Objective: To determine the roles of nitric oxide, endothelin-1 and phosphatidylinositol 3-kinase (PI3-kinase) in acute responses of isolated rat skeletal muscle arterioles to insulin. Methods: Rat cremaster first order arterioles were separated from surrounding tissue, cannulated in a pressure myograph and responses to insulin (4 μU/ml–3.4 mU/ml) were studied without intraluminal blood or flow. Results: Insulin alone did not significantly affect arteriolar diameter. Non-selective antagonism of endothelin receptors, with PD-142893, uncovered insulin-induced vasodilatation (25±8% from baseline at 3.4 mU/ml), which was abolished by inhibition of NO synthesis with NG-nitro-l-arginine (l-NA). Inhibition of NO synthesis alone uncovered insulin-induced vasoconstriction at physiological concentrations (21±5% from baseline diameter at 34 μU/ml), which was abolished by PD-142893. The NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) inhibited insulin-induced vasoconstriction during NOS inhibition, even at a concentration that did not elicit vasodilatation itself. Inhibition of PI3-kinase, an intracellular mediator of insulin-induced NO production, with wortmannin, also uncovered insulin-induced vasoconstriction (13±3% from baseline at 34 μU/ml) that was abolished by PD-142893. Conclusions: Insulin induces both nitric oxide and endothelin-1 activity in rat cremaster first-order arterioles. This study demonstrates for the first time that vasoconstrictive effects of physiological concentrations of insulin during inhibition of NOS activity are mediated by endothelin and that insulin induces endothelin-1-mediated vasoconstriction in isolated skeletal muscle arterioles during inhibition of PI3-kinase. These findings support the hypothesis of altered microvascular reactivity to insulin in conditions of diminished PI3-kinase activity, a prominent feature of insulin resistance.
Time for primary review 27 days.
Resistance to the blood-glucose-lowering effect of insulin is associated with hypertension , microvascular dysfunction  and increased incidence of ischemic heart disease . However, the role of hyperinsulinemia in the vascular complications of insulin resistance is still unclear.
On the one hand, acute vasodilatation in response to insulin has been shown in the human forearm and human skeletal muscle [4–7], the rat hindlimb [8–10] and isolated rat skeletal muscle arterioles [11,12]. Nitric oxide, produced by the endothelium, plays an important role in these responses to insulin in humans [4,5,13,14] and in rats [9,11,12,15]. Insulin, at pharmacological concentrations, stimulates nitric oxide production within minutes in cultured endothelial cells through activation of phosphatidylinositol 3-kinase (PI3-kinase) . On the other hand, some studies have failed to show insulin-induced vasodilatation  and no direct evidence exists of insulin-induced NO production at physiological concentrations [16,18,19]. Therefore, the role of NO in effects of insulin on vascular tone is not completely clear.
While attenuated activation of PI3-kinase by insulin is a prominent feature of insulin resistance  and defective PI3-kinase-dependent activation of eNOS by insulin has been implicated in the pathogenesis of hypertension in insulin-resistant states , the implications of PI3-kinase inhibition for vascular reactivity to insulin are unknown.
During inhibition of NOS  and during hyperglycemia  some investigators have even reported an acute vasoconstrictor effect of insulin, suggesting that insulin has vasoconstrictive effects in addition to effects mediated by nitric oxide. Recent evidence suggests that in vivo, insulin at physiological concentrations stimulates the activity of both NO and endothelin (ET-1), a potent endogenous vasoconstrictor . In addition, insulin acutely enhances the production of ET-1 by cultured human endothelial cells  and rat microvessels  in vitro. However, since evidence of ET-1-mediated vasoconstriction in response to insulin is lacking, the functional role of ET-1 in vascular responses to insulin is still unclear.
Skeletal muscle tissue is the location of 80–90% of insulin-induced glucose uptake  and has been recognized as one of the primary sites of insulin's vascular actions in vivo [4,23]. However, the role of endothelin-1 in acute responses of the skeletal muscle microvasculature to insulin is unknown.
The aims of this study were to determine the role of nitric oxide and endothelin-1 in acute responses of rat skeletal muscle arterioles to insulin and to elucidate the role of PI3-kinase. To study effects of insulin on skeletal muscle arterioles without effects of surrounding tissue or blood flow, we used the isolated first-order arteriole of the rat cremaster muscle as a model.
