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Insulin resistance does not impair contractile responses of cerebral arteries
Life Sciences 77 (2005) 2262 – 2272 www.elsevier.com/locate/lifescie
Insulin resistance does not impair contractile responses of cerebral arteries Steve A. SimandleT, Benedek Erdo¨s, James A. Snipes, Allison W. Miller, David W. Busija Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, United States Received 11 November 2004; accepted 31 January 2005
Abstract Insulin resistance (IR) impairs endothelium-mediated vasodilation in cerebral arteries as well as K+ channel function in vascular smooth muscle. Peripheral arteries also show an impaired endothelium-dependent vasodilation in IR and concomitantly show an enhanced contractile response to endothelin-1 (ET-1). However, the contractile responses of the cerebral arteries in IR have not been examined systematically. This study examined the contractile responses of pressurized isolated middle cerebral arteries (MCAs) in fructose-fed IR and control rats. IR MCAs showed no difference in pressure-mediated (80 mmHg) vasoconstriction compared to controls, either in time to develop spontaneous tone (control: 61 F 3 min, n = 30; IR: 63 F 2 min, n = 26) or in the degree of that tone (control: 60 min: 33 F 2%, n = 22 vs. IR 60 min: 34 F 3%, n = 17). MCAs treated with ET-1 (10 8.5 M) constrict similarly in control (53 F 3%, n = 14) and IR (53 F 3%, n = 14) arteries. Constrictor responses to U46619 (10 6 M) are also similar in control (48 F 9%, n = 8) and IR (42 F 5%, n = 6) MCAs as are responses to extraluminal uridine 5Vtriphosphate (UTP; 10 4.5 M) (control: 35 F 7%, n = 11 vs. IR: 38 F 3%, n = 10). These findings demonstrate that constrictor responses remain intact in IR despite a selective impairment of dilator responses and endothelial and vascular smooth muscle K+ channel function in cerebral arteries. Thus, it appears that the increased susceptibility
Abbreviations: COX, cyclooxygenase; ET-1, endothelin-1; ETRA, endothelin receptor subtype A; ETRB, endothelin receptor subtype B; IR, insulin resistance; MCAs, middle cerebral arteries; NE, norepinephrine; NO, nitric oxide; STZ, streptozotocin; TXA2, thromboxane; UTP, uridine 5V-triphosphate. T Corresponding author. Department of Pediatrics, Wake Forest University Health Sciences, 1053 Hanes Bldg., Medical Center Blvd., Winston-Salem, NC 27157, United States. Tel.: +1 336 713 5190. E-mail address: [email protected] (S.A. Simandle). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.01.028
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to cerebrovascular abnormalities associated with IR and diabetes (including cerebral ischemia, stroke, vertebrobasilar transient ischemic attacks) is not due to an enhanced vasoreactivity to constrictor agents. D 2005 Elsevier Inc. All rights reserved. Keywords: Insulin resistance; Endothelin; Cerebral arteries
Introduction Insulin resistance (IR) is a major health problem and is associated with vascular dysfunction, coronary artery disease, and stroke (Abbott et al., 1987; Bressler et al., 1996; Fontbonne et al., 1991; Howard et al., 1996) with IR being an independent risk factor for stroke (Abbott et al., 1987; Barrett-Connor and Khaw, 1988). Unfortunately, the mechanisms by which IR and hyperinsulinemia lead to vascular dysfunction are not well understood. Possible mechanisms that have received attention are the impairment of endothelial function or the enhancement of constrictor responses. Both IR humans and animals show an impaired endothelium-dependent vasodilation (Erdos et al., 2002b; Miller et al., 1998, 1999; Steinberg et al., 1996; Verma et al., 1996). Previous studies utilizing the fructose-fed model of IR have documented an impaired endothelium-dependent vasodilation as defined by a decreased response to bradykinin or acetylcholine in small mesenteric, coronary, and cerebral arteries. In mesenteric arteries, the impaired dilation has been shown to be related to a nitric oxide (NO)and prostacyclin-independent relaxing factor that activates KCa channels (Miller et al., 2001). In cerebral arteries, this dysfunction is the result of reduced cyclooxygenase (COX)-dependent relaxation while NOmediated responses remain intact (Erdos et al., 2002b). Furthermore, IR alters the function of endothelial and vascular smooth muscle KATP and KCa channel activity in cerebral arteries, thereby affecting the control of resting vascular tone and the mediation of dilator stimuli (Erdos et al., 2002a). Thus, the mechanisms underlying vascular dysfunction in IR appear to vary depending on which vascular bed is studied. Several reports have implicated endothelin-1 (ET-1) in the pathenogenesis of insulin-induced vascular dysfunction and hypertension. In peripheral arteries, the vasoconstrictor response to ET-1 is enhanced in IR due to an enhanced expression of ET-1 receptors in addition to the underlying endothelial dysfunction (Katakam et al., 2001). Furthermore, in models of Type I diabetes, chronic ET-1 antagonism has been shown to prevent alterations in myogenic tone and to restore endothelium-dependent vasomotion in cerebral arteries (Dumont et al., 2003). However, the contractile responses of the cerebral arteries in a pre-diabetic model of IR have not been examined. Thromboxane A2 (TXA2) has also been implicated in diabetic vascular dysfunction. An increase in synthesis, secretion, and action of TXA2, as well as ET-1 (Dandona et al., 2003), has been reported in diabetics. A potent vasoconstrictor, TXA2 can activate platelet aggregation and, in diabetic patients, can be derived from both activated platelets as well as the vessel wall (Sobol and Watala, 2000). Accordingly, the effects of TXA2 may be mediated by its actions on platelets and/or its mediation of vascular reactivity. As with ET-1, responses of TXA2 on cerebrovascular reactivity have not been examined in a model of IR. The purpose of the present study was to examine the effects of IR on vasoconstrictor responses in the rat middle cerebral artery (MCA) and also to test the hypothesis that MCAs from IR rats show enhanced vasoconstrictor responses. The constrictor responses to ET-1 and U46619, a stable TXA2 analog, were examined in isolated, pressurized MCAs from control and IR rats. Also, the constrictor responses of
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control and IR MCAs to uridine 5V-triphosphate (UTP) were examined. We used a well-established model in which IR was induced by the feeding of a high fructose diet. Fructose-fed rats are normoglycemic and normotensive, but are hyperinsulinemic (Katakam et al., 1998).
