INTRODUCTION

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TYPE II DIABETES MELLITUS impacts ~11 million Americans and is a potent risk factor for the development of peripheral vascular disease, a debilitating condition impacting nearly 60 million Americans (1). Over the last 10 years, the incidence of type II diabetes mellitus has increased more than 70% in 30- to 39-yr-old Americans and has become increasingly common among American children (25). Due to a nonfunctional leptin receptor gene (11), the obese Zucker rat (OZR) provides a valuable animal model for examining the effects of type II (non-insulin-dependent) diabetes. As a result of an extreme increase in food consumption, OZR rapidly develop numerous pathological conditions that are highly relevant to public health issues challenging Western society; among these conditions are type II diabetes, moderate hypertension, and obesity (15, 26, 28). Although the progression of these pathologies has been well documented, the effects of these conditions developing concurrently on peripheral vascular function remain unknown.

In OZR, previous studies (14, 29, 32) have indicated that intestinal and mesenteric microvessel dilation to nitric oxide (NO)-dependent stimuli was significantly reduced compared with responses in lean Zucker rat (LZR) control animals. This impaired dilation may have been related to either an reduced sensitivity of vessels to NO (31) or to a reduction in the bioavailability of NO in OZR compared with LZR. Reduction in the bioavailability of NO in OZR may be due to an increase in vascular superoxide levels (2, 21). The goal of the present study was to test the hypothesis that NO-mediated dilation is impaired in skeletal muscle distal arterioles of diabetic OZR compared with responses determined in LZR. Furthermore, we also hypothesized that an increased level of vascular oxidant stress underlies this impaired NO-dependent dilator reactivity of skeletal muscle microvessels through reductions in the bioavailability of NO.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. All experiments used 13- to 15-wk-old male LZR (Harlan; body weight, 358 ± 12 g; mean arterial pressure, 125 ± 4.1 mmHg; [glucose] in blood, 138 ± 15 mg/dl) and OZR (Harlan; body weight, 604 ± 16 g; mean arterial pressure, 159 ± 5.8 mmHg; [glucose] in blood, 486 ± 48 mg/dl) fed standard rat chow and tap water ad libitum. Rats (n = 16 per group) were housed in an animal care facility at the Medical College of Wisconsin that was approved by the American Association for the Accreditation of Laboratory Animal Care, and all protocols were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. Rats were anesthetized with an injection of pentobarbital sodium (60 mg/kg ip), and a carotid artery was cannulated for determination of arterial pressure.

3

Inhibition of prostanoid and NO production. To assess the role of prostanoid or NO release from the endothelium in regulating flow-induced dilation of in situ arterioles, the cremaster muscle was treated the cyclooxygenase inhibitor indomethacin (10⁶ M) or the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME; 10⁴ M), respectively (9). Both inhibitors were added to the PSS superfusate.

Dilator agonists. In addition, the dilation of in situ cremasteric arterioles from LZR and OZR were assessed in response to 1) acetylcholine (10⁹-10⁶ M), a receptor-mediated endothelium-dependent dilator agonist; 2) sodium nitroprusside (10⁹-10⁶ M), an endothelium-independent NO donor; 3) aprikalim (10⁹-10⁶ M), an activator of ATP-sensitive potassium (K_ATP) channels; and 4) Ca²⁺-free PSS containing 10³ M adenosine, to produce complete relaxation of the microvessel and determine the maximum (passive) arteriolar diameter (10).

Scavenging of oxidative free radicals. To determine the extent to which increased oxidant stress levels might underlie alterations to NO-dependent dilation of in situ skeletal muscle arterioles between LZR and OZR, the cremaster muscle from both groups was treated with the oxidative free radical scavengers polyethylene glycol-superoxide dismutase (PEG-SOD; 200 U/ml, Sigma) and catalase (80 U/ml, Sigma). Treatment of tissues with these substances has been previously demonstrated to significantly lower oxidant stress levels within skeletal muscle and vascular tissues (13, 22).

Measurement of superoxide production. Vascular superoxide levels in LZR and OZR were assessed using the dihydroethidine (DHE) microfluorography assay as described previously (5, 12, 24). Briefly, DHE is a lipophilic cell-permeable dye that is rapidly oxidized to ethidium in the presence of free radical superoxide. The produced ethidium is fixed by intercalation into nuclear DNA, thus giving an indication of oxidant stress within cells under investigation. In LZR and OZR, the aortas were removed from all animals, cleared of surrounding connective tissue, cut into segments, and placed in warm PSS (37°C) for 30 min. After this period, segments were incubated for 30 min in either control PSS or in PSS containing PEG-SOD (200 U/ml) and catalase (80 U/ml). DHE (Molecular Probes) was then added to the PSS for 30 min, followed by a final 30-min wash in DHE-free PSS. Segments were split longitudinally and placed endothelium side down on a PSS-moistened coverslip. The medial smooth muscle layer was visualized using confocal laser microscopy (Odyssey Systems) and acquired using Metamorph Image Acquisition software (Universal Imaging; West Chester, PA). Acquired images were analyzed for fluorescent intensity using National Institutes of Health Image software.