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and the local ethics committee for animal experiments approved procedures. Forty-nine male Wistar rats weighing 318±9 g were anesthetized with pentobarbital sodium (Nembutal; 70 mg/kg i.p.) and ketamine (25 mg/kg i.m.). The right cremaster muscle was isolated as described earlier . Briefly, the right cremaster muscle was exposed by a ventral incision of the scrotal sac and cleared from connective tissue. The muscle was opened by an incision after which the testis was removed. The cremaster was then excised and pinned to a dissecting dish containing 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (for composition see Section 2.2) at 5 °C. A first-order arteriole (passive inner diameter 150–200 μm) was carefully dissected from the surrounding muscle and a 1–2-mm long segment was transferred to a pressure myograph. The myograph consisted of a vessel chamber and a video camera mounted on a microscope connected to an electronic measurement system to monitor inner arteriolar diameters continuously. The vessel chamber contained two glass cannulas (outer diameter of tip ∼100 μm), a circular heating coil, and a thermistor. The vessel chamber was filled with MOPS buffer (see below) and sealed with a glass cover.
The vessel segment was mounted on one cannula and secured with a single strand of a 20-μm suture. Then, by raising the perfusion pressure to 5 mmHg, the vessel was flushed gently to remove blood cells. The other side of the vessel was then mounted on the second cannula and also secured with a suture. The second cannula was then connected to a column filled with MOPS buffer. Pressure inside the vessel was set at 65 mmHg, which has been reported to be within the normal range for first-order arterioles in the cremaster muscle of anesthetized rats . Vessels were stretched to their in vivo length and temperature was raised gradually to 34 °C, the in vivo temperature of the cremaster muscle. Experiments were performed under no-flow conditions without superfusion, and vessels were exposed to vasoactive substances by adding them directly to the vessel chamber.
Prior to experiments, vessel segments were superfused with MOPS buffer for at least 15 min. Segments were then equilibrated for at least 30 min after the end of the washout period. Arterioles included in this study had to fulfil three criteria. First, arterioles that showed signs of leakage were excluded from the study. Second, the arterioles had to develop a level of spontaneous tone corresponding to a reduction in diameter of 40–60% of the passive diameter. Finally, the arterioles had to dilate for a prolonged period of time (>6 min) in response to the endothelium-dependent vasodilator acetylcholine (ACh, 0.1 μM) before and after the experiment as a measure of endothelial integrity and stability. Steady-state responses to acetylcholine are reported. At the end of each experiment the passive (maximally dilated) diameter was assessed by adding papaverine (0.1 mM). One or more segments were obtained from one animal. If multiple segments from the same rat were used, each segment was included in a different group (e.g., insulin and control).
Acute effects of insulin on the diameter of cremaster first-order arterioles were studied by cumulatively adding four concentrations of insulin to the vessel bath and recording diameter changes during the first 30 min after each concentration step. Since part of the insulin is lost by adhesion to the bath, a parallel series of experiments was performed in which samples were taken from the bath and insulin concentrations were determined by radioimmunoassay. The measured insulin concentrations were 4±0.7, 34±6, 272±44 μU/ml and 3.4±0.3 mU/ml. Since insulin concentrations measured in vivo in the rat range between 3 and 300 μU/ml [26,27], the first three concentrations are considered physiological, and the last concentration pharmacological. Effects of insulin on vessel diameter are plotted against these measured insulin concentrations.
The first group of vessel segments (n = 9) was treated with insulin alone. To study the roles of endothelin-1 and nitric oxide synthesis in responses to insulin, the second group of vessels (n = 8) was treated with the non-selective ET-1 receptor antagonist PD-142893 (3 μM), a third group (n = 6) was pre-treated for 30 min with an inhibitor of NO synthase, 0.1 mM N-nitro-l-arginine (l-NA) and a fourth group (n = 5) was treated with a combination of both. To study interference of NO with insulin-induced changes in vessel diameter during blockade of NO synthesis, a fifth group of vessel segments (n = 4) was treated with l-NA and a nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), before addition of insulin. SNAP was added after l-NA at a concentration (40–80 nM) that reversed l-NA-induced vasoconstriction, but did not raise the vessel diameter above the diameter before the addition of l-NA. At a ratio of 750:1 between the concentration of SNAP and the resulting NO concentration with the assumption of a linear relationship between the two concentrations , the concentration of NO can be estimated at 50–100 pM.
