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CARDIOVASCULAR

A Nonthiazolidinedione Peroxisome Proliferator-Activated Receptor Agonist Reverses Endothelial Dysfunction in Diabetic (db/db^-/-) Mice

Smooth Muscle Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada (A.G.H., W.B.W., M.P., Y.J., D.S., T.J.A., C.R.T.); and Department of Metabolic Disorders, Merck Research Laboratories, Rahway, New Jersey (J.P.B.)

Received March 16, 2005; accepted September 29, 2005.

	Abstract

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We have previously reported that endothelium-dependent relaxation to acetylcholine is impaired in small mesenteric arteries from spontaneously diabetic (db/db) mice. The objective of the present study was to examine the effects of treatment of the db/db and the insulin-resistant ob/ob mice with the PPAR agonist 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH). In the db/db model, an 8-week treatment with COOH (30 mg/kg/day) reduced plasma glucose from 48.0 ± 2.5 (untreated) to 12.6 ± 1.1 mM. In contrast, plasma glucose was not elevated in untreated ob/ob mice. Relaxation of small mesenteric arteries mediated by acetylcholine was impaired in the untreated db/db diabetic mice (51.7 ± 7.4% maximal relaxation, n = 6) but not in the ob/ob mice (70.8 ± 8.6% maximal relaxation, n = 3). This impairment was reversed with COOH treatment (86.9 ± 0.4% maximal relaxation, n = 5). Malondialdehyde was elevated in plasma from diabetic db/db mice (13.9 ± 1.1 versus 12.0 ± 0.7 µmol/ml); however, when normalized to total cholesterol, no significant differences in the ratio of lipid peroxidation in plasma were identified. Western blot analysis and quantitative polymerase chain reaction for eNOS was performed on the isolated mesenteric vessels and revealed no differences in the relative levels of eNOS expression in diabetic and control animals; in addition, treatment with COOH had no significant effect on eNOS levels in either group. In summary, endothelial dysfunction and hyperglycemia were completely normalized in COOH-treated db/db mice. In contrast, nonhyperglycemic ob/ob mice exhibited normal vasodilatory responses to acetylcholine and, consequently, COOH treatment had no effect on endothelial function.

A significant advance in our understanding of vascular disease in type 2 diabetes has been the development of mouse models. Two such examples of models of insulin resistance are the db/db and ob/ob mouse lines (Coleman, 1982; Leibel, 1997). The db/db mouse is an extensively studied mouse model that spontaneously develops characteristics of type 2 diabetes, including obesity, early insulin resistance-producing hyperinsulinemia, and an eventual -cell secretory defect, marked hyperglycemia, and lipid abnormalities (Hofmann et al., 2002). The db/db phenotype has been linked to a mutation in the leptin receptor of these animals (Leibel, 1997). The ob/ob mouse is similar to the db/db mouse in the development of obesity, hyperinsulinemia, and insulin resistance (Haluzik et al., 2004); however, hyperglycemia is not prominent. The ob mutation arose spontaneously in the leptin gene of the C57BL/6J mouse (Zhang et al., 1994).

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily and have multiple metabolic and cardiovascular effects (Hsueh and Bruemmer, 2004). Several ligands for the PPAR isoform are clinically available. Thiazolidinedione agonists for PPAR receptors act as insulin-sensitizing agents and are thought to have uniquely beneficial effects on vascular function in diabetics (van Wijk and Rabelink, 2004). Thiazolidinediones have been shown to prevent the development of atherosclerosis in several experimental models (Li, 2000; Aizawa et al., 2001; Collins et al., 2001). In addition, the thiazolidinedione rosiglitazone has been reported to improve endothelial function in diabetic mice independently of improvements in metabolic dysfunction (Bagi et al., 2004). COOH is a PPAR agonist that bears a carboxylic acid pharmacophore in place of the thiazolidinedione moiety found in the PPAR agonists currently in clinical use. It has previously been shown to act as an insulin sensitizer in a pharmacological manner similar to the thiazolidinediones (Berger et al., 2001; Laplante et al., 2003).

