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Chapitre 2 : Effets d’un traitement avec un antioxydant, le tempol, sur la dysfonction endothéliale et vasculaire ainsi que sur la résistance à l’insuline induite par un régime riche en graisse et en sucrose (HFHS) chez le rat

Table des matières

Ce projet a pour but d’étudier et de caractériser la réactivité vasculaire de l’aorte, la fonction endothéliale et les actions métaboliques de l’insuline chez l’animal résistant à l’insuline avec un surplus de poids suite à un traitement chronique et préventif de 4 semaines avec un antioxydant. La consommation quotidienne de nourriture, le gain de poids, la glycémie, l’insulinémie, la triglycéridémie ainsi que les AGL sériques ont été mesurés. Les expérimentations ont porté sur la réactivité vasculaire de l’aorte, l’expression de la protéine eNOS dans le muscle squelettique et l’aorte ainsi que la nitrotyrosination des protéines dans l’aorte et l’expression de l’ET-1 dans le lit mésentérique. De plus, le transport du glucose stimulé par l’insuline dans le muscle squelettique, les débits sanguins régionaux, la conductance vasculaire ainsi que le taux d’infusion du glucose durant un clamp euglycémique hyperinsulinémique ont été mesurés. Nos résultats indiquent clairement la présence d’une dysfonction endothéliale et d’une diminution de la sensibilité à l’insuline chez les rats nourris avec le régime HFHS durant 4 semaines. Par contre, le traitement préventif avec le tempol améliore ou normalise ces paramètres.

Cet article sera soumis prochainement pour publication dans la revue scientifique British Journal of Pharmacology.

Title: Effect of treatment with tempol on endothelial and vascular dysfunctions and insulin resistance induced by a high fat high sucrose diet in rats.

Authors: Mylène Badeau (MSc)1, Frédéric Bourgoin (MSc)1, Sébastien Mélançon (MSc)1, Maryse Pitre (MSc)1, André Nadeau (MD, FRCPC)2, Richard Larivière3 (PhD) and Hélène Bachelard* (PhD)1

From: Department of Medicine and Lipid Research Unit1 and Diabetes Research Unit2, from CHUL Research Center, Research Center of L’Hôtel Dieu de Québec3, Centre Hospitalier Universitaire de Québec (CHUQ), Laval University, Quebec, Canada.

Running title: Tempol and metabolic and vascular dysfuntions.

*The author to whom correspondence should be sent:

Dr. Hélène Bachelard

Lipid Research Unit

CHUL Research Center, CHUQ

2705 blvd. Laurier

Ste-Foy, (Quebec)

Canada, G1V 4G2.

Phone number: (418) 656-4141 (ext.: 48253)

Fax number: (418) 654-2759

E-mail: helene.bachelard@crchul.ulaval.ca

Phone number: (418) 656-4141 (ext.: 48253)

This study was undertaken to investigate the effect of treatment with an antioxidant, tempol, on vascular reactivity, endothelial function and insulin metabolic and vascular actions in a rat model acquired insulin resistance and overweight, the high fat high sucrose-fed rat (HFHS). Male Sprague-Dawley rats were randomly divided in four groups to receive a regular rat chow-diet, in the absence or presence of daily treatment with tempol (1,5 mM/kg), or a HFHS diet, in the absence or presence of treatment with tempol for 4 weeks. In a first series of experiments, the effects of treatment with tempol were examined on plasma levels of glucose, insulin, triglyceride and free fatty acid, body weight, vascular endothelium function, endothelial nitric oxide synthase(eNOS) protein expression in vascular and muscular tissues, endothelin content in vascular tissues, tissue abundance of nitrotyrosine, and insulin-stimulated skeletal muscle glucose transport. In a second series of experiments, new groups of tempol-treated or vehicle-treated HFHS- and chow-fed rats had pulsed Doppler flow probes and intravascular catheters implanted to determine blood pressure, heart rate and regional blood flows. Insulin sensitivity was assessed during a euglycemic hyperinsulinemic clamp performed in conscious rats. Treatment with tempol was found to prevent several of the alterations produced by the HFHS diet. Notably, we found that tempol treatment prevented the increases in plasma levels of glucose and insulin noted in the HFHS-fed rats, and the development of insulin resistance, as demonstrated during a euglycemic hyperinsulinemic clamp. Tempol treatment was also found to prevent the rise in blood pressure, the reduction in endothelium-dependent vasorelaxation, the decrease in eNOS protein expression in skeletal muscle and vascular endothelium, and the rise in vascular concentration of endothelin associated with the diet. Furthermore, tempol was found to potentiate the enhancing effect of L-NAME on the contracting responses to phenylephrine, and to prevent the impairment in the insulin-mediated renal and skeletal muscle vasorelaxing responses, and in insulin-stimulated glucose transport activity in skeletal muscles. Finally, tempol-treatment in HFHS-fed rats was found to prevent the increased formation of nitrotyrosine in vascular tissues. Together, these findings point toward an enhanced ROS-mediated inactivation and sequestration of NO, and its possible contribution to the impaired endothelium-dependent relaxation and impaired insulin-mediated renal and skeletal muscle vasodilator responses noted in our HFHS-fed rats. Furthermore, our results indicate that antioxidative agents, could be useful to prevent the metabolic and hemodynamic abnormalities resulting from 4 weeks consumption of a HFHS diet in rats.

In the Western hemisphere, the incidence of insulin resistance and its complications has been growing at an alarming rate and is reaching epidemic proportions (Keller & Lemberg, 2003; Meigs, 2003; Seidell, 2000) . While insulin resistance plays a critical role in the pathogenesis of type 2 diabetes, it also represents a major underlying abnormality driving cardiovascular disease. The expression “insulin resistance syndrome” or metabolic syndrome, refers to insulin resistance as a common denominator of the syndrome and is associated with a clustering of cardiovascular risk factors currently encountered in affected individual, including hypertension, hyperlipidemia, hyperinsulinemia, hypertriglyceridemia and obesity. Among these cardiovascular risk factors, upper body obesity, has been pointed out as a primary contributor to acquired insulin resistance as increasing adiposity is correlated with impaired insulin action (DeFronzo & Ferrannini, 1991; Haffner et al ., 1988) . Lifestyle factors that contribute to obesity have also been shown to affect insulin action independently and adversely. Recent studies by Barnard et al (Barnard et al ., 1993; Barnard et al ., 1998; Barnard & Wen, 1994) have indicated that the metabolic syndrome can be induced in rats by feeding a high-fat refined-sugar diet, whereas it can be controlled in humans by feeding a low-fat complex-carbohydrate diet combined with aerobic exercise.

Insulin is characteristically recognized for its ability to stimulate glucose uptake into insulin-sensitive tissues. However, in addition to its effects on glucose metabolism, insulin was shown to vasodilate skeletal muscle vasculature, through a nitric oxide (NO)–mediated mechanism (Scherrer et al ., 1994; Steinberg et al ., 1994) , in insulin-sensitive, but not insulin-resistant subjects (Baron et al ., 1995) . The vasodilating action of insulin was confirmed by us in conscious rats (Gaudreault et al ., 2001; Pitre et al ., 1996) , and by others in humans and experimental animals over a range of physiological insulin concentrations and by using different techniques (Anderson et al ., 1991; Clark et al ., 2001; Cleland et al ., 1999; Liang et al ., 1982; Vincent et al., 2002; Vollenweider et al ., 1993) . The vasodilator effect of insulin was suggested to play a physiological role in its glucose lowering action, by increasing glucose delivery to metabolically active tissue (Baron et al ., 1995) . Earlier studies from our laboratory have clearly shown the blood flow regulatory effect of insulin in rats and its paradoxical effect in insulin resistant rats (Pitre et al ., 1996; Santuré et al ., 2002) . Indeed, endothelial dysfunction, initially described as an impaired endothelium-dependent vasodilation to acetylcholine, and defects in insulin stimulation of blood flow (BF), has been described in several states of insulin resistance, including hypertension, obesity and type 2 diabetes (Higashi et al ., 1997; Hogikyan et al ., 1998; Laakso et al ., 1992; Laakso et al ., 1990; Steinberg et al ., 1996) , and has been related to a decrease in bioavailability of NO. In keeping with these, we recently found that a high sucrose or a combined high fat high sucrose (HFHS) diet in rats induces insulin resistance, which was associated with a reduced eNOS protein expression in skeletal muscle and vascular tissues, and abnormal endothelium-dependent vasorelaxation (Bachelard et al ., 2005; Santuré et al ., 2002) . Concomitant alterations were also noted in the insulin-mediated skeletal muscle glucose uptake and vasodilation in this animal model. Others laboratories have reported endothelial dysfunction and hypertension in rats fed a high-fat, refined-carbohydrate diet Reil et al Roberts et al .Roberts et al .2000. We suggested that a reduced production of NO, consecutive to a decreased expression and/or activity of the enzyme NOS, could be responsible for the complex interaction between endothelial dysfunction, and abnormal insulin-mediated vascular responses and glucose uptake (Bachelard et al ., 2005; Santuré et al ., 2002) . However, beside the fact that NO production could be impaired, an increased degradation of NO by chemical interactions with locally formed reactive oxygen species (ROS), such as the superoxide anion (O2-) (McIntyre et al ., 1999) , could also be an important factor that contribute to impair endothelial vasomotor function by reducing bioavailability of the endogenous nitrovasodilator NO. Indeed, we and others have observed an increased formation of nitrotyrosine in vascular tissues isolated from HFHS-fed rats, which is indicative of oxidative stress (Bachelard et al ., 2005; Roberts et al ., 2002) . In the presence of O2-, the half-life of NO is significantly reduced by the rapid formation of peroxinitrite, a powerful oxidizing agent (Beckman & Koppenol, 1996). O2- is a major limiting factor of NO availability leading to alteration of the vascular effects of NO on blood pressure and cell growth (Raij & Baylis, 1995) . Increasing evidence suggest that an exaggerated production of endothelial ROS is involved in endothelial dysfunction and hypertension (Akpaffiong & Taylor, 1998; Kitiyakara & Wilcox, 1998; McIntyre et al ., 1999) .