To determine the role of PI3-kinase in the microvascular responses to insulin, a sixth group (n = 6) of vessel segments was treated for 30 min with wortmannin (50 nM), a PI3-kinase inhibitor, prior to insulin addition. This concentration of wortmannin has been shown to inhibit insulin-induced PI3-kinase activity by approximately 85% in rat adipocytes . To study the role of endothelin in these responses, responses to insulin were studied in the presence of both wortmannin and PD-142893 in another group (n = 5).
Effects of the different inhibitors on basal vessel diameter were investigated in time-matched control groups in which insulin solvent (MOPS buffer+0.1% BSA) was added to the bath at the same time points as the insulin additions in the other experiments. For comparison, diameter changes after the first, second, third and fourth addition of solvent are given the same x-axis coordinates as the diameter changes after the first, second, third and fourth concentration step of insulin.
Steady-state responses are reported as mean changes in diameter from baseline (in percent)±standard error of mean (S.E.M.). The baseline diameter is defined as the arteriolar diameter just before addition of insulin. Therefore, responses are corrected for diameter changes during pre-treatment. Differences between two means were assessed by an unpaired, two-tailed Mann–Whitney U-test, and differences were considered statistically significant if P<0.05. Diameter changes during pre-treatment were tested against a hypothetical value of 0 with a Wilcoxon signed-rank test. Non-parametric tests were chosen because of the small sample size and because percentages are not likely to be normally distributed.
MOPS buffer consisted of (in mM) 145 NaCl, 4.7 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.2 NaH2PO4, 11.2 dextrose, 2.0 pyruvate, 0.02 EDTA and 3.0 MOPS. Bovine serum albumin (BSA) was added to insulin stock solutions at 0.1% (m/v) to reduce loss of insulin by adhesion to the vessel bath. pH of the buffer was set at 7.4. All salts and MOPS were of analytic grade and purchased from Merck (Darmstadt, Germany). Pyruvate, l-NNA, SNAP, BSA, papaverine, wortmannin and acetylcholine were obtained from Sigma (St. Louis, MO). Human insulin (Velosulin®) was obtained from Novo Nordisk, The Netherlands. The non-selective endothelin receptor antagonist PD-142893 was obtained from Kordia (Leiden, The Netherlands)
The glucose concentration used has been reported as optimal for the culture of isolated vessel segments [11,29]. Since it is essentially a hyperglycemic concentration, acute effects of insulin alone at a euglycemic glucose concentration, 5.5 mM, were studied in a separate series of four experiments. Acute responses to insulin were virtually identical at the two glucose concentrations used (Fig. 1).
3.1 General characteristics
Passive intraluminal diameters of vessel segments averaged 181±3 μm (n = 63) when pressurized to 65 mmHg. During the equilibration period all vessel segments developed spontaneous tone ranging from 40 to 60% of their passive diameters, reducing the diameter by 93±2 μm (51±1%) to 88±2 μm. When stimulated with the endothelium-dependent vasodilator ACh (0.1 μM) at the start of the experiment, all vessels dilated by more than 10% for more than 6 min. Responses of untreated control vessels (n = 6) to acetylcholine at the beginning and the end of experiments averaged 35±13 and 45±10% (P = 0.59), indicating vessel stability in this setup.
3.2 Endothelin receptor blockade reveals a nitric oxide-mediated vasodilator effect of insulin at high physiological to pharmacological concentrations
Insulin alone (n = 9) did not induce significant changes in vessel diameter compared to time-matched controls (n = 6; Fig. 1). In contrast, pre-treatment with the non-selective endothelin receptor antagonist PD-142893 uncovered a concentration-dependent vasodilatory effect of insulin (Fig. 2A). While PD did not affect vessel diameter in the pre-treatment period (Table 1), vasodilatation in response to insulin in the PD-treated group (n = 8) was significantly different from controls (n = 6) at 272 μU/ml (13±5 vs. −5±5% from baseline, P = 0.02) and 3.4 mU/ml (25±8 vs. −4±3% from baseline, P = 0.01). The vasodilatation was significantly different from the group treated with insulin alone at 272 μU/ml (13±5 vs. −1±5% from baseline, P = 0.03), indicating interference of endothelin receptor blockade with effects of insulin. There were no significant differences between controls treated with PD-142893 and controls without PD (not shown).