Materials and methods The Animal Care and Use Committee of Wake Forest University Health Sciences approved all procedures. Male Sprague–Dawley rats were obtained at 6 weeks of age and randomized into one of two groups: control (n = 30) and IR (n = 26). Control animals received standard rat chow. Animals in the IR group were fed a fructose-rich diet containing 66% fructose, 22% casein, and 12% lard plus essential vitamins and minerals. This diet has been utilized often and is well characterized. Rats on the fructose diet exhibit normal weight, normal fasting glucose levels, and hyperinsulinemia (Katakam et al., 2001, 1998, 1999; Miller et al., 2001, 2002). After a 4 week diet treatment, the rats were anesthetized with pentobarbital sodium (50 mg kg 1 ip) and anticoagulated with heparin sodium (500 U ip). Determination of vascular reactivity After anesthesia, the rats were decapitated and the brain was removed immediately. The brain was placed into ice cold oxygenated modified Kreb’s–Ringer bicarbonate solution (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 harvested with the aid of a dissecting microscope. A section of the MCA (approximately 2 mm in length) was transferred to a vessel chamber and mounted and secured between two glass micropipettes with 10-0 opthalamic suture. This vessel chamber was then transferred to an inverted light microscope stage which was connected to a video dimension analyzer (Living Systems Instrumentation, Burlington, VT). Output from the video dimension analyzer was sent to both a video monitor (for visualization of the vessel) and a strip chart recorder (Kipp and Zonen) for constant recording of the intraluminal diameter of the vessel. The lumen of the MCA was filled with Kreb’s solution through the micropipettes by closing the outflow cannula and attaching the inflow cannula to an elevated reservoir to maintain a constant pressure of 80 mmHg while oxygenated (20% O2, 5% CO2, 75% N2) Kreb’s solution, maintained at 37 8C, was continuously circulated through the vessel bath. Drugs were added abluminally into the bath solution and only one concentration–response response experiment was performed per artery. After mounting and pressurization, the MCAs developed spontaneous tone to c 40% of the initial diameter over the course of about one hour. Experimental protocols were not initiated until spontaneous tone was stable for at least 15 min. Concentration–response experiments were performed using ET-1 (10 11 to 10 8.5 M), U46619 (10 10 to 10 6 M), and UTP (10 8 to 10 4.5 M). Maximal responses to ET-1 were stable after 20 min, for U46619, 8 min, and with UTP, 10 min; diameters were measured at these times. Determination of ET-1 receptor protein Two groups of rats were used for Western blot analysis of ETRA and ETRB protein levels using a method adapted from Liu et al. (1997). Control (n = 6) and IR (n = 6) rats were anesthetized as described above. After decapitation, the brain was removed and placed in ice-cold Kreb’s solution. Due to the paucity of tissue available from the MCAs alone, other arteries of the brain (basilar, anterior cerebral,
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and posterior cerebral arteries) were also dissected, cleaned of adherent tissue, and flushed with Kreb’s solution. The arteries from each individual animal were combined together in homogenizing buffer (in mM: 2 EDTA; 2 EGTA; 250 sucrose; 50 MOPS; 0.1 PMSF; and leupeptin, antipain, and aprotinin at 5 Ag/ml). These vessels were processed intact and every effort was made to preserve the endothelium. Protein concentration was determined using the DC Protein assay (BioRad) using bovine serum albumin as a standard. Loading buffer (Laemmli Sample Buffer; BioRad) was added 1 : 1 to the homogenates and the samples were heated for 5 min at 90 8C in a dry bath and then centrifuged at 10,000 g for 1 min. Equal amounts of protein (1.8 Ag) were loaded into 10% SDS-PAGE gels. After transfer to a PVDF membrane (BioRad), the blots were blocked for 1h with Chemiblocker (Chemicon) and then primary antibody (ETRA or ETRB; Alamone Labs) was added at a dilution of 1 : 500. After washing (3 5 min TBST) secondary antibody (goat anti-rat IgG; Jackson Immunochemicals) was added at a 1 : 60,000 dilution and incubated for 1h. After washing (2 4 min TBST; 2 4 min TBS) the blots were exposed to ECL reagent (Pierce Chemical) and developed using Hyperfilm (Amersham) and a Kodak developer. Densitometric analysis was conducted using ImageJ (NIH) software. Drugs The fructose rich diet was obtained from Harlan Teklad (Indianapolis, IN, USA). The thromboxane analog, U46619, was purchased from Cayman Chemical (Ann Arbor, MI, USA). The antibodies for ETRA and ETRB were bought from Alamone Labs (Jerusalem, Israel). The other drugs used, including ET-1, UTP, and various reagents, were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Data analysis All data are presented as means F SE. The magnitude of spontaneous vascular tone was calculated as a percentage of preconstricted diameter according to the equation: % constriction ¼
DA DV T100 DA
ð1Þ
where DA is the diameter upon development of spontaneous tone at 80 mmHg and DV is the diameter after drug treatment. The concentration–response curves were evaluated at each concentration for differences between treated and untreated arteries from control and IR groups with ANOVA followed by Tukey’s post hoc test using SigmaStat (SPSS, INC.) software. Differences in maximal diameter and preconstricted diameter and data from Western blotting were evaluated by t-test. The criterion for significance was set at p b 0.05.