Therefore, in the lights of these previous findings, we undertook the present study in HFHS-fed rats, a rat model with acquired insulin resistance and obesity, to characterize the effect of a preventive treatment with an antioxidant agent, the superoxide dismutase (SOD) mimetic tempol, on endothelial NO bioavailability, vascular endothelium function and insulin metabolic and vascular actions. Tempol proved to be a relatively stable low molecular weight compound with non-immunogenic properties and low toxicity, and that can freely crosses cell membrane (Zollner et al ., 1997). In a previous study, a significant improvement in endothelium-dependent vasodilation was shown in diabetic afferent arterioles treated with tempol (Schnackenberg & Wilcox, 2001). In this study, the rats were challenged with the HFHS diet, in the absence or in the presence of treatment with tempol, for a period of 4 weeks. The effects of treatment with tempol on vascular responses to euglycemic infusion of insulin, and insulin sensitivity were determined during a euglycemic hyperinsulinemic clamp carried out in conscious, unrestrained rats, chronically instrumented with pulsed Doppler flow probes and intravascular catheters. In vitro studies were also performed in isolated tissues to further examine the effect of tempol on insulin-mediated glucose transport activity in skeletal muscles, vascular reactivity, NO bioavailability, nitrotyrosine formation and endothelin-1 (ET-1) protein content in vascular tissues.

Animal and feeding protocol

All surgical and experimental procedures were approved by the Animal Care and Handling Committee of Laval University. The research and the care of animals conformed to the Guide for the Care and Use of Laboratory Animals as adopted by the National Institute of Health. Male Sprague-Dawley (n=101) aged 5 weeks and initially weighing 175-230 g were purchased from Charles River (St-Constant, Canada) and were housed individually in stainless steel cages. They were placed in a temperature-controlled room (22 ± 1°C) on a 12 hours/12 hours light/dark (lights on at 06:00) and had free access to tap water. The animals were randomly divided into two groups. One group of rats (n=46) was fed standard laboratory rat chow (Teklad Global 18% Protein Rodent Diet, 2018) and the other (n=55) was fed a purified HFHS diet. The composition of the diets is given in Table 1. At the beginning of each diet, one group of HFHS-fed rats (n=27) and one group of chow-fed rats (n=26) received by oral gavage the SOD mimetic agent, tempol, at a dose of 1.5 mmol kg-1 day-1 for all the duration of the diet. The two other groups of rats (one group of HFHS-fed rats (n=28) and one group of chow-fed rats (n=20)) did not receive tempol, but were treated with the vehicle (phosphate buffer saline (PBS) 0.2 mM, pH 7.4) also by oral gavage. The animals were allowed to acclimate to their environmental conditions and diet for 4 weeks before the experiments were initiated. During that time, the animals had free access to water and the diet. Body weight and food intake were recorded every two day.

Triacylglycerol, nonesterified fatty acid, glucose and insulin measurements.

Plasma glucose, insulin, triglycerides and free fatty acids concentration were measured in tempol-treated HFHS-fed rats (n=27) and chow-fed rats (n=26) and compared to untreated HFHS-fed rats (n=28) and chow-fed rat (n=20). Plasma glucose concentration was determined using a Beckman glucose analyzer (Beckman Instruments, Palo Alto, CA). Insulin level was measured by radioimmunoassay (RIA) using porcine insulin standards and polyethylene glycol for separation (Desbuquois & Aurbach, 1971). Plasma triglycerides were assayed by an enzymatic method (Kohlmeier, 1986) using a reagent kit from Roche Diagnostics (Basel, Switzerland), which allowed correction for free glycerol. Free fatty acids were measured enzymatically using commercially available kits (Wako Chemicals, Dallas, USA).

Vascular reactivity

Following the settling-in period, the rats from each dietary group (tempol-treated HFHS-fed rats (n=19), tempol-treated chow-fed rats (n=18), untreated HFHS-fed rats (n=17) and untreated chow-fed rats (n=15)) were anaesthetized (sodium pentobarbital, 75 mg/kg i.p.) and then killed by cardiac puncture. Blood was collected, placed in untreated polypropylene tubes, and kept on ice until centrifuged (1,500 x g, 4oC, 15 minutes). The separated plasma was stored at -20oC until later biochemical determinations. The thoracic aorta from each rat was quickly excised and placed into a ice-cold Kreb-Henseleit solution, containing (mM): 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.18 MgSO4, 1.18 KH2PO4, 2.5 CaCl2, 5.5 Glucose; pH 7.4. The fat and connective tissue were trimmed from the aorta, and the aorta was cut into 3-4 mm rings. The rings were mounted under 2 g resting tension on stainless steel hooks in 15-ml organ baths filled with the Krebs-Henseleit buffer and gassed with 95% O2 and 5% CO2 at 37oC. Tension was recorded with a Grass force transducer on a four-channel multi-pen recorder (Grass Instruments, Quincy, Mass., U.S.A.). The rings were allowed to equilibrate in the chamber for 1 h, during which time the incubation medium was changed at 15-min intervals. In some experiments, the NOS inhibitor, L-NAME (100 µM), was added at the beginning of the equilibration period and was present for all the duration of the experiments. At the end of the equilibration, contractions were elicited by 18 mM KCl. After a 30-minutes washout period, contractions were elicited by 10-6 M phenylephrine. While the rings were contracted with phenylephrine, endothelium-dependent dilator responses to 10-5 M carbachol were obtained to ensure that the endothelium of each vascular ring was functioning. After washout of the phenylephrine and carbachol, a cumulative dose-response curve to phenylephrine (10-9-10-5M) was obtained. After a washout period, rings were precontracted with phenylephrine (10-6M) and cumulative relaxation curves to carbachol (10-8-10-4 M), and then to SNP (10-10-10-7M) were quantified to assess endothelium-dependent and endothelium-independent ability of the smooth muscle to relax in each experimental group. After a washout period, cumulative dose-response curves to phenylephrine (10-9-10-5M) were obtained in the absence and then in the presence of insulin (150 nM) to assess the attenuating action of insulin on contractions induced by phenylephrine. Each curve was separated by a 30-minute washout period. The contracting curves to phenylephrine were expressed in gram of tension, while the relaxation curves to carbachol and SNP were expressed as the percentage changes from the contraction induced by phenylephrine.

Glucose transport activity in isolated rat skeletal muscles

The effect of tempol treatment on glucose transport activity in isolated skeletal muscles was characterized in HFHS-fed rats (n=9) and chow-fed rats (n=11) and compared to untreated HFHS-fed rats (n=12) and chow-fed rats (n=10). Basal and insulin-stimulated glucose utilization were examined in isolated soleus and extensor digitorum longus (EDL) skeletal muscles from overnight fasted rats. Glucose transport was measured by use of the glucose analogue [3H]-2-deoxy-D-glucose, according to the method developed by Hansen et al (Hansen et al ., 1994) and as previously described (Santuré et al ., 2002). The rats were anaesthetized with a mixture of ketamine-xylazine (100 and 10mg kg-1, respectively, i.p.). Soleus and EDL muscles were dissected out and rapidly cut into 20-30 mg strips and incubated for 30 min at 30°C in a shaking waterbath into 25 ml flasks containing 3 ml of oxygenated Krebs-Ringer bicarbonate (KRB) buffer supplemented with 8 mM glucose, 32 mM mannitol and 0.1% BSA (RIA grade). Flasks were gassed continuously with 95% O2/5% CO2 throughout the experiment. Then, the rats were killed by decapitation and vascular tissues (descending thoracic aorta and mesenteric vascular bed) and gastrocnemius skeletal muscles were quickly removed, clamp-frozen and stored at –80°C for further analysis (see below).