|% Change in||No. of||P value|
|% Change in||No. of||P value|
The vasodilatory effects of insulin during endothelin receptor blockade were abolished by NOS inhibition (Fig. 2B), indicating that nitric oxide mediates the insulin-induced vasodilatation. Pre-treatment with the NOS inhibitor l-NA for 30 min resulted in a constriction of 6% from baseline both in the presence and absence of the non-selective endothelin receptor antagonist PD-142893 (Table 1). When corrected for this constriction, there were no significant differences between controls and l-NA-treated vessels. In contrast, l-NA abolished insulin/PD-induced vasodilatation at 272 μU/ml (13±5 vs. −8±4% from baseline, P = 0.01) and 3.4 mU/ml (25±8 vs. −8±6% from baseline, P = 0.01)
3.3 Inhibition of nitric oxide synthesis reveals a endothelin-mediated vasoconstrictor effect of insulin at physiological concentrations
During blockade of nitric oxide synthesis with l-NA, vessel segments treated with insulin (n = 6) constricted more than time-matched controls (n = 8) at insulin concentrations of 4 μU/ml (−17±5 vs. −6±3% from baseline, P = 0.04; Fig. 3) and 34 μU/ml (−21±5 vs. −6±4% from baseline, P = 0.008). At insulin concentrations of 272 μU/ml and 3.4 mU/ml, there were no significant differences between insulin-treated vessels and time-matched controls. Constrictions started within 5 min after insulin addition and reached a maximum after 20–25 min of incubation.
The insulin-induced vasoconstriction in the presence of l-NA was abolished by pre-treatment with PD-142893, a non-selective inhibitor of endothelin-1 receptors (Fig. 3). PD-142893/l-NA-treated vessels (n = 5) showed attenuated constriction compared to l-NA-treated vessels in the presence of 34 μU/ml of insulin (−5±3% from baseline in vessels treated with l-NA and PD versus −21±5% from baseline in vessels treated with l-NA, P = 0.009).
3.4 Nitric oxide inhibits insulin-induced vasoconstriction
SNAP, a donor of nitric oxide, reversed insulin-induced vasoconstriction in the presence of l-NA (Fig. 4A) but did not produce net vasodilatation at the concentrations used (Table 1). Constriction in response to l-NA alone was not significantly altered in SNAP/l-NA-treated segments (n = 4) compared to l-NA-treated segments (−1±3 vs. −6±2% from baseline), but abolished insulin-induced vasoconstriction at 4 μU/ml (0±1 vs. −17±5% from baseline, P = 0.02) and 34 μU/ml (−1±4 vs. −21±5% from baseline, P = 0.01).
3.5 Inhibition of PI3-kinase uncovers insulin-induced, endothelin-mediated vasoconstriction
Pre-treatment of vessel segments (n = 11) with wortmannin, an inhibitor of PI3-kinase, resulted in a constriction of 3±2% of the baseline diameter (P = 0.16; Table 1). In the presence of wortmannin, segments (n = 6) constricted by 13±3% from baseline after addition of insulin (34 μU/ml), whereas time-matched controls (n = 5) constricted by 3±2% from baseline (Fig. 4B; insulin vs. solvent: P = 0.01). Additional treatment with the non-selective ET-1 receptor antagonist PD-142893 (n = 5) abolished this insulin-induced vasoconstriction (Fig. 4B; P = 0.03).
The aims of this study were to determine the role of nitric oxide and endothelin-1 in acute responses to insulin of rat skeletal muscle arterioles and to elucidate the role of PI3-kinase in those responses. The principal findings of this study are that (1) insulin alone does not induce significant diameter changes; (2) endothelin-1 receptor blockade reveals a nitric oxide-dependent vasodilator effect of insulin, at high physiological to pharmacological concentrations; (3) physiological concentrations of insulin induce endothelin-1-mediated vasoconstriction during impairment of nitric oxide synthesis, which disappears at high physiological to pharmacological concentrations; and (4) insulin at a physiological concentration induces endothelin-1-mediated vasoconstriction during inhibition of PI3-kinase.