Results Spontaneous tone Maximal intraluminal diameter of the MCAs did not differ between groups: 222 F 5 Am (n = 30) control vs. 227 F 4 Am (n = 26) IR; p N 0.05. Spontaneously developed tone in response to 80 mmHg was also similar between groups (Fig. 1). At 1 h, the MCAs constricted to 33 F 2% and 34 F 3% of maximal
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Percent Contraction (%)
60 Control
50
Insulin Resistant 40 30 20 10 0 0
10
20
30
40
50
60
Time (minutes) Fig. 1. Spontaneously developed tone. Vessels were maintained at an intraluminal pressure of 80 mmHg and developed tone over the course of about an hour. Percent constriction is from maximal dilation. Both control and IR arteries developed tone similarly and, on average, over the same amount of time. Control n = 30; IR n = 26.
diameter in the control and IR groups, respectively. Additionally, the mean time required to develop spontaneous tone did not differ between groups (61 F 3 min control vs. 63 F 2 min IR; p N 0.05). Contractile response to ET-1 The cumulative dose–responses of control and IR MCAs to ET-1 are shown in Fig. 2. Both control and IR arteries constricted in response to ET-1 to just over 50% of the preconstricted diameter.
Percent Contraction (%)
60 50 40 30 20 Control 10
Insulin Resistant
0 -11
-10
-3.5
-9
-8.5
ET-1 (log M) Fig. 2. Cumulative dose–response curves to endothelin-1 (ET-1) in isolated, pressurized middle cerebral arteries of control (open circle) and insulin resistant (closed square) rats. Both control and IR arteries respond similarly to ET-1, p N 0.05. Control n = 14; IR n = 14.
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Interestingly, both the control and IR MCAs constricted similarly at each dose of ET-1, such that no significant differences at any dose were evident. Although the IR MCAs tended to be more responsive at the lower doses of ET-1 that were examined, this effect was not statistically different from control. Determination of protein levels of ETRA and ETRB Western blot analysis of ET-1 receptor subtypes A (ETRA) and B (ETRB) revealed no differences in protein content between control and IR arteries. Semi-quantitative densitometric analysis of Western blots for ETRA show that these receptors are expressed to a similar extent in control and IR rats: ETRA, control 76 F 3 arbitrary units (au) vs. IR 71 F 2 au. Likewise for ETRB, no differences were apparent between the groups (control 113 F 7 au vs. IR 114 F 3 au). Due to the paucity of tissue available from the MCAs, other arteries of the brain were also used in Western blot analysis. As such, it cannot be ruled out that certain vessels expressed more or less of the ETRA or ETRB proteins and that these segmental differences were lost upon homogenization. However, that is probably not the case, as the results of the Western blot analysis are in line with the in vitro vessel reactivity experiments on the MCA. Contractile response to U46619 The cumulative dose–response curves of control and IR MCAs to U46619 are shown in Fig. 3. Both control and IR arteries constricted in response to U46619 to about 45% of the preconstricted diameter. Both control and IR MCAs constricted similarly to each dose of U46619 and at no point were the groups significantly different. Contractile response to UTP The cumulative dose–response curves of control and IR MCAs to UTP are shown in Fig. 4. Both control and IR arteries constricted in response to UTP to almost 40% of the preconstricted diameter. Both
Percent Contraction (%)
60 Control
50
Insulin Resistant 40 30 20 10 0 -10
-9
-8
-7
-6
U46619 (log M) Fig. 3. Cumulative dose–response curves to U46619 in isolated, pressurized middle cerebral arteries of control (open circle) and insulin resistant (closed square) rats. Both control and IR arteries respond similarly to U46619, p N 0.05. Control n = 8; IR n = 6.
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Percent Contraction (%)
Control 50 Insulin Resistant 40 30 20 10 0 -10 -8
-7
-6
-5
-4.5
UTP (log M) Fig. 4. Cumulative dose–response curves to uridine 5V-triphosphate (UTP) in isolated, pressurized middle cerebral arteries of control (open circle) and insulin resistant (closed square) rats. Both control and IR arteries respond similarly to UTP, p N 0.05. Control n = 10; IR n = 11.
the control and IR MCAs constricted similarly at each dose of UTP, such that no significant differences at any dose were evident. Although the responses of IR MCAs tended to be greater than controls, at no time did they become significantly different.
Discussion The principle finding of this study is that MCAs from IR rats show no differences in contractile responses whether induced by pressure, ET-1, UTP, or U46619, a thromboxane analog. Furthermore, the cerebrovasculature of the IR animals show no changes in ETRA or ETRB protein levels. These findings suggest that the brain is protected from the enhanced contractile response to ET-1 that is seen in the peripheral vasculature (Katakam et al., 2001) in this model of IR. Thus, increased risk for stroke related complications in the context of IR is likely due to the inability of the cerebral arteries to dilate, and not to an enhanced response to constrictor stimuli. Pressure-induced vasoconstriction or myogenic tone is an integral part of cerebrovascular regulation. In this study, a constant intraluminal pressure of 80 mmHg caused a constriction of about 40% of the maximal diameter in MCAs from both control and IR animals. Also, MCAs from IR rats as well as those from controls, took the same amount of time, just over one hour, to achieve full spontaneous tone. These results differ from similar studies using a model of Type I diabetes. Zimmermann et al. (1997) used streptozotocin (STZ) to induce diabetes in female Sprague–Dawley rats and found that diabetic cerebral arteries showed more vasoconstriction to the same amount of intraluminal pressure than did control arteries. Dumont et al. (2003) also examined MCA responses to graded pressure increases in STZ-induced diabetic rats and demonstrated that diabetic MCAs constricted more than control arteries. These researchers went on to illustrate that bosentan, an ETR antagonist, normalized the diabetic responses. While the present study did not use graded pressure increases, the results presented here show no such discrepancy between control and IR arteries at the