Endothelial dysfunction can be identified by the reduction in endothelium-dependent vasodilator response to acetylcholine (ACh). Although the endothelium has many additional functions beyond the control of vascular tone, this reduced response to ACh serves as an important indicator of vascular dysfunction and has been closely associated with diabetes in both humans and animals (De Vriese et al., 2000; Pannirselvam et al., 2003). There is increasing evidence of a prognostic link between endothelial dysfunction, as defined by the vasodilator response to ACh, and the later development of vascular complications in both diabetic and nondiabetic populations (Verma et al., 2003; Mancini, 2004); however, the cellular basis for endothelial dysfunction remains unknown.

We have previously demonstrated the development of endothelial dysfunction in the db/db mouse model of type 2 diabetes (Pannirselvam et al., 2002, 2003). In the present study, we compared endothelial function in small mesenteric arteries (SMA) from hyperglycaemic db/db mice with SMA from obese but not overtly hyperglycemic ob/ob mice and assessed the influence of chronic treatment with the experimental nonthiazolidinedione PPAR agonist COOH. Our objectives were 2-fold: first, to determine whether correction of endothelial dysfunction by PPAR agonists extended to nonthiazolidinedione members of this class of agents and, second, to examine potential molecular targets within the vasculature that may be involved in mediating endothelial dysfunction and the response to the PPAR class of drugs.

	Materials and Methods

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Drug Treatment. Male C57BL/KsJ-lepr^db/lepr^db (db/db) mice (18 animals), male C57BL/6J-lep^ob/lep^ob (ob/ob) mice (nine animals), and respective age-matched controls (db/+ and ob/+) were obtained from Jackson Laboratories (Bar Harbor, ME). At 8 weeks of age, each type of animal was divided into three equal groups. Group 1 received 30 mg/kg/day of COOH (2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid) in powder chow (Carley et al., 2004) for 8 weeks. Group 2 received regular powdered chow (ProLab RMH 2500/5P14; PMI International, Brentwood, MO) for 8 weeks. Group 3 received 30 mg/kg/day of COOH for 8 weeks and then were crossed over to untreated feed for a further 5 weeks. At the end of the treatment period, the animals were killed by cervical dislocation in accordance with a protocol approved by Animal Care Committee at the University of Calgary.

Experimental Protocols. Second- and third-order mesenteric arteries were cut into 2-mm rings and mounted on a Mulvany-Halpern myograph (Mulvany and Halpern, 1977). After a 45-min equilibration period in carbogen (95% O₂, 5% CO₂) aerated Krebs' solution, the vascular reactivity to the adrenoceptor agonist phenylephrine (PE) was studied. After a 30-min stabilization period, endothelium-dependent vascular relaxation to ACh was recorded in preparations contracted with a submaximal concentration of PE (EC_75–80). A repeated 30-min stabilization period was followed with measurement of relaxation to sodium nitroprusside in similarly contracted SMA tissues from db/db, ob/ob, and respective controls. PE-induced contractions were normalized to the percentage of 120 mM KCl-induced contraction.

Plasma glucose, triglyceride, and cholesterol were assayed using commercial kits (Sigma-Aldrich, St. Louis, MO). Samples of mesenteric arteries and aorta were flash-frozen and later used for RNA extraction.

Quantitative PCR. Total RNA was extracted from mesenteric arteries and aortic tissue using an RNeasy Mini Kit with on-column DNase treatment (QIAGEN, Valencia, CA), and first-strand cDNA was subsequently synthesized using a Superscript RT Kit (QIAGEN). Real-time PCR primers were designed (-actin F, 5'-ACGGCCAGGTCATCACTATTG-3'; -actin R, 5'-CCAAGAAGGAAGGCTGGAAAAGA-3'; eNOS F, 5'-CAACGCTACCACGAGGACA-3'; and eNOS R, 5'-CTCCTGCAAAGAAAAGCTCTGG-3') and analyzed in positive and negative control PCR reactions with SYBR Green (QIAGEN) at eight annealing temperatures ranging from 52 to 62°C. Melt-curve analysis was performed to visualize primer specificity by revealing the presence or absence of primer dimers. For further verification, 1.0 µl of the PCR reaction was analyzed on a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) using a DNA 500 LabChip kit. Sequencing was performed on products from successful reactions. PCR efficiency was determined by performing real-time PCR on serial dilutions of mouse heart cDNA (94.6% for -actin, 96.7% for eNOS). Real-time PCR reactions were carried out using 2.0 µl of first-strand cDNA in a total reaction volume of 25 µl containing 1x QuantiTect SYBR Green Supermix (QIAGEN) and a 0.25 µM concentration of forward and reverse primers. PCR reactions were hot-started (95°C for 15 min) and then exposed to 40 cycles of 94°C for 0.25 min, 55.7°C for 0.5 min, and 72°C for 0.5 min where fluorescence data collection occurred during each extension phase. Melt-curve analysis was again performed following cycling as a method of validation. The -fold relative to -actin for eNOS was calculated using the 2^-CT method (Livak and Schmittgen, 2001).

