Insulin-resistance (IR) impairs agonist-induced relaxation in cerebral arteries, but little is known about its effect on constrictor mechanisms. We examined the vascular responses of the basilar artery (BA) and its side branches in anesthetized Zucker lean (ZL) and IR Zucker obese (ZO) rats using a cranial window technique. Endothelin-1 (ET-1) constricted the BAs in both the ZL and ZO rats, but there was no significant difference between the two groups (ZL: 36 ± 8%; ZO: 33 ± 3% at 10−8 M). Inhibition of the ETA receptors by BQ-123 slightly increased the diameters of the BAs, with no difference shown between the ZL (6 ± 1%) and ZO (5 ± 3%) rats. Expressions of the ETA receptors and ET-1 mRNA examined by immunoblot analysis and RT-PCR, respectively, were also similar in the ZL and ZO groups. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), and the thromboxane A2 (TxA2) mimetic U-46619 constricted the BAs, but similarly to ET-1, there was no significant difference between the ZL and ZO groups (10−6 M PDBu: ZL: 33 ± 2%; ZO: 32 ± 4%; and 10−7 M U-46619: ZL: 23 ± 1%; ZO: 19 ± 2%). Inhibition of Rho-kinase with Y-27632 induced dilation of the BAs, and these responses were also comparable in the ZL and ZO rats (ZL: 39 ± 4%; ZO: 38 ± 2% at 10−5 M). In contrast, nitric oxide-dependent relaxation to bradykinin was significantly reduced in the ZO rats (10−6 M: 10 ± 3%) compared with ZLs (29 ± 7%, P < 0.01). These findings indicate that vasoconstrictor responses of the BA mediated by ET-1, TxA2, PKC, and Rho-kinase are not affected by IR.
- protein kinase C
- rho kinase
insulin resistance (IR) and type 2 diabetes increase the prevalence of cerebrovascular events, and IR patients with stroke are subject to more severe progression, slower recovery, and higher mortality (14, 25, 28, 35). As we showed previously in dietary (5–7) and genetic models (8) of IR, these adverse effects of IR are probably due to a reduced ability of the cerebral arteries to respond to dilator stimuli. Fructose-rich diet-induced IR leads to the impairment of vascular smooth muscle cell (VSMC) K+ channel-dependent relaxation in isolated cerebral arteries, which also affects endothelial cyclooxygenase-mediated dilator pathways (5–7). Also, both K+ channel-mediated relaxation and endothelial nitric oxide (NO)-dependent dilations are impaired in the basilar artery (BA) of the leptin receptor knockout (fa/fa) Zucker obese rats (8). However, the effect of IR on cerebrovascular constrictor mechanisms has not yet been investigated, despite the fact that numerous findings in the peripheral circulation indicate a possible role of altered vasoconstrictor mechanisms in the pathology of IR-induced vascular dysfunction.
Endothelin-1 (ET-1)-mediated vascular responses are augmented in mesenteric arteries and aorta of IR rats due to increased production of ET-1 and overexpression of the ETA receptor (15, 17). ET-1 serum levels are higher in human subjects with syndrome X compared with healthy individuals, and ET-1 production can be elevated by simultaneous increases in insulin and triglyceride levels (31). Furthermore, resistin, a possible mediator of IR, promotes endothelial cell activation and elevated ET-1 release in human saphenous vein endothelial cell culture (39). Thus increased amounts of ET-1 released from the endothelium or upregulation of the ETA-receptor in the VSMC could result in protein kinase C (PKC) activation, elevated intracellular Ca2+ concentration ([Ca2+]i), enhanced Ca2+ sensitivity, and increased vascular tone (11, 29, 37, 42). These effects might antagonize decreases in VSMC [Ca2+]i elicited by dilator stimuli, and the increased PKC activity, either directly or by enhancing production of reactive oxygen species, can lead to reduced responsiveness to NO and K+ channel-mediated relaxation (1, 3, 22).