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physiologically relevant 80 mmHg intraluminal pressure. The difference between this study and those of Zimmermann et al. (1997) and Dumont et al. (2003) may lie in the fact that the animals in this study were neither Type I diabetic animals nor Type II, but were pre-diabetic or IR animals. It is possible that Type I diabetes affects the cerebral arteries while the cerebral arteries in IR are not yet similarly affected. The data in this study show that MCAs from IR rats show no differences from control MCAs in response to ET-1. This is the first report to examine the constrictor responses of the cerebral vasculature in a model of IR. Other studies using fructose feeding to induce IR and looking at different vascular beds show differing results. Juan et al. (1998), using a 40 day diet of fructose feeding, presented evidence that the feeding led to an enhanced response to ET-1 in the thoracic aorta. Navarro-Cid et al. (1995), using a perfusion model of the mesenteric vascular bed, showed that four weeks of fructose feeding did not enhance responsiveness to ET-1. Additionally, Verma et al. (1997) using a nine week fructose feeding program demonstrated that while there was more ET-1 in the mesenteric arteries, those arteries showed a decreased maximal response and a decreased sensitivity to ET-1. Prolonged fructose feeding is known to induce hypertension; therefore comparisons to studies that utilize fructose feeding beyond four weeks are difficult. As such, a more relevant study is one which uses the same experimental model, such as that conducted by Katakam et al. (2001) who also examined vessels in vitro using an arteriograph system. The fructose feeding model used here, and by Katakam et al. (2001) has been shown to precede hypertensive and hyperglycemic responses (Katakam et al., 1998, 1999). Katakam et al. (2001) showed that mesenteric arteries from IR animals had a decreased EC50 in response to ET-1 despite showing the same E max as controls. That is, although the IR vessels constricted similarly to control vessels at maximal doses of ET-1, they were more responsive at sub-maximal doses. The data presented here suggest that the cerebral vasculature is not affected by the same increase in ET-1 sensitivity as seen in the mesenteric circulation (Katakam et al., 2001). In another study also using this model, Erdos et al. (2002b) examined dilator responses of MCAs in response to bradykinin. Preconstriction of the MCAs in that study was accomplished by the same dose of ET-1 for both control and IR arteries, highlighting the fact that both control and IR MCAs constricted similarly to the same dose of ET-1. This finding supports our results; the dose used by Erdos et al. (2002b) is within the range used in this study. Further supportive evidence comes from the results of Western blotting for ETRA and ETRB protein. Katakam et al. (2001) demonstrated that the enhanced ET-1 response they saw in the mesentery was associated with an increase in 125[I]-ET-1 binding. In a like manner, our analysis of ET-1 receptor (ETR) protein in brain vessels demonstrated that IR animals show no difference in ETRA protein levels from control; likewise ETRB protein levels did not differ between the groups. Because the endothelium was not removed from these vessels, ETRB receptors on the endothelium will contribute to higher ETRB protein levels. However, these protein levels were similar for both control and IR animals. The lack of an enhanced vasoconstrictor response to ET-1 in isolated control and IR MCAs coupled with the lack of a difference in ETRA and ETRB protein illustrates that the cerebral vasculature is protected from the increase in ET-1 sensitivity seen in the mesenteric circulation. The experiments with U46619, a stable TXA2 analog, again show no differences between control and IR arteries. Vasoconstrictor responses to U46619 were similar for control and IR MCAs at every dose examined (10 10 to 10 6 M). This also suggests that the cerebrovascular vasoconstrictor responses are not altered in IR. Despite the lack of a difference in vasoactivity, these studies do not
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address other actions of TXA2. For example, it remains to be addressed whether TXA2 activity on platelet aggregation varies in this model of IR from that seen in diabetic populations. The data presented here demonstrates that both control and IR MCAs constrict similarly to UTP. UTP is a P2Y receptor agonist and P2Y receptors are a G protein-coupled receptor population that responds to extracellular nucleotides. The P2Y receptor is present in the vascular smooth muscle of vessels and mediates vasoconstriction when applied abluminally (Wang et al., 2002). In the rat, ATP and UTP are equipotent against the P2Y receptor (Wang et al., 2002) and its use here allows the examination of a separate endogenous vasoconstrictor whose signal transduction pathway is distinct from ET-1 and TXA2. The abluminal application of UTP in this study resulted in a degree of constriction that was not significantly different between control and IR MCAs at any dose of UTP used (10 8 to 10 4.5 M). These results show that another vasoconstrictor agent with a signaling pathway distinct from ET-1 and TXA2 results in similar degrees of vasoconstriction for both control and IR MCAs. Other studies utilizing IR or diabetic arterial blood vessels show similar results with various constrictor agents. Verma et al. (1997) presented evidence that fructose feeding of rats for 9 weeks did not affect mesenteric arterial response to norepinephrine (NE). Navarro-Cid et al. (1995) illustrated that 4 weeks of fructose feeding in male Sprague–Dawley rats had no significant effect on constrictor responses of perfused mesenteric vessels in response to angiotensin II or phenylephrine. Additionally, Verma et al. (1996) gave further evidence that 4 weeks of fructose feeding did not affect mesenteric arterial response to NE using arterial rings. Indeed, in models of STZ-induced Type I diabetes, the mesenteric vascular bed and the hamster cheek pouch microcirculation show no altered responses to NE (Furman and Sneddon, 1993; Mayhan et al., 1999) nor to a thromboxane analogue, U-46619 (Mayhan et al., 1999). The present results show that, in the cerebral circulation of IR rats, vasoconstriction to ET-1 is not augmented and vasoconstriction to other constrictor agents, U46619 and UTP, is also unaltered by IR. Taken together, these data suggest that vasoconstrictor responses are, in large part, unaffected by IR or diabetes. Furthermore, the results presented here show that any abnormality in vasoconstrictor action in IR is limited to peripheral vascular beds (Miller et al., 2002) and does not affect the cerebrovasculature.
Conclusion The results of the present study show that vasoconstrictor responses of the cerebral circulation are not altered by IR induced by fructose feeding. Contractile responses of IR MCAs to pressure, ET-1, UTP, and U46619 do not differ from control responses. Thus, it appears that the increased susceptibility to cerebrovascular abnormalities associated with IR and diabetes (including cerebral ischemia, stroke, vertebrobasilar transient ischemic attacks (Abbott et al., 1987; Biller and Love, 1993)) is not due to an enhanced vasoreactivity to constrictor agents.
Acknowledgements This work is supported by grants from NIH (HL-30260, HL-50587, HL-65380, HL-66074) and AHA Bugher Foundation Award (0270114Z). B. Erdo¨s was supported by a Hungarian National Eo¨tvo¨s Fellowship.