Western Blotting. Homogenates of thoracic aorta were prepared from flash-frozen tissue in lysis buffer containing 100 mM Hepes (pH 7.4), 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 0.1% Tween 20, 1% Triton X-100, and protease inhibitor cocktail (Sigma-Aldrich). Approximately 50 mg of tissue was combined with 0.1 ml of lysis buffer, ground with a micropestle, and subjected to three rapid freeze/thaw cycles to disrupt tissue. Once completely lysed, solution was centrifuged at 13,000 rpm for 10 min to remove insoluble material. Protein concentration was determined for each sample using a Bio-Rad protein analysis kit (Hercules, CA). Equal amounts of protein were resolved under reducing conditions on an 8% SDS polyacrylamide gel. Immunoblotting was performed with a polyclonal antibody to eNOS (BD Biosciences Transduction Laboratories, Lexington, KY) at a dilution of 1:500 in nonfat milk/Tris buffer. The membrane was subsequently probed with a secondary antirabbit antibody conjugated to horseradish peroxidase at a dilution of 1:1000 and developed with chemiluminescence (Pierce, Rockford, IL). The membrane was then exposed to X-ray film (Eastman Kodak Co., Rochester, NY), which was subsequently developed.

Statistical Analysis. In all experiments, n equals the number of animals used in the protocol. Values are mean ± S.E.M. Statistical significance of difference between means of different groups were performed using either Student's t test or two-way analysis of variance with Bonferroni post hoc test. A value of P < 0.05 was considered statistically significant.

	Results

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The monogenic murine models of type 2 diabetes used in this study have mutations in the leptin receptor (db/db) and leptin molecule (ob/ob) on genetic backgrounds predisposed to development of insulin resistance (C57BL/KsJ and C57BL/6J, respectively). Loss of the leptin response axis in both ob/ob and db/db mice leads to chronic hyperphagia and resultant obesity. We observed increased body weights in db/db mice (mean 51.5 versus 30 g db/+; p < 0.05) and ob/ob mice (mean 60.7 versus 30.2 g ob/+; p < 0.05) at 16 weeks of age (Table 1). This obesity was accompanied by marked hyperglycemia in untreated db/db mice (48.0 ± 2.5 mM) compared with untreated control db/+ mice (12.3 ± 0.8 mM) (Fig. 1). In contrast, the ob/ob mouse displayed no significant change in plasma glucose at 16 weeks of age (16.6 ± 2.4 versus 13.8 ± 2.0 mM in control ob/+ mice). Both the db/db and ob/ob mice showed an increase in total cholesterol relative to their aged matched controls: 2.77 versus 1.81 mM in the db/db versus db/+ and 4.30 versus 2.06 mM in the ob/ob versus ob/+ (Table 1). Likewise, triglycerides were elevated in db/db mice (2.11 versus 1.18 mM) but not in ob/ob mice (1.60 versus 1.53 mM) relative to control animals.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Plasma glucose measurements from mice prior to sacrifice. n = 5 for db mouse types and n = 3 for ob mouse types. db/db mice had significantly elevated plasma glucose, which was reduced while on COOH treatment. Error bars represent mean ± S.E.M. *, P < 0.0001.