4.1 Effects of insulin alone
In our series of experiments insulin did not induce significant vasodilatation within 30 min. This finding is in agreement with some studies in humans [14,17,18], isolated rat aorta  and rat mesentery , but differs from reports of acute insulin-induced vasodilatation in humans [4–7] and in rats [8,10,21,30]. In rat skeletal muscle first-order arterioles, some investigators have reported acute insulin-induced vasodilatation in vitro [11,12], while others found no acute effect of insulin in these arterioles in vivo . Vasodilating effects of physiological concentrations of insulin may differ between different vascular beds  or may be localized in arterioles of the higher branching orders . Finally, recent reports have suggested that insulin induces ET-1 activity to attenuate nitric oxide-mediated vasodilatation [14,15,19].
When comparing our results with those of in vivo studies, some practical aspects of our setup have to be considered. First, abluminal administration of insulin, a 6-kDa protein, might attenuate endothelium-dependent effects of insulin. However, responses were observed at low concentrations of insulin in our experiments and vasoconstrictive effects as well as NO-dependent vasodilating effects started within minutes. Moreover, pilot experiments showed no effect of insulin alone and approximately 15% constriction in response to insulin in the presence of wortmannin when administered to the vessel lumen. These observations argue against penetration of insulin into the vessel as a factor influencing our results. Second, interactions of insulin with effects of blood components or blood flow on vascular tone have not been investigated in this study. While blood flow is a well-known stimulator of nitric oxide production, NO has a much shorter half-life in vivo than in vitro because of an abundance of NO scavengers. Therefore, the role of NO may be a different one in vivo. Finally, small effects of vasoactive substances on arteriolar diameter in vivo may have large effects on flow, for static flow through a blood vessel is related to the fourth power of its radius. For example, a diameter change of 10% in a blood vessel, at constant pressure, would result in a 46% increase in flow.
4.2 Roles of NO and ET-1 in effects of insulin on microvascular tone
Our results support a balance between vasoconstrictive and vasodilatory effects of insulin, with functional roles for nitric oxide and endothelin in both processes. In agreement with recent reports, inhibition of endothelin activity revealed an acute, nitric oxide-dependent vasodilatory effect of insulin [14,19]. The finding of mediation of insulin's vasodilatory effects by NO is in agreement with earlier reports [5,13,16]. In the mesenteric  and coronary beds , some evidence exists for a role of KCa channels in insulin-induced vasodilatation, and our results do not exclude a role for those channels. In other experiments, a role for vasodilatory prostaglandins has been proposed in the aorta . However, since insulin does not induce vasodilatation in the absence of both endothelin and NO activity, a dilating mechanism in our setup independent of NO is not very likely.
In addition, inhibition of NO synthesis uncovered acute, insulin-induced, ET-1-mediated vasoconstriction at physiological insulin concentrations (4–34 μU/ml). The role of endothelin in insulin-induced vasoconstriction may include stimulation of ET-1 release by insulin, reported in cultured endothelial cells , or insulin-induced upregulation of expression of ET-1 receptors on vascular smooth muscle cells , or both. Since stimulation of ET-1 release by insulin, in contrast to the effect on ET-1 receptor expression, occurs within 1 h, ET-1 release probably plays a greater role than receptor expression in insulin-induced vasoconstriction.
The vasoconstrictive effect of insulin was diminished at the higher concentrations used (0.272–3.4 mU/ml), suggesting an effect of insulin aside from NO and ET-1 at these concentrations. This effect could be an attenuation by insulin of endothelin-induced calcium influx, which has been reported at high insulin concentrations .
The endothelin receptor subtype mediating the vasoconstrictive effects of insulin in our setup remains elusive. While the type A endothelin receptor (ETA) is mainly present on smooth muscle cells and mediates vasoconstriction, the type B receptor is present on both endothelial cells, stimulating synthesis of NO, and smooth muscle cells, stimulating vasoconstriction. In isolated cremaster arterioles of lower branching orders of normal rat, ETA receptors are likely the predominant mediators of endothelin-induced vasoconstriction, since this constriction could be fully inhibited by the ETA-antagonist in another study .
4.3 Interaction between ET-1 and NO in microvascular responses to insulin?
One might hypothesize that the sum of insulin's separate effects on NO and ET-1 determines its net acute effect on microvascular tone. However, the different course of dose–response relations of insulin-induced constriction and dilatation (Figs. 2 and 3) argue against a simple balance between two independent effects. First, endothelin-dependent vasoconstriction is maximal at 34 μU/ml of insulin (Fig. 3), but blocking endothelin receptors does not reveal insulin-induced dilatation at that concentration (Fig. 2). Second, maximal NO-dependent vasodilatation occurs at a concentration of insulin of 3.4 mU/ml (Fig. 2), but NOS inhibition does not reveal insulin-induced vasoconstriction at that concentration (Fig. 3). The first point suggests that NO interferes with insulin-induced vasoconstriction, even at insulin concentrations insufficient to induce vasodilatation. In agreement with this, exogenous nitric oxide inhibited insulin-induced vasoconstriction in our study. This effect occurred in the absence of insulin-induced production of nitric oxide, at concentrations of nitric oxide which do not invoke net vasodilatation. This attenuation of insulin-induced, endothelin-1-dependent vasoconstriction may involve inhibition of endothelin production  or endothelin post-receptor signaling .
The second problem follows from the interaction of ET-1 receptor blockade with insulin-induced vasodilatation, at insulin concentrations that do not induce ET-1-dependent vasoconstriction during NOS inhibition. This can be explained by direct interaction of ET-1 with NO-induced vasodilatation, supported by a report of interaction of ET-1 with activation of guanylate cyclase by NO in rat cremaster arterioles .
In conclusion, our results support dual roles of both NO and ET-1 in the effects of insulin on vascular tone, inhibiting each other's effects as well as mediating insulin-induced vasodilatation and vasoconstriction, respectively.
4.4 Role of PI3-kinase in microvascular effects of insulin
Impaired activation of PI3-kinase in skeletal muscle tissue by insulin is an important feature of insulin resistance in humans  and rats , Moreover, activation of PI3-kinase by insulin is specifically impaired in the vasculature of Zucker rats . Although the involvement of PI3-kinase in insulin-induced NO production has been well-documented in recent years  it is unknown whether PI3-kinase mediates insulin's vasoconstrictor effects. In our experiments, insulin-induced vasoconstriction at 34 μU/ml in the presence of wortmannin, an inhibitor of PI3-kinase. In analogy with the insulin-induced vasoconstriction observed during NOS inhibition, this insulin-induced vasoconstriction in the presence of wortmannin is also mediated by endothelin (Fig. 4B). Yet, the insulin-induced vasoconstriction at 34 μU/ml was more pronounced during NOS inhibition than during inhibition of PI3-kinase. This can be explained by the fact insulin's stimulation of NOS is not totally dependent on PI3-kinase activation , but possibly by calcium-dependent mechanisms as well. Overall, these results suggest that the balance between vasodilator and vasoconstrictor effects of insulin is altered during inhibition of insulin-induced, PI3-kinase-dependent activation of NOS.
In summary, a balance of nitric oxide and endothelin-1 determines acute responses of isolated rat cremaster first-order arterioles to insulin. While endothelin receptor blockade reveals nitric oxide-mediated dilatation at pharmacological concentrations of insulin, insulin induces endothelin-mediated vasoconstriction at physiological concentrations during inhibition of nitric oxide synthesis. Nitric oxide has a dual role in these responses, inhibiting endothelin-mediated vasoconstriction as well as mediating insulin-induced vasodilatation. Inhibition of PI3-kinase reveals a vasoconstrictor effect of insulin, indicating that insulin's vasodilator and vasoconstrictor effects are differentially regulated. To our knowledge, this is the first functional evidence that physiological concentrations of insulin induce endothelin-mediated vasoconstriction during inhibition of nitric oxide and that PI3-kinase inhibition reveals insulin-induced vasoconstriction. These data support the hypotheses that the role of ET-1 in vascular effects of insulin is enhanced during diminished production of nitric oxide and that activation of PI3-kinase is an important determinant of vascular responses to insulin.