In addition to the changes in VSMC [Ca2+]i, which determine the activity of the myosin light chain (MLC) kinase (MLCK), vascular tone is also controlled by the MLC phosphatase (MLCP), whose activity is predominantly regulated by the RhoA/Rho-kinase (ROK) pathway (reviewed in Ref. 37). RhoA/ROK is activated by several vasoconstrictor agents including catecholamines, ET-1, thromboxane, histamine, and serotonin, and ROK activation leads to MLCP inhibition and increased Ca2+ sensitivity. Insulin inhibits ROK activity via the NO-cGMP pathway (33), and this effect is abolished in type 2 diabetic rats (34), resulting in increased MLC phosphorylation and VSMC contraction. Such an increase in ROK activity in the IR cerebral arteries could explain both diminished NO and K+ channel-mediated dilation. It could antagonize the inhibitory effect of the NO-cGMP pathway on ROK, and, by increasing Ca2+ sensitivity, the decrease in [Ca2+]i after K+ channel-dependent hyperpolarization and reduced Ca2+ entry through the voltage-dependent Ca2+ channels would result in a reduced vascular relaxation.
Thus to examine whether IR indeed modifies vasoconstrictor mechanisms of the cerebral arteries, we evaluated vascular responses of the basilar artery (BA) and its side branches (SBR) in vivo with the use of a well-established genetic model for IR, the (fa/fa) Zucker obese (ZO) and its control counterpart the Zucker lean (ZL) rats. We determined the vasoconstrictor effects of ET-1, the PKC activator phorbol 12,13-dibutyrate (PDBu), and the thromboxane A2 (TxA2) mimetic U-46619, as well as vascular responses after inhibition of ETA, PKC, and ROK with BQ-123, Ro31–8220, and Y-27632, respectively. Furthermore, we examined whether IR alters the expression of the ETA receptor in the cerebral arteries and whether it influences ET-1 mRNA levels in the endothelial cells of cerebral microvessels. To verify that, similarly to our earlier investigations, endothelial dysfunction is present in the ZO rats, we tested vascular responses to bradykinin.
MATERIALS AND METHODS
Animal preparation, measurement of vascular responses.
The experimental protocol was approved by the Animal Care and Use Committee at Wake Forest University. Experiments were performed on 12-wk-old male ZL and ZO rats (Harlan, Indianapolis, IN). Animals were fed standard rat chow and drank tap water ad libitum. The ZO rats at this age are overweight and IR as indicated by normal fasting glucose concentration and significantly higher fasting insulin, total cholesterol, and triglyceride levels compared with their ZL counterparts (8).
Rats were anesthetized with pentobarbital sodium (70–80 mg/kg ip, supplemented with 10–20 mg·kg−1·h−1 iv) and were ventilated through the trachea with a mixture of room air and O2. Depth of anesthesia was regularly monitored by applying pressure to a paw. If changes in heart rate or blood pressure were observed, additional pentobarbital was administered. A catheter was placed in a femoral artery to measure systemic arterial blood pressure and to obtain arterial blood samples, whereas a femoral vein was cannulated for infusion of supplemental anesthetics. Arterial blood gases during the experiments were pH 7.41 ± 0.01, Pco2 36 ± 1 mmHg, Po2 112 ± 3 mmHg in the ZL rats; and pH 7.42 ± 0.01, Pco2 37 ± 2 mmHg, Po2 110 ± 2 mmHg in the ZO rats.
A ventral craniotomy was performed over the brain stem, as described previously (9), and the cranial window was superfused with artificial cerebrospinal fluid (CSF) at a rate of 3 ml/min. The CSF, which contained (in mM) 2.95 KCl, 132 NaCl, 3.69 dextrose, 1.7 CaCl2, 0.64 MgCl2, and 23.2 NaHCO3, was bubbled with 5% CO2 in N2 and maintained at 37–38°C. Gas tensions of the CSF sampled from the cranial window were pH 7.37 ± 0.01, Pco2 35 ± 2 mmHg, and Po2 96 ± 4 mmHg in both the ZL and ZO groups. Changes in diameters of the BA and SBRs were observed at a sampling rate of 0.5 Hz with a microscope equipped with a charge-coupled device camera connected to a personal computer and analyzed using the Scion Image Software (Scion, Frederick, MD). Baseline diameters of the BAs and SBRs were similar in the ZL and ZO groups: 237 ± 3 μm and 248 ± 4 μm (BA) and 91 ± 6 μm and 106 ± 4 μm (SBR), respectively. Concentration-dependent vascular responses were evaluated in response to ET-1, BQ-123, U-46619, PDBu, Ro31–8220, or Y-27632. At the end of each study, animals were killed with 150–200 mg/kg pentobarbital sodium.