Treatment with COOH, a PPAR receptor agonist, completely corrected the hyperglycemia seen in the db/db mice (from 48–13 mM; Fig. 1) and significantly decreased cholesterol in the control db/+ group and triglyceride levels in all groups of animals with the exception of the control db/+ group (Table 1). When COOH was discontinued and animals consumed a normal diet for an additional 5 weeks (crossover, Fig. 1), the db/db mice became hyperglycemic again, indicating that continued treatment with COOH was required to maintain normoglycemia. No significant differences in glucose levels were noted in the ob/ob mice with treatment; however, these animals are relatively euglycemic without treatment. Malondialdehyde, a marker of lipid peroxidation, was elevated in plasma from the db/db mice (13.9 ± 1.1 versus 9.0 ± 1.2 µmol/ml in db/+) and ob/ob mice (22.9 ± 5.2 versus 8.7 ± 1.2 µmol/ml in ob/+); however, when these results were normalized to total cholesterol in serum, no significant differences in the amount of malondialdehyde per unit cholesterol were seen between treated and untreated animals (Table 1).

Endothelial and vascular function was analyzed using the Mulvany-Halparen wire myograph technique on second- and third-order SMA from each of the animals. Data are presented in summary graph form. Phenylephrine applied in cumulative concentrations until maximal contractions were achieved (Fig. 2A) showed no significant difference between either treated or untreated db/db or ob/ob mice. In contrast, there was an impairment of the relaxation of the SMA mediated by acetylcholine (Fig. 2B) in the untreated db/db diabetic mice (51.7 ± 7.4% maximal relaxation, pEC50 6.75, n = 6) versus their age-matched control animals (db/+; 86.1 ± 5.6% maximal relaxation, pEC50 6.33 n = 6; P < 0.05). This impairment (endothelial dysfunction) was reversed in the COOH-treated db/db animals (86.9 ± 0.4% maximal relaxation, pEC50 6.87, n = 5). The ob/ob mouse model did not demonstrate a statistically significant impairment of ACh-mediated relaxation (70.9% maximal relaxation, pEC50 6.51; Fig. 2B) when compared with its control littermates (69.5% maximal relaxation, pEC50 6.14; Fig. 2B); the experimental number in the ob group was smaller (n = 3). No significant differences between any of the groups were observed in relaxation of SMA by the endothelium-independent agent sodium nitroprusside (Fig. 2C).

View larger version (26K): [in this window] [in a new window]   Fig. 2. A, phenylephrine induced contraction of SMA from db and ob mice. The PE-induced contraction was normalized to 120 mM KCl. The data are expressed as mean ± S.E.M. There are no significant differences between treated and untreated animals observed. B, maximal acetylcholine induced relaxation in SMA precontracted with phenylephrine. The data are expressed as mean ± S.E.M. *, P < 0.005 db/db and db/db crossover compared with db/db-treated. C, sodium nitroprusside-induced relaxation following contraction with phenylephrine. The data are expressed as mean ± S.E.M. No significant differences between treated and untreated animals are observed.

Quantitative PCR was performed on flash-frozen samples of both aorta and mesenteric arteries for each of the animals to determine whether COOH had an effect on eNOS gene transcription. No significant differences were observed in eNOS gene expression between treated and untreated animals in either the db/db or ob/ob mouse models when results were normalized to -actin expression (Fig. 3). Expression of eNOS in the ob/+ and ob/ob mouse aorta and mesenteric arteries demonstrated statistically significant elevation in mean level of message compared with their db/+ and db/db counterparts. The relatively lower level of eNOS expression per unit -actin observed in the aortic tissues is likely accounted for by the much increased quantity of smooth muscle in aortic samples relative to the eNOS-synthesizing endothelial cell layer, thus decreasing the relative amount of eNOS (endothelial cell derived) per unit -actin (endothelial and smooth muscle cell derived).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Quantitative PCR for eNOS mRNA in aorta and mesentery isolated from animal groups as labeled. n = 5 in db mice and n = 3 in ob mouse types. Error bars represent mean ± 1 S.D.

View larger version (68K): [in this window] [in a new window]   Fig. 4. Western blot of eNOS protein in thoracic aorta. Aortic tissue homogenates were loaded for equivalent total protein. A single band is observed at 140 kDa in all samples. Left lane shows positions of molecular mass standards. Second and third lanes show aortic tissue from db/+ animals untreated (c-) and treated (c+) with COOH. Third and fourth lanes show aortic tissue from db/db animals: untreated (d-) and treated (d+) with COOH.