After the initial incubation, muscles were incubated for 30 min in oxygenated KRB buffer in the absence or presence of insulin (Humulin R) at 4 different concentrations (0.002, 0.02, 0.2 and 2 mU/ml). Muscles were next washed for 10 min at 29°C in 3 ml of KRB buffer containing 40 mM mannitol, and 0.1% BSA. Muscles were then incubated for 20 min at 29°C in 3 ml KRB buffer containing 8 mM [3H]-2-deoxy-D-glucose (2.25 µCi/ml), 32 mM [14C]-mannitol (0.3 µCi/ml), 2 mM sodium pyruvate, and 0.1% BSA. Insulin was present throughout the wash and uptake incubations (if it was present in the previous incubation medium). After the incubation, muscles were rapidly blotted at 4°C, clamp frozen and stored at –80°C until processed. Muscles were processed by boiling for 10 min in 1 ml of water. Extracts were transferred to an ice bath, vortexed and then centrifuged at 1000 x g. Triplicate 200 µl aliquots of the muscle extract supernatant and of the incubation medium were counted for radioactivity using a Wallac 1409 counter. [3H]-2-deoxy-D-glucose uptake rates were corrected for extracellular trapping using [14C]-mannitol (Hansen et al ., 1994).

Determination of eNOS protein expression and nitrotyrosine formation in thoracic aorta

Protein expression of eNOS and nitrotyrosine formation in thoracic aorta from untreated and tempol-treated chow-fed and HFHS-fed rats were determined by Western blot analysis. The aorta of each rat was individually homogenized in 5 volume of homogenization buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1mM EGTA, 10% glycerol, 1mM DTT, 50 mM NaF, 5 mM Na pyrophosphate, 1% Triton X-100, 0.1 mg.ml-1 phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors cocktail (P-8340, Sigma-Aldrich, Oakville, Ont. Canada). The homogenates were centrifuged at 10,000x g for 10 minutes at 4oC to remove non-homogenized material (crude homogenate). Protein concentrations of the supernatant were determined by the bicinchoninic acid method (Pierce), using BSA as the standard.

Protein samples (45 µg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, proteins were electrophoretically transferred (100 V, 2 hours) to a polyvinyldene difluoride (PVDF) filter membrane. Then, the PVDF membranes were incubated for 1 hour at room temperature with buffer 1 (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.04 % Igepal, and 0.02 % Tween-20) containing 5 % BSA, followed by overnight incubation at 4°C with the specific primary antibodies. Monoclonal eNOS antibodies were purchased from Transduction Laboratories (Lexington, KY, USA), and monoclonal nitrotyrosine antibodies were obtained from Calbiochem (San Diego, CA, USA). Antibodies were diluted 1:500 for both eNOS and nitrotyrosine in buffer I containing 1% BSA. Monoclonal α-tubullin antibodies purchased from Sigma-Aldrich (Oakville, Ont., Canada) were used to standardize the results. Dilution of α-tubullin antibodies was 1:5000 in buffer I containing 1% BSA. Then, PVDF membranes were washed for 45 min in buffer I (at room temperature), followed by 1 hour incubation with anti-mouse immunoglobulin G (1:10000 dilution) conjugated to horseradish peroxidase (Amersham, Ont., Canada) in buffer I containing 5 % non-fat milk. After PVDF membranes were washed for 45 minutes in buffer I (at room temperature) the immunoreactive bands were detected by the enhanced chemiluminescence method (PerkinElmer Life Sciences). Autoradiographs were analyzed by laser scanning densitometry using a tabletop Agfa scanner (Arcus II, Etobicoke, Ont., Canada) and quantified with the NIH Image program (http://rsb.info.nih.gov/nih-image/Default.html).

Determination of eNOS protein expression in gastrocnemius muscle by Western blot analysis

Approximately 200 mg of gastrocnemius muscle from tempol-treated HFHS-fed (n=8) and chow-fed (n=7) and untreated HFHS-fed (n=7) and chow-fed rats (n=5) were homogenized in 1 ml of homogenization buffer and centrifuged at 10,000x g for 10 minutes at 4oC, as described above. Protein concentrations of the supernatant were determined by the bicinchoninic acid method (Pierce), using BSA as the standard. Muscle lysates (2 mg protein) were used to purify eNOS enzyme using 2’5’-ADP-Sepharose resin as described (Perreault & Marette, 2001) . Briefly, the muscle extracts were incubated (2 h, 4 ˚C) with 5 mg (dry weight) of 2’,5’-ADP Sepharose (4B) beads (Amersham Pharmacia) equilibrated in PBS. ADP Sepharose beads were collected by centrifugation and washed 3 times with PBS containing 2% Triton X-100. The beads were then boiled 10 min in Laemmli buffer , centrifuged at 6000x g for 1 min, and subjected to western blot analysis. Thus, samples of muscle homogenates were separated on 6% SDS-PAGE, and immunoblotting was performed as described above.

Determination of eNOS protein level in thoracic aorta and localization by immunofluorescence analysis using confocal microscopy.

The thoracic aortas of tempol-treated chow-fed (n=5) and HFHS-fed rats (n=5) and of untreated chow-fed (n=5) and HFHS-fed rats (n=5)were individually immersed in Optimal Cutting Tissue Embedding Medium (Tissue-teck, Sakura Finetek, Torrance, CA, USA), quickly frozen and stored at -80˚C. Tissue sections (7 µm) were collected and mounted on glass slides. The tissue sections were fixed in acetone at -20˚C for 10 minutes and washed in phosphate-buffered saline (PBS). They were then incubated in the blocking solution containing 10% bovine serum albumin diluted in PBS at room temperature for 45 minutes. The sections were then incubated overnight at 4˚C with 1:200 diluted primary monoclonal antibody for eNOS (Transduction Laboratories, Lexington, KY, USA) and the Alexa Fluor 488 conjugated Phalloidine (Molecular Probes, Burlington, ON, Canada) diluted 1:150 in the blocking solution, which interacts with smooth muscle actin filaments. The sections were then washed three times with PBS for 10 minutes each time. All sections were then incubated with Alexa Fluor-594 rabbit anti-mouse IgG (H+L) diluted 1:1000 (Molecular Probes, Burlington, ON, Canada). After rinsing in PBS, tissue slices were mounted on coverslips. Immunofluorescence was detected and localized by confocal microscope (MRC-1024 Confocal System, BioRad, CA, USA) combined with the Laser Sharp image acquisition software 3.2. Comparison of the fluorescence levels between groups was performed, under the same conditions of laser exposure, using the Metamorph software (Molecular Devices Corporation, Downington, PA, USA).

Measurement of ir-ET-1 in vascular tissues

Frozen tissues (mesenteric vascular bed) were homogenized twice with a Tissue-Tearor for 15 seconds in 2 ml ice-cold extraction solution (1 m HCl, 1% acetic acid, 1% TFA and 1% NaCl), as previously performed (Santuré et al ., 2002). The homogenate was centrifuged at 3000 x g for 30 minutes at 4°C. The supernatant was then collected, and 100 µl of [125I]ET-1 (about 1000 cpm) was added prior to extraction on a C18 Sep-Pak column. The Sep-Pak column was activated with 4 ml 60% acetonitrile and 0.1% TFA, then rinsed twice with 10 ml 0.1% TFA. After sample loading, the column was washed twice with 10 ml 0.1% TFA and the ir-ET-1 fraction was eluted with 3 ml 60 % acetonitrile and 0.1 % TFA, then counted in a gamma counter (recovery is 90-95%). The sample extracts were dried overnight in a Speed-Vac and reconstituted in 500µl RIA buffer. Aliquots of 100 and 200 µl of extracted samples or 200 µl of standards (ET-1, Peninsula Laboratories) were added to 100 µl of anti-ET-1 antibody and the final reaction volume adjusted to 300 µl with RIA buffer. After a 24 hour incubation period at 4°C, 100 µl of [125I]ET-1 (15 000 cpm.) in RIA buffer was added and the tubes were incubated for an additional 24-hour at 4°C. Bound and free radioactivity were separated by the second antibody method. After a 2-hour incubation period at room temperature, 0.5 ml RIA buffer was added and the tubes were centrifuges at 2500 x g for 20 minutes at 4°C. The supernatant was then discarded and the pellet was counted in a gamma counter. Ir-ET-1 concentrations were corrected for losses in extraction.

