Tetrahydrobiopterin rescues impaired responses of cerebral resistance arterioles during type 1 diabetes

Abstract

Our goal was to test the hypothesis that administration of tetrahydrobiopterin (BH₄) would improve impaired endothelial nitric oxide synthase–dependent dilation of cerebral arterioles during type 1 diabetes. In addition, we examined the influence of BH₄ on levels of superoxide in brain tissue. In vivo diameter of cerebral arterioles in nondiabetic and diabetic rats was measured in response to endothelial nitric oxide synthase–dependent agonists (acetylcholine and adenosine 5′-diphosphate) and an endothelial nitric oxide synthase–independent agonist (nitroglycerine) before and during application of BH₄ (1.0 µM). We also measured levels of superoxide from cortex tissue in nondiabetic and diabetic rats under basal states and during BH₄. Acetylcholine and adenosine 5′-diphosphate dilated cerebral arterioles in nondiabetic rats, but this vasodilation was significantly impaired in diabetic rats. In contrast, nitroglycerine produced similar vasodilation in nondiabetic and diabetic rats. Application of BH₄ did not enhance vasodilation in nondiabetic rats but improved impaired cerebral vasodilation in diabetic rats. Basal superoxide levels were increased in cortex tissue from diabetic rats, and BH₄ reduced these levels to that found in nondiabetic rats. Thus, BH₄ is an important mediator of endothelial nitric oxide synthase–dependent responses of cerebral arterioles in diabetes and may have therapeutic potential for the treatment of cerebral vascular disease.

Keywords

Acetylcholine adenosine 5′-diphosphate rats brain superoxide

Introduction

Type 1 and type 2 diabetes mellitus impair endothelial nitric oxide synthase (eNOS)- and neuronal nitric oxide synthase (nNOS)-dependent relaxation/dilation of large and small cerebral arteries/arterioles.^1–10 Many mechanisms have been proposed to account for impaired eNOS- and nNOS-dependent responses of cerebral arteries/arterioles during diabetes, including the production of a cyclooxygenase constrictor substance,⁹ activation of protein kinase C^11,12 and an increase in oxidative stress via activation of multiple cellular pathways.^8,13–17

Activation of nitric oxide synthases (NOS) has the ability to produce nitric oxide and superoxide.^18,19 The production of nitric oxide requires arginine as a substrate and several co-factors [nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), calcium and tetrahydrobiopterin (BH₄)] for normal activity.²⁰ Several studies have shown that diabetes leads to a decrease in levels of BH₄ within endothelium of peripheral blood vessels.^21,22 In addition, it appears that the biosynthesis of BH₄ in the brain is reduced in diabetes.²³ This loss of BH₄ would then lead to an uncoupling of eNOS and favour the formation of superoxide over that for nitric oxide.

Many previous studies have shown that acute or chronic supplementation with BH₄ can improve/restore impaired eNOS-dependent responses of large and small peripheral arteries/arterioles.^22,24–29 In contrast to studies of peripheral arteries/arterioles, there is little information regarding the influence of BH₄ on reactivity of cerebral blood vessels during diabetes. A study by Katakam et al.² found that impaired relaxation of isolated cerebral arteries in Zucker obese rats (a model of insulin resistance) in response to insulin could be partially reversed by pretreatment with BH₄, suggesting a reduction in BH₄ bioavailability leading to eNOS uncoupling. However, there is no information available regarding the influence of BH₄ on responses of cerebral resistance arterioles to eNOS-dependent agonists during type 1 diabetes. Thus, the first goal of this study was to characterize the influence of acute treatment with BH₄ on eNOS-dependent and eNOS-independent responses of cerebral arterioles. Given that oxidative stress is an important component in impaired responses of cerebral arterioles during diabetes, our second goal was to determine the influence of BH₄ on superoxide levels in cortex tissue obtained from nondiabetic and diabetic rats.

Methods

Induction of diabetes

All procedures involving rats were reviewed and approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (200–220 g) were divided randomly into nondiabetic and diabetic groups. All rats had access to food and water ad libitum. One group of rats was injected with streptozotocin [50–60 mg/kg intraperitoneally (IP)] to induce diabetes. The second group of rats (nondiabetic) was injected with vehicle (sodium citrate buffer). Blood samples, for measurement of blood glucose concentration, were obtained 3–5 days after injection of streptozotocin or vehicle and on the day of the experiment.