	Discussion

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We studied two monogenic mouse models of obesity and metabolic dysfunction in this study. The diabetic db/db mouse is a well accepted model of type 2 diabetes (Coleman, 1982), whereas the ob/ob mouse is a model of obesity and insulin resistance without overt hyperglycemia (Coleman, 1982; Haluzik et al., 2004). Both of the mouse models studied developed metabolic dysfunction as a result of monogenic mutations in the leptin endocrine axis; the db/db mouse was deficient in the ObRb leptin receptor 2B, and the ob/ob mouse was deficient in the leptin molecule itself. However, it is the polygenic background of these animals that largely contributes to the development of insulin resistance (Haluzik et al., 2004). The db/db mouse on the C57BL/KsJ background develops marked insulin resistance and then declining insulin levels because of a pancreatic -cell secretory defect, producing severe hyperglycemia by 16 weeks of age. The ob/ob mouse, on the C57BL/6J background, exhibits marked obesity and lipid abnormalities without significant hyperglycemia at the same age of 16 weeks. Serum insulin levels have been previously been reported for the C57/BL/KsJ db/db mouse (Hofmann et al., 2002) showing increases in circulating insulin levels relative to control animals until approximately 14 weeks of age and then a progressive decline. These data are in accordance with other published data regarding the db/db and ob/ob mice on the C57BL/KsJ and C57BL/6J backgrounds, respectively (Coleman, 1982; Haluzik et al., 2004). However, only the db/db mouse model demonstrated marked endothelial dysfunction in the SMA. This suggests that endothelial dysfunction in the diabetic db/db animals is more closely linked to hyperglycemia than to alterations in the lipid profile.

The marked hyperglycemia in untreated db/db mice was normalized by chronic (8 weeks) treatment with COOH. This glucose-lowering effect of COOH is consistent with the observation that insulin-stimulated glucose uptake was enhanced in cardiomyocytes from COOH-treated db/db mice (Carley et al., 2004). Remarkably, the endothelial dysfunction, which characterized SMA from untreated db/db mice, was completely normalized by COOH treatment. Thus, our study suggests that the prevention of endothelial dysfunction with COOH treatment may be secondary to the improvement of the metabolic profile of the db/db animals, most probably because of correction of chronic hyperglycemia. However, Bagi et al. (2004) have reported that a 1-week treatment of db/db mice with rosiglitazone reduced oxidative stress and reversed endothelial dysfunction in the coronary arteries of db/db mice without any significant action on the metabolic abnormalities, thus indicating that the improvement of endothelial function, at least with rosiglitazone, is independent of changes in insulin sensitivity. Further studies are required to investigate the direct effects of PPAR activation on endothelial function.

The key source of NO for NO-mediated relaxation in the endothelium is thought to be the enzyme eNOS. Whereas post-translational modification and cofactors play a major role in the regulation of this enzyme, its production in the endothelium is critical to normal endothelial function (Verma et al., 2003; Andrews et al., 2005). Using quantitative PCR techniques and Western blotting, we examined eNOS message and protein levels in endothelium of arterial tissue. Although we found significant differences in eNOS expression between the db/db and ob/ob mouse models, we did not observe an effect of COOH on eNOS gene expression or protein content. This suggests that COOH does not directly affect eNOS gene transcription. However, the increased expression of eNOS in the ob/ob mice, relative to that determined in db/db and control mice, may have a protective effect inhibiting the development of endothelial dysfunction in these animals that may result from the dyslipidemic state of the ob/ob mouse.

PPAR activation stimulates fatty acid storage in adipose tissue. Its activation results in both an increase in adipocyte number and fatty acid influx into adipocytes and a remodeling of adipose tissue (Ferre, 2004). In addition, COOH has been shown to alter the distribution of adiposity in rodents (Laplante et al., 2003). In this study, we note that additional weight was gained by those animals treated with COOH (Table 1). Thus, the glucose lowering effect of PPAR agonists due to insulin sensitization may be due largely to adipose tissue actions, a proposition supported by studies performed with tissue-specific PPAR null mice (Evans et al., 2004). Although it is tempting to ascribe all of the actions of COOH to its role in regulating adipose tissue metabolism, PPAR agonists have pleiotropic effects, including stimulation of insulin-dependent glucose transporter GLUT4, up-regulation of the angiopoietin-related gene peroxisome proliferator-activated receptor- angiopoietin-related gene and adiponectin, as well as direct actions in vascular smooth muscle (Bruemmer et al., 2003a,b) The antiatherosclerotic actions of the PPAR agonists (see Berger et al., 2005) are also suggestive that these drugs are also targeting specific endothelial cell genes.