Characterization of IR in the ZO rats.
In some experiments, systemic arterial blood pressure was measured, and blood samples were taken after 12 h of fasting from awake rats through an implanted femoral artery catheter. Plasma insulin and glucose levels were measured using a rat insulin ELISA kit (Crystal Chem, Chicago, IL) and Trinder reagent (Sigma, St. Louis, MO), respectively. Triglyceride and total cholesterol were measured by automatic analyzer (Technicon RA-1000).
Protein samples of cerebral arteries (BA and middle cerebral arteries) from ZL and ZO rats were prepared as described previously (24). An equal amount of protein for each sample was separated by 10% SDS-PAGE, transferred onto a polyvinylidine difluoride membrane, and blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20. Blots were incubated overnight at 4°C with anti-ETA (BD Transduction Laboratories, 1:1,000) and anti β-actin (Sigma, 1:2,500) antibodies. The bound antibodies were detected by chemiluminescence.
Total RNA was obtained from isolated cerebrocortical microvessels using SV Total RNA Isolation System (Promega, Madison, WI), and RT-PCR experiments were carried out as described previously (18). We used a microvessel preparation to examine ET-1 expression, because this method provides relatively pure endothelial cells that can also be used for cell culturing (19). Expression of ET-1 mRNA was analyzed using specific primers (sense primer: 5′-CTCGCTCTATGTAAGTCATGG-3′; antisense primer: 5′-GCTCCTGCTCCTCCTTGATG-3′); the expected length of the RT-PCR product was 500 base pairs. β-Actin primers were also included in the RT-PCR reaction to normalize our RT-PCR results with an expected product length of 285 base pairs.
ET-1, BQ-123, PDBu, Ro31–8220, U-46619, and bradykinin were purchased from Sigma (St. Louis, MO), while Y-27632 was obtained from Calbiochem (San Diego, CA). Drugs were dissolved in CSF, except for PDBu, which was dissolved in DMSO and CSF. The same concentration of DMSO alone had no effect on vessel diameter.
All data are expressed as means ± SE. The magnitudes of vascular responses were calculated as a percentage of baseline diameter, which was measured for 1 min immediately before application of drugs. Effects of treatments and differences between ZL and ZO rats were evaluated using analysis of variance, followed by Tukey's post hoc test. The criterion for significance was P < 0.05.
Characterization of IR in the ZO rats.
At 12 wk of age, ZO rats were significantly heavier than ZLs [455 ± 6 g (n = 29) vs. 312 ± 5 g (n = 27, P < 0.01)]. Mean arterial blood pressure and fasting glucose levels were similar in the ZL and ZO rats, whereas insulin was one magnitude higher in the ZO rats compared with ZLs. Total cholesterol and triglyceride levels were also significantly elevated in the ZO group (Table 1).
ET-1 induced dose-dependent constrictions in the BAs and SBRs in both the ZL and ZO rats without significant differences between the two groups. For example, the diameters of the BAs of the ZL (n = 10) and ZO (n = 11) rats decreased by 36 ± 8% and 33 ± 3% at 10−8 M ET-1 [not significant (NS), Fig. 1A], whereas the same concentration of ET-1 constricted the SBRs by 70 ± 6% and 64 ± 6% in the ZL and ZO rats, respectively (NS). BQ-123 (10−7 M), the inhibitor of the ETA receptor, also induced comparable responses in the ZL (n = 5) and ZO (n = 5) rats, the diameter of the BAs increased by 6 ± 1% and 5 ± 3%, whereas the SBRs dilated by 4 ± 2% and 5 ± 2%, respectively (NS). The same concentration of BQ-123 reversed the ET-1-induced constriction to ETB receptor-mediated dilation in both groups indicating that the ETA receptor inhibition was effective. In the presence of 10−7 M BQ-123, 10−8 M ET-1 relaxed the ZL and ZO BAs by 49 ± 6% (n = 5) and 53 ± 8% (n = 5), respectively.