Surgical preparation

In vivo experiments were performed in additional groups of tempol-treated HFHS-fed rats (n=6) and chow-fed rats (n=8) and untreated HFHS-fed (n=9) and chow-fed rats (n=20). Two weeks after the beginning of the feeding diets performed in the presence or in the absence of treatment with tempol, the rats from each group were given subcutaneous injection of buprenorphine (0.05 mg kg-1) and were anaesthetized (sodium pentobarbital, 75 mg/kg i.p.) and had pulsed Doppler flow probes implanted to monitor changes in renal, mesenteric and hindquarter blood flows, according to the method developed by Gardiner and Bennett (Gardiner & Bennett, 1988) and as previously described in detail (Pitre et al ., 1996). After surgery, the rats were given subcutaneous injections of buprenorphine (0.05 mg kg-1) and returned to their cages. The chow or HFHS diet (with or without treatment with tempol) continued during postsurgical recovery, and the latter was deemed satisfactory by the resumption of growth and normalization of 24-h food intake. At least 7 days later, the rats were re-anaesthetized as describe previously. The leads of the implanted probes were soldered to a microconnector (Microtech Inc.), and two separate catheters were implanted in the right jugular vein (for glucose and insulin infusions) and one catheter in the distal abdominal aorta via the left femoral artery (for measurement of blood pressure and heart rate). The catheters were tunneled subcutaneously to emerge at the same point as the probe wires. The rats were given subcutaneous injections of buprenorphine (0.05 mg kg-1) and returned to their cages. The diet and treatment continued during this second postsurgical recovery. Experiments began at least 72 h after this last surgical step in conscious, unrestrained animals with free access to water but not food.

Euglycemic hyperinsulinemic clamp studies

The rats were deprived of food for 12-14 h before the glucose clamp study. Before each experiment, blood glucose and plasma insulin were determined and the resting heart rate, blood pressure, and regional blood flows were recorded over 30 minutes in couscious, quiet and unrestrained rats. A first group of tempol-treated HFHS-fed (n=6)and chow-fed rats (n=8)and untreated HFHS-fed (n=9) and chow-fed rats (n=9)received insulin at a rate of 4 mU kg-1min-1. The insulin solution was diluted to the appropriate concentration in saline (0.9% NaCl) containing 0.2% BSA to prevent the adsorption of insulin to the glassware and plastic surfaces. In control experiments, a second group of untreated chow-fed rats (n=11) was infused with saline-0.2% bovine serum albumin (BSA) instead of insulin and dextrose to match approximately the saline load delivered during the clamp studies. The control animals were treated in the same way as the groups receiving insulin. After basal measurements of blood glucose and plasma insulin, the euglycemic hyperinsulinemic clamp was then carried out over 2 h while heart rate, blood pressure, and blood flows were measured continuously as previously described (Pitre et al ., 1996) .

Analytic methods .

Blood samples for plasma glucose and insulin determinations in the basal state and during insulin infusion were obtained, placed in untreated polypropylene tubes, and centrifuged with an Eppendorf microcentrifuge (Minimax, International Equipment Company). The plasma was stored at -20oC until assay. The glucose concentration of the supernatant was measured by the glucose oxidase method (Richterich & Dauwalder, 1971) using a glucose analyzer (Technicon RA-XT), and the plasma insulin level was measured by radioimmunoassay (RIA) using porcine insulin standards and polyethylene glycol for separation (Desbuquois et al ., 1971).

Data analysis

Values are expressed as the mean ± SE; n is the number of observations. Data were analyzed for statistical significance by an analysis of variance (ANOVA). Post-hoc comparisons were made using Fisher’s test. A p value < 0.05 was taken to indicate a significant difference.

Body weight and metabolic changes

Table 2 illustrates the initial and final body weights and the average daily food intake measured in our four experimental groups. The data indicate that the four groups of rats displayed comparable initial body weight. After the four weeks of feeding, the untreated and tempol-treated HFHS-fed rats appeared significantly heavier than their chow-fed counterparts, which is likely a consequence for their higher food energy intake when compared to the chow-fed groups. However the tempol-treated HFHS-fed group was slightly but significantly lighter than the untreated HFHS-fed group, despite that tempol treatment had no effect on daily food intake. Table 3 shows the effects of the long-term diets and treatment with tempol on plasma levels of glucose, insulin, triglycerides and nonesterified fatty acids. These results indicate that the HFHS diet contributes to significantly increase plasma levels of glucose and insulin when compared to their chow-fed counterparts. The combination of higher glycemia and higher insulinemia found in HFHS-fed rats is indicative of glucose intolerance and insulin resistance. In the group of HFHS-fed rats treated with tempol, we noted that the plasma levels of glucose and insulin were not different than those measured in both groups of chow-fed rats. Furthermore, plasma levels of triglycerides and nonesterified fatty acids were higher in the HFHS-fed group than in the chow-fed groups. Tempol treatment had no significant effect on these values in both HFHS- and chow-fed rats.

Vascular reactivity

The effects of the HFHS diet and tempol treatment versus the normal chow diet on vascular reactivity were evaluated in vitro using aortic rings isolated from our four experimental groups (Figs 1 to 4). We found that the contractions induced by increasing concentrations of phenylephrine were not significantly altered by the HFHS diet or by the treatment with tempol when compared to the chow diet only (Figure 1A). The addition of L-NAME in the incubation medium was found to significantly enhance the contracting responses to phenylephrine in the four groups of rats (Figure 1B). Interestingly, the enhancing effect of L-NAME was found to be significantly higher in the group of HFHS-fed rats treated with tempol than in the untreated HFHS-fed group. This tendency did not reach the level of significance in the chow-fed group treated with tempol when compared to the untreated chow-fed group. Furthermore, we observed that the endothelium-dependent relaxations induced by increasing concentrations of carbachol were slightly but significantly lower in the aortic rings from the HFHS-fed rats than those from the chow-fed group (Figure 2). Treatment with tempol was found to significantly increase the relaxing response to carbachol in HFHS-fed rats, while there was no effect of tempol in the chow-fed group. The addition of L-NAME in the incubation medium was found to completely abrogate the vasorelaxing effects of carbachol in the four experimental groups. The evaluation of the endothelium-independent vasorelaxation by using SNP in cumulative concentrations revealed a similar ability of the HFHS-fed rat vascular smooth muscles to relax than those from chow-fed rats (Figure 3). Treatment with tempol had no effect on the vascular responses to SNP in both groups of treated rats.

The addition of insulin in the incubation medium tends to attenuate the contracting responses to phenylephrine in the untreated HFHS-fed group. However, this tendency did not reach the level of significance, and was not observed in the HFHS-fed group treated with tempol (Figure 4A). The addition of L-NAME in the incubation medium completely reverses the attenuating effect of insulin in the untreated HFHS-fed group. Interestingly, in the group of tempol-treated HFHS-fed rats, we noted that in the presence of L-NAME and insulin, the contracting response to phenylephrine was significantly higher than that observed in aortic rings of untreated HFHS-fed incubated in the absence or presence of L-NAME and/or insulin. Figure 4B shows that the addition of insulin in the incubation medium of aortic rings isolated from untreated and tempol-treated chow-fed rats significantly attenuate the contracting responses to phenylephrine. These attenuating effects of insulin were both abolished in the presence of L-NAME. Indeed, in aortic rings isolated from the tempol-treated chow-fed rats, we found that in the presence of L-NAME and insulin, the contracting responses to phenylephrine were enhanced to levels significantly higher than those observed from untreated chow-fed rats incubated in the absence or presence of L-NAME and/or insulin.

Effect of HFHS diet and treatment with tempol on eNOS protein expression in thoracic aorta and skeletal muscle.

We examined the expression of eNOS protein in thoracic aorta and gastrocnemius skeletal muscle isolated from our four experimental groups, and determined whether eNOS protein contents were affected by the diet and treatment with tempol. Equivalent amounts of proteins from vascular or muscle lysates were resolved on SDS-PAGE and immunoblotting was done using an eNOS specific antibody. Western blot analysis showed that eNOS proteins were detectable in vascular and muscle tissues from every group of rats. The proteins migrated as a single band of about 140 000 Mr. Immunoreactivity of eNOS was quantified by scanning densitometry and the mean data are presented (Figures 5 and 6). The results indicate that eNOS protein expression was significantly lower in thoracic aorta and gastrocnemius muscle from HFHS-fed rats than from both groups of chow-fed rats. Treatment with tempol in HFHS-fed rats had no detectable effect on eNOS protein expression in vascular tissue when compared to the untreated HFHS-fed group, while in gastrocnemius muscle, tempol treatment was found to significantly enhance eNOS protein expression to levels comparable to those measured in both groups of chow-fed rats. Using confocal microscopy, we found a significant reduction (2-3 folds) in eNOS immunofluorescence level in the endothelium of thoracic aorta from HFHS-fed rats when compared to the chow-fed groups (untreated and treated with tempol) (Figure 7). This reduction was no longer observed in the endothelium of thoracic aorta from HFHS-fed rats treated with tempol.