Reactive oxygen species (ROS) are a family of molecules, including molecular oxygen and its derivatives, which are produced in all aerobic cells. NO is an important tonic inhibitory factor for controlling mitochondrial respiration, and thus a decrease in eNOS activity (or NO bioavailability) will result in an increase in superoxide production by mitochondria. Brownlee and colleagues (2000) have argued, based on studies with cultured endothelial cells, that mitochondria are the source of ROS and observed that uncoupling oxidative phosphorylation in bovine aortic endothelial cells that had been treated with high-glucose prevents the sequelae of hyperglycemia. Unfortunately, intervention studies in humans with antioxidants (notably vitamin(s) C and/or E) have provided confusing and conflicting results. Data from studies with the db/db mouse indicate that acute incubation with indomethacin, a nonselective inhibitor of COX, and SQ29548, a selective thromboxane receptor antagonist, significantly attenuated the enhanced contraction to -adrenoceptor agonists in the SMA; as a result, enhanced thromboxane generation may also contribute to vascular dysfunction in diabetes (Pannirselvam et al., 2005). Thus, we examined lipid peroxidation products in the serum of our animals using an assay for malondialdehyde. Malondialdehyde is derived from peroxidation of fatty acid chains and has been proposed to provide an approximation of oxidative stress (Nielsen et al., 1997). Although plasma malondialdehyde levels were higher in the db/db and ob/ob animals compared with controls, we found no significant differences in the amount of malondialdehyde per unit cholesterol. We chose to undertake the normalization to serum cholesterol to account for the significant differences in serum lipid content among the different animals. Our assumption is that a greater content of fatty acids in the serum results in an increase in malondialdehyde levels independent of changes in oxidative stress. Although this normalization suggests that there is not a marked elevation in overall oxidative stress in these murine models, alondialdehyde levels provide only a gross measure of overall oxidative stress in the animals and do not rule out the possibility of an increase in oxidative stress within the endothelium itself (Halliwell and Whiteman, 2004). We have reported, based on the use of dihydroethidium as a fluorescent dye indicator of oxidative stress, that intracellular superoxide levels are elevated in in situ frozen unfixed sections of SMA from 16-week male (untreated) db/db mice (Pannirselvam et al., 2005), thus suggesting that it is the elevated intracellular oxidative stress that may determine endothelial dysfunction.

The development of peripheral edema is a major side effect limiting the usefulness of currently available PPAR agonists, affecting approximately 5% of patients on a thiazolidinedione and 15% of patients who combine thiazolidinediones with insulin therapy (Mudaliar et al., 2003; King and Levi, 2004). Thus, there has been great interest in the development of new PPAR ligands that minimize these side effects. We have demonstrated that the nonthiazolidinedione PPAR agonist COOH reverses the endothelial dysfunction that develops in the db/db mouse model of type 2 diabetes, without any change in eNOS expression. In addition, we demonstrated that endothelial dysfunction does not occur in the nonhyperglycemic ob/ob mouse model and that COOH-treated ob/ob mice show no change in endothelial function. This selective modulatory effect of COOH upon endothelial function may be a reflection of the reversal of hyperglycemia in db/db mice.

	Footnotes

 
This work was supported by operating grants from the Canadian Diabetes Association and Canadian Institutes for Health Research (CIHR) (to T.J.A. and C.R.T.) and a CIHR fellowship (to A.G.H.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.086397.

ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; COOH, 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid; SMA, small mesenteric arteries; PCR, polymerase chain reaction; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; SQ29548, [1S-[1,2(Z),3,4]]-7-[3[[2-[(phenylamino)carbonyl-[hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid]; ACh, acetylcholine; PE, phenylephrine.

¹ Current affiliation: Departments of Physiology and Medicine, University of Toronto, Ontario, Canada.

² Current affiliation: School of Medical Sciences, Royal Melbourne Institute of Technology University, Bundoora, Victoria, Australia.

Address correspondence to: Dr. Andrew G. Howarth, Rm. 82A, HMRB, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada, T2N 4N1. E-mail: aghowart{at}ucalgary.ca

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