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Insulin resistance does not impair contractile responses of cerebral arteries Steve A. SimandleT, Benedek Erdo¨s, James A. Snipes, Allison W. Miller, David W. Busija Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, United States Received 11 November 2004; accepted 31 January 2005
Abstract Insulin resistance (IR) impairs endothelium-mediated vasodilation in cerebral arteries as well as K+ channel function in vascular smooth muscle. Peripheral arteries also show an impaired endothelium-dependent vasodilation in IR and concomitantly show an enhanced contractile response to endothelin-1 (ET-1). However, the contractile responses of the cerebral arteries in IR have not been examined systematically. This study examined the contractile responses of pressurized isolated middle cerebral arteries (MCAs) in fructose-fed IR and control rats. IR MCAs showed no difference in pressure-mediated (80 mmHg) vasoconstriction compared to controls, either in time to develop spontaneous tone (control: 61 F 3 min, n = 30; IR: 63 F 2 min, n = 26) or in the degree of that tone (control: 60 min: 33 F 2%, n = 22 vs. IR 60 min: 34 F 3%, n = 17). MCAs treated with ET-1 (10 8.5 M) constrict similarly in control (53 F 3%, n = 14) and IR (53 F 3%, n = 14) arteries. Constrictor responses to U46619 (10 6 M) are also similar in control (48 F 9%, n = 8) and IR (42 F 5%, n = 6) MCAs as are responses to extraluminal uridine 5Vtriphosphate (UTP; 10 4.5 M) (control: 35 F 7%, n = 11 vs. IR: 38 F 3%, n = 10). These findings demonstrate that constrictor responses remain intact in IR despite a selective impairment of dilator responses and endothelial and vascular smooth muscle K+ channel function in cerebral arteries. Thus, it appears that the increased susceptibility
Abbreviations: COX, cyclooxygenase; ET-1, endothelin-1; ETRA, endothelin receptor subtype A; ETRB, endothelin receptor subtype B; IR, insulin resistance; MCAs, middle cerebral arteries; NE, norepinephrine; NO, nitric oxide; STZ, streptozotocin; TXA2, thromboxane; UTP, uridine 5V-triphosphate. T Corresponding author. Department of Pediatrics, Wake Forest University Health Sciences, 1053 Hanes Bldg., Medical Center Blvd., Winston-Salem, NC 27157, United States. Tel.: +1 336 713 5190. E-mail address: [email protected] (S.A. Simandle). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.01.028
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to cerebrovascular abnormalities associated with IR and diabetes (including cerebral ischemia, stroke, vertebrobasilar transient ischemic attacks) is not due to an enhanced vasoreactivity to constrictor agents. D 2005 Elsevier Inc. All rights reserved. Keywords: Insulin resistance; Endothelin; Cerebral arteries
Introduction Insulin resistance (IR) is a major health problem and is associated with vascular dysfunction, coronary artery disease, and stroke (Abbott et al., 1987; Bressler et al., 1996; Fontbonne et al., 1991; Howard et al., 1996) with IR being an independent risk factor for stroke (Abbott et al., 1987; Barrett-Connor and Khaw, 1988). Unfortunately, the mechanisms by which IR and hyperinsulinemia lead to vascular dysfunction are not well understood. Possible mechanisms that have received attention are the impairment of endothelial function or the enhancement of constrictor responses. Both IR humans and animals show an impaired endothelium-dependent vasodilation (Erdos et al., 2002b; Miller et al., 1998, 1999; Steinberg et al., 1996; Verma et al., 1996). Previous studies utilizing the fructose-fed model of IR have documented an impaired endothelium-dependent vasodilation as defined by a decreased response to bradykinin or acetylcholine in small mesenteric, coronary, and cerebral arteries. In mesenteric arteries, the impaired dilation has been shown to be related to a nitric oxide (NO)and prostacyclin-independent relaxing factor that activates KCa channels (Miller et al., 2001). In cerebral arteries, this dysfunction is the result of reduced cyclooxygenase (COX)-dependent relaxation while NOmediated responses remain intact (Erdos et al., 2002b). Furthermore, IR alters the function of endothelial and vascular smooth muscle KATP and KCa channel activity in cerebral arteries, thereby affecting the control of resting vascular tone and the mediation of dilator stimuli (Erdos et al., 2002a). Thus, the mechanisms underlying vascular dysfunction in IR appear to vary depending on which vascular bed is studied. Several reports have implicated endothelin-1 (ET-1) in the pathenogenesis of insulin-induced vascular dysfunction and hypertension. In peripheral arteries, the vasoconstrictor response to ET-1 is enhanced in IR due to an enhanced expression of ET-1 receptors in addition to the underlying endothelial dysfunction (Katakam et al., 2001). Furthermore, in models of Type I diabetes, chronic ET-1 antagonism has been shown to prevent alterations in myogenic tone and to restore endothelium-dependent vasomotion in cerebral arteries (Dumont et al., 2003). However, the contractile responses of the cerebral arteries in a pre-diabetic model of IR have not been examined. Thromboxane A2 (TXA2) has also been implicated in diabetic vascular dysfunction. An increase in synthesis, secretion, and action of TXA2, as well as ET-1 (Dandona et al., 2003), has been reported in diabetics. A potent vasoconstrictor, TXA2 can activate platelet aggregation and, in diabetic patients, can be derived from both activated platelets as well as the vessel wall (Sobol and Watala, 2000). Accordingly, the effects of TXA2 may be mediated by its actions on platelets and/or its mediation of vascular reactivity. As with ET-1, responses of TXA2 on cerebrovascular reactivity have not been examined in a model of IR. The purpose of the present study was to examine the effects of IR on vasoconstrictor responses in the rat middle cerebral artery (MCA) and also to test the hypothesis that MCAs from IR rats show enhanced vasoconstrictor responses. The constrictor responses to ET-1 and U46619, a stable TXA2 analog, were examined in isolated, pressurized MCAs from control and IR rats. Also, the constrictor responses of
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control and IR MCAs to uridine 5V-triphosphate (UTP) were examined. We used a well-established model in which IR was induced by the feeding of a high fructose diet. Fructose-fed rats are normoglycemic and normotensive, but are hyperinsulinemic (Katakam et al., 1998).
Materials and methods The Animal Care and Use Committee of Wake Forest University Health Sciences approved all procedures. Male Sprague–Dawley rats were obtained at 6 weeks of age and randomized into one of two groups: control (n = 30) and IR (n = 26). Control animals received standard rat chow. Animals in the IR group were fed a fructose-rich diet containing 66% fructose, 22% casein, and 12% lard plus essential vitamins and minerals. This diet has been utilized often and is well characterized. Rats on the fructose diet exhibit normal weight, normal fasting glucose levels, and hyperinsulinemia (Katakam et al., 2001, 1998, 1999; Miller et al., 2001, 2002). After a 4 week diet treatment, the rats were anesthetized with pentobarbital sodium (50 mg kg 1 ip) and anticoagulated with heparin sodium (500 U ip). Determination of vascular reactivity After anesthesia, the rats were decapitated and the brain was removed immediately. The brain was placed into ice cold oxygenated modified Kreb’s–Ringer bicarbonate solution (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 harvested with the aid of a dissecting microscope. A section of the MCA (approximately 2 mm in length) was transferred to a vessel chamber and mounted and secured between two glass micropipettes with 10-0 opthalamic suture. This vessel chamber was then transferred to an inverted light microscope stage which was connected to a video dimension analyzer (Living Systems Instrumentation, Burlington, VT). Output from the video dimension analyzer was sent to both a video monitor (for visualization of the vessel) and a strip chart recorder (Kipp and Zonen) for constant recording of the intraluminal diameter of the vessel. The lumen of the MCA was filled with Kreb’s solution through the micropipettes by closing the outflow cannula and attaching the inflow cannula to an elevated reservoir to maintain a constant pressure of 80 mmHg while oxygenated (20% O2, 5% CO2, 75% N2) Kreb’s solution, maintained at 37 8C, was continuously circulated through the vessel bath. Drugs were added abluminally into the bath solution and only one concentration–response response experiment was performed per artery. After mounting and pressurization, the MCAs developed spontaneous tone to c 40% of the initial diameter over the course of about one hour. Experimental protocols were not initiated until spontaneous tone was stable for at least 15 min. Concentration–response experiments were performed using ET-1 (10 11 to 10 8.5 M), U46619 (10 10 to 10 6 M), and UTP (10 8 to 10 4.5 M). Maximal responses to ET-1 were stable after 20 min, for U46619, 8 min, and with UTP, 10 min; diameters were measured at these times. Determination of ET-1 receptor protein Two groups of rats were used for Western blot analysis of ETRA and ETRB protein levels using a method adapted from Liu et al. (1997). Control (n = 6) and IR (n = 6) rats were anesthetized as described above. After decapitation, the brain was removed and placed in ice-cold Kreb’s solution. Due to the paucity of tissue available from the MCAs alone, other arteries of the brain (basilar, anterior cerebral,
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and posterior cerebral arteries) were also dissected, cleaned of adherent tissue, and flushed with Kreb’s solution. The arteries from each individual animal were combined together in homogenizing buffer (in mM: 2 EDTA; 2 EGTA; 250 sucrose; 50 MOPS; 0.1 PMSF; and leupeptin, antipain, and aprotinin at 5 Ag/ml). These vessels were processed intact and every effort was made to preserve the endothelium. Protein concentration was determined using the DC Protein assay (BioRad) using bovine serum albumin as a standard. Loading buffer (Laemmli Sample Buffer; BioRad) was added 1 : 1 to the homogenates and the samples were heated for 5 min at 90 8C in a dry bath and then centrifuged at 10,000 g for 1 min. Equal amounts of protein (1.8 Ag) were loaded into 10% SDS-PAGE gels. After transfer to a PVDF membrane (BioRad), the blots were blocked for 1h with Chemiblocker (Chemicon) and then primary antibody (ETRA or ETRB; Alamone Labs) was added at a dilution of 1 : 500. After washing (3 5 min TBST) secondary antibody (goat anti-rat IgG; Jackson Immunochemicals) was added at a 1 : 60,000 dilution and incubated for 1h. After washing (2 4 min TBST; 2 4 min TBS) the blots were exposed to ECL reagent (Pierce Chemical) and developed using Hyperfilm (Amersham) and a Kodak developer. Densitometric analysis was conducted using ImageJ (NIH) software. Drugs The fructose rich diet was obtained from Harlan Teklad (Indianapolis, IN, USA). The thromboxane analog, U46619, was purchased from Cayman Chemical (Ann Arbor, MI, USA). The antibodies for ETRA and ETRB were bought from Alamone Labs (Jerusalem, Israel). The other drugs used, including ET-1, UTP, and various reagents, were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Data analysis All data are presented as means F SE. The magnitude of spontaneous vascular tone was calculated as a percentage of preconstricted diameter according to the equation: % constriction ¼
DA DV T100 DA
ð1Þ
where DA is the diameter upon development of spontaneous tone at 80 mmHg and DV is the diameter after drug treatment. The concentration–response curves were evaluated at each concentration for differences between treated and untreated arteries from control and IR groups with ANOVA followed by Tukey’s post hoc test using SigmaStat (SPSS, INC.) software. Differences in maximal diameter and preconstricted diameter and data from Western blotting were evaluated by t-test. The criterion for significance was set at p b 0.05.
Results Spontaneous tone Maximal intraluminal diameter of the MCAs did not differ between groups: 222 F 5 Am (n = 30) control vs. 227 F 4 Am (n = 26) IR; p N 0.05. Spontaneously developed tone in response to 80 mmHg was also similar between groups (Fig. 1). At 1 h, the MCAs constricted to 33 F 2% and 34 F 3% of maximal
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Percent Contraction (%)
60 Control
50
Insulin Resistant 40 30 20 10 0 0
10
20
30
40
50
60
Time (minutes) Fig. 1. Spontaneously developed tone. Vessels were maintained at an intraluminal pressure of 80 mmHg and developed tone over the course of about an hour. Percent constriction is from maximal dilation. Both control and IR arteries developed tone similarly and, on average, over the same amount of time. Control n = 30; IR n = 26.
diameter in the control and IR groups, respectively. Additionally, the mean time required to develop spontaneous tone did not differ between groups (61 F 3 min control vs. 63 F 2 min IR; p N 0.05). Contractile response to ET-1 The cumulative dose–responses of control and IR MCAs to ET-1 are shown in Fig. 2. Both control and IR arteries constricted in response to ET-1 to just over 50% of the preconstricted diameter.
Percent Contraction (%)
60 50 40 30 20 Control 10
Insulin Resistant
0 -11
-10
-3.5
-9
-8.5
ET-1 (log M) Fig. 2. Cumulative dose–response curves to endothelin-1 (ET-1) in isolated, pressurized middle cerebral arteries of control (open circle) and insulin resistant (closed square) rats. Both control and IR arteries respond similarly to ET-1, p N 0.05. Control n = 14; IR n = 14.
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Interestingly, both the control and IR MCAs constricted similarly at each dose of ET-1, such that no significant differences at any dose were evident. Although the IR MCAs tended to be more responsive at the lower doses of ET-1 that were examined, this effect was not statistically different from control. Determination of protein levels of ETRA and ETRB Western blot analysis of ET-1 receptor subtypes A (ETRA) and B (ETRB) revealed no differences in protein content between control and IR arteries. Semi-quantitative densitometric analysis of Western blots for ETRA show that these receptors are expressed to a similar extent in control and IR rats: ETRA, control 76 F 3 arbitrary units (au) vs. IR 71 F 2 au. Likewise for ETRB, no differences were apparent between the groups (control 113 F 7 au vs. IR 114 F 3 au). Due to the paucity of tissue available from the MCAs, other arteries of the brain were also used in Western blot analysis. As such, it cannot be ruled out that certain vessels expressed more or less of the ETRA or ETRB proteins and that these segmental differences were lost upon homogenization. However, that is probably not the case, as the results of the Western blot analysis are in line with the in vitro vessel reactivity experiments on the MCA. Contractile response to U46619 The cumulative dose–response curves of control and IR MCAs to U46619 are shown in Fig. 3. Both control and IR arteries constricted in response to U46619 to about 45% of the preconstricted diameter. Both control and IR MCAs constricted similarly to each dose of U46619 and at no point were the groups significantly different. Contractile response to UTP The cumulative dose–response curves of control and IR MCAs to UTP are shown in Fig. 4. Both control and IR arteries constricted in response to UTP to almost 40% of the preconstricted diameter. Both
Percent Contraction (%)
60 Control
50
Insulin Resistant 40 30 20 10 0 -10
-9
-8
-7
-6
U46619 (log M) Fig. 3. Cumulative dose–response curves to U46619 in isolated, pressurized middle cerebral arteries of control (open circle) and insulin resistant (closed square) rats. Both control and IR arteries respond similarly to U46619, p N 0.05. Control n = 8; IR n = 6.
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Percent Contraction (%)
Control 50 Insulin Resistant 40 30 20 10 0 -10 -8
-7
-6
-5
-4.5
UTP (log M) Fig. 4. Cumulative dose–response curves to uridine 5V-triphosphate (UTP) in isolated, pressurized middle cerebral arteries of control (open circle) and insulin resistant (closed square) rats. Both control and IR arteries respond similarly to UTP, p N 0.05. Control n = 10; IR n = 11.
the control and IR MCAs constricted similarly at each dose of UTP, such that no significant differences at any dose were evident. Although the responses of IR MCAs tended to be greater than controls, at no time did they become significantly different.
Discussion The principle finding of this study is that MCAs from IR rats show no differences in contractile responses whether induced by pressure, ET-1, UTP, or U46619, a thromboxane analog. Furthermore, the cerebrovasculature of the IR animals show no changes in ETRA or ETRB protein levels. These findings suggest that the brain is protected from the enhanced contractile response to ET-1 that is seen in the peripheral vasculature (Katakam et al., 2001) in this model of IR. Thus, increased risk for stroke related complications in the context of IR is likely due to the inability of the cerebral arteries to dilate, and not to an enhanced response to constrictor stimuli. Pressure-induced vasoconstriction or myogenic tone is an integral part of cerebrovascular regulation. In this study, a constant intraluminal pressure of 80 mmHg caused a constriction of about 40% of the maximal diameter in MCAs from both control and IR animals. Also, MCAs from IR rats as well as those from controls, took the same amount of time, just over one hour, to achieve full spontaneous tone. These results differ from similar studies using a model of Type I diabetes. Zimmermann et al. (1997) used streptozotocin (STZ) to induce diabetes in female Sprague–Dawley rats and found that diabetic cerebral arteries showed more vasoconstriction to the same amount of intraluminal pressure than did control arteries. Dumont et al. (2003) also examined MCA responses to graded pressure increases in STZ-induced diabetic rats and demonstrated that diabetic MCAs constricted more than control arteries. These researchers went on to illustrate that bosentan, an ETR antagonist, normalized the diabetic responses. While the present study did not use graded pressure increases, the results presented here show no such discrepancy between control and IR arteries at the
