this basis alone, one could postulate that endothelin acts to impair insulin action by antagonizing this vascular component of insulin action. Many lines of evidence suggest that endothelin may contribute to insulin resistance and vascular dysfunction. In vitro, sustained exposure of insulin-responsive cells to endothelin 1 (ET-1) induces insulin resistance (1416). Molecular studies have shown that this effect is mediated by effects of endothelin on a number of components of the insulin signaling cascade, including heterologous desensitization of insulin signaling by a Gαq/11-mediated pathway (14), impaired stimulation of phosphoinositol-3 kinase by insulin receptor substrate (IRS)-1 and IRS-2 (14,17), and impairments in downstream plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and actin cytoskeleton interactions (18).

Endothelin induces insulin resistance in isolated rat soleus muscle strips (16), and studies of whole organisms have found similar effects of exogenous endothelin. In normal conscious rats, bolus intraperitoneal injection of ET-1 raises endogenous glucose and insulin levels and reduces whole-body responses to infused insulin (19). Sustained exposure to ET-1 (delivered continuously by osmotic minipump for 5 days) induces whole-body insulin resistance and was associated with reduced skeletal muscle glucose uptake with evidence for impaired insulin signaling in muscle (16). In humans, ET-1 infusion induces insulin resistance as measured by hyperinsulinemic-euglycemic clamp (20,21).

The actions of exogenous endothelin on metabolism have not been exclusive to skeletal muscle beds. Effects of ET-1 on splanchnic glucose production independent of glucagon or insulin appear to be a component of the effects of endothelin on insulin sensitivity (22), and using the minimal model approach, one group found that ET-1 infusion produced impairment in the acute pancreatic response to glucose rather than inducing peripheral insulin resistance (23). How these observations relate to the circumstance of endogenous states of insulin resistance is unknown.

The metabolic effects of endothelin antagonists have begun to be evaluated in animal models of insulin resistance. In the Goto-Kakizaki rat, a model of type 2 diabetes, antagonism of the ET(A) receptor improved glucose metabolism sufficiently to significantly reduce hyperglycemia (24). Bosentan, a mixed antagonist of type A and B endothelin receptors, was found to improve insulin action in mouse models of physiological stress and insulin deficiency (25). Atrastentan, an ET(A) receptor selective agent, improved fasting and meal-stimulated insulin levels in insulin-resistant Zucker fatty rats (26). No studies have been published of effects of endothelin antagonists on insulin sensitivity in human subjects with endogenous insulin resistance. We have therefore undertaken studies of the effects of endothelin antagonism on insulin-stimulated whole-body and skeletal muscle glucose uptake in lean and obese/insulin-resistant humans.

Subjects were recruited through newspaper advertisement and classified as either lean or obese according to BMI cut points of ≥26 kg/m2 for men or ≥28 kg/m2 for women. Exclusion criteria included hypertension (systolic blood pressure >140 mmHg diastolic blood pressure >90 mmHg) or antihypertensive therapy, elevated serum lipids (total cholesterol >5.2 mmol/l, LDL >2.3 mmol/l, or triglycerides >2.0 mmol/l), biochemical evidence of renal or hepatic dysfunction, and significant underlying medical conditions. The use of a peroxisome proliferator–activated receptor γ agonist within the previous 6 months, prior evidence of retinopathy, neuropathy, and nephropathy, was exclusion criteria. All subjects underwent a standard 75-g oral glucose tolerance test to screen for diabetes and had body composition assessed by dual-energy X-ray absorptiometry measurement. This study was approved by the local institutional review board, and all subjects gave written informed consent. All procedures were performed in accordance with institutional guidelines.

Technique.

All studies were performed after an overnight fast. A 6F sheath (Cordis, Miami, FL) was placed into the right femoral vein to allow the insertion of a custom-designed 5F double-lumen thermodilution catheter (Baxter Scientific, Edwards Division, Irvine, CA) to measure leg blood flow (LBF). The right femoral artery was cannulated with a 5.5F double-lumen catheter to allow simultaneous infusion of substances and invasive blood pressure monitoring via a vital signs monitor (Spacelabs, Redmond, WA). All hemodynamic measurements were obtained with the subjects in the supine position in a quiet temperature-controlled room. Basal LBF and mean arterial pressure (MAP) measurements were obtained after ≥30 min of rest after the insertion of the catheters. Femoral vein thermodilution curves were used to measure rates of LBF, calculated by integration of the area under the curve, using a cardiac output computer (model 9520A; American Edwards Laboratories). Initially, 24 basal LBF measurements were obtained at ∼30-s intervals. During subsequent drug infusions, the mean of 10 measurements was taken. Invasively determined MAP was recorded with every other LBF determination.

Protocol.

Vascular responses were measured as changes in LBF as above, 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 BQ123 (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 using an intrafemoral arterial infusion rate of 0.6 mg/min (1 μmol/min) (7), and this rate was used in the current study protocol. NG-monomethyl-l-arginine (l-NMMA) (Clinalfa, Basel, Switzerland), a competitive antagonist of l-arginine, was infused at 16 mg/min into the femoral artery to block NO generation by NO synthase. This is the standard infusion rate used in our laboratory (8,27), chosen on the basis that it provides near-maximal effect across all populations. Hyperinsulinemic-euglycemic insulin clamps were performed using a high insulin exposure (300 mU · m−2 · min−1 administered systemically via an antecubital vein) to provide an unequivocal stimulus to vascular and tissue insulin actions in both lean and obese/insulin-resistant subjects.

The protocol is presented diagrammatically in Fig. 1. Subjects were studied in random sequence on two occasions, receiving insulin alone on one occasion and insulin with concurrent BQ123 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 co-infusion of l-NMMA.

Laboratory.

Blood for serum glucose determinations was put in untreated polypropylene tubes and centrifuged using an Eppendorf microcentrifuge (Brinkman, Westbury, NY). The glucose concentration of the supernatant was then measured by the glucose oxidase method using a glucose analyzer (Model 2300; Yellow Springs Instruments, Yellow Springs, OH). Blood for determination of plasma insulin was collected in heparinized tubes, processed immediately, and frozen at −20°C. Insulin determinations were made using a dual-site radioimmune assay, specific for human insulin and with cross-reactivity with proinsulin <0.2%. The lower detection limit is 0.56 pmol/l, and in our laboratory, the inter- and intra-assay coefficients of variation are 4.1 and 2.6%, respectively. Total serum nitrate (NOx) was determined by chemiluminescence (NO analyzer; Sievers, Denver, CO) after a Greiss reaction to oxidize NO and NO2 to NO3. NOx flux was calculated as venous NOx times LBF and used as an index of net NO production. Endothelin levels were determined by ELISA (R&D Systems, Minneapolis, MN). Standard methodologies for cholesterol and triglyceride determinations were performed through our local hospital’s clinical laboratory.

Statistical analysis.

Data that were not normally distributed were normalized through logarithmic transformations before analysis. Comparisons between and within groups were performed by t tests, ANOVA, and repeated-measures ANOVA as appropriate. Statistical significance was accepted at a level of P < 0.05. Population descriptive statistics are presented as means ± SD; otherwise, results are presented as means ± SE.

The subject characteristics are presented in Table 1. We studied nine lean and seven obese subjects. As expected, obese subjects had higher BMI, waist circumference, and insulin levels. Obese subjects had marginally higher glucose and triglyceride levels, not statistically different from lean subjects. One obese subject was found to be diabetic on oral glucose tolerance testing (fasting glucose 5.0 mmol/l, 2-h glucose value of 12.8 mmol/l). The results presented below include this subject; analyses excluding this subject were not materially different. No differences across groups in the lipid profiles were seen.

Whole-body glucose uptake.

The whole-body response of the obese subjects to insulin was reduced compared with the lean subjects, as expected (Fig. 2; P < 0.001). Concurrent exposure to BQ123 produced a nonsignificant increase in whole-body glucose uptake in obese subjects, with no such increase seen in lean subjects (Fig. 2; means ± SE, 11 ± 13% change in whole-body glucose uptake in obese subjects and −4 ± 9% change in lean subjects). This response was not significantly different across treatment groups. Expressing whole-body glucose uptake per kilogram lean body mass did not materially change this observation (lean, 10.2 ± 0.67 mg · kg−1 fat free mass · min−1 without BQ123, 10.6 ± 0.9 with BQ123; obese, 5.3 ± 0.4 without BQ123, 5.8 ± 0.2 with BQ123), and again no effect of BQ123 to modulate this response was evident in either group.

Leg glucose uptake.

Leg glucose uptake was calculated using the Fick equation as LBF multiplied by the arteriovenous (AV) glucose difference (glucose extraction). Changes in these inter-related parameters are presented in Figs. 35. Increases in LBF in response to insulin were seen in both subject groups, but as expected, this response was reduced among obese subjects (Fig. 3, P < 0.001 comparing groups). Concurrent exposure to BQ123 augmented the leg vascular response to insulin in obese subjects, correcting the deficit in LBF response (Fig. 3, left, P = 0.03 comparing change in LBF insulin vs. insulin plus BQ123); this effect of BQ123 was not statistically greater in obese than lean subjects (P = 0.07 by repeated-measures ANOVA). As expected, given that both insulin and BQ123 are vasodilators, blood pressure dropped in response to both treatments, with a greater effect seen with combined insulin and BQ123 (change in MAP: insulin, −5.9 ± 1.6 mmHg lean and −7.5 ± 2.3 obese; insulin plus BQ123, −9.1 ± 1.6 lean and −14.1 ± 6.9 obese; P = 0.01 comparing insulin vs. insulin plus BQ123, NS comparing the effect across groups). Expressing the vascular response as leg vascular conductance (LVC) to adjust for these blood pressure effects again revealed significant differences in the vascular response with BQ123 (change in LVC: insulin, 1.1 ± 0.3 units lean and 0.5 ± 0.2 obese; insulin plus BQ123, 1.6 ± 0.5 lean and 2.1 ± 0.5 obese; P = 0.03 for BQ123 effect, NS across groups).

Glucose extraction, reflected in the AV glucose difference, was modestly increased in obese subjects during concurrent BQ123 exposure compared with insulin alone, although this change did not reach statistical significance (Fig. 4; P = 0.13). In lean subjects, a modest, nonsignificant reduction in extraction was seen. By repeated-measures ANOVA, this differential response across groups was statistically significant (P = 0.03).

Combined, these effects of BQ123 produced a marked increase in insulin-stimulated leg glucose uptake in obese but not lean subjects (Fig. 5; P = 0.04 comparing BQ123 effect across groups). This amounted to a 112 ± 41% increase in leg glucose uptake in obese subjects. This increase was achieved through effects of BQ123 to augment both insulin-stimulated LBF and insulin-stimulated glucose uptake.

Effects on endothelin.

There were no evident baseline differences in circulating levels of ET-1 between the groups (Table 1). We did not see an effect of insulin to augment systemic levels of ET-1 (steady-state levels 0.89 ± 0.38 and 0.89 ± 0.48 pg/ml in lean and obese subjects, respectively; NS vs. pretreatment levels and NS comparing groups). There was also no evident effect of insulin to augment ET-1 levels specifically in the femoral venous circulation, with or without concurrent BQ123 (not shown).

Effects on NO.

This protocol included two measures of NO responses. First, the change in leg vascular resistance after exposure to l-NMMA (a competitive antagonist of NO synthase) provides a measure of bioavailable NO (Fig. 3, bottom). Second, NOx flux was measured, providing an independent measure of total nitrate production by the leg. Under conditions of insulin stimulation alone, LBF fell significantly in both groups (P < 0.001), without a difference in this effect across groups. NOx flux similarly fell significantly (lean 5.3 ± 1.2 μmol/min steady state vs. 2.8 ± 0.6 l-NMMA; obese 5.6 ± 1.4 steady state vs. 3.0 ± 0.9 l-NMMA, P < 0.001), without a significant difference across groups in this response. Reductions in LBF with l-NMMA were also seen under conditions of insulin plus BQ123 (Fig. 3, bottom right). Under these conditions, the response among lean subjects was not different from insulin alone, but among obese subjects, we observed augmented vasoconstrictor responses to l-NMMA (P = 0.015 comparing effect of BQ123 on l-NMMA response across groups). The NOx responses were parallel, although less statistically powerful (lean 5.6 ± 1.4 steady state vs. 3.4 ± 0.6 l-NMMA; obese 8.9 ± 4.3 steady state vs. 5.0 ± 2.4 l-NMMA, P = 0.02 for l-NMMA effect, NS comparing effect across groups).

Metabolic response to l-NMMA.

The reductions in LBF with l-NMMA resulted in increased glucose extraction (i.e., an increased AV glucose difference), evident particularly among lean subjects (Fig. 4, P < 0.001 for all subjects, P = 0.02 comparing subject groups under conditions with and without l-NMMA). The addition of BQ123 produced a differential response across subject groups (P = 0.01), allowing an augmentation of extraction among obese subjects.

Combined with changes in LBF, these changes in glucose extraction produced markedly different leg glucose uptake responses to l-NMMA depending on the prior exposure to BQ123 (Fig. 5, right). Overall, insulin-stimulated leg glucose uptake was reduced by l-NMMA (P < 0.001, ∼33% reduction in lean subjects and ∼39% reduction in obese subjects). The prior addition of BQ123 resulted in higher steady-state leg glucose uptake in obese subjects, as noted above. From this shifted starting point, there was a reduction after the addition of l-NMMA, with a significant difference across groups (P < 0.001 for steady state vs. l-NMMA, P = 0.006 comparing this effect across groups). Nonetheless, the residual insulin-stimulated leg glucose uptake after l-NMMA treatment was augmented by BQ123 in obese subjects (P < 0.001 comparing left vs. right panels).

Using BQ123, an antagonist of the type A endothelin receptor, we have found a significant contribution of endogenous endothelin to insulin action in obese humans. Through effects to augment insulin-stimulated LBF and insulin-stimulated glucose extraction, a marked increase in leg glucose uptake in response to insulin was observed with endothelin antagonism. No such effect was seen in lean control subjects. Much smaller effects were seen at the level of whole-body glucose metabolism, not reaching statistical significance comparing across or within groups. Overall, these findings suggest that endogenous endothelin contributes to insulin resistance in human obesity/insulin resistance through combined vascular and nonvascular effects in skeletal muscle beds.

This observation places endothelin in a very select group of modulators of vascular tone which are able to also affect tissue metabolism. Vasoconstriction with inhibitors of NO synthase reliably reduces both flow and tissue glucose uptake as previously reported (27,28) and recapitulated here. Catecholamines fall into two camps, either stimulating or impairing metabolism (conceptually, activating either “nutritive” or “non-nutritive” patterns of tissue perfusion) (29,30). By corollary, nonspecific vasodilation is not sufficient in and of itself to augment metabolism (31,32). Even endothelium-dependent vasodilation alone is not sufficient to exert this effect. Rather, this appears to reflect agonist-specific metabolic responses, and until this report, only cholinergic vasodilator agonists and the vasoconstrictors noted above had been observed to enhance the effects of insulin through vascular actions (33,34). Therefore, our observations imply that the actions of endothelin in skeletal muscle vasculature are via mechanisms which support the actions of insulin to redistribute perfusion into nutritive blood flow patterns.

Endothelin and insulin resistance.

The first suggestion that endothelin interacted with insulin resistance came from observations that states of insulin resistance were associated with increased circulating levels of ET-1 polycystic ovarian syndrome (16). Also, improvements in insulin resistance through physiological or pharmacological means are associated with reductions in circulating endothelin (5,6,35,36).

Subsequent experimental studies have shown that the application of exogenous endothelin to intact skeletal muscle (16), intact rats (16,19), and humans can induce impairments in insulin-stimulated glucose uptake (20,21) through mechanisms that involve the type A endothelin receptor (21). However, effects at multiple levels beyond skeletal muscle have been described, including effects on splanchnic glucose production independent of glucagon or insulin (22), effects on hepatic and renal blood flow (37), and effects on islet function (38). These observations highlight the importance of the current observations, which clearly show a significant and relevant effect of endothelin action in skeletal muscle on insulin action.

The current results provide the first data demonstrating a contribution of endothelin to endogenous insulin resistance in humans. This observation is concordant with prior work demonstrating such an effect in the Goto-Kakizaki rat, a model of type 2 diabetes (24), fatty Zucker rats (26), and insulin-deficient rats (25).

It is not obvious that systemic infusions of endothelin appropriately mimic the increased endogenous endothelin activity that is seen in obesity and diabetes. The major source of endothelin is believed to be the endothelial cells lining the vessel wall, but this is a largely paracrine function with the majority of secreted endothelin acting in the underlying tissue rather than being released into the circulation. Therefore, although circulating levels are increased and likely exert actions at a distance, these actions a priori would be expected to represent a minority of the overall effects. To date, little has been done to clarify the balance of these actions in states of endogenous insulin resistance. The contribution of the current study to this discussion lies in the demonstration of a clear and important effect, including a significant vascular effect, within skeletal muscle. Definitive studies regarding the contributions of endothelin to the overall balance of metabolism, including in particular effects on splanchnic glucose metabolism, will be needed to better delineate this issue.

Vascular versus cellular effects of endothelin.

The current understanding of the mechanisms of action of insulin and cellular interactions of insulin with endothelin supports an expectation for both vascular and nonvascular effects of endothelin antagonism on insulin action. The discovery of endothelin as a vasoconstrictor agent produced by the endothelium prompted initial explorations of its role as a modulator of vascular function (39). The net tissue actions of insulin in skeletal muscle depend in part on the capacity of the vasculature to vasodilate in response to insulin (12,40,41), with this vasodilation actively redistributing blood flow in support of the increased metabolism (13). This vascular action of insulin is impaired in states of insulin resistance (9,42,43), and we and others have previously shown that increased endogenous endothelin contributes to vascular dysfunction in obesity and diabetes (7,8). Based on these observations, we had an a priori expectation that endothelin antagonism would improve insulin-stimulated vasodilation and therefore improve net insulin action in skeletal muscle. This was in fact seen in the current study.

A number of molecular studies have described interactions of endothelin and insulin at the level of insulin signaling. After ET-1 receptor binding, the ET(A) G protein-coupled receptor activates phospholipase Cβ. This, in turn, increases the formation of inositol triphosphate and diacylglycerol, leading to an increase in cytosolic Ca2+ and activation of protein kinase C (PKC) (44,45). Acute exposure of insulin-responsive cells to this endothelin stimulates GLUT4 translocation and glucose transport in the absence of insulin (46). Sustained exposure of insulin-responsive cells to ET-1, on the other hand, induces insulin resistance (14,16,17). In vascular smooth muscle cells and adipocytes, ET-1 causes insulin resistance by a PKC-dependent mechanism (14,17). Endothelin has also been found to impair insulin action through interactions with downstream signaling events at the plasma membrane involving PIP2 and actin cytoskeleton components of the insulin response (18), suggesting that ET-1 negatively impacts cytoskeletal structure important for insulin function. From this perspective, the question of effects of endothelin antagonism on glucose extraction as distinct from effects on blood flow is of interest. We observed an increase in insulin-stimulated glucose extraction with BQ123 in obese subjects. Given that impairing insulin-stimulated vasodilation with l-NMMA induced a 30–40% reduction in leg glucose uptake through effects on both blood flow and glucose extraction, concordant with prior reports of this effect (27,47), we cannot attribute the observed increase in glucose extraction to correction of the vascular response alone. Therefore we surmise that this observation reflects actions of BQ123 on the tissue (i.e., cellular) response to insulin over and above the vascular effects. Together, these effects provided an ∼100% increase in leg glucose uptake, not all of which was lost on blocking the NO-mediated vasodilation of insulin. Overall, we interpret these observations as evidence in support of both vascular and nonvascular effects of BQ123 to improve insulin action in human obesity/insulin resistance.

Endothelin and NO are mutually antagonistic at multiple subcellular levels (48,49). This system appears to function with NO and ET-1 as mutual antagonists (50). Under conditions of health, this can produce a tightly regulated balance between these major determinants of vascular tone (8,51,52), and we have recently reported that endothelin antagonism can augment bioavailable NO in obesity (8). In the present study, we performed two unrelated measures of the contribution of NO to the vascular response observed under insulin stimulation with and without endothelin antagonism. The vasoconstriction seen in response to NO synthase antagonism provides a measure of bioactive NO, and the direct measurement of NO flux provides a measure of NO production. Both parameters were increased under insulin stimulation, although the NOx values in particular were highly variable after exposure to BQ123. The changes in LBF are concordant with our prior observations that endothelin antagonism can allow augmented expression of bioavailable NO in obese subjects (8). Reductions in blood flow with l-NMMA were associated with augmented glucose extraction, more prominent among lean than obese subjects, with an effect of BQ123 to restore this effect in obese subjects (Fig. 4). Overall, the net reduction in glucose uptake after l-NMMA was of a similar magnitude to insulin-only conditions but in the setting of an increased starting point, such that the post–l-NMMA levels were significantly higher than without BQ123. These findings suggest that despite changes in NO bioavailability, an independent effect of endothelin antagonism to support or restore glucose metabolism is evident in obese subjects.

Limitations.

A high dose of insulin used was chosen to ensure that both insulin-mediated vasodilation and augmentation of tissue glucose uptake were evident in both subject groups. However, with this exposure, the responses seen in lean subjects are expected to be near maximal. This limits any possible additional effect of endothelin antagonism to augment the actions of insulin in the lean subjects. Therefore, although the current results provide convincing evidence for an enhancement of these actions of insulin in obese/insulin-resistant subjects, the lack of such an effect in the lean subjects does not exclude contributions of endothelin to insulin action in normal physiology as well.

The current studies use direct intrafemoral arterial exposure of the endothelin antagonist and provide clear effects within the directly perfused skeletal muscle bed. The observed systemic effects therefore reflect changes in whole-body metabolism due to these changes in the directly exposed muscle bed, plus other possible effects in other tissue beds that experience secondary systemic exposure. This balance of effects is not the same as that which would be achieved, for example, with an orally available endothelin antagonist. Therefore questions of insulin-sensitizing effects of systemically or orally delivered endothelin antagonists will need to be specifically investigated.

FIG. 1.

Study schema. Subjects were studied on two occasions in random sequence, receiving a systemic hyperinsulinemic-euglycemic clamp (300 mU · m−2 · min−1) alone or in combination with intrafemoral arterial ET(A) antagonism (BQ123, 1 μmol/min). The NO synthase antagonist l-NMMA was infused at the end of each study (16 mg/min). Arrows indicate stages where vascular and hemodynamic measurements were taken.

FIG. 1.

Study schema. Subjects were studied on two occasions in random sequence, receiving a systemic hyperinsulinemic-euglycemic clamp (300 mU · m−2 · min−1) alone or in combination with intrafemoral arterial ET(A) antagonism (BQ123, 1 μmol/min). The NO synthase antagonist l-NMMA was infused at the end of each study (16 mg/min). Arrows indicate stages where vascular and hemodynamic measurements were taken.

FIG. 2.

Whole-body metabolic response to endothelin antagonism. Changes within each group did not reach significance, and there was no difference across groups in the response to BQ123.

FIG. 2.

Whole-body metabolic response to endothelin antagonism. Changes within each group did not reach significance, and there was no difference across groups in the response to BQ123.

FIG. 3.

Modulation of LBF responses to insulin by endothelin antagonism. Insulin-stimulated increases in LBF were seen in both groups (A), with significant reduction in this response after the addition of l-NMMA (B). BQ123 augmented the vasodilator response to insulin in obese but not lean subjects (P = 0.03 for BQ effect by subject group, repeated-measures ANOVA). The reduction in LBF with l-NMMA was also augmented in obese but not lean subjects. For comparisons within each study, the response did not differ across subject groups except under insulin-stimulated conditions with BQ123, where the response to l-NMMA was greater in obese than lean subjects (P = 0.015).

FIG. 3.

Modulation of LBF responses to insulin by endothelin antagonism. Insulin-stimulated increases in LBF were seen in both groups (A), with significant reduction in this response after the addition of l-NMMA (B). BQ123 augmented the vasodilator response to insulin in obese but not lean subjects (P = 0.03 for BQ effect by subject group, repeated-measures ANOVA). The reduction in LBF with l-NMMA was also augmented in obese but not lean subjects. For comparisons within each study, the response did not differ across subject groups except under insulin-stimulated conditions with BQ123, where the response to l-NMMA was greater in obese than lean subjects (P = 0.015).

FIG. 4.

Modulation of AV glucose difference under insulin stimulation by endothelin antagonism. Statistics in the figure represent the interaction term for a difference in the observed response to BQ123 across subject groups by repeated-measures ANOVA. l-NMMA significantly changed this parameter compared with the preceding study stages (left vs. right panels, P < 0.001 by repeated-measures ANOVA), driven by a significant increase in AV difference in lean subjects (P = 0.002), with a significant difference in this response across subject groups (P = 0.017).

FIG. 4.

Modulation of AV glucose difference under insulin stimulation by endothelin antagonism. Statistics in the figure represent the interaction term for a difference in the observed response to BQ123 across subject groups by repeated-measures ANOVA. l-NMMA significantly changed this parameter compared with the preceding study stages (left vs. right panels, P < 0.001 by repeated-measures ANOVA), driven by a significant increase in AV difference in lean subjects (P = 0.002), with a significant difference in this response across subject groups (P = 0.017).

FIG. 5.

Modulation of insulin-stimulated leg glucose uptake by endothelin antagonism. Statistics in the figure represent the interaction term for a difference in the observed response to BQ123 across subject groups by repeated-measures ANOVA. The effect of BQ123 on leg glucose uptake was different across groups (left), with a significant increase seen in obese subjects but no significant change seen in lean subjects. The addition of l-NMMA resulted in a reduction in leg glucose uptake in both groups (P = 0.007 comparing left vs. right panels), but overall, obese subjects experienced an augmented post–l-NMMA leg glucose uptake under BQ123-treated conditions (P < 0.001).

FIG. 5.

Modulation of insulin-stimulated leg glucose uptake by endothelin antagonism. Statistics in the figure represent the interaction term for a difference in the observed response to BQ123 across subject groups by repeated-measures ANOVA. The effect of BQ123 on leg glucose uptake was different across groups (left), with a significant increase seen in obese subjects but no significant change seen in lean subjects. The addition of l-NMMA resulted in a reduction in leg glucose uptake in both groups (P = 0.007 comparing left vs. right panels), but overall, obese subjects experienced an augmented post–l-NMMA leg glucose uptake under BQ123-treated conditions (P < 0.001).

TABLE 1

Subject characteristics

LeanObese
n 
Age (years) 37 ± 7 33 ± 7 
Race (African American/Caucasian) 5/4 6/1 
BMI (kg/m221.8 ± 2.8 34.3 ± 4.8* 
Fat (%) 16.7 ± 5.6 35.7 ± 5.5* 
Waist (cm) 72.5 ± 2.9 92.7 ± 8.3* 
Glucose (mmol/l) 5.2 ± 1.4 6.0 ± 2.0 
Insulin (pmol/l) 48.2 ± 32.6 199.8 ± 238.9* 
ET-1 (pg/ml) 1.05 ± 0.50 1.01 ± 0.39 
Systolic blood pressure (mmHg) 93 ± 6 107 ± 35 
Diastolic blood pressure (mmHg) 72 ± 15 67 ± 7 
Cholesterol (mmol/l) 3.4 ± 0.7 3.5 ± 1.0 
LDL (mmol/l) 2.0 ± 0.5 2.5 ± 0.7 
HDL (mmol/l) 0.9 ± 0.1 1.0 ± 0.3 
Triglycerides (mmol/l) 1.1 ± 0.5 1.6 ± 1.0 
LeanObese
n 
Age (years) 37 ± 7 33 ± 7 
Race (African American/Caucasian) 5/4 6/1 
BMI (kg/m221.8 ± 2.8 34.3 ± 4.8* 
Fat (%) 16.7 ± 5.6 35.7 ± 5.5* 
Waist (cm) 72.5 ± 2.9 92.7 ± 8.3* 
Glucose (mmol/l) 5.2 ± 1.4 6.0 ± 2.0 
Insulin (pmol/l) 48.2 ± 32.6 199.8 ± 238.9* 
ET-1 (pg/ml) 1.05 ± 0.50 1.01 ± 0.39 
Systolic blood pressure (mmHg) 93 ± 6 107 ± 35 
Diastolic blood pressure (mmHg) 72 ± 15 67 ± 7 
Cholesterol (mmol/l) 3.4 ± 0.7 3.5 ± 1.0 
LDL (mmol/l) 2.0 ± 0.5 2.5 ± 0.7 
HDL (mmol/l) 0.9 ± 0.1 1.0 ± 0.3 
Triglycerides (mmol/l) 1.1 ± 0.5 1.6 ± 1.0 

Data are means ± SD.

*

P < 0.05 across groups.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

K.J.M. was supported in part by an American Diabetes Association Junior Faculty Award. This work was supported by the Indiana University General Clinical Research Center (National Institutes of Health Grant M01-RR-00750) and directly by the National Institutes of Health (DK-42469).

The expert technical assistance and hyperinsulinemic clamp expertise of Paula Robinett, RN, is gratefully acknowledged.

1.
Caballero AE, Arora S, Saouaf R, Lim SC, Smakowski P, Park JY, King GL, LoGerfo FW, Horton ES, Veves A: Microvascular and macrovascular reactivity is reduced in subjects at risk for type 2 diabetes.
Diabetes
48
:
1856
–1862,
1999
2.
Mangiafico RA, Malatino LS, Santonocito M, Spada RS: Plasma endothelin-1 concentrations in non-insulin-dependent diabetes mellitus and nondiabetic patients with chronic arterial obstructive disease of the lower limbs.
Int Angiol
17
:
97
–102,
1998
3.
Irving RJ, Noon JP, Watt GC, Webb DJ, Walker BR: Activation of the endothelin system in insulin resistance.
QJM
94
:
321
–326,
2001
4.
Piatti P, Fragasso G, Monti LD, Caumo A, Van Phan C, Valsecchi G, Costa S, Fochesato E, Pozza G, Pontiroli AE, Chierchia S: Endothelial and metabolic characteristics of patients with angina and angiographically normal coronary arteries: comparison with subjects with insulin resistance syndrome and normal controls.
J Am Coll Cardiol
34
:
1452
–1460,
1999
5.
Ferri C, Bellini C, Desideri G, Baldoncini R, Properzi G, Santucci A, De Mattia G: Circulating endothelin-1 levels in obese patients with the metabolic syndrome.
Exp Clin Endocrinol Diabetes
105
:
38
–40,
1997
6.
Diamanti-Kandarakis E, Spina G, Kouli C, Migdalis I: Increased endothelin-1 levels in women with polycystic ovary syndrome and the beneficial effect of metformin therapy.
J Clin Endocrinol Metab
86
:
4666
–4673,
2001
7.
Mather K, Mirzamohammadi B, Lteif A, Steinberg H, Baron A: Endothelin contributes to basal vascular tone and endothelial dysfunction in human obesity and type 2 diabetes mellitus.
Diabetes
51
:
3517
–3523,
2002
8.
Mather KJ, Lteif A, Steinberg HO, Baron AD: Interactions between endothelin and nitric oxide in the regulation of vascular tone in obesity and diabetes.
Diabetes
53
:
2060
–2066,
2004
9.
Baron AD, Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G: Insulin-mediated skeletal muscle vasodilation contributes to both insulin sensitivity and responsiveness in lean humans.
J Clin Invest
96
:
786
–792,
1995
10.
Vincent MA, Dawson D, Clark AD, Lindner JR, Rattigan S, Clark MG, Barrett EJ: Skeletal muscle microvascular recruitment by physiological hyperinsulinemia precedes increases in total blood flow.
Diabetes
51
:
42
–48,
2002
11.
Coggins M, Lindner J, Rattigan S, Jahn L, Fasy E, Kaul S, Barrett E: Physiologic hyperinsulinemia enhances human skeletal muscle perfusion by capillary recruitment.
Diabetes
50
:
2682
–2690,
2001
12.
Steinberg HO, Brechtel G, Johnson A, Fineberg N, Baron AD: Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent: a novel action of insulin to increase nitric oxide release.
J Clin Invest
94
:
1172
–1179,
1994
13.
Vincent MA, Barrett EJ, Lindner JR, Clark MG, Rattigan S: Inhibiting NOS blocks microvascular recruitment and blunts muscle glucose uptake in response to insulin.
Am J Physiol Endocrinol Metab
285
:
E123
–E129,
2003
14.
Ishibashi KI, Imamura T, Sharma PM, Huang J, Ugi S, Olefsky JM: Chronic endothelin-1 treatment leads to heterologous desensitization of insulin signaling in 3T3–L1 adipocytes.
J Clin Invest
107
:
1193
–1202,
2001
15.
Jiang ZY, Lin YW, Clemont A, Feener EP, Hein KD, Igarashi M, Yamauchi T, White MF, King GL: Characterization of selective resistance to insulin signaling in the vasculature of obese Zucker (fa/fa) rats.
J Clin Invest
104
:
447
–457,
1999
16.
Wilkes JJ, Hevener A, Olefsky J: Chronic endothelin-1 treatment leads to insulin resistance in vivo.
Diabetes
52
:
1904
–1909,
2003
17.
Jiang ZY, Zhou QL, Chatterjee A, Feener EP, Myers MG Jr, White MF, King GL: Endothelin-1 modulates insulin signaling through phosphatidylinositol 3-kinase pathway in vascular smooth muscle cells.
Diabetes
48
:
1120
–1130,
1999
18.
Strawbridge AB, Elmendorf JS: Phosphatidylinositol 4,5-bisphosphate reverses endothelin-1-induced insulin resistance via an actin-dependent mechanism.
Diabetes
54
:
1698
–1705,
2005
19.
Juan CC, Fang VS, Huang YJ, Kwok CF, Hsu YP, Ho LT: Endothelin-1 induces insulin resistance in conscious rats.
Biochem Biophys Res Commun
227
:
694
–699,
1996
20.
Ottosson-Seeberger A, Lundberg JM, Alvestrand A, Ahlborg G: Exogenous endothelin-1 causes peripheral insulin resistance in healthy humans.
Acta Physiol Scand
161
:
211
–220,
1997
21.
Ahlborg G, Lindstrom J: Insulin sensitivity and big ET-1 conversion to ET-1 after ETA- or ETB-receptor blockade in humans.
J Appl Physiol
93
:
2112
–2121,
2002
22.
Ahlborg G, Ottosson-Seeberger A, Hemsen A, Lundberg JM: Big ET-1 infusion in man causes renal ET-1 release, renal and splanchnic vasoconstriction, and increased mean arterial blood pressure.
Cardiovasc Res
28
:
1559
–1563,
1994
23.
Teuscher AU, Lerch M, Shaw S, Pacini G, Ferrari P, Weidmann P: Endothelin-1 infusion inhibits plasma insulin responsiveness in normal men.
J Hypertens
16
:
1279
–1284,
1998
24.
Balsiger B, Rickenbacher A, Boden PJ, Biecker E, Tsui J, Dashwood M, Reichen J, Shaw SG: Endothelin A-receptor blockade in experimental diabetes improves glucose balance and gastrointestinal function.
Clin Sci (Lond)
103 (Suppl. 48)
:
430S
–433S,
2002
25.
Said SA, Ammar el SM, Suddek GM: Effect of bosentan (ET(A)/ET(B) receptor antagonist) on metabolic changes during stress and diabetes.
Pharmacol Res
51
:
107
–115,
2005
26.
Berthiaume N, Wessale JL, Opgenorth TJ, Zinker BA: Metabolic responses with endothelin antagonism in a model of insulin resistance.
Metabolism
54
:
735
–740,
2005
27.
Baron AD, Zhu JS, Marshall S, Irsula O, Brechtel G, Keech C: Insulin resistance after hypertension induced by the nitric oxide synthesis inhibitor L-NMMA in rats.
Am J Physiol
269
:
E709
–E715,
1995
28.
Kapur S, Bedard S, Marcotte B, Cote CH, Marette A: Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action.
Diabetes
46
:
1691
–1700,
1997
29.
Rattigan S, Dora KA, Colquhoun EQ, Clark MG: Inhibition of insulin-mediated glucose uptake in rat hindlimb by an alpha-adrenergic vascular effect.
Am J Physiol
268
:
E305
–E311,
1995
30.
Clark AD, Youd JM, Rattigan S, Barrett EJ, Clark MG: Heterogeneity of laser Doppler flowmetry in perfused muscle indicative of nutritive and nonnutritive flow.
Am J Physiol Heart Circ Physiol
280
:
H1324
–H1333,
2001
31.
Natali A, Quinones GA, Pecori N, Sanna G, Toschi E, Ferrannini E: Vasodilation with sodium nitroprusside does not improve insulin action in essential hypertension.
Hypertension
31
:
632
–636,
1998
32.
Meneilly GS, Battistini B, Floras JS: Lack of effect of sodium nitroprusside on insulin-mediated blood flow and glucose disposal in the elderly.
Metabolism
49
:
373
–378,
2000
33.
Mahajan H, Richards SM, Rattigan S, Clark MG: Local methacholine but not bradykinin potentiates insulin-mediated glucose uptake in muscle in vivo by augmenting capillary recruitment.
Diabetologia
47
:
2226
–2234,
2004
34.
Baron AD, Tarshoby M, Hook G, Lazaridis EN, Cronin J, Johnson A, Steinberg HO: Interaction between insulin sensitivity and muscle perfusion on glucose uptake in human skeletal muscle: evidence for capillary recruitment.
Diabetes
49
:
768
–774,
2000
35.
Pontiroli AE, Pizzocri P, Koprivec D, Vedani P, Marchi M, Arcelloni C, Paroni R, Esposito K, Giugliano D: Body weight and glucose metabolism have a different effect on circulating levels of ICAM-1, E-selectin, and endothelin-1 in humans.
Eur J Endocrinol
150
:
195
–200,
2004
36.
Lavrencic A, Salobir BG, Keber I: Physical training improves flow-mediated dilation in patients with the polymetabolic syndrome.
Arterioscler Thromb Vasc Biol
20
:
551
–555,
2000
37.
Ahlborg G, Weitzberg E, Lundberg JM: Circulating endothelin-1 reduces splanchnic and renal blood flow and splanchnic glucose production in humans.
J Appl Physiol
79
:
141
–145,
1995
38.
De Carlo E, Milanesi A, Martini C, Maffei P, Sicolo N, Scandellari C: Endothelin-1 and endothelin-3 stimulate insulin release by isolated rat pancreatic islets.
J Endocrinol Invest
23
:
240
–245,
2000
39.
Haynes WG, Ferro CJ, O’Kane KPJ, Somerville D, Lomax CC, Webb DJ: Systemic endothelin receptor blockade decreases peripheral vascular resistance and blood pressure in humans.
Circulation
93
:
1860
–1870,
1996
40.
Vincent MA, Montagnani M, Quon MJ: Molecular and physiologic actions of insulin related to production of nitric oxide in vascular endothelium.
Curr Diab Rep
3
:
279
–288,
2003
41.
Scherrer U, Randin D, Vollenweider P, Vollenweider L, Nicod P: Nitric oxide release accounts for insulin’s vascular effects in humans.
J Clin Invest
94
:
2511
–2515,
1994
42.
Cleland SJ, Petrie JR, Shinichiro U, Elliott HL, Connell JMC: Insulin-mediated vasodilation and glucose uptake are functionally linked in humans.
Hypertension
33
:
554
–558,
1999
43.
Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G, Baron AD: Obesity/insulin resistance is associated with endothelial dysfunction: implications for the syndrome of insulin resistance.
J Clin Invest
97
:
2601
–2610,
1996
44.
Inoue A, Yanagisawa M, Takuwa Y, Mitsui Y, Kobayashi M, Masaki T: The human preproendothelin-1 gene: complete nucleotide sequence and regulation of expression.
J Biol Chem
264
:
14954
–14959,
1989
45.
Opgenorth TJ: Endothelin receptor antagonism.
Adv Pharmacol
33
:
1
–65,
1995
46.
Imamura T, Ishibashi K, Dalle S, Ugi S, Olefsky JM: Endothelin-1-induced GLUT4 translocation is mediated via Galpha(q/11) protein and phosphatidylinositol 3-kinase in 3T3–L1 adipocytes.
J Biol Chem
274
:
33691
–33695,
1999
47.
Baron AD, Brechtel-Hook G, Johnson A, Cronin J, Leaming R, Steinberg HO: Effect of perfusion rate on the time course of insulin-mediated skeletal muscle glucose uptake.
Am J Physiol
271
:
E1067
–E1072,
1996
48.
Flowers MA, Wang Y, Stewart RJ, Patel B, Marsden PA: Reciprocal regulation of endothelin-1 and endothelial constitutive NOS in proliferating endothelial cells.
Am J Physiol
269
:
H1988
–H1997,
1995
49.
Goligorsky MS, Tsukahara H, Magazine H, Andersen TT, Malik AB, Bahou WF: Termination of endothelin signaling: role of nitric oxide.
J Cell Physiol
158
:
485
–494,
1994
50.
Levin ER: Endothelins as cardiovascular peptides.
Am J Nephrol
16
:
246
–251,
1996
51.
Ahlborg G, Lundberg JM: Nitric oxide-endothelin-1 interaction in humans.
J Appl Physiol
82
:
1593
–1600,
1997
52.
Cardillo C, Kilcoyne CM, Cannon RO III, Panza JA: Interactions between nitric oxide and endothelin in the regulation of vascular tone of human resistance vessels in vivo.
Hypertension
35
:
1237
–1241,
2000