Experimental procedures. In all preparations, dilator reactivity of in situ cremaster muscle arterioles to acetylcholine, sodium nitroprusside, and aprikalim was evaluated as described above. Subsequently, experiments were completed to determine whether a difference in the dilation of arterioles to elevated wall shear rate exists between LZR and OZR and to determine the mediators of this response. As such, cremasteric arteriolar dilation in response to parallel occlusion was assessed in both rat groups under control conditions, after treatment of muscle with L-NAME or indomethacin, and after simultaneous application of both inhibitors.

Mathematical and statistical analyses. Blood flow volume through the daughter vessel was calculated from erythrocyte velocity and vessel radius (4) as follows

(1)

where F is the volume of blood flow (in nl/s), V is the centerline erythrocyte velocity (in mm/s), and r is the vessel radius (in µm). Wall shear rate (WSR; in s¹) was calculated as outlined by Koller and Kaley (17) as follows

(2)

where V_M is the mean flow velocity in the daughter vessel (calculated by dividing the optical Doppler measurement of centerline velocity by 1.6 to correct for the parabolic flow pattern in the vessel) and D is the vessel diameter.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Table 1 presents data describing the diameter and wall shear rate of in situ cremasteric arterioles from LZR and OZR under the experimental conditions of the present study. There was no difference in resting diameter of skeletal muscle arterioles of LZR and OZR under control conditions. Treatment of the cremaster muscle with L-NAME reduced arteriolar diameter in LZR only. Application of PEG-SOD and catalase increased diameter of arterioles in OZR only, with no effect in LZR. Furthermore, the passive diameter of vessels in OZR was significantly less than that in LZR, an effect that caused a significantly decreased level of calculated active tone in arterioles of OZR. Calculated wall shear rate in arterioles of OZR was not different from that in LZR under any conditions in the present study.

Data describing the dilation of cremasteric arterioles from LZR and OZR in response to challenge with acetylcholine, sodium nitroprusside, and aprikalim are presented in Fig. 1. In cremasteric arterioles of OZR, dilator responses to acetylcholine (Fig. 1A) and sodium nitroprusside (Fig. 1B) were reduced across the agonist concentration range compared with responses determined in LZR. In contrast, dilator responses to the K_ATP channel agonist aprikalim were not different between microvessels of LZR and OZR (Fig. 1C).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   The response of in situ cremaster muscle distal arterioles of lean Zucker rats (LZR) and obese Zucker rats (OZR) after application of increasing concentrations of acetylcholine (A), sodium nitroprusside (B), and aprikalim (C). n = 1 arteriole/rat from 6 animals. *P < 0.05 vs. LZR. , slope coefficient.

The dilation of cremasteric arterioles (Fig. 2A) and the changes in microvessel wall shear rate (Fig. 2B) in LZR and OZR during parallel arteriolar occlusion are presented in Fig. 2. In response to elevated wall shear rate, cremasteric arterioles of LZR underwent a significant dilation, rapidly restoring wall shear rate to preocclusion values. In contrast, arterioles of OZR did not dilate in response to elevated wall shear rate, thus impairing the ability of the microvessel to regulate wall shear rate.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   The change in arteriolar diameter (A) and wall shear rate (B) for in situ cremasteric arterioles of LZR and OZR after 120 s of physical occlusion of a parallel arteriole. n = 1 arteriole/rat from 6 animals. *P < 0.05 vs. LZR.

Figure 3 presents data describing the effects of treatment of in situ cremaster muscles of LZR and OZR with L-NAME or indomethacin on flow-induced dilation and the ability of the arteriole to regulate wall shear rate. In LZR, treatment of vessels with L-NAME significantly impaired arteriolar dilation after parallel occlusion (Fig. 3A), compromising the ability of the vessel to regulate shear levels (Fig. 3B). Treatment of the cremaster muscle of LZR with indomethacin had no effect on arteriolar responses to parallel occlusion. Furthermore, treatment of vessels of OZR with either L-NAME or indomethacin had no discernible effect on either flow-induced dilation (Fig. 3C) or the ability of the vessel to regulate wall shear rate (Fig. 3D).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   The change in arteriolar diameter (A and C) and wall shear rate (B and D) for in situ cremasteric arterioles of LZR (A and B) and OZR (C and D) after 120 s of physical occlusion of a parallel arteriole. Data are presented for arterioles under control conditions and after treatment of the cremaster muscle with either 104 M N-nitro-L-arginine methyl ester (L-NAME) or 106 M indomethacin. n = 1 arteriole/rat from 5 animals. *P < 0.05 vs. control.