Effect of HFHS diet and treatment with tempol on nitrotyrosine expression in vascular tissue

The effects of the diet and treatment with tempol versus the normal chow diet on nitrotyrosine formation were evaluated by Western immunoblot analysis in the thoracic aortas isolated from our four experimental groups. The results indicate a higher level of nitrosylated proteins in vascular tissue isolated from HFHS-fed rats than from untreated and tempol-treated chow-fed rats (Figure 8).Four weeks of preventive treatment with tempol was found to cause a significant decrease in nitrotyrosine expression in HFHS-fed rats. Indeed, the level of expression measured in vascular tissues of HFHS-fed rats treated with tempol was similar to those observed in both groups of chow-fed rats.

ET-1 protein content in vascular tissues.

Figure 9 shows the effects of HFHS diet and treatment with tempol on immunoreactive ET-1 (ir-ET-1) concentration in vascular tissues. Higher concentrations of ir-ET-1 were noted in the mesenteric arterial bed isolated from the HFHS-fed rats than in the untreated or tempol-treated chow-fed groups. Chronic and preventive treatment with tempol in HFHS-fed rats was found to prevent the increases in ir-ET-1 concentrations in the vascular tissue. In these animals the level of ir-ET-1 measured was comparable to those obtained in both groups of chow-fed rats.

Effect of HFHS diet and treatment with tempol on [ 3 H]-2-deoxy-D-glucose uptake in isolated skeletal muscles.

The effects of the diets and treatment with tempol on basal and insulin-stimulated glucose transport activities in isolated soleus and EDL muscles are shown in Figure 10. Thus, in both skeletal muscles we found that HFHS-feeding or tempol treatment had no influence on basal glucose transport activity compared with that observed in chow-fed rats. However, in the presence of insulin, we found a significantly smaller glucose transport activity in soleus (at doses of 0.02 and 0.2 mU/ml of insulin) and EDL muscles (at doses of 0.02, 0.2 and 2 mU/ml of insulin) isolated from HFHS-fed rats than in those isolated from chow-fed rats. Tempol treatment in HFHS-fed rats was found to significantly improve insulin-mediated glucose transport activity in both skeletal muscles.

Haemodynamic responses to insulin infusion during the euglycemic hyperinsulinemic clamp.

The baseline values (prior to any i.v. infusion) for cardiovascular variables are listed in Table 4 for the four groups of rats. We found that, the basal heart rate and mean blood pressure in HFHS-fed rats were higher than in the three other groups of rats. Furthermore, we found that the HFHS-fed rats had a smaller basal superior mesenteric flow than in the chow-fed rats, while no differences were noted between the two other groups of tempol-treated rats and the untreated chow-fed rats. Moreover, there were no significant differences in basal renal or hindquarter flows or vascular conductances between the four groups of rats. However a significantly higher superior mesenteric vascular conductance was noted in the chow-fed rats when compared with the others groups of rats.

Figure 11 shows that insulin infusion at a rate of 4 mU kg-1 min-1 in a group of untreated or tempol-treated chow-fed rats caused significant increases in renal and hindquarter blood flows, but ithad no effect on heart rate, mean arterial blood pressure, or superior mesenteric flow compared with measurements following a control infusion of vehicle (saline-0.2% BSA). These responses were associated with slight but significant increases in renal and hindquarter vascular conductances, while no significant change was noted in superior mesenteric vascular conductance (Figure 12). In HFHS-fed rats, the same infusion of insulin had no effect on heart rate, mean arterial blood pressure or renal or hindquarter flows, while a significant decrease in superior mesenteric flow was observed when compared with the effects of control infusion of saline-0.2% BSA (Figure 11). The renal, superior mesenteric and hindquarter blood flow responses to insulin differed significantly from those observed in both groups of chow-fed rats. Furthermore, in HFHS-fed rats the euglycemic infusion of insulin elicited a significant fall in superior mesenteric vascular conductance, but had no consistent effect on renal or hindquarter vascular conductances when compared with the effects of control infusion of saline-0.2% BSA (Figure 12).These responses differed significantly from those seen in untreated and tempol-treated chow-fed rats, in which insulin produced a marked vasodilation in renal and hindquarter vascular beds, but had no effect in the superior mesenteric vascular bed. Treatment with tempol in HFHS-fed rats was found to restore the increases in hindquarter flows and vascular conductances, and to prevent the decreases in superior mesenteric flows and vascular conductances elicited by insulin in untreated HFHS-fed rats. Furthermore, in tempol-treated HFHS-fed rats, we observed that euglycemic infusion of insulin tend to increase renal flow and vascular conductance, but this tendency did not reach the level of significance.

Responses during Euglycemic Hyperinsulinemic Clamp.

Table 5 shows that, in the fasting state, basal arterial plasma glucose and insulin levels were similar in the four groups of rats studied. During the euglycemic hyperinsulinemic clamp, which was performed at an insulin infusion rate of 4 mU kg-1 min-1, we found that fasting plasma insulin levels in the four groups rose acutely and achieved similar plateaus, whereas normal plasma glucose levels were maintained in every group of rats. However, we noted that the average glucose infusion rate required to maintain euglycemia during the last hour of the clamp (GIR60-120) was significantly lower in the HFHS-fed rats than in the three others groups of rats.

The HFHS diet was shown to cause several metabolic and vascular alterations in the rat. Four weeks treatment with tempol was found to prevent several of these alterations. Notably, 1- The increases in plasma levels of glucose and insulin noted in the HFHS-fed rats, were not observed in the group of HFHS-fed rats treated with tempol, indicating that tempol treatment would contribute to prevent glucose intolerance and insulin resistance induced by the diet, which was indeed demonstrated during a euglycemic hyperinsulinemic clamp. Moreover, a slight but significant reduction in body weight intake was noted after the four weeks treatment with tempol, when compared with the untreated HFHS-fed group. However, tempol treatment had no effect on the rise in plasma levels of triglyceride and free fatty acid noted in the untreated HFHS-fed group. 2-Tempol treatment was found to prevent the rise in blood pressure, the reduction in endothelium-dependent vasorelaxation, the decrease in eNOS protein expression in skeletal muscle and the fall in immunofluorescence level in blood vessel endothelium displayed in the untreated HFHS-fed group. Furthermore, tempol treatment in HFHS-fed rats was found to potentiate the enhancing effect of L-NAME on the contracting responses to phenylephrine, when compared to the untreated HFHS-fed rats. 3- Treatment with tempol was also found to prevent the impairment in the insulin-mediated vascular responses. Thus, the renal and skeletal muscle vasodilator responses to insulin were not altered in HFHS-fed rats treated with tempol. Moreover, in these rats insulin did not caused significant superior mesenteric vasoconstrictor effect, as observed in the untreated HFHS-fed rats. Furthermore, tempol treatment in those animals was found to improve the reduced insulin-stimulated glucose transport activity in skeletal muscles. 4- The increased concentration of ET-1 observed in vascular tissues isolated from untreated HFHS-fed rats was not seen in tempol-treated rats, and 5- Tempol-treatment was found to prevent the increased formation of nitrotyrosine in vascular tissues from HFHS-fed rats. 6- Tempol treatment in chow-fed rats had no detectable effect on metabolic or vascular parameters or on metabolic or vascular responses to insulin. Together, these findings point toward an enhanced ROS-mediated inactivation and sequestration of NO, and its possible contribution to the impaired endothelium-dependent relaxation and impaired insulin-mediated renal and skeletal muscle vasodilator responses noted in our HFHS-fed rats. Furthermore, our results indicate that anti-oxidative agents, such as a SOD mimetic, could be useful to prevent the metabolic and haemodynamic abnormalities resulting from 4 weeks consumption of a HFHS diet in rats.

Effect of treatment with tempol on the alterations induces by the HFHS diet on some biochemical parameters.

