INTRODUCTION

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INSULIN RESISTANCE IS A risk factor for stroke in humans (21, 27); however, the underlying mechanisms have not yet been clarified. One possible factor in the elevated risk of stroke may be the impairment of endothelium-mediated relaxation in cerebral arteries (5). In our previous study (5), we demonstrated that responses to endothelium-dependent dilator agents are reduced in the middle cerebral arteries (MCAs) of insulin-resistant (IR) animals as a result of impaired cyclooxygenase (COX)-dependent relaxation, whereas nitric oxide (NO)-mediated responses remain intact.

ATP-dependent and Ca²⁺-activated K⁺ channels (K_ATP and K_Ca channels, respectively) are important targets of mediators released from the endothelium including those that are produced by COX (8, 11, 25, 29). In addition, it has been shown previously that insulin resistance alters the function of these ion channels in mesenteric and coronary arteries and leads to diminished endothelium-dependent relaxation (13, 15, 22, 23). Therefore, we hypothesized that insulin resistance affects the K_ATP and K_Ca channel functions in cerebral arteries and ultimately leads to altered vascular regulation. No previous studies have addressed this issue with regard to the cerebral circulation.

In this study, the functions of K_ATP and K_Ca channels were examined in isolated MCAs of control and IR rats. We studied the roles of these ion channels in the regulation of spontaneous vascular tone as well as in endothelium-dependent dilation induced by bradykinin (BK). In addition, we assessed the function of K_ATP channels with the subtype-specific channel opener cromakalim. We used a well-established model in which insulin resistance was induced by the feeding of a high-fructose diet. Fructose-fed rats within the conditions of the current study are characterized as having hyperinsulinemia and dyslipidemia (i.e., hypertriglyceridemia and decreased levels of high-density lipoprotein) yet are normotensive and normoglycemic (14, 23).

	METHODS

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The experimental protocol was approved by the Animal Care and Use Committee at Wake Forest University School of Medicine. Male Sprague-Dawley rats were obtained at 6 wk of age and randomized into one of two groups: control (n = 35) and IR (n = 33). Animals in the IR group were fed a fructose-rich diet that contained 66% fructose, 22% casein, and 12% lard plus essential vitamins and minerals (Teklad Labs; Madison, WI), whereas control animals received standard rat chow. After a 4-wk diet treatment, the rats (in a fasting state) were anesthetized with pentobarbital sodium (50 mg/kg ip) and anticoagulated with heparin sodium (500 U ip).

Biochemical measurements. To confirm that fructose-fed rats were indeed insulin resistant, we measured plasma insulin and glucose levels after 12 h of fasting using a rat-insulin ELISA kit (Crystal Chem; Chicago, IL) and Trinder reagent (Sigma; St. Louis, MO), respectively. Fasting glucose levels were similar between the two groups (control rats, 117 ± 2; IR rats, 134 ± 3 mg/dl), whereas insulin was markedly increased in the IR rats (control rats, 649 ± 87; IR rats, 1,781 ± 331 pg/ml; P < 0.05). The mean body weight was similar in the two groups of animals (control, 311 ± 7; IR rats, 313 ± 5 g; P = not significant).

Determination of vascular reactivity. Methods have been described previously in detail (5). Briefly, after each rat was decapitated, the brain was immediately removed and placed in cold, oxygenated modified Krebs-Ringer bicarbonate solution [that contained (in mM) 119 NaCl, 4.7 KCl, 24 NaHCO₃, 1.18 KH₂PO₄, 1.17 MgSO₄, 0.026 EDTA, 1.6 CaCl₂, and 5.5 glucose]. Both MCAs were carefully harvested, and a section of MCA (~2 mm in length) was transferred to a vessel chamber and mounted and secured between two glass micropipettes. The vessel chamber was transferred to an inverted light-microscope stage, and a video dimension analyzer (Living Systems Instrumentation; Burlington, VT) was used to measure the intraluminal diameter. Oxygenated (20% O₂-5% CO₂-75% N₂) Krebs solution maintained at 37°C was continuously circulated through the vessel bath, and the lumen of the artery was also filled with Krebs solution through the micropipettes. The outflow cannula was clamped off while the inflow cannula was connected to an elevated reservoir to maintain a constant intraluminal pressure of 80 mmHg. After the MCAs were mounted and pressurized, the MCAs from either control or IR rats developed spontaneous tone by constricting to ~70% of the initial diameter over the course of 1 h. Drugs were added abluminally into the bath solution, and only one concentration-response experiment was performed per artery.

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Chemicals. BK, L-NAME, TEA, IBTX, glibenclamide, and ET-1 were obtained from Sigma, whereas cromakalim was obtained from Tocris Cookson. All drugs were dissolved in Krebs solution except for cromakalim, which was dissolved in DMSO and Krebs solution. The same concentration of DMSO alone had no effect on vessel diameter.

Data analysis. All data are expressed as means ± SE. The magnitudes of spontaneous vascular tone and treatment-induced vasoconstriction were calculated as percentages of maximal intraluminal diameter, whereas vascular relaxations were calculated as percentages of preconstriction. Maximal diameter was measured as the initial diameter immediately after pressurization; for control, maximal diameter was also measured at the end of the experiments after complete relaxation with sodium nitroprusside (3 × 10⁴ M). It was shown previously in the same experimental model that the initial diameter of the arteries is the same as the maximal diameter that was obtained in Ca²⁺-free solution (30). The concentration-response curves were evaluated at each concentration for differences between treated and untreated arteries from control and IR groups using ANOVA and subsequent Tukey's post hoc test. Changes in spontaneous vascular tone in control and IR MCAs after administration of specific K⁺ channel blockers were compared using Student's t-test. The criterion for significance was P < 0.05.

	RESULTS

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The maximal intraluminal diameter of the MCAs did not differ between the two groups (control, 227 ± 2, n = 58; IR, 223 ± 2 µm, n = 54). The spontaneously developed tone was also similar in that the MCAs constricted to 72 ± 1% and 74 ± 1% of the initial diameter in the control and IR groups, respectively.

Blockade of the K_Ca channels with TEA significantly augmented the spontaneous vascular tone of the arteries from both control and IR rats by an additional 10.5 ± 1.3% (n = 10) and 8.0 ± 1.1% (n = 10), respectively (Fig. 1; difference not significant). The selective BK_Ca-channel blocker IBTX induced similar vasoconstriction in the control MCAs (13.3 ± 2.3%, n = 6) but changed the vascular diameter only by 3.1 ± 0.9% (n = 8) in IR arteries (difference, P < 0.01). Inhibition of the K_ATP channels with glibenclamide did not alter the vascular tone in either of the two groups. Vascular diameter decreased by 1.7 ± 0.5% (n = 8) and 1.2 ± 0.5% (n = 7) in the control and IR arteries, respectively (Fig. 1).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Effects of tetraethylammonium chloride (TEA), iberiotoxin (IBTX), and glibenclamide (GLIB) on spontaneously developed vascular tone. TEA constricted significantly both the control (C) and insulin-resistant (IR) arteries. IBTX induced significant vasoconstriction in the control middle cerebral arteries (MCAs) but had only a weak effect on the IR arteries. Glibenclamide had no effect on the spontaneous vascular tone. *P < 0.05, constriction compared with spontaneous vascular tone.

BK induced concentration-dependent relaxation in both the control and IR MCAs; however, responses were significantly reduced in the IR group (Fig. 2). Maximum relaxation to BK was 42 ± 4% in control MCAs (10⁶ M, n = 9) and only 19 ± 2% in the IR arteries (10⁶ M, n = 10; P < 0.01). As we have shown previously (5), in control MCAs, relaxation to BK is mediated by COX and NO, and the responses can be abolished by the combination of L-NAME and indomethacin. In contrast, the already-reduced dilation in IR MCAs depends entirely on NO and can be eliminated by L-NAME alone. However, relaxation to sodium nitroprusside was similar in the control and IR arteries.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Cumulative dose-response curves to bradykinin (BK) in MCAs of control and IR rats. BK-induced dilation is significantly reduced in IR MCAs. *P < 0.01 compared with control group.

In control arteries, blockade of K_Ca channels with either the nonselective K_Ca channel blocker TEA or the selective BK_Ca channel blocker IBTX reduced the dilation to BK (Fig. 3A). Maximum relaxations to BK were 19 ± 2 (n = 6) and 22 ± 1% (n = 6) in the presence of TEA and IBTX, respectively. In contrast, in IR arteries, neither TEA nor IBTX pretreatment had any effect on the already diminished BK-induced vascular responses (Fig. 3B). Maximum relaxations were 20 ± 3% (n = 7) and 23 ± 3% (n = 8) in the presence of TEA and IBTX, respectively. The combination of L-NAME and TEA completely blocked the BK-induced dilation in both the control and IR MCAs: the maximum changes in diameter were 3 ± 2% (n = 7) and 1 ± 4% (n = 4), respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Cumulative dose-response curves to BK in MCAs of control (A) and IR (B) rats. Application of TEA or IBTX reduced the BK-induced relaxation significantly in control MCAs but had no effect in IR arteries. Combination of N-nitro-L-arginine methyl ester (L-NAME) and TEA completely abolished the BK-induced dilation in both control and IR MCAs. Increased vascular tone induced by endothelin-1 (ET-1) had no effect on responses to BK. aP < 0.05, treated vs. untreated groups; bP < 0.05 compared with arteries treated with TEA alone.

To examine whether the changes in BK-induced relaxations after K_Ca channel blockade were not simply the consequence of the increased vascular tone, we constricted the arteries to a similar level by applying the appropriate amount of ET-1 without blocking the K_Ca channels. Thus the vascular tone of MCAs from control and IR rats was augmented by 12.5 ± 2.6% (n = 4) and 12.0 ± 2.2% (n = 4), respectively, but responses to BK did not differ significantly from those of the untreated arteries (Fig. 3). Maximum relaxations to BK were 35 ± 5% (n = 4) and 23 ± 3% (n = 4) in the control and IR arteries, respectively.

Blockade of the K_ATP channels with glibenclamide also reduced the dilation to BK in the control MCAs [maximum relaxation was 21 ± 3% (n = 8); Fig. 4A]. In contrast, glibenclamide had no effect on BK-induced responses in the IR arteries [maximum relaxation to BK after glibenclamide administration was 19 ± 4% (n = 7); Fig. 4B]. The combination of L-NAME and glibenclamide completely abolished BK-induced dilation in both the control and IR arteries. Maximum changes in diameter were 3 ± 2% (n = 6) and 1 ± 2% (n = 4), respectively. Coadministration of TEA and glibenclamide had no additional effect in either the control or IR MCAs. Relaxation to BK was similar to the responses after TEA or glibenclamide administration alone. Maximum dilations were 18 ± 3% (n = 6) and 23 ± 1% (n = 4) in the control and IR arteries, respectively (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Cumulative dose-response curves to BK in MCAs of control (A) and IR rats (B). Application of glibenclamide reduced the BK-induced relaxation significantly in control MCAs but had no effect in IR arteries. Combination of L-NAME and glibenclamide completely abolished the BK-induced dilation in both control and IR MCAs. Coadministration of glibenclamide and TEA had no additional effect in either group of arteries. aP < 0.05, treated vs. untreated groups; bP < 0.05 compared with arteries treated with glibenclamide alone.

Finally, the function of the K_ATP channels was examined with the specific channel opener cromakalim. Cromakalim induced dose-dependent dilation in both the control and IR MCAs; however, the relaxation was markedly reduced in the IR arteries (Fig. 5). Maximum dilations to cromakalim were 48 ± 3% (10⁵ M, n = 6) and 19 ± 2% (10⁵ M, n = 6) in the control and IR arteries, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Cumulative dose-response curves to cromakalim in MCAs of control and IR rats. Cromakalim-induced dilation was significantly reduced in IR MCAs compared with controls. *P < 0.01 compared with control group.

	DISCUSSION

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The major finding of this study is that the functions of both K_Ca and K_ATP channels are altered in the cerebral arteries of IR rats. K_Ca channels are involved in the control of spontaneous vascular tone, and the blockade of these ion channels leads to significant vasoconstriction. Although the spontaneous vascular tone and the vasoconstriction induced by nonspecific blockade of the K_Ca channels are similar in the control and IR groups, it seems that the role of BK_Ca channels in the regulation of resting vascular tone is diminished in IR arteries compared with controls. Moreover, in control arteries, K_ATP and K_Ca channels contribute to the endothelium-dependent relaxation elicited by BK; however, in IR arteries, the relaxation mediated by these ion channels is missing. Dilation to the K_ATP channel opener cromakalim is also reduced in IR MCAs compared with control arteries. Thus insulin resistance alters the function of K_ATP and K_Ca channels in the regulation of both basal and stimulated vascular control mechanisms.

K_Ca channels play a key role in buffering pressure-induced constriction of small cerebral arteries (4, 9, 18). Thus in control arteries, both TEA (a nonspecific K_Ca channel blocker) and IBTX (a specific BK_Ca channel blocker) induced significant vasoconstriction of similar magnitude. These data are in accordance with previous studies (4, 9, 18) that suggest that under normal conditions, the BK_Ca channel is the major K_Ca channel subtype that regulates vascular tone. In contrast, specific blockade of the BK_Ca channels in IR arteries does not lead to significant vasoconstriction in addition to the pressure-induced tone. These data indicate that the role of BK_Ca channels in buffering the pressure-induced constriction is reduced in insulin resistance. The decreased K⁺ efflux through the BK_Ca channels would be expected to result in increased vascular tone. However, there was no significant difference in the resting tone of arteries from the two experimental groups. This could be due to a compensatory increase in the contribution of other K_Ca channels, because TEA, which also blocks the intermediate- and small-conductance K_Ca channels in addition to the BK_Ca channels, induced a similar vasoconstriction in the IR arteries compared with the control arteries. Thus it is possible that these subtypes of the K_Ca channel family, which are probably not affected by insulin resistance, compensate for the loss of BK_Ca channel function. It should be kept in mind that the dysfunction of BK_Ca channels might be compensated in this way in isolated arteries, but in vivo, the control of vascular tone is more complex and particularly in certain pathological conditions such as hypertension, the role of BK_Ca channels is more important (18). In these cases, alteration in BK_Ca channel function may have a more significant impact.

K_ATP and K_Ca channels play an important role in the mediation of numerous dilator stimuli in the cerebral arteries including endothelium-dependent relaxation (for review, see Refs. 6 and 7). BK, for example, has been shown to activate these ion channels in several circulatory beds including cerebral circulation (10, 16, 24, 28). In a previous study, we showed that in isolated MCAs of normal rats, BK-induced relaxation is mediated by NO and COX derivates (5). Products of COX [i.e., prostacyclin or reactive oxygen species (ROS)] are well-known openers of K_ATP and K_Ca channels (8, 11, 25, 29), and the data of the present study show that the NO-independent, COX-mediated responses to BK require the opening of these K⁺ channels. In control arteries, glibenclamide reduced the magnitude of relaxation to BK and in combination with L-NAME led to complete blockade of BK-induced responses. Similarly, both TEA and IBTX diminished dilation to BK, and the combined blockade of NO synthesis and K_Ca channels completely abolished the BK-induced dilation. Furthermore, the fact that IBTX and TEA had similar effects suggests that the dominant K_Ca channel subtype in this mechanism is the BK_Ca channel.

Although BK has been shown to activate either K_Ca or K_ATP channels (10, 16, 24, 28) depending on the species and the circulatory area examined, it was a surprising finding of the present study that in isolated rat MCAs, these two types of K⁺ channels are both involved in the mediation of BK-induced dilation and contribute to the same regulatory pathway. Combined blockade of K_ATP and K_Ca channels with TEA and glibenclamide did not further inhibit the BK-induced relaxations. Previously, Schubert et al. (25) reported that relaxation to the prostacyclin analog iloprost involves the activation of both types of K⁺ channels, and the combined blockade of the K_ATP and K_Ca channels provides no additional effects. However, the mechanisms that connect the activation of these two ion channels remain to be clarified.

Despite this uncertainty regarding the mechanisms that induce the simultaneous activation of K_ATP and K_Ca channels in control arteries, it is evident that in IR arteries, neither type of K⁺ channel plays a role in the BK-induced relaxation. The already reduced dilations in IR MCAs were not affected by the application of glibenclamide, TEA, or IBTX. The combined blockade of K_ATP and K_Ca channels was also ineffective. Thus the present data and our previous findings (5) demonstrate that BK-induced dilation is reduced in insulin resistance, and this appears to be due to impaired K⁺ channel activation by COX metabolites leaving BK-induced relaxation entirely dependent on NO.

It is not clear at this time what kinds of mechanisms are responsible for the insulin resistance-induced impairment of this pathway. Theoretically, both the reduced endothelial release of COX products and the dysfunction of vascular smooth muscle K⁺ channels activated by these mediators can explain our findings. However, several facts suggest that the function of K_ATP and BK_Ca channels is altered in insulin resistance. The data of the present study demonstrate that relaxation to cromakalim is significantly reduced in IR arteries compared with control MCAs, which indicates that K_ATP channel mediated dilation was impaired even when the ion channels were stimulated by a specific channel opener. Although a similar specific agonist for the K_Ca channels is not available, the fact that BK_Ca channel function in the regulation of resting tone is diminished in IR MCAs indicates that these ion channels are also affected by insulin resistance. Moreover, insulin resistance impairs the function of K_ATP and BK_Ca channels, probably via the same mechanisms, in other circulatory beds such as small coronary and mesenteric arteries, which also leads to reduced endothelium-mediated dilation (13, 15, 22, 23).

Further investigation is needed to reveal how the changes in insulin signaling affect the function of these vascular smooth muscle cell K⁺ channels. One possibility is that insulin resistance increases the production of ROS in the vascular tissues, which influences the structure and activity of these ion channels. The following evidence supports this hypothesis. First, superoxide production has been shown to be elevated in aortas of fructose-fed rats due to the activation of endothelial NADH/NADPH oxidase (12) and the dysfunction of endothelial NO synthase caused by abnormal biopterin metabolism (26). Second, K⁺ channel mediated relaxations are impaired in pathophysiological conditions when excessive ROS production occurs, for example, during ischemia-reperfusion (3, 20), brain injury (1, 2), or hyperglycemia (17, 19). Thus the impairment of K_ATP and K_Ca channel mediated vascular responses in IR cerebral arteries could also be the consequence of elevated ROS production.

In conclusion, results of the present study indicate that high-fructose-diet-induced insulin resistance impairs the function of BK_Ca and K_ATP channels in rat MCAs, and the dysfunction of these K⁺ channels influences the control of resting vascular tone as well as the mediation of endothelium-dependent relaxation. The altered cerebrovascular regulation may be responsible for the increased risk of cerebrovascular events in insulin resistance.