Immunoblots using isolated cerebral arteries and antibodies against the ETA receptors showed that the expression of this protein is not affected by IR in the ZO rats compared with ZLs (Fig. 1B). The densities of the immunoreactive bands normalized to β-actin were 0.99 ± 0.03 and 1.13 ± 0.03 in the ZL and ZO samples (n = 6; NS). In addition, RT-PCR experiments using isolated cerebrocortical microvessels showed that expression of the ET-1 mRNA was also similar in the ZL and ZO rats; densities of the bands normalized to β-actin were 2.79 ± 0.04 and 2.65 ± 0.10, respectively (NS).
Activation of PKC by PDBu resulted in similar vascular responses in the ZL (n = 6) and ZO (n = 6) rats. For example, at 10−6 M concentration, the BAs constricted by 33 ± 2% and 32 ± 4% in the ZL and ZO rats, respectively (NS, Fig. 2), whereas the same concentration of PDBu constricted the SBRs by 33 ± 6% and 28 ± 2% (NS). Inhibition of PKC by Ro31–8220 (5 μM) abolished the PDBu-induced constrictions completely (changes in BA diameter to 10−6 M PDBu in the presence of Ro31–8220 were 1 ± 1% and 0 ± 1% in the ZL and ZO rats, respectively), but did not have any effect on the resting vascular tone either in the ZL or in the ZO rats when applied alone. The diameters of the BAs and SBRs changed by 4 ± 1% and 0 ± 1% and by 3 ± 1% and 1 ± 1% in the ZL (n = 5) and ZO (n = 6) groups, respectively (NS).
Application of U-46619 induced concentration-dependent constriction in the BAs and SBRs, but again the responses were similar in the ZL and ZO groups. For example, at 10−7 M U-46619, the BAs of the ZL (n = 7) and ZO (n = 9) rats constricted by 23 ± 1% and 19 ± 2% (NS; Fig. 3), whereas the SBRs constricted by 23 ± 3% and 22 ± 2% (NS), respectively.
Inhibition of Rho-kinase with Y-27632 induced comparable vasodilation in the two experimental groups. For example, the BAs of the ZL (n = 8) and ZO (n = 7) rats relaxed by 39 ± 4% and 38 ± 2% at 10−5 M Y-27632 (NS; Fig. 4), whereas the SBRs dilated by 53 ± 4% and 45 ± 2% in the ZL and ZO rats, respectively (NS).
To verify that despite unaltered constrictor responses, endothelial dysfunction is present in the ZO rats used in this study, we tested vascular responses to bradykinin. Bradykinin-induced relaxation was significantly reduced in the ZO rats compared with ZLs. For example, the BAs of the ZL (n = 6) and ZO (n = 7) rats relaxed by 29 ± 7% and 10 ± 3% at 10−6 M bradykinin (P < 0.01; Fig. 5), whereas the SBRs dilated by 47 ± 12% and 17 ± 3%, respectively (P < 0.01). Bradykinin-induced dilation was mediated by NO, because in the presence of an NO synthase inhibitor [NG-nitro-l-arginine methyl ester, 10 μM] bradykinin-induced responses were almost completely eliminated in all experimental groups; relaxation of the BAs in the ZL and ZO rats was 5 ± 2% (n = 6) and 4 ± 2% (n = 6), whereas SBRs dilated by 11 ± 1% and 6 ± 1%, respectively.