In the present study, the rats fed with a HFHS diet for 4 weeks were significantly heavier than their chow-fed counterparts, and developed insulin resistance, that was associated with significant changes in several biochemical parameters, namely, increases in plasma insulin, glucose, triglyceride and free fatty acid. This HFHS rat model closely resembles certain features of the nondiabetic human insulin-resistant state of obesity and glucose intolerance (Kaufman et al ., 1993; Reaven, 1991; Reaven, 1988). Treatment with tempol in HFHS-fed rats was found to prevent, or at least, attenuate the appearance of several of these features. It significantly improved insulin sensitivity, prevented the rises in plasma insulin and glucose, and reduced body weight intake. Previous studies using other antioxidant agents, such as α-lipoic acid or linoleic acid in fructose-fed rats (Thirunavukkarasu et al ., 2004; Thirunavukkarasu and Anuradha, 2004) or obese Zucker rats (Teachey et al ., 2003) have reported similar improvement in plasma levels of insulin and glucose and in insulin sensitivity. Although we did not characterize the mechanisms of these effects, a previous study carried out by Lee and collaborators demonstrated that α-lipoic acid could increase insulin sensitivity by activating AMP-activated protein kinase, and then, increasing insulin-stimulated glucose uptake in skeletal muscle (Lee et al ., 2005).

Effect of the HFHS diet and tempol treatment on blood pressure

In this study, using untreated and tempol-treated chow- and HFHS-fed rats chronically instrumented with intra-arterial catheters and pulsed Doppler flow probes to directly and continuously record intra-arterial blood pressure, heart rate, and regional blood flows in conscious rats, we noted that the HFHS feeding, and the concomitant induction of insulin resistance, led to a significant rise in blood pressure and heart rate. These findings agree with those of previous studies in which inducing insulin resistance with a high fat and/or high sucrose or fructose diet in rats led to insulin resistance and a rise in blood pressure (Barnard et al ., 1998; Dobrian et al ., 2000; Hulman & Falkner, 1994; Katakam et al ., 1998; Reaven & Ho, 1991; Roberts et al ., 2000; Srinivasan et al ., 2004; Vrana et al ., 1993; Yoshioka et al ., 2000) . Treatment with tempol in HFHS-fed rats was found to prevent the rises in blood pressure and heart rate. It is likely that the improved endothelial function noted in our tempol-treated HFHS-fed rats, when compared with the untreated HFHS-fed group, could have contributed to normalize blood pressure at a level similar to that found in the chow-fed group. In the present study, we found that the vasorelaxing response to carbachol, a muscarinic agonist and an endothelium-dependent vasodilator, was significantly improved in tempol-treated HFHS-fed rats, as compared to the untreated HFHS-fed group.

Among the other possible mechanisms by which tempol treatment might have contributed to prevent the rise in blood pressure in HFHS-fed rats, there is the attenuation of the diet-induced insulin resistance and hyperinsulinemia. Indeed, previous studies have indicated that in diet-induced insulin resistance, hyperinsulinemia could be the trigger for the development of endothelial dysfunction and subsequent hypertension (Katakam et al ., 1998; Reil et al ., 1999), as both, insulin resistance and hyperinsulinemia, appear to precede hypertension (Barnard et al ., 1998). Indeed, in addition to its metabolic effects,it is now well recognized that insulin has pressor, as well as depressor (eg vasodilator) effects on the cardiovascular system that could elevate or reduce blood pressure, depending on their relative importance (Anderson & Mark, 1993; Hall, 1993).Therefore, by attenuating the diet-induced insulin resistance and the rise in plasma levels of insulin and its pressor effects, tempol treatment could have contributed to prevent the rise in blood pressure in HFHS-fed rats. However, the effect of insulin on blood pressure are complex, and there is considerable controversy about whether insulin is capable of exerting sustained effects on cardiovascular and renal function that are necessary to cause chronic hypertension. Further, studies are required to clarify this point.

Effect of diet and tempol treatment on vascular responses to insulin

In addition to the diet effects on resting blood pressure and heart rate, we found that the vascular responses to insulin were significantly altered by the diet. Indeed, in untreated HFHS-fed rats, the euglycemic infusion of insulin elicited a vasoconstrictor effect in the superior mesenteric vascular bed, while the renal and hindquarter vasodilator responses previously observed in the chow-fed rats, were absent in the HFHS-fed rats. Four weeks treatment with tempol was found to prevent the deleterious effect of the HFHS diet on the vascular responses to insulin. The mechanism underlying impaired vascular responses to insulin in the HFHS-fed group remains unclear. However, given that hyperinsulinemia and simple carbohydrate feeding in the rat are known to increase sympathetic activity (Anderson et al ., 1991; Fournier et al ., 1986) , it is thus likely that the altered vascular response to insulin was caused by a general enhanced vasoconstrictor effect of insulin in HFHS-fed rats due to an abnormal sympathetic overactivity in response to hyperinsulinemia. Thus, if this is the case, tempol treatment could have contributed to prevent the alteration of the vascular responses to insulin by attenuating the rises in plasma levels of insulin and glucose, and then by preventing abnormal sympathetic overactivity in response to hyperinsulinemia.

Alternatively or additionally, as a role for endothelium-derived NO has been shown in the mediation of insulin vasodilator effect (Scherrer et al., 1994; Steinberg et al., 1994) , it is likely that an improved production of NO, consecutive to an up-regulation of eNOS protein in the vasculatures of tempol-treated HFHS-fed rats, might have contributed, at least in part, to prevent the deleterious effect of the HFHS diet on the vascular responses to insulin. In agreement with that, our results clearly indicate a decreased abundance of eNOS protein expression in skeletal muscles and thoracic aortas from untreated HFHS-fed rats, when compared to their chow-fed counterpart. However, in HFHS-fed rats treated with tempol, we observed an increased abundance of eNOS protein expression in skeletal muscles, and in eNOS immunofluorescence level in the endothelium of thoracic aorta, using confocal microscopy. Although we did not measure the level of expression of iNOS protein, it is possible that the induction of iNOS by the HFHS diet could have contributed to the reduction in eNOS protein expression noted in our untreated HFHS-fed rats. Indeed, previous studies have clearly shown an increased expression and/or activity of iNOS protein in cells (aorta, white adipose tissue and skeletal muscle) from high fat-fed rats or mice (Noronha et al., 2005; Perreault & Marette, 2001), and it has been reported that iNOS protein can reduced eNOS protein expression in endothelial cells (Frutos et al., 1999). Furthermore, Polytarchou and Papadimitriou demonstrated that a 48 hours incubation tempol can prevent the increased expression and/or activity of iNOS in blood cell lysates of chicken embyo chorioallantoic membrane (Polytarchou & Papadimitriou, 2004). Interestingly, in tempol-treated HFHS-fed rats, we noted an improved endothelium-dependent vasorelaxation response to carbachol in isolated aortic rings, when compared to the untreated HFHS-fed group. The vascular smooth muscle contracting response to phenylephrine, and the response to the endothelium-independent vasodilator, sodium nitroprussiate, did not differ between the four groups of rats, indicating that the ability of vascular smooth muscle to contract or relax in response to exogenous NO was not impaired by the diet or influenced by the treatment with tempol. This might suggest that the HFHS diet selectively impaired endothelium-dependent vasodilation, which would be selectively improved by the treatment with tempol. These findings agree with previous data from this laboratory and others showing impaired endothelium-mediated relaxation and depressed eNOS protein expression in obese subjects and patients with type 2 diabetes, and in animal models with insulin resistance (Cohen, 1993; McVeigh et al., 1992; Pitre et al., 1996; Roberts et al., 2005; Roberts et al., 2003; Santuré et al., 2002; Steinberg et al., 1996), and studies demonstrating improvement in vascular endothelium function with antioxidants (Keaney et al., 1994 ; Jiang et al., 2003 ; Böger et al., 1998).

Effect of diet and tempol treatment on oxidative stress

In this study, by using an anti-oxidative agent we sought to evaluate the possibility that an increase in endothelial production of reactive oxygen species (ROS), such as the superoxide anion, leading to NO scavenging and then limiting the NO-dependent vasodilator capacity at the level of the vascular smooth muscle, might have also contributed to the altered vascular responses noted in the group of HFHS-fed rats. In this regard, superoxide anion rapidly reacts with and inactivates NO to produce peroxynitrite, a potent cytotoxic reactive nitrogen species that subsequently reacts with proteins, lipids, and DNA (Halliwell, 1997). Peroxynitrite reacts with tyrosine residues to form nitrotyrosine, a stable footprint of NO oxidation by ROS that can be detected in tissues. In the present study, we found that HFHS consumption significantly increases nitrotyrosine formation in vascular tissues. Interestingly, four weeks preventive treatment with tempol in these rats was found to significantly attenuate the increases in nitrotyrosine formation. Thus, our findings highly suggest that enhanced ROS-mediated inactivation and sequestration of NO might have contributed to the impaired insulin-mediated vasodilator responses in renal and skeletal muscle vascular beds of HFHS-fed rats, and to the reduction in the endothelium-dependent vasorelaxation also noted in these animals. Our findings agree with those of two recent studies carried out in female Fischer rats and demonstrating that long-term consumption of a high-fat, refined carbohydrate diet (for 2 months and longer) induces endothelial dysfunction, impaired endothelium-dependent relaxation, a marked accumulation of nitrotyrosine in several tissues and oxidant/antioxidant imbalance (Roberts et al., 2005; Roberts et al., 2000) .