Figure 4 presents data describing vascular oxidant stress levels in LZR and OZR. As determined using DHE microfluorography, baseline levels of superoxide anion in aortic smooth muscle LZR (Fig. 4A) were substantially less than those in OZR (Fig. 4B). Treatment of aortas of OZR with PEG-SOD and catalase lowered superoxide levels to values that were not distinguishable from those determined in LZR under control conditions (Fig. 4D), although this procedure had no discernible effect on vascular oxidant stress levels in LZR (Fig. 4C). These data are presented graphically as arbitrary units in Fig. 4F.

View larger version (77K): [in this window] [in a new window]   Fig. 4.   Vascular oxidant stress levels in aortas from LZR and OZR assessed using dihydroethidine microfluorography. Representative data are presented for LZR and OZR under control conditions (A and B, respectively) and after treatment of vessels from LZR and OZR with polyethylene glycol-superoxide dismutase (PEG-SOD) and catalase (C and D, respectively). All images are color matched (color standard; E). Fluorescent intensities in A-D are presented as arbitrary units (F). *P < 0.05 vs. LZR (control); P < 0.05 vs. OZR (control).

Data describing the dilation of cremasteric arterioles from OZR after challenge with acetylcholine and sodium nitroprusside under control conditions and in response to treatment of the muscle with PEG-SOD and catalase are presented in Fig. 5. Application of oxidative free radical scavengers significantly increased dilator responses of distal arterioles of OZR to acetylcholine [Fig. 5, A (percent rest diameter) and C (percent maximum possible dilation)] and sodium nitroprusside [Fig. 5, B (percent rest diameter) and D (percent maximum possible dilation)] compared with responses determined under control conditions. In contrast, dilation of cremasteric arterioles of LZR to these agonists after treatment with PEG-SOD and catalase was not altered from responses determined under control untreated conditions (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   The dilation of in situ cremaster muscle distal arterioles of OZR under control conditions and after treatment of the cremaster muscle with the oxidative free radical scavengers PEG-SOD and catalase in response to challenge with increasing concentrations of acetylcholine or sodium nitroprusside. The dilation of arterioles to the agonists are normalized to either the control arteriolar diameter (A: acetylcholine; B: sodium nitroprusside) or to the maximum possible dilation of the arteriole, determined during superfusion of the cremaster muscle with Ca2+-free physiological saline solution (C: acetylcholine; D: sodium nitroprusside). n = 1 arteriole/rat from 5 animals. *P < 0.05 vs. control responses. No significant differences were determined in the response of cremasteric arterioles of LZR to the agonists between control conditions and after treatment of the cremaster muscle with PEG-SOD and catalase.

The effects of treatment of the cremaster muscle with oxidative free radical scavengers on flow-induced dilation and wall shear rate after parallel occlusion are presented in Fig. 6. The flow-induced dilation of cremasteric arterioles of LZR after treatment with PEG-SOD and catalase was not altered from responses determined under control conditions (data not shown). In contrast, treatment of the cremaster muscle of OZR with free radical scavengers significantly increased skeletal muscle arteriolar dilation in response to elevated wall shear rate (Fig. 6A), thus enhancing the ability of the microvessel to restore wall shear rate toward normal preocclusion levels (Fig. 6B).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   The changes in arteriolar diameter (A) and wall shear rate (B) for in situ cremasteric arterioles of OZR after 120 s of physical occlusion of a parallel arteriole. Data are presented for arterioles under control conditions and after treatment of the cremaster muscle with PEG-SOD and catalase. n = 1 arteriole/rat from 5 animals. *P < 0.05 vs. control. No significant differences were determined in dilation or wall shear rate of cremasteric arterioles of LZR in response to parallel arteriolar occlusion between control conditions and after treatment of the cremaster muscle with PEG-SOD and catalase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Given that a recent study (25) has demonstrated that the incidence of type II diabetes mellitus has increased dramatically in 30- to 39-yr-old Americans and has become increasingly common among American children, ongoing investigation into clinically relevant animals models is of paramount importance. As such, OZR, with a nonfunctional leptin receptor gene (11) and ensuing elevations in caloric intake, provide a critically important animal model for study owing to the rapid development of type II diabetes mellitus and hypertension in parallel with obesity (15, 26, 28). However, as yet there has been little attempt to determine basic characteristics of skeletal muscle microvessels of OZR. To address this paucity in the literature, the present study was designed to determine whether alterations to NO-dependent dilator reactivity of skeletal muscle distal arterioles of OZR exist compared with responses in LZR and to determine the mechanisms that contribute to any alterations in these responses.