Preparation of animals

Rats were prepared for studies at 2–3 months after injection of streptozotocin or vehicle. Rats were anaesthetized (thiobutabarbital sodium; Inactin; 100 mg/kg IP), and a tracheotomy was performed. The animals were ventilated mechanically with room air and supplemental oxygen. A catheter was inserted into a femoral vein for injection of supplemental anaesthesia (20–30 mg/kg). A femoral artery was cannulated for measurement of arterial pressure and to obtain a blood sample for the measurement of blood glucose concentration and arterial blood gases.

After these initial procedures, a craniotomy was performed over the left parietal cortex as described previously.³⁰ Briefly, the rat was placed in a head holder, and an incision was made in the skin to expose the skull. The skin was retracted with sutures and served as a ‘well’ for the suffusion fluid. A cranial window was made in the bone, and the dura was incised to expose the cerebral microcirculation. The cranial window was continuously suffused with a bicarbonate buffer that was bubbled with 95% nitrogen and 5% carbon dioxide. The temperature of the suffusate was maintained at 37°C. The cranial window was connected via a three-way valve to a syringe pump, which allowed for infusion of agonists into the suffusate. This method maintained a constant pH, pCO₂ and pO₂ of the suffusate during the infusion of agents.

Reactivity of cerebral arterioles

The cranial window was suffused for 30–45 min before testing responses to the agonists. In each rat, we examined responses of the largest pial arteriole exposed by the craniotomy to eNOS-dependent agonists [acetylcholine, 1 and 10 µM and adenosine 5′-diphosphate (ADP), 10 and 100 µM] and a NOS-independent agonist (nitroglycerine, 1 and 10 µM). After an initial examination of vascular reactivity to the agonists, the cranial window was suffused with BH₄ (1 µM) for 60 min prior to and during a second application of the agonists. The diameter of cerebral arterioles was measured using a video image shearing monitor.^31,32 Baseline diameter was measured immediately prior to application of agonists and during application of agonists at 1-min intervals for 5 min. Steady-state responses to agonists were reached within 3 min after exposure to the drug, and the diameter of cerebral arterioles returned to baseline within 5 min after application of agonist was discontinued. All agonists were prepared using the bicarbonate buffer.

Measurement of superoxide levels

Superoxide levels were measured by lucigenin-enhanced chemiluminescence as described previously.^15,33 In separate groups of nondiabetic (n = 8) and diabetic (n = 6) rats, the brain was removed and placed in a Krebs/HEPES buffer (pH 7.4). Samples of cortex tissue, cut from brains of nondiabetic and diabetic rats, were placed in polypropylene tubes containing 5 µM lucigenin. The tubes were then read in a Sirius/FB15 luminometer (Berthold Detections Systems), which reports relative light units (RLU) emitted over a 30-s interval for 5 min. Levels of superoxide reported are the value of tissue plus lucigenin-containing buffer minus background (lucigenin-containing buffer without tissue) and are normalized for tissue weight (RLU/min/mg tissue). We measured the levels of superoxide under basal conditions and following a 30-min pretreatment of cortex tissue with BH₄.

Statistical analysis

Analysis of variance (ANOVA) with Fisher’s test for significance was used to compare reactivity of cerebral arterioles to the agonists before and during treatment with BH₄, and superoxide levels under basal conditions and during treatment with BH₄. Unpaired t-tests were used to compare baseline diameter of cerebral arterioles, blood pressure, blood glucose concentration and body weight. A p-value of 0.05 was considered to be significant.

Results

Control conditions

Baseline diameter of cerebral arterioles was 44 ± 3 µm in nondiabetic rats and 51 ± 1 µm in diabetic rats (p < 0.05). Blood pressure was 106 ± 5 mmHg in nondiabetic rats and 104 ± 7 mmHg in diabetic rats (p > 0.05). On the day of the experiment, blood glucose concentration was 105 ± 9 mg/dL in nondiabetic rats and 393 ± 25 mg/dL in diabetic rats (p < 0.05). Body weight was 454 ± 22 g in nondiabetic rats and 245 ± 30 g in diabetic rats (p < 0.05).

Responses in nondiabetic and diabetic rats

Acetylcholine and ADP dilated cerebral arterioles in nondiabetic and diabetic rats (Figure 1). However, the magnitude of vasodilation was significantly less in diabetic rats compared to nondiabetic rats (Figure 1). In contrast, nitroglycerine produced similar dose-related dilation of cerebral arterioles in nondiabetic and diabetic rats (Figure 1). Thus, impaired responses of cerebral arterioles to acetylcholine and ADP in diabetic rats cannot be explained by nonspecific impairment of vasodilation during diabetes.