Effect of diet on ET-1

On the other hand, the impaired vascular reactivity noted in our HFHS-fed rats could be related to an exaggerated production of endothelium-derived contracting factors, for instance ET-1, consecutive to a decrease in NO production, as NO is known to exert inhibitory effect on ET-1 synthesis (Aliev et al., 1998; Boulanger & Luscher, 1991; Mather et al., 2004; Onoue et al., 1999; Richard et al., 1995) . Consistent with this is the demonstration here that the mesenteric arteries from the HFHS-fed rats contained a greater amount of ET-1 protein than the chow-fed rats, while tempol treatment was found to reduce this amount to levels comparable to those measured in both groups of chow-fed rats. It is thus likely that, by improving eNOS protein expression and NO production, tempol treatment would contribute to attenuate ET-1 production and thus preventing the effect of the HFHS diet on vascular endothelial function and reactivity. Indeed, insulin is known to stimulate gene expression of vascular ET-1 in vitro, and to modulate ET-1 production and release, both in vivo and in vitro (Frank et al., 1992; Hu et al., 1993; Verma et al., 1995; Wolpert et al., 1993). Elevated vascular expression of ET-1 and ETA receptor mRNA and increased plasma levels of ET-1 have been reported in fructose-fed rats (Juan et al., 1998), and we recently reported increased ET-1 concentration in the mesenteric arteries isolated from sucrose-fed rats (Santuré et al., 2002). A previous study carried out in type 2 diabetic subjects demonstrated that 15 days treatment with a dietary antioxidant supplement (N-acetylcysteine, vitamin E and vitamin C) effectively reduced the levels of ET-1 in arterial blood (Neri et al., 2005). Therefore, it is possible that hyperinsulinemia and/or insulin resistance may have created an environment resulting in activation of the ET system (Hopfner et al., 1998; Juan et al., 2004; Katakam et al., 2001; Piatti et al., 1996), and that treatment with tempol contributes to prevent this induction.

Insulin vascular effects and insulin sensitivity

In the present study, tempol treatment was found to prevent the reduction in whole-body insulin sensitivity together with the alteration in the normal hindquarter vasodilator response to insulin noted in the untreated HFHS-fed rats. Given that, on a quantitative basis, skeletal muscle was pointed as the predominant site of insulin-stimulated glucose disposal, and as the major tissue responsible for postprandial hyperglycemia in insulin resistant states (Baron et al ., 1988; Baron et al ., 1993; DeFronzo et al ., 1985), we believe that the beneficial effect of treatment with tempol on the vascular responses to insulin would have contributed to attenuate the development of insulin resistance by improving delivery of glucose and insulin to muscle beds. Although some debate took place over the physiologic relevance of the vascular effects of insulin (Baron et al ., 1991; Yki-Järvinen & Utriainen, 1998), a number of recent lines of evidence further support the concept of a significant functional relationship between insulin’s metabolic and vascular actions. Thus, using higher resolution methods, recent studies have reported the vascular effects of insulin at physiological concentrations and over a time course similar to that observed for whole-body glucose uptake (Clark et al ., 2001; Vincent et al ., 2002). At these concentrations, insulin was found to increase muscle perfusion by recruiting microvascular beds and redistributing flow preferentially to areas with high rates of glucose uptake, suggesting that at least regionally, the metabolic and haemodynamic effects of insulin are coupled (Clark et al ., 2001; Vincent et al ., 2002). Interestingly, it was shown that whenthe action of insulin on total blood flow (Baron & Clark, 1997) or capillary recruitment (Rattigan et al ., 1999) is prevented in vivo , itconcomitantly induces anacute state of insulin resistance.

The endothelium-derived NO has been proposed as the mediator coupling the vasodilator action of insulin to its glucoregulatory action (Baron, 1996). Indeed, striking parallels between metabolic insulin-signaling pathways and pathways related to its vasodilator actions have been reported, suggesting that the vascular endothelium could be a physiological target of insulin that couples regulation of glucose metabolism with haemodynamics (Zeng et al., 2000; Zeng & Quon, 1996). Moreover, it was recently reported that the targeted disruption of NO production, using a eNOS mouse knockout models, induces insulin resistance (Shankar et al., 2000) and reduces insulin stimulation of muscle blood flow (Duplain et al., 2001). These data provide genetic evidence that the enzyme NOS plays a role in modulating insulin sensitivity and carbohydrate metabolism (Shankar et al., 2000), as well as in mediating insulin vascular action, and confirm earlier observations obtained with acute pharmacological antagonism of NOS activity in humans (Baron et al., 1995; Scherrer et al., 1994; Steinberg et al., 1994) and in rats (Pitre et al., 2000; Shankar et al., 1998). Taken together, these findings further support the concept of a significant functional relationship between insulin’s metabolic and vascular actions, possibly at the endothelial level (Clark et al., 1995; Cleland et al., 1999; Duplain et al., 2001).

Effect of diet and tempol treatment on glucose transport activity in isolated muscles

The soleus and EDL muscles were obtained from untreated and tempol-treated chow- and HFHS-fed rats. We found that, compared with the chow diet, the HFHS diet caused resistance of skeletal muscle glucose transport to stimulation by insulin, suggesting that there is an intrinsic defect induces by the diet in the tissue itself, and that vascular reactivity impairment would not be the sole factor counting for insulin resistance. Similar reduction in the ability of insulin to stimulate glucose uptake in muscles was reported in rats fed a high fat and/or high sucrose diet (Barnard et al ., 1998; Hansen et al ., 1998; Kim et al ., 1999; Santuré et al ., 2002; Singh et al ., 2003; Youngren et al ., 2001). Treatment with tempol was found to prevent the effect of the HFHS diet on glucose transport activity stimulated by insulin. In support with our findings, some previous studies carried out in rats model of obesity and/or type 2 diabetes have also reported an improvement in insulin-stimulated glucose transport following treatment with antioxidants (Bitar et al., 2004; Saengsirisuwan et al., 2004; Teachey et al., 2003). The extent to which the HFHS diet and then the preventive treatment with tempol have contributed to alter or improve, respectively, insulin-stimulated glucose transport activity by altering the total cellular content of GLUT 4 proteins, the translocation process of GLUT 4 to the cell surface, or its intrinsic activity remain to be verified.

In conclusion, metabolic insulin resistance has been identified as a major risk factor for cardiovascular morbidity and mortality. Loss of the modulatory role of the endothelium may be a critical and initiating factor in the development of cardiovascular disease. Our findings in HFHS-fed rats, provide further evidence for the importance of eNOS gene in linking metabolic and vascular disease, and indicate the ability of a Westernized diet to induce oxidative stress, which would likely cause enhanced ROS-mediated inactivation and sequestration of NO, contributing to metabolic and vascular endothelium dysfunction. The present results also indicate that anti-oxidative agents, such as a SOD mimetic, could be useful to prevent several of the metabolic and haemodynamic abnormalities resulting from 4 weeks consumption of a HFHS diet in rats.

The authors wish to thank Marie Tremblay for her expert assistance for insulin and glucose plasma analysis. We thank Geneviève Pilon and Patrice Dallaire from André Marette’s laboratory for their professional support and judious advices in the western blot method analysis. We also thank Nadia Chbinou from Richard Larivière’s laboratory for her experimental support in confocal microscopy method. This work was supported by grants to Dr. H. Bachelard from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Quebec. F. Bourgoin was supported by a studentship from the Association Diabète Québec.

Akpaffiong, M. & Taylor, A. (1998). Antihypertensive and vasodilator actions of antioxidants in spontaneously hypertensive rats. Am. J. Hypertens. , 11, 1450-1460.

Aliev, G., Bodin, P. & Burnstock, G. (1998). Free radical generators cause changes in endothelial and inducible nitric oxide synthases and endothelin-1 immunoreactivity in endothelial cells from hyperlipidemic rabbits. Mol. Genet. Metab. , 63, 191-197.

Anderson, E.A., Hoffman, R.P., Balon, T.W., Sinkey, C.A. & Mark, A.L. (1991). Hyperinsulinemia produces both sympathetic neural activation and vasodilatation in normal humans. J. Clin. Invest. , 87, 2246-2252.