The initial observation of the present study was that dilator responses of in situ cremaster muscle distal arterioles of OZR to endothelium-dependent (acetylcholine; Fig. 1A) or endothelium-independent (sodium nitroprusside; Fig. 1B) NO-mediated dilator agonists or stimuli (elevated wall shear rate; Figs. 2 and 3) were severely impaired compared with responses determined in LZR control animals. In contrast, dilator responses to direct activation of K_ATP channels, the ion channel previously suggested as mediating NO-induced dilation in skeletal muscle microvessels (3, 6, 27), produced comparable dilator responses of these vessels between OZR and LZR (Fig. 1C). While our observation of an impaired acetylcholine-induced dilator reactivity of the skeletal muscle vasculature is in agreement with previous studies (2, 20, 21) using in situ skeletal muscle preparations in OZR and our observation of an impaired flow-induced microvessel dilation is in agreement with a previous study (14) in intestinal arterioles, to our knowledge these are the first observations of an impaired response to either challenge with NO or of a preserved dilator reactivity of skeletal muscle arterioles to direct activation of K_ATP channels mediating NO-dependent vasodilation.

At least three primary mechanisms could potentially contribute to the reduced NO-dependent dilation of skeletal muscle arterioles, each of which having been previously hypothesized as occurring in tissues of diabetic hypertensive OZR: 1) impaired production and/or release of NO by the microvessel endothelium in response to an appropriate agonist and/or stimulus (19); 2) an impaired vascular smooth muscle responsiveness to NO released from the endothelium (31); and/or 3) a reduced bioavailability of NO, potentially due to increased scavenging by oxidative free radicals, thus preventing the full manifestation of the vasodilator effects of released NO (2, 20, 21). The results of the current study support the third mechanism, elevation in superoxide reduced the bioavailability of NO.

On the basis of the results shown in Fig. 4, vascular oxidant stress levels in OZR, assessed using DHE microfluorography (5, 12, 24), were elevated compared with levels determined in LZR (Fig. 4). These observations are in agreement with those of previous studies (2, 19, 20) and support hypotheses recently suggested for the skeletal muscle circulation (21). The dramatic increase in vascular oxidant stress levels in OZR provides compelling evidence in support of the hypothesis that a reduced bioavailability of NO through scavenging by oxidative free radicals may represent the predominant mechanism underlying the impaired dilation of skeletal muscle microvessels to the challenges in the present study. Also evident in Fig. 4 was that treatment of aortas with the oxidative free radical scavengers PEG-SOD and catalase, while having no discernible effect on oxidant stress levels in LZR, reduced these levels in OZR to such an extent that they were not distinguishable from LZR.

After normalization of vascular oxidant stress levels subsequent to treatment of tissues with PEG-SOD and catalase, the dilator reactivity of in situ cremaster muscle arterioles of OZR to acetylcholine (Fig. 5A) and sodium nitroprusside (Fig. 5B) was increased to levels that were comparable with those determined in LZR under control conditions. In addition, the NO-dependent dilator stimulus of elevated wall shear rate was also enhanced in OZR after application of PEG-SOD and catalase, such that the ability of the microvessel to regulate wall shear rate was restored to levels that were comparable with those of LZR under control conditions (Fig. 6). These observations suggest that neither impaired agonist- or stimulus-induced endothelial production of NO nor an impaired vascular smooth muscle sensitivity to NO may be a dominant mechanism underlying the impaired dilation of cremasteric arterioles of OZR to the NO-dependent agonists or stimuli of the present study. Rather, these data suggest that scavenging of endothelium-derived NO by superoxide anion may be occurring, with the potential conversion to the much less biologically active substance peroxynitrate before the NO can effect a response in the vascular smooth muscle (29). However, it should be noted that these data do not rule out a potential contribution of impaired endothelial release of, or vascular smooth muscle sensitivity to, NO that was due to elevated oxidant stress levels that may have also been alleviated after treatment of the cremaster muscle with PEG-SOD and catalase.

In conclusion, the results of the current study describe a significant impairment in NO-mediated dilation of skeletal muscle arterioles in diabetic OZR. The compromised ability of these microvessels to dilate to NO occurred concomitantly with increased vascular oxidant tone. Furthermore, concentrations of free radical scavengers that ameliorated the increased superoxide levels also restored reactivity of microvessels to NO-dependent stimuli. We conclude that skeletal muscle perfusion may be impaired in obese diabetic patients via elevations in tissue oxidant stress. Targeting oxidant pathways therapeutically may by of substantial benefit to populations at risk for peripheral vascular disease.