Figure 1.

Responses of cerebral arterioles to acetylcholine, ADP and nitroglycerine in nondiabetic (n = 5) and diabetic (n = 6) rats. Values are mean ± SE.

Responses following BH₄

Topical application of BH₄ (1.0 µM) did not alter baseline diameter cerebral arterioles in nondiabetic rats (42 ± 2 µm before vs 42 ± 3 µm during suffusion with BH₄) or diabetic rats (52 ± 2 µm before vs 49 ± 2 µm during suffusion with BH₄).

Topical application of BH₄ did not alter dilation of cerebral arterioles in response to acetylcholine, ADP or nitroglycerine in nondiabetic rats (Figure 2). In contrast, topical application of BH₄ enhanced dilation of cerebral arterioles to acetylcholine and ADP, but not to nitroglycerine, in diabetic rats (Figure 2).

Figure 2.

Responses of cerebral arterioles to acetylcholine, ADP and nitroglycerine in nondiabetic and diabetic rats before and during topical application of BH₄. Values are mean ± SE.

Superoxide levels

Baseline levels of superoxide were significantly higher in cortex tissue from diabetic than in cortex tissue from nondiabetic rats (Figure 3). Treatment of cortex tissue from nondiabetic rats with BH₄ did not alter superoxide levels. In contrast, treatment of cortex tissue from diabetic rats with BH₄ produced a significant decrease in superoxide levels (Figure 3).

Figure 3.

Superoxide levels in brain tissue from nondiabetic (n = 8) and diabetic (n = 6) rats under basal conditions and during exposure to BH₄. Values are mean ± SE.

Discussion

There are two major new findings of this study. First, we found that acute treatment with BH₄ can improve impaired eNOS-dependent responses of cerebral arterioles during type 1 diabetes. Second, we found that BH₄ reduced basal superoxide levels in cortex tissue from type 1 diabetic rats. Thus, it appears that a deficiency of BH₄ during type 1 diabetes leads to an uncoupling of eNOS to favour the production of superoxide, which then contributes to impaired eNOS-dependent dilation of cerebral arterioles observed in diabetic rats.

Responses to acetylcholine and ADP

Acetylcholine and ADP were used to examine eNOS-dependent responses of cerebral arterioles. Studies have shown that dilation of large and small cerebral blood vessels in response to acetylcholine is dependent upon the synthesis/release of nitric oxide, presumably via the activation of eNOS.^9,34–38 These previous studies found that acetylcholine produced dose-related dilatation of the basilar artery and cerebral (pial) arterioles that was inhibited by enzymatic inhibitors of NOS (L-NMMA and L-NNA). Previous studies also have shown that ADP dilates cerebral arterioles via the synthesis/release of nitric oxide, presumably via activation of eNOS.^38–41 Although, others^42,43 have suggested that relaxation of the rat middle cerebral artery to purines^42,43 and dilation of pial arterioles in mice⁴⁴ are related, in part, to the synthesis/release of nitric oxide and to synthesis/release of an endothelium-derived hyperpolarizing factor(s) to activate potassium channels. However, studies by others^45–49 have suggested that activation of potassium channels does not play a significant role in dilatation of cerebral arterioles to the agonists used in this study. Based upon these previous findings, we suggest that acetylcholine and ADP are appropriate agonists to evaluate eNOS-dependent dilatation of cerebral arterioles.

Consideration of previous studies

Several studies have examined the influence of exogenous treatment with BH₄ or its precursor, sepiapterin, on responses of large and small peripheral blood vessels during type 1 and type 2 diabetes. Pieper²⁴ reported that relaxation of the aorta from type 1 diabetic rats in response to acetylcholine could be restored to that observed in nondiabetic rats by acute exposure to BH₄. Shinozaki et al.^22,50 found that impaired eNOS-dependent relaxation of the aorta in insulin-resistant rats (high fructose diet) could be restored by acute treatment with BH₄. Pannirselvam et al.^27,29 reported that acute and chronic treatment of db/db mice with BH₄ or sepiapterin restored eNOS-dependent responses of mesenteric arterioles. Bagi and Koller⁵¹ found impaired flow-mediated dilation of isolated arterioles from the gracilis muscle in type 1 diabetes rats, and that acute treatment with sepiapterin could reduce this impairment. Finally, Alp et al.⁵² found that transgenic mice with endothelial-targeted overexpression of guanosine triphosphate-cyclohydrolase I, the rate-limiting enzyme in BH₄ synthesis, prevented impaired eNOS-dependent relaxation of the aorta during type 1 diabetes. In addition to studies using animal models of diabetes, studies examining the influence of BH₄ have also been conducted using human subjects with diabetes. Heitzer et al.²⁵ found that flow-mediated dilation was impaired in humans with type 2 diabetes, and acute treatment with BH₄ could improve this impairment. In addition, a study by Ihlemann et al.²⁶ found that an oral glucose challenge in human subjects impaired flow-mediated dilation and this could be reversed by acute treatment with BH₄. Thus, it appears that BH₄ is an important mediator of eNOS regulation of vascular function in both animals and humans with type 1 and type 2 diabetes.