The present study demonstrates that despite the consistent findings that IR impairs dilator responsiveness in the cerebral arteries, constrictor mechanisms are unaffected in the cerebral circulation of IR ZO rats. Application of ET-1 or the inhibition of the ETA receptor induces similar vascular responses in the ZL and IR ZO rats, and the expressions of ET-1 mRNA and the ETA receptor protein are also comparable in the two experimental groups. Furthermore, constrictor responses to activation of PKC and to the application of a TxA2 mimetic are also similar, whereas the inhibition of ROK results in comparable dilator responses in the examined cerebral arteries.
Similarly to other circulatory beds, vascular tone of the cerebral arteries is controlled by a finely tuned balance between dilator and constrictor stimuli. Whereas the mediation of these antagonistic mechanisms is rather complex, in the end they manifest in either changes in [Ca2+]i or changes in Ca2+ sensitivity of the VSMCs (37). Previous results indicated that in IR, the balance between dilator and constrictor mechanisms of the cerebral arteries might be disturbed because of the impairment of NO and K+ channel-mediated relaxation (5–8). However, several findings indicated that constrictor mechanisms may also be altered in IR (15–17).
For example, in the peripheral circulation, one of the most important effects of IR on constrictor mechanisms is the augmentation of ET-1-induced responses. ET-1 causes enhanced constriction in mesenteric arteries and aorta of fructose-fed IR rats compared with controls due to increased production of ET-1 and overexpression of ETA receptors in these arteries (15, 17). Furthermore, IR-induced inflammatory mechanisms in the vascular wall involve activation of endothelial cells, which also results in increased ET-1 production (10, 38). Subjects diagnosed with syndrome X have elevated serum ET-1 levels compared with healthy individuals (31), and resistin, a recently described adipokine that has been suggested to play a role in the development of IR, also induces ET-1 release from human endothelial cells (39). Because ET-1 activates PKC in the VSMCs (11, 29), and previous results in ZO rats showed that increased PKC activity is partially responsible for IR-induced cerebrovascular impairment (8), ET-1 seemed to be a likely candidate to link IR and vascular dysfunction in the cerebral circulation. The results of the present study, however, indicate that in contrast with the peripheral circulation, ET-1-mediated vascular responses are unaffected by IR in the cerebral arteries. ET-1- and BQ-123-induced changes in BA, and SBR diameters were similar in the ZL and ZO groups, which indicates that both the responsiveness of VSMCs to ET-1 and the basal ET-1 release from the endothelium are unaltered by IR. In addition, we found no difference in the expression of ET-1 mRNA and ETA receptor protein between the two experimental groups. Thus it seems that despite the similarities between the impairment of cerebral and peripheral circulation, IR affects these circulatory beds differently, and the previously observed PKC activation in the ZO cerebral arteries is probably not caused by ET-1.
Moreover, inhibition of PKC did not alter resting vascular diameter even in the ZO rats, which indicates that despite the increased PKC activity in the ZO cerebral arteries, similarly to ZL or normal Sprague-Dawley rats (2), PKC does not play a significant role in the control of resting vascular tone. Combining these data with those previous findings that PKC inhibition reverses only endothelial NO-mediated responses but not VSMC K+ channel-dependent relaxation of ZO BAs (8) provides evidence that PKC may be activated only in the endothelial cells of the IR cerebral arteries, but not in the VSMCs. Presently we are unable to measure PKC activity selectively in the different cell types of these small arteries and cannot provide direct evidence for this hypothesis. However, the finding that activation of PKC by PDBu induces similar constrictor responses in the ZL and ZO rats indicates that PKC-mediated control mechanisms are intact in the VSMCs.