Anderson, E.A. & Mark, A.L. (1993). The vasodilator action of insulin. Implications for insulin hypothesis of hypertension. Hypertension , 21, 136-141.

Bachelard, H., Badeau, M., Bourgoin, F., Nadeau, A. & Larivière, R. (2005). Insulin resistance and vascular function in high fat high sucrose (HFHS)-fed rats. Diabetes , 54, A429.

Barnard, R.J., Faria, D.J., Menges, J.E. & Martin, D.A. (1993). Effects of a high-fat, sucrose diet on serum insulin and related atherosclerotic risk factors in rats. Atherosclerosis , 100, 229-236.

Barnard, R.J., Roberts, C.K., Varon, S.M. & Berger, J.J. (1998). Diet-induced insulin resistance precedes other aspects of the metabolic syndrome. J. Appl. Physiol. , 84, 1311-.

Barnard, R.J. & Wen, S.J. (1994). Exercise and diet in the prevention and control of the metabolic syndrome. Sport Med. , 18, 218-228.

Baron, A.D. (1996). The coupling of glucose metabolism and perfusion in human skeletal muscle. The potential role of endothelium-derived nitric oxide. Diabetes , 45, S105-S109.

Baron, A.D., Brechtel, G., Wallace, P. & Edelman, S.V. (1988). Rates and tissue sites of non-insulin and insulin mediated glucose uptake in humans. Am. J. Physiol. , 255, E769-E774.

Baron, A.D., Brechtel-Hook, G., Johnson, A. & Hardin, D. (1993). Skeletal muscle blood flow. A possible link between insulin resistance and blood pressure. Hypertension , 21, 129-135.

Baron, A.D. & Clark, M.G. (1997). Role of bloof flow in the regulation of muscle glucose uptake. Ann. Rev. Nutr. , 17, 487-499.

Baron, A.D., Laakso, M., Brechtel, G. & Edelman, S.V. (1991). Mechanism of insulin resistance in insulin-dependent diabetes mellitus: A major role for reduced skeletal muscle blood flow. J. Clin. Endocrinol. Metab. , 73, 637-643.

Baron, A.D., Steinberg, H.O., Chaker, H., Leaming, R., Johnson, A. & Brechtel, G. (1995). Insulin-mediated skeletal muscle vasodilation contributes to both insulin sensitivity and responsiveness in lean humans. J. Clin. Invest. , 96, 786-792.

Beckman, J.S. & Koppenol, W.H. (1996). Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am. J. Physiol. , 271, C1424-C1437.

Bitar, M.S., Wahid, S., Pilcher, C.W.T., Al-Saleh, E. & Al-Mulla, F. (2004). α-lipoic acid mitigates insulin resistance in Goto-Kakizaki rats. Horm. Metab. Res . 36 , 542-549.

Böger, R.H., Bode-Böger, S.M., Phivthong-ngam, L., Brandes, R.P., Schwedhelm, E., Mügge, A., Böhme, M., Tsikas, D. & Frölich, J.C. (1998). Dietary L-arginine and α-tocopherol reduce vascular oxidative stress and preserve endothelial function in hypercholesterolemic rabbits via different mechanisms. Atherosclerosis , 141 , 31-43.

Boulanger, C. & Luscher, T.F. (1991). Release of endothelin from the porcine aorta: inhibition by endothelium-derived nitric oxide. J. Clin. Invest. , 92, 587-590.

Clark, A.D.H., Barrett, E.J., Rattigan, S., Wallis, M.G. & Clark, M.G. (2001). Insulin stimulated laser Doppler signal by rat muscle in vivo, consistent with nutritive flow recruitment. Clin. Sci. , 100, 283-290.

Clark, M.G., Colquhoun, E.Q., Rattigan, S., Dora, K.A., Eldershaw, T.P., Hall, J.L. & Ye, J. (1995). Vascular and endocrine control of muscle metabolism. Am. J. Physiol. , 268, E797-E812.

Cleland, S.J., Petrie, J.R., Ueda, S., Elliott, H.L. & Connell, J.M.C. (1999). Insulin-mediated vasodilation and glucose uptake are functionally linked in humans. Hypertension , 33, 554-558.

Cohen, R.A. (1993). Dysfunction of vascular endothelium in diabetes mellitus. Circulation , 87, V67-V76.

DeFronzo, R.A. & Ferrannini, E. (1991). Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care , 14, 173-194.

DeFronzo, R.A., Gunnarson, R., Bjorkman, O., Olson, M. & Wahren, J. (1985). Effect of insulin on peripheral and splanchnic glucose metabolism in non-insulin-dependent (type II) diabetes mellitus. J. Clin. Invest. , 76, 149-155.

Desbuquois, B. & Aurbach, G.D. (1971). Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J. Clin. Endocr. Metab. , 37, 732-738.

Dobrian, A.D., Davies, M.J., Prewitt, R.L. & Lauterio, T.J. (2000). Development of hypertension in a rat model of diet-induced obesity. Hypertension , 35, 1009-1015.

Duplain, H., Burcelin, R., Sartori, C., Cook, S., Egli, M., Lepori, M., Vollenweider, P., Pedrazzini, T., Nicod, P., Thorens, B. & Scherrer, U. (2001). Insulin resistance, hyperinsulinemia, and hypertension in mice lacking endothelial nitric oxide synthase. Circulation , 104, 342-345.

Fournier, R.D., Chieuh, C.C., Kopin, I.J., Knapka, J.J., Dipette, D. & Preuss, H.G. (1986). Refined carbohydrate increases blood pressure and catecholamine excretion in SHR and WKY. Am. J. Physiol. , 250, E381-E385.

Frank, H.J., Levin, E.R., Hu, R.M. & Pedram, A. (1992). Insulin stimulates endothelin binding and action on cultured vascular smooth muscle cells. Endocrinology , 133, 1092-1097.

de Frutos, T., de Miguel, L.S., Garcia-Duran, M., Gonzalez-Fernandez, F., Rodriguez-Feo, J.A., Monton, M., Guerra, J., Farre, J., Casado, S. & Lopez-Farre, A. (1999). NO from smooth muscle cells decreases NOS expression in endothelial cells: role of TNF-alpha. Am. J. Physiol ., 277 , H1317-H1325.

Gardiner, S.M. & Bennett, T. (1988). Regional haemodynamic responses to adrenoceptor antagonism in conscious rats. Am. J. Physiol. , 255, H813-H824.

Gaudreault, N., Santuré, M., Pitre, M., Nadeau, A., Marette, A. & Bachelard, H. (2001). Effects of insulin on regional blood flow and glucose uptake in Wistar and Sprague Dawley rats. Metabolism , 50, 65-73.

Haffner, S.M., Fong, D. & Zuda, H.P. (1988). Hyperinsulinemia, upper body obesity and cardiovascualr risk factors in non-diabetics. Metabolism , 37, 336-345.

Hall, J.E. (1993). Hyperinsulinemia: a link between obesity and hypertension? Kidney Int. , 43, 1402-1417.

Halliwell, B. (1997). What nitrates tyrosine? Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vitro? FEBS Lett. , 411, 157-160.

Hansen, P.A., Gulve, E.A. & Holloszy, J.O. (1994). Suitability of 2-deoxyglucose for in vitro measurement of glucose transport activity in skeletal muscle. J. Appl. Physiol. , 76, 979-985.

Hansen, P.A., Han, D.H., Marshall, B.A., Nolte, L.A., Chen, M.M., Mueckler, M. & Holloszy, J.O. (1998). A high-fat diet impairs insulin stimulation of glucose transport in muscle. J. Biol. Chem. , 273, 26157-26163.

Higashi, Y., Oshima, T., Sasaki, N., Ishioka, N., Nakano, Y., Ozono, R., Yoshimura, M., Ishibashi, K., Matsuura, H. & Kajiyama, G. (1997). Relationship between insulin resistance and endothelium-dependent vascular relaxation in patients with essential hypertension. Hypertension , 29, 280-285.

Hogikyan, R.V., Galecki, A.T., Pitt, B., Halter, J.B., Greene, D.A. & Supiano, M.A. (1998). Specific impairment of endothelium-dependent vasodilation in subjects with type 2 diabetes independent of obesity. J. Clin. Endocrinol. Metab. , 83, 1946-1952.

Hopfner, R.L., Hasnadka, R.V., Wilson, T.W., McNeill, J.R. & Gopalakrishnan, V. (1998). Insulin increases endothelin-1 evoked intracellular free calcium response by increased ETA receptor expression in rat aortic smooth muscle cells. Diabetes , 47, 937-944.

Hu, R.M., Levin, E.R., Pedram, A. & Frank, H.J.L. (1993). Insulin stimulates production and secretion of endothