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(Circulation. 1995;92:2911-2918.)
© 1995 American Heart Association, Inc.

Articles

Effect of Insulin on Acetylcholine-Induced Vasodilation in Normotensive Subjects and Patients With Essential Hypertension

Stefano Taddei, MD; Agostino Virdis, MD; Paola Mattei, MD; Andrea Natali, MD; Ele Ferrannini, MD; Antonio Salvetti, MD

From I Clinica Medica, University of Pisa, and the Metabolism Unit, CNR Institute of Clinical Physiology (A.N., E.F.), Pisa, Italy.

Correspondence to Dr Stefano Taddei, I Clinica Medica, University of Pisa, Via Roma 67, 56100 Pisa, Italy.

	Abstract

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Background The present study was designed to directly test the vasodilation action of insulin and its relation to endothelium-dependent mechanisms.

Methods and Results In 18 normotensive subjects and 27 patients with untreated mild to moderate essential hypertension, we studied the effect of intrabrachial insulin on the changes in forearm blood flow (strain-gauge plethysmography) induced by intrabrachial acetylcholine (at doses of 0.15, 0.45, 1.5, 4.5, and 15 µg · min^-1 · dL^-1), an endothelium-dependent vasodilator, or sodium nitroprusside (at doses of 1, 2, and 4 µg · min^-1 · dL^-1), an endothelium-independent vasodilator. Local hyperinsulinemia (deep venous plasma insulin, 48±6 and 51±5 µU/mL in control subjects and hypertensive patients, respectively) did not affect basal forearm blood flow and stimulated forearm glucose extraction (control subjects, 3±1% to 11±2%, P<.001; hypertensive patients, 3±1% to 6±1%, P<.001; P<.01 for the between-group difference). In both normotensive and hypertensive subjects, insulin significantly potentiated acetylcholine-induced vasodilation, whereas it did not alter the vasodilatory response to sodium nitroprusside. N^G-monomethyl-L-arginine, an inhibitor of endothelial nitric oxide synthesis, blunted insulin-induced facilitation of acetylcholine vasodilation in normotensive but not in hypertensive subjects. In contrast, in hypertensive patients but not in normotensive control subjects, the potentiation of the vascular response to acetylcholine induced by local hyperinsulinemia was abolished by intrabrachial ouabain, an inhibitor of Na⁺-K⁺ pump.

Conclusions In healthy humans and essential hypertensive patients alike, local physiological hyperinsulinemia per se does not increase forearm blood flow but potentiates the vasodilation induced by acetylcholine regardless of metabolic insulin resistance. This effect is endothelium-dependent because it is not seen with nitroprusside and is related to the L-arginine–nitric oxide pathway in normotensive subjects and to smooth muscle cell hyperpolarization in essential hypertensive patients.

Key Words: insulin • acetylcholine • blood flow • endothelium-derived factors

	Introduction

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Although the possibility that insulin may have vasoactive properties has been recognized for many years, the recent demonstration^{1 2} that essential hypertension is associated with insulin resistance has rekindled interest in the vascular effects of the hormone. In both animals^{3 4} and humans,^{5 6} systemic hyperinsulinemia decreases peripheral vascular resistance by causing some degree of limb (leg or forearm) vasodilation. This finding is in contrast to the negative results of several studies that tested the vasodilating effects of the local administration of insulin in the human forearm.^{7 8 9 10} This conflict has cast doubt on a direct vasodilating action of the hormone and has raised the possibility that insulin has different hemodynamic effects according to the route of administration, systemic or local.

Endothelial cells play a key role in modulating vascular tone through the production of relaxing substances, the most important of which are the EDRF nitric oxide, a product of the degradation of L-arginine into citrulline, a still-unidentified EDHF, and prostacyclin.¹¹ It recently was documented that in normotensive humans, leg¹² vasodilation induced by methacholine, an endothelium-dependent vasodilator,¹¹ is enhanced during systemic hyperinsulinemia, whereas the vasodilating effect of sodium nitroprusside, an SMC relaxant,¹³ is not affected. Thus, these observations suggest that in healthy subjects the vasorelaxant effects of systemic hyperinsulinemia are dependent, at least in part, on the endothelium. However, as mentioned above, the vascular effects of systemic and local insulin can be different. In particular, systemic insulin appears to be sympathoexcitatory,^{3 5} probably by direct action on the central nervous system.¹⁴

Therefore, the present study using the perfused forearm technique was undertaken to test whether local hyperinsulinemia modulates endothelial function in healthy humans and in patients with essential hypertension and, if so, by what mechanism.

	Methods

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Subjects
The study group included 27 patients with mild to moderate, uncomplicated essential hypertension and 18 healthy subjects matched for sex, age, and body weight (Table 1). The hypertensive patients either were untreated or had stopped antihypertensive therapy at least 2 weeks before the study. The study protocol was approved by the Institutional Ethics Committee; informed consent was obtained from all subjects before their participation in the study.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Study Subjects

Experimental Procedure
All studies were performed at 8AM, after an overnight fast, with the subjects lying supine in a quiet, air-conditioned room (22°C to 24°C). A polyethylene cannula (21 gauge, Abbot) was inserted into the brachial artery under local anesthesia (2% lidocaine). The cannula was connected through stopcocks to a pressure transducer (model MS20, Electromedics) for the determination of systemic mean arterial blood pressure (one third pulse pressure plus diastolic pressure) and heart rate (model VSM1, Physiocontrol) and for intra-arterial infusions. In 16 of the 18 normotensive subjects and 24 of the 27 hypertensive patients, another cannula (6 cm long) was advanced into an ipsilateral deep forearm vein retrogradely. FBF was measured in both forearms (experimental and contralateral) by strain-gauge venous occlusion plethysmography (LOOSCO, GL LOOS).¹⁵ Circulation to the hand was excluded 1 minute before each measurement of FBF by inflation of a pediatric cuff around the wrist at suprasystolic pressure. Earlier work determined the sensitivity and reproducibility of the method.¹⁶ Forearm volume was measured by the water displacement technique. Infusion rates of drugs were normalized to 100 mL tissue by alteration of the drug concentration in the solvent while the pump flow rate was kept constant. The drugs used were infused through separate ports through three-way stopcocks at concentrations that had no systemic effects.

Experimental Design
Protocol A
In 6 healthy subjects and 8 hypertensive patients, the effect of insulin on endothelium-dependent and endothelium-independent vasodilation was assessed by a dose-response curve to intra-arterial acetylcholine¹¹ (at infusion rates of 0.15, 0.45, 1.5, 4.5, and 15 µg · min^-1 · dL^-1 forearm tissue, 5 minutes for each dose) and sodium nitroprusside¹³ (at infusion rates of 1, 2, and 4 µg · min^-1 · dL^-1, 5 minutes for each dose). Drugs were administered both under control conditions (ie, during the intrabrachial infusion of saline at 0.2 mL/min) and in the presence of an intrabrachial infusion of insulin. The insulin infusion rate was calculated in each subject (from the mean basal FBF and forearm volume) to produce increments in local arterial plasma insulin level of approximately 60 µU/mL (420 pmol/L) and was started 20 minutes before each drug dose response (insulin prime). For each insulin infusion, simultaneous arterial and venous samples were obtained before the start and at the end of the 20-minute prime for the measurement of plasma insulin and glucose concentrations. The sequence of infusion of acetylcholine and sodium nitroprusside was randomized, and 1 hour of washout was allowed between each dose-response curve. Fig 1 shows the experimental designs.

View larger version (29K): [in this window] [in a new window]   Figure 1. Plots showing experimental protocol. The time scale is not linear, but real experimental times are indicated. Ach indicates acetylcholine; Np, sodium nitroprusside; and Ouab, ouabain.

Protocol B
To test whether insulin increases the endothelium-dependent release of nitric oxide, we used the arginine analogue L-NMMA, which antagonizes the synthesis of nitric oxide from L-arginine in a competitive manner.^{11 17} In 6 normotensive subjects and 7 hypertensive patients, the dose-response curve to intra-arterial acetylcholine at the same doses as in protocol A was performed according to the following design: during saline (0.2 mL/min), in the presence of insulin as in protocol A, in the presence of intra-arterial L-NMMA (100 µg · min^-1 · dL^-1 started 5 minutes before acetylcholine and continued throughout), and finally in the presence of simultaneous infusions of insulin and L-NMMA. Again, 1 hour of washout was allowed between each dose-response curve.

Protocol C
Finally, to assess whether insulin-induced hyperpolarization can affect the vascular response to acetylcholine infusion, in another group of 6 normotensive subjects and 7 essential hypertensive patients, protocol B was modified by replacing L-NMMA with ouabain¹⁸ infused intrabrachially at 0.72 µg · min^-1 · dL^-1. Because the insulin-induced hyperpolarizing effect is quite rapid in onset,¹⁹ ouabain was infused 20 minutes before acetylcholine was added (ie, it was coinfused with insulin).

Protocol D
To rule out the possible interference caused by L-NMMA– or ouabain-induced vasoconstriction on the response to acetylcholine, in an additional 4 normotensive subjects and 4 hypertensive patients, we tested the effect of norepinephrine infused into the brachial artery at doses (0.015 and 0.05 µg · min^-1 · dL^-1) titrated to induce an FBF decrement comparable to that obtained with ouabain (35%) or L-NMMA (50%). The experimental design was similar to protocol C or B, with L-NMMA or ouabain replaced with norepinephrine.

Analytical Procedures
Insulin was measured in plasma by a standard radioimmunoassay method (INSKIT, Sorin). Plasma glucose was assayed by an enzymatic method (glucose oxidase, Beckman Glucose Analyzer).

Drugs
Acetylcholine hydrochloride (Farmigea SpA), L-NMMA (Clinalfa AG), insulin (Actrapid), ouabain (Ouabaine Arnaud), and sodium nitroprusside (Malesci) were obtained from commercially available sources and diluted freshly to the desired concentration by the addition of normal saline. To avoid adsorption to syringe and connecting line, insulin was dissolved in saline plus 1% albumin. Sodium nitroprusside was dissolved in a dextrose solution and protected from light by aluminum foil.

Data Analysis
Forearm glucose balance was obtained as the product of the AV plasma glucose concentration gradient and forearm plasma flow. Plasma flow rates were calculated to be the product of FBF and (1-hematocrit). Glucose extraction ratio was computed as the ratio of the AV difference to the arterial concentration. Because mean arterial blood pressure did not change significantly during the study, data were analyzed in terms of changes in FBF. Because L-NMMA and ouabain altered resting FBF, data were analyzed as percent increase from baseline. Contralateral FBF changed slightly but not significantly during the experimental period. Nonetheless, to account for these changes, data also were expressed as the I/C ratio. Results are expressed as mean±SEM. Statistical analysis was done by two- or three-way ANOVA. Differences were considered to be statistically significant only when P<.05.

	Results

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Table 1 summarizes the characteristics of the normotensive subjects and essential hypertensive patients. Except for arterial blood pressure, there were no significant differences between the two study groups.

Twenty minutes of intrabrachial insulin preinfusion raised deep venous plasma insulin concentrations into the physiological range of postcibal values (normotensive subjects, from 10±1 to 48±6 µU/mL, P<.001; hypertensive patients, from 10±1 to 51±5 µU/mL, P<.001) and caused a decrease in deep venous plasma glucose levels (normotensive subjects, from 4.45±0.09 to 4.06±0.10 mmol/L, P<.01; hypertensive patients, from 4.52±0.06 to 4.32±0.07 mmol/L, P<.05). In both groups, arterial plasma glucose levels did not change during the experiment. Forearm glucose extraction was stimulated by insulin administration in both control subjects (from 3.3±0.8% to 11.3±1.5%, P<.001) and hypertensive patients (from 2.7±0.5% to 6.0±1.1%, P<.001). This degree of insulin-stimulated forearm glucose extraction was significantly (P<.01) greater in control subjects than in hypertensive patients.

Effect of Insulin on Vasodilatory Response to Acetylcholine and Sodium Nitroprusside (Protocol A)
In normotensive subjects under control conditions, acetylcholine and sodium nitroprusside led to similar forearm vasodilation (FBF rose from 3.5±0.2 to a maximum of 24.9±1.9 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 7.1±0.8] and from 3.6±0.3 to 22.1±1.7 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 6.0±0.6], respectively; see Fig 2). Insulin administration did not alter basal FBF (3.6±0.2 versus 3.7±0.2 mL · min^-1 · dL^-1 [I/C, 1.0±0.1 versus 1.1±0.1], P=NS) but significantly (P<.01) increased acetylcholine-induced vasodilation (from 3.7±0.2 to a maximum of 34.9±3.3 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 10.1±1.2], P<.01 versus saline), whereas it did not alter the response to sodium nitroprusside (from 3.7±0.3 to 21.7±1.8 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 5.7±0.6], P=NS versus saline).

View larger version (19K): [in this window] [in a new window]   Figure 2. Plots showing increase in FBF induced by intra-arterial acetylcholine (top) and sodium nitroprusside (bottom) under control conditions (, saline infusion at 0.2 mL/min) and in the presence of local hyperinsulinemia () in normotensive subjects (n=6, left) and hypertensive subjects (right). Data are shown as mean±SEM and expressed as percent increase above basal. Asterisks denote significant differences between infusions with and without insulin (P<.05 or less).

In essential hypertensive patients, the vasodilating effect of acetylcholine (from 3.4±0.4 to a maximum of 16.8±3.1 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 4.7±0.4]) was significantly (P<.05) less than in normotensive control subjects. Nevertheless, the FBF response to acetylcholine was significantly potentiated by insulin (from 3.5±0.5 to a maximum of 25.1±4.1 mL · min^-1 · dL^-1 [I/C, from 1.1±0.1 to 7.4±0.8], P<.01 versus saline). This facilitatory effect of insulin on acetylcholine-induced vasodilation was not statistically different between normotensive subjects and essential hypertensive patients (Fig 3). The response to sodium nitroprusside (from 3.3±0.4 to 18.7±3.1 mL · min^-1 · dL^-1 [I/C, from 1±0.1 to 6.1±0.7]) was not statistically different from that observed in the control subjects, and insulin likewise failed to affect the vasodilatory response to sodium nitroprusside (from 3.4±0.4 to 18.1±3.1 mL · min^-1 · dL^-1 [I/C, from 0.94±0.1 to 5.3±0.4], P=NS versus saline; Fig 2). As in control subjects, insulin administration did not alter basal FBF.

View larger version (13K): [in this window] [in a new window]   Figure 3. Bar graph showing the potentiation of acetylcholine-induced vasodilation by insulin in normotensive subjects (, n=22) and essential hypertensive patients (, n=31). Data are shown as mean±SEM and expressed as percent increase of acetylcholine-induced vasodilation in the presence of insulin compared with acetylcholine-induced vasodilation under control conditions. The facilitatory effect of insulin on acetylcholine-induced vasodilation was not statistically different between the two groups.

L-NMMA Infusion (Protocol B)
In this group of normotensive control subjects, acetylcholine-dependent vasodilation (from 3.6±0.3 to 25.5±1.9 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 6.9±0.7]) again was significantly (P<.01) increased by the simultaneous infusion of insulin (from 3.7±0.3 to 35.9±2.3 mL · min^-1 · dL^-1 [I/C, from 1.1±0.1 to 10.6±1.3]; see Fig 4). L-NMMA infusion caused a decrease in basal FBF (from 3.8±0.3 to 2.0±0.2 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 0.5±0.3], P<.01) and significantly blunted the vasodilating effect of acetylcholine both under control conditions (from 2.0±0.2 to 7.7±2.2 mL · min^-1 · dL^-1 [I/C, from 0.5±0.3 to 2.2±0.3], P<.001 versus saline) and during insulin administration (from 2.1±0.3 to 15.5±3.4 mL · min^-1 · dL^-1 [I/C, from 0.6±0.4 to 4.3±0.5], P<.01 versus insulin alone). Of note is that at the two lower rates of acetylcholine infusion, L-NMMA abolished the potentiating effect of insulin.

View larger version (19K): [in this window] [in a new window]   Figure 4. Plots showing acetylcholine-induced increases in FBF in the absence () and presence () of hyperinsulinemia under control conditions (top) and in the presence of L-NMMA (100 µg · min-1 · dL-1, bottom) in normotensive subjects (n=6, left) and essential hypertensive patients (n=7, right). Data are shown as mean±SEM and expressed as percent increase above basal. Asterisks denote significant differences between infusion with and without insulin (P<.05 or less).

Different results were obtained in patients with essential hypertension. Acetylcholine infusion again caused a dose-dependent vasodilation (from 3.2±0.5 to 18.1±2.6 mL · min^-1 · dL^-1 [I/C from 1.0±0.1 to 6.1±0.7]), which was significantly (P<.01) increased by insulin (from 3.1±0.4 to 23.9±3.1 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 7.9±0.9], P<.001 versus saline). L-NMMA infusion caused a decrease in basal FBF (from 3.1±0.4 to 2.3±0.3 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 0.8±0.1], P<.01), which was significantly (P<.01) smaller than that observed in normotensive control subjects. Moreover, L-NMMA failed to blunt the response to acetylcholine either when infused alone (from 2.3±0.3 to 11.8±2.4 mL · min^-1 · dL^-1 [I/C, from 0.8±0.1 to 5.6±0.6], P=NS versus saline) or when coinfused with insulin (from 2.3±0.5 to 18.7±3.3 mL · min^-1 · dL^-1 [I/C, from 0.7±0.1 to 8.5±0.9], P=NS versus saline).

To rule out the possibility of insufficient L-NMMA blockade, in another group of 5 hypertensive patients, the L-NMMA dose was doubled (200 µg · min^-1 · dL^-1). Nonetheless, the nitric oxide synthase antagonist did not affect the response to acetylcholine either under control conditions or in the presence of insulin (Table 2, protocol 1).

View this table: [in this window] [in a new window]   Table 2. Protocol Results

Ouabain Infusion (Protocol C)
In this group of control subjects, vasodilation to acetylcholine (from 3.9±0.4 to 25.3±2.1 mL · min^-1 ·dL^-1 [I/C, from 1.0±0.1 to 6.6±0.7]) was still significantly (P<.001) increased by the simultaneous infusion of insulin (from 3.7±0.3 to 34.1±4.6 mL · min^-1 · dL^-1 [I/C, from 9.5±0.1 to 9.2±1.1], P<.001 versus saline; Fig 5). Ouabain administration decreased basal FBF (from 3.7±0.5 to 2.6±0.4 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 0.6±0.5], P<.01) but failed to affect the response to acetylcholine infused either alone (from 2.6±0.4 to 17.3±2.7 mL · min^-1 · dL^-1 [I/C, from 0.6±0.5 to 4.7±0.6], P=NS versus acetylcholine during saline infusion) or during insulin administration (FBF, from 3.0±0.5 to 24.7±4.1 mL · min^-1 · dL^-1 [I/C, from 0.7±0.1 to 6.2±0.7], P=NS versus acetylcholine during saline and insulin infusion).

View larger version (20K): [in this window] [in a new window]   Figure 5. Plots showing acetylcholine-induced increase in FBF in the absence () and presence () of hyperinsulinemia under control conditions (top) and in the presence of ouabain (0.72 µg · min-1 · dL-1, bottom) in normotensive subjects (n=6, left) and essential hypertensive patients (n=7, right). Data are shown as mean±SEM and expressed as percent increase above basal. Asterisks denote significant differences between infusion with and without insulin (P<.05 or less).

In the essential hypertensive patients, the response to acetylcholine (from 2.8±0.5 to 16.5±3.6 mL · min^-1 ·dL^-1 [I/C, from 1.0±0.1 to 6.3±0.7]) was again potentiated by insulin (from 2.8±0.5 to 23.5±4.3 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 8.7±1.0], P<.001 versus saline). Ouabain infusion decreased basal FBF (from 3.0±0.5 to 2.1±0.3 mL · min^-1 · dL^-1 [I/C, from 1.0±0.1 to 0.7±0.4], P<.01), did not alter the response to acetylcholine (from 2.1±0.4 to 13.1±3.2 mL · min^-1 · dL^-1 [I/C, from 0.7±0.4 to 4.4±0.5], P=NS versus acetylcholine during saline infusion), but abolished the potentiating effect of insulin on acetylcholine-induced vasodilation (FBF, from 2.2±0.5 to 12.8±2.6 mL · min^-1 · dL^-1 [I/C, from 0.7±0.1 to 4.5±0.5], P=NS versus acetylcholine during saline and insulin infusion).

Protocol D
To rule out the possible interference caused by L-NMMA–induced vasoconstriction on the response to acetylcholine, in 4 additional normotensive subjects norepinephrine was infused intrabrachially at a rate of 0.05 µg · min^-1 · dL^-1. FBF decreased from 2.6±0.1 to 1.4±0.1 µg · min^-1 · dL^-1 (I/C, from 0.96±0.1 to 0.52±0.3), a vasoconstrictor effect comparable to L-NMMA (50%), but neither vasodilation to acetylcholine nor the facilitating effect of local hyperinsulinemia on the latter was altered (Table 2, protocol 2). To further exclude any interference of ouabain-induced vasoconstriction on the response to acetylcholine, in another 4 hypertensive subjects norepinephrine (0.015 µg · min^-1 · dL^-1), while causing an FBF decrease comparable to that obtained with ouabain (35%) (FBF, from 3.2±0.3 to 2.1±0.3 mL · min^-1 · dL^-1 [I/C, from 1.1±0.1 to 0.7±0.4], failed to alter either vasodilation to acetylcholine or the potentiating effect of local hyperinsulinemia; Table 2, protocol 3).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Although it is generally accepted that systemic hyperinsulinemia lowers peripheral vascular resistances, in vivo studies that evaluated the direct vascular action of the hormone (ie, the forearm vasodilatory response to intrabrachial insulin) gave contradictory results. A few reports showed a direct relaxant effect of insulin (however, in two of these studies, a different technique—dye dilution—was used),^{20 21 22} several other studies did not confirm this finding.^{7 8 9 10} Clearly, this discrepancy may be related to methodological problems or differences in experimental design. Interestingly, a recent report by Neahring et al²³ confirmed the original finding of Andres et al²⁰ that the vehicle habitually used to dissolve insulin (saline plus albumin) can cause vasodilation, an effect probably related to the presence of albumin. Thus, it is conceivable that at least part of the direct insulin-induced vasodilation observed by some authors may be aspecific. In the present study, after intrabrachial insulin infusion in a total of 18 normotensive subjects and 22 essential hypertensive patients, we did not find significant modifications in mean FBF (normotensive subjects, 3.3±0.4 versus 3.5±0.5 mL · min^-1 · dL^-1; essential hypertensive patients, 3.1±0.2 versus 3.2±0.3 mL · min^-1 · dL^-1), although in some patients (3 of 18 normotensive subjects and 4 of 22 essential hypertensive patients) FBF did increase modestly.

The aim of the current study was to evaluate the effect of insulin on endothelium-dependent vasodilation in humans. We chose essential hypertension because vascular⁶ and metabolic¹ actions of systemic hyperinsulinemia appear to be different in this condition. Indeed, in the present series, forearm glucose extraction caused by local insulin was significantly lower in the hypertensive patients than in control subjects despite the short preinfusion time. We found that forearm vasodilation to acetylcholine, an endothelium-dependent vasodilator, is potentiated by forearm hyperinsulinemia in both normotensive subjects and patients with essential hypertension. Because the response to sodium nitroprusside, a direct SMC relaxant, was not altered by insulin, a nonspecific enhancement of vascular SMC responsiveness to vasodilators was excluded. Importantly, the potentiating effect of insulin on the response to acetylcholine was similar in the two groups of patients (Fig 3), indicating that in essential hypertensive patients the action of the hormone on endothelial function is intact even in the presence of metabolic insulin resistance.

With regard to the mechanism(s) involved in the insulin-mediated potentiation of the response to acetylcholine, in normotensive control subjects inhibition of nitric oxide production by L-NMMA blunted the response to acetylcholine itself and abolished the potentiating effect of insulin at low doses of the muscarinic agonist (and reduced it at the higher rates). This indicates that the facilitating action of the hormone on endothelium-dependent vasodilation is, at least in part, mediated by activation of the L-arginine nitric oxide pathway. That L-NMMA canceled the effect of insulin only at low doses of acetylcholine can be explained by the fact that the L-arginine analogue is a competitive inhibitor, its action being curtailed by increasing concentrations of the substrate. Thus, Steinberg et al¹² and Scherrer et al²⁴ were able to abolish the vasodilation induced by systemic insulin by using a higher dose of L-NMMA. Furthermore, the extent of nitric oxide activation may be different with local insulin plus acetylcholine versus systemic insulin administration.

In contrast to the healthy control subjects, in essential hypertensive patients L-NMMA did not alter the effect of insulin on acetylcholine-induced vasodilation. It is important to note that in the hypertensive patients the constrictor effect of L-NMMA infusion was blunted compared with that in normotensive control subjects. In addition, even the control subjects' acetylcholine dose-response curves were flatter than those in normotensive subjects and were not altered by L-NMMA administration tested at two different doses. These findings, although not universally confirmed,²⁵ are in line with previous observations showing that both basal²⁶ and receptor-activated^{27 28 29} release of nitric oxide is impaired in essential hypertension. Therefore, the facilitating effect of insulin on acetylcholine-mediated vasodilation must also be exerted through mechanisms other than from the L-arginine–nitric oxide pathway.

Further support to this hypothesis is offered by our studies with ouabain. In fact, in the patients with essential hypertension, the inhibitor of the Na⁺-K⁺ pump prevented the facilitating effect exerted by insulin, although it did not alter acetylcholine-induced vasodilation under control conditions. It is noteworthy that in normotensive subjects ouabain did not affect either the control response to acetylcholine or the enhancing effect of insulin. Taken together, these results suggest that insulin-induced potentiation of endothelium-dependent vasodilation acts partially through different mechanisms in normotensive subjects and essential hypertensive patients. Direct evidence bearing on the mechanism by which ouabain counters insulin potentiation of acetylcholine vasodilation is not available. Evidence from animal studies indicates that both acetylcholine³⁰ and insulin^{20 31} hyperpolarize cell membranes, an effect mediated by the Na⁺-K⁺ pump and antagonized by ouabain.^{20 30} At least in certain tissues, muscarinic agonists and other endothelial stimulants can activate the endothelium to produce a diffusible substance, ie, EDHF, which hyperpolarizes the membrane of SMCs.^{30 32 33} Thus, it is possible to hypothesize that in essential hypertensive patients insulin-induced potentiation of acetylcholine-induced vasodilation is to be mediated by SMC hyperpolarization. In summary, the vasodilation induced by acetylcholine usually is predominantly mediated by nitric oxide and therefore is inhibited by L-NMMA. This pathway is defective in essential hypertension and is not antagonized by L-NMMA. Therefore, other pathways must exist, one of which, when acted on by insulin, involves cell hyperpolarization because it is blocked by ouabain. Insulin per se is not a vasodilator despite the presence of specific receptors on both endothelial cell³⁴ and SMC³⁵ membranes. However, at some intracellular level (perhaps calcium³⁶ ) in endothelial cells, insulin synergizes the vasodilating action of acetylcholine. This response is not impaired in essential hypertension, but in contrast to the normal situation, it is dependent more heavily on hyperpolarization than on nitric oxide production. Thus, in hypertension the ouabain-inhibitable pathway is amplified by insulin, possibly because it is hypertrophic relative to the classic nitric oxide–dependent pathway. Whether this imbalance is a compensatory adjustment or a primary defect and whether it bears any relation to the metabolic insulin resistance associated with essential hypertension remain to be investigated.

	Selected Abbreviations and Acronyms

 

EDHF = endothelium-derived hyperpolarizing factor EDRF = endothelium-derived relaxing factor FBF = forearm blood flow I/C = ratio ofinfused to contralateral FBF L-NMMA = NG-monomethyl-L-arginine SMC = smooth muscle cell<\/.>

	Acknowledgments

 
The authors thank Moreno Rocchi for artwork.

Received December 15, 1994; revision received June 9, 1995; accepted July 5, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Ferrannini E, Buzzigoli G, Bonadonna R, Giorico MA, Oleggini M, Graziadei L, Pedrinelli R, Brandi L, Bevilacqua S. Insulin resistance in essential hypertension. N Engl J Med. 1987;317:350-357. [Abstract]
   
2. Bonora E, Zavaroni I, Alpi O, Pezzarossa A, Bruschi F, Dall'Aglio E, Guerra L, Coscelli C, Butturini U. Relationship between blood pressure and plasma insulin in non-obese and obese non-diabetic subjects. Diabetologia. 1987;30:719-723. [Medline] [Order article via Infotrieve]
   
3. Hall JE, Brands MW, Kivlighn SD, Mizelle HL, Hildebrandt DA, Gaillard CA. Chronic hyperinsulinemia and blood pressure: interaction with catecholamines? Hypertension. 1990;15:519-527. [Abstract/Free Full Text]
   
4. Liang C-S, Doherty JU, Faillace R, Maekawa K, Arnold S, Gavras H, Hood WB. Insulin infusion in conscious dogs: effects on systemic and coronary hemodynamics, regional blood flows, and plasma catecholamines. J Clin Invest. 1982;69:1321-1336.
   
5. Anderson EA, Hoffman RP, Balon TW, Sinkey CA, Mark AL. Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans. J Clin Invest. 1991;87:2246-2252.
   
6. Baron AD, Brechtel-Hook G, Johnson A, Hardin D. Skeletal muscle blood flow: a possible link between insulin resistance and blood pressure. Hypertension. 1993;21:129-135. [Abstract/Free Full Text]
   
7. Ferrannini E, Taddei S, Santoro D, Natali A, Boni A, Del Chiaro D, Buzzigoli G. Independent stimulation of glucose metabolism and Na⁺-K⁺ exchange by insulin in the human forearm. Am J Physiol. 1988;255:E953-E958. [Abstract/Free Full Text]
   
8. Natali A, Buzzigoli G, Taddei S, Santoro D, Cerri M, Pedrinelli R, Ferrannini E. Effects of insulin on hemodynamics and metabolism in human forearm. Diabetes. 1990;39:490-500. [Abstract]
   
9. Lembo G, Rendina V, Iaccarino G, Lamenza F, Volpe M, Trimarco B. Insulin reduces reflex forearm sympathetic vasoconstriction in healthy humans. Hypertension. 1993;21:1015-1019. [Abstract/Free Full Text]
   
10. Sakai K, Imaizumi T, Masaki H, Takeshita A. Intra-arterial infusion of insulin attenuates vasoreactivity in human forearm. Hypertension. 1993;22:67-73. [Abstract/Free Full Text]
    
11. Luscher TF, Vanhoutte PM. The Endothelium: Modulator of Cardiovascular Function. Boca Raton, Fla: CRC Press; 1990:1-228.
    
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 decrease nitric oxide release. J Clin Invest. 1994;94:1172-1179.
    
13. Schultz KD, Schultz K, Schultz G. Sodium nitroprusside and other smooth muscle relaxants increase cyclic GMP levels in rat ductus deferens. Nature. 1977;265:750-751. [Medline] [Order article via Infotrieve]
    
14. Vollenweider P, Tappy L, Randin D, Schneiter P, Jequier E, Nicod P, Scherrer U. Differential effects of hyperinsulinemia and carbohydrate metabolism on sympathetic nerve activity and muscle blood flow in humans. J Clin Invest. 1993;92:147-154.
    
15. Whitney RJ. The measurement of volume changes in human limbs. J Physiol (Lond). 1953;121:1-27.
    
16. Pedrinelli R, Taddei S, Graziadei L, Salvetti A. Vascular responses to ouabain and norepinephrine in low and normal renin hypertension. Hypertension. 1986;8:786-792. [Abstract/Free Full Text]
    
17. Vallance P, Coller J, Moncada S. Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet. 1989;2:997-1000. [Medline] [Order article via Infotrieve]
    
18. Tonomura Y. (Na⁺,K⁺)ATPase in the plasma membrane. In: Energy-Transducing ATPase: Structure and Kinetics. Cambridge, UK: Cambridge University Press; 240-281.
    
19. Iannaccone ST, Kai-Xun LI, Sperelakis N, Lathrop DA. Insulin-induced hyperpolarization in mammalian skeletal muscle. Am J Physiol. 1989;256:C368-C374. [Abstract/Free Full Text]
    
20. Andres R, Baltzan MA, Cader G, Zierler KL. Effect of insulin on carbohydrate metabolism and on potassium in the forearm of man. J Clin Invest. 1961;41:108-115.
    
21. Gelfand RA, Barrett EJ. Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man. J Clin Invest. 1987;80:1-6.
    
22. Creager MA, Liang CS, Coffman JD. Beta adrenergic-mediated vasodilator response to insulin in the human forearm. J Pharmacol Exp Ther. 1985;235:709-714. [Abstract/Free Full Text]
    
23. Neahring JM, Stepniakowski K, Greene AS, Egan BM. Insulin does not reduce forearm -vasoreactivity in obese hypertensive or lean normotensive men. Hypertension. 1993;22:584-590. [Abstract/Free Full Text]
    
24. Scherrer U, Randin D, Vollenweider P, Vollenweider L, Nicod P. Nitric oxide release accounts for insulin's vascular effects in humans. J Clin Invest. 1994;94:2511-2515.
    
25. Cockcroft JR, Chowienczyk PJ, Benjamin N, Ritter JM. Preserved endothelium-dependent vasodilation in patients with essential hypertension. N Engl J Med. 1994;330:1036-1040. [Abstract/Free Full Text]
    
26. Calver A, Collier J, Moncada S, Vallance P. Effect of local intra-arterial N^G-monomethyl-L-arginine in patients with essential hypertension: the nitric oxide dilator mechanism appears abnormal. J Hypertens. 1992;10:1025-1031. [Medline] [Order article via Infotrieve]
    
27. Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation. 1993;87:1468-1474. [Abstract/Free Full Text]
    
28. Taddei S, Mattei P, Virdis A, Sudano I, Ghiadoni L, Salvetti A. Effect of potassium on vasodilation to acetylcholine in essential hypertension. Hypertension. 1994;23:485-490. [Abstract/Free Full Text]
    
29. Panza JA, Garcìa CE, Kilcoyne CM, Quyyumi A, Cannon RO III. Impaired endothelium-dependent vasodilation in patients with essential hypertension: evidence that nitric oxide abnormality is not localized to a single signal transduction pathway. Circulation. 1995;91:1732-1738. [Abstract/Free Full Text]
    
30. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol. 1988;93:515-524.[Medline] [Order article via Infotrieve]
    
31. Moore RD, Rabovsky JL. Mechanism of insulin action on resting membrane potential of frog skeletal muscle. Am J Physiol. 1978;235:C249-C254.
    
32. Beny JL, Brunet PC. Electrophysiological and mechanical effects of substance P and acetylcholine on rabbit aorta. J Physiol (Lond). 1988;398:277-289. [Abstract/Free Full Text]
    
33. Nakashima M, Mombouli JV, Taylor AA, Vanhoutte PM. Endothelium-dependent hyperpolarization caused by bradykinin in human coronary arteries. J Clin Invest. 1993;92:2867-2871.
    
34. King GL, Goodman AD, Buzney S, Moses A, Kahn CR. Receptors and growth-promoting effects of insulin and insulinlike growth factors on cells from bovine retinal capillaries and aorta. J Clin Invest. 1985;75:1028-1036.
    
35. Pfeiffe B, Ditschuneit H. Effect of insulin on growth of cultured human arterial smooth muscle cells. Diabetologia. 1981;20:155-158. [Medline] [Order article via Infotrieve]
    
36. Rasmussen H, Rasmussen JE. Calcium as intracellular messenger: from simplicity to complexity. Curr Top Cell Regul. 1990;31:1-109.[Medline] [Order article via Infotrieve]
    

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