Intact regulation of VSMC Ca2+ sensitivity in the IR cerebral arteries is indicated by our findings that constrictor responses to U-46619 and relaxation to Y-27632 were similar in the ZL and ZO rats. Ca2+ sensitivity of the VSMCs is determined by the activity of MLCP, which by dephosphorylating the regulatory light chain of myosin counteracts the Ca2+- and MLCK-induced MLC phosphorylation and constriction (37). Because ROK inhibits MLCP, activation of the RhoA/ROK pathway induces Ca2+ sensitization and increased vascular tone (21, 32, 40). We tested this regulatory mechanism using two different approaches. We examined constrictor responses of the BA and its SBRs to the TxA2 mimetic U-46619, because TxA2-induced constriction is thought to be mediated primarily by the G12/13-RhoA/ROK pathway as opposed to increases in [Ca2+]i (12, 32). Furthermore, we tested the effect of the inhibition of basal ROK activity by Y-27632. The finding that both activation and inhibition of the RhoA/ROK pathway led to similar vascular responses in the examined arteries of ZL and ZO rats suggests that this regulatory pathway is also protected from the adverse cerebrovascular effects of IR. This is an interesting discrepancy between the peripheral and cerebral circulation, because previous studies reported increased ROK activity in aorta VSMCs of type 2 diabetic rats, due to the lack of insulin's NO-cGMP pathway-mediated inhibition on ROK (33, 34).
We do not know why the examined vasoconstrictor mechanisms are affected by IR only in the peripheral circulation, whereas in the cerebral circulation they remain unaltered, but one possible explanation might be that the ability of arteries to properly constrict and prevent blood perfusion fluctuations due to changes in arterial blood pressure is much more important in the cerebral circulation than in the peripheral circulation. Thus the regulation of constrictor mechanisms in the cerebral vasculature is more resistant to the IR-induced pathological stimuli. It is also possible, however, that vasoconstrictor responses elicited by other vasoactive agents or observed in different experimental conditions are altered in IR despite the revealed intact PKC- and ROK-mediated regulation of vascular tone in our experimental setup. For example, using the same animal model of IR, Karagiannis et al. (16) found that isolated BAs constricted significantly more in response to serotonin in the ZO rats compared with ZLs. However, in these experiments, 36-wk-old animals were studied rather than 12-wk-old ZO rats as were studied in our experiments, and the 36-wk-old ZO rats were also hypertensive. Furthermore, in streptozotocin-treated type 1 diabetic rats, pressure-induced vascular tone of cerebral arteries was found to be augmented compared with controls (43); however, in the same rat model, constrictor responses of the BA to serotonin, arginine vasopressin, ET-1, and U-46619 were found to be unaltered (26, 27). In addition, we cannot exclude the possibility that results obtained in vivo in anesthetized animals are biased by the anesthetics used, because barbiturates were shown to influence regulatory pathways related to NO, VSMC K+ channels, or voltage-dependent Ca2+ channels (13, 23, 41).
Effects of other pathological disease states on cerebrovascular constrictor responsiveness also vary considerably depending on the experimental model used. For example, ischemia-reperfusion was shown to reduce myogenic reactivity and serotonin-induced vasoconstriction in isolated middle cerebral arteries (4), whereas during subarachnoid hemorrhage, oxyhemoglobin increases vascular tone of the cerebral arteries by activating ROK and PKC (40). Traumatic brain injury and chronic hypertension were also shown to augment constrictor responses of cerebral arteries by enhancing ET-1-mediated vasoconstriction, and increasing the activity of L-type Ca2+ channels and ROK (20, 36). In contrast, constrictor responses to increases in transmural pressure or to PDBu are impaired in hypertensive Dahl salt-sensitive rats after stroke (30).
In summary, we conclude that, in contrast with the peripheral circulation, ET-1-, TxA2-, PKC-, or ROK-mediated vasoconstrictor mechanisms are protected in the cerebral vasculature of IR rats; thus the increased risk of cerebrovascular events in IR is more likely due to reduced dilator responsiveness than to inappropriate vasoconstrictor mechanisms in the cerebral arteries.
This research was supported by National Institutes of Health Grants HL-30260, HL-65380, HL-66074, DK-62372, and HL-77731; AHA Bugher Foundation Award 0270114N; and the Hungarian OTKA (T 29169, T 90665, T 97334, T 37885, T37386). B. Erdös was also supported by a Hungarian National Eötvös scholarship.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2004 the American Physiological Society