The mechanisms for the effect of BH₄ or sepiapterin on peripheral vascular function during diabetes appear to be related to the effects of BH₄ on oxidative stress and nitric oxide production/bioavailability. Previous studies have shown that reduced levels of BH₄ during diabetes, due to oxidative degradation, lead to the formation of dihydrobiopterin (BH₂).^21,22,50 BH₂ can then compete for binding to eNOS at the BH₄ binding site and thus promotes eNOS uncoupling and a loss of nitric oxide production/bioavailability.^20,53,54 Many of the studies discussed above have found increases in oxidative stress and decrease in nitric oxide levels during diabetes that appear to be restored towards that seen in nondiabetic models by treatment with BH₄ and sepiapterin.

Consideration of present study

With regard to the cerebral circulation, a very little information exists regarding the influence of BH₄ on cerebral vascular function. Katakam et al.² have reported that acute treatment of middle or posterior cerebral arteries with sepiapterin improved relaxation in response to insulin in Zucker obese rats. In addition, Hamon et al.²³ have reported reduced levels of BH₄ in the brain of type 1 diabetic rats, but no function studies were conducted. This study, to the best of our knowledge, is the first to determine the influence of BH₄ on eNOS-dependent responses of cerebral resistance arterioles in vivo during type 1 diabetes. We found that acute treatment with BH₄ rescued impaired eNOS-dependent responses of cerebral arterioles observed during type 1 diabetes. Surprisingly, we did not observe an effect of BH₄ on baseline diameter of cerebral arterioles. Although we did not expect that BH₄ would alter baseline diameter of cerebral arterioles in nondiabetic rats, we did consider the possibility that BH₄ would increase baseline diameter in type 1 diabetic rats. However, this was not the case. Our findings are similar to that reported by others for cerebral and peripheral arteries.^2,29,51 The precise reason(s) for a lack of an effect of BH₄ on baseline diameter in diabetic animals remain unclear.

In addition to our findings regarding cerebral vascular function, we found that basal levels of superoxide were increased in cortex tissue from diabetic rats, and that these levels could be reduced by exogenous treatment with BH₄. This finding may suggest that impaired responses of cerebral arterioles during type 1 diabetes may be related to a decrease in the bioavailability of BH₄ leading to an increase in the levels of superoxide through the uncoupling of eNOS.

We suggest that the findings from this study may have important therapeutic implications for the treatment of cerebral vascular diseases observed in diabetic patients. Others have reported that BH₄, when administered peripherally (oral, intravenous and intraperitoneal) to monkeys,⁵⁵ rats⁵⁶ and humans,⁵⁷ has the ability to cross the blood–brain barrier and enter the central nervous system. Thus, it is conceivable that treatment of type 1 diabetic subjects with BH₄, by rescuing cerebral vascular reactivity, could then decrease the risk of cerebral vascular diseases, including stroke and cognitive dysfunction, associated with type 1 diabetes.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

This study was supported by the National Heart, Lung, and Blood Institute Grant HL-079587.

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Tetrahydrobiopterin rescues impaired responses of cerebral resistance arterioles during type 1 diabetes

Abstract

Keywords

Introduction

Methods

Induction of diabetes

Preparation of animals

Reactivity of cerebral arterioles

Measurement of superoxide levels

Statistical analysis

Results

Control conditions

Responses in nondiabetic and diabetic rats

Responses following BH4

Superoxide levels

Discussion

Responses to acetylcholine and ADP

Consideration of previous studies

Consideration of present study

Footnotes

Declaration of conflicting interests

Funding

References

Responses following BH₄