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physiologically relevant 80 mmHg intraluminal pressure. The difference between this study and those of Zimmermann et al. (1997) and Dumont et al. (2003) may lie in the fact that the animals in this study were neither Type I diabetic animals nor Type II, but were pre-diabetic or IR animals. It is possible that Type I diabetes affects the cerebral arteries while the cerebral arteries in IR are not yet similarly affected. The data in this study show that MCAs from IR rats show no differences from control MCAs in response to ET-1. This is the first report to examine the constrictor responses of the cerebral vasculature in a model of IR. Other studies using fructose feeding to induce IR and looking at different vascular beds show differing results. Juan et al. (1998), using a 40 day diet of fructose feeding, presented evidence that the feeding led to an enhanced response to ET-1 in the thoracic aorta. Navarro-Cid et al. (1995), using a perfusion model of the mesenteric vascular bed, showed that four weeks of fructose feeding did not enhance responsiveness to ET-1. Additionally, Verma et al. (1997) using a nine week fructose feeding program demonstrated that while there was more ET-1 in the mesenteric arteries, those arteries showed a decreased maximal response and a decreased sensitivity to ET-1. Prolonged fructose feeding is known to induce hypertension; therefore comparisons to studies that utilize fructose feeding beyond four weeks are difficult. As such, a more relevant study is one which uses the same experimental model, such as that conducted by Katakam et al. (2001) who also examined vessels in vitro using an arteriograph system. The fructose feeding model used here, and by Katakam et al. (2001) has been shown to precede hypertensive and hyperglycemic responses (Katakam et al., 1998, 1999). Katakam et al. (2001) showed that mesenteric arteries from IR animals had a decreased EC50 in response to ET-1 despite showing the same E max as controls. That is, although the IR vessels constricted similarly to control vessels at maximal doses of ET-1, they were more responsive at sub-maximal doses. The data presented here suggest that the cerebral vasculature is not affected by the same increase in ET-1 sensitivity as seen in the mesenteric circulation (Katakam et al., 2001). In another study also using this model, Erdos et al. (2002b) examined dilator responses of MCAs in response to bradykinin. Preconstriction of the MCAs in that study was accomplished by the same dose of ET-1 for both control and IR arteries, highlighting the fact that both control and IR MCAs constricted similarly to the same dose of ET-1. This finding supports our results; the dose used by Erdos et al. (2002b) is within the range used in this study. Further supportive evidence comes from the results of Western blotting for ETRA and ETRB protein. Katakam et al. (2001) demonstrated that the enhanced ET-1 response they saw in the mesentery was associated with an increase in 125[I]-ET-1 binding. In a like manner, our analysis of ET-1 receptor (ETR) protein in brain vessels demonstrated that IR animals show no difference in ETRA protein levels from control; likewise ETRB protein levels did not differ between the groups. Because the endothelium was not removed from these vessels, ETRB receptors on the endothelium will contribute to higher ETRB protein levels. However, these protein levels were similar for both control and IR animals. The lack of an enhanced vasoconstrictor response to ET-1 in isolated control and IR MCAs coupled with the lack of a difference in ETRA and ETRB protein illustrates that the cerebral vasculature is protected from the increase in ET-1 sensitivity seen in the mesenteric circulation. The experiments with U46619, a stable TXA2 analog, again show no differences between control and IR arteries. Vasoconstrictor responses to U46619 were similar for control and IR MCAs at every dose examined (10 10 to 10 6 M). This also suggests that the cerebrovascular vasoconstrictor responses are not altered in IR. Despite the lack of a difference in vasoactivity, these studies do not
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address other actions of TXA2. For example, it remains to be addressed whether TXA2 activity on platelet aggregation varies in this model of IR from that seen in diabetic populations. The data presented here demonstrates that both control and IR MCAs constrict similarly to UTP. UTP is a P2Y receptor agonist and P2Y receptors are a G protein-coupled receptor population that responds to extracellular nucleotides. The P2Y receptor is present in the vascular smooth muscle of vessels and mediates vasoconstriction when applied abluminally (Wang et al., 2002). In the rat, ATP and UTP are equipotent against the P2Y receptor (Wang et al., 2002) and its use here allows the examination of a separate endogenous vasoconstrictor whose signal transduction pathway is distinct from ET-1 and TXA2. The abluminal application of UTP in this study resulted in a degree of constriction that was not significantly different between control and IR MCAs at any dose of UTP used (10 8 to 10 4.5 M). These results show that another vasoconstrictor agent with a signaling pathway distinct from ET-1 and TXA2 results in similar degrees of vasoconstriction for both control and IR MCAs. Other studies utilizing IR or diabetic arterial blood vessels show similar results with various constrictor agents. Verma et al. (1997) presented evidence that fructose feeding of rats for 9 weeks did not affect mesenteric arterial response to norepinephrine (NE). Navarro-Cid et al. (1995) illustrated that 4 weeks of fructose feeding in male Sprague–Dawley rats had no significant effect on constrictor responses of perfused mesenteric vessels in response to angiotensin II or phenylephrine. Additionally, Verma et al. (1996) gave further evidence that 4 weeks of fructose feeding did not affect mesenteric arterial response to NE using arterial rings. Indeed, in models of STZ-induced Type I diabetes, the mesenteric vascular bed and the hamster cheek pouch microcirculation show no altered responses to NE (Furman and Sneddon, 1993; Mayhan et al., 1999) nor to a thromboxane analogue, U-46619 (Mayhan et al., 1999). The present results show that, in the cerebral circulation of IR rats, vasoconstriction to ET-1 is not augmented and vasoconstriction to other constrictor agents, U46619 and UTP, is also unaltered by IR. Taken together, these data suggest that vasoconstrictor responses are, in large part, unaffected by IR or diabetes. Furthermore, the results presented here show that any abnormality in vasoconstrictor action in IR is limited to peripheral vascular beds (Miller et al., 2002) and does not affect the cerebrovasculature.
Conclusion The results of the present study show that vasoconstrictor responses of the cerebral circulation are not altered by IR induced by fructose feeding. Contractile responses of IR MCAs to pressure, ET-1, UTP, and U46619 do not differ from control responses. Thus, it appears that the increased susceptibility to cerebrovascular abnormalities associated with IR and diabetes (including cerebral ischemia, stroke, vertebrobasilar transient ischemic attacks (Abbott et al., 1987; Biller and Love, 1993)) is not due to an enhanced vasoreactivity to constrictor agents.
Acknowledgements This work is supported by grants from NIH (HL-30260, HL-50587, HL-65380, HL-66074) and AHA Bugher Foundation Award (0270114Z). B. Erdo¨s was supported by a Hungarian National Eo¨tvo¨s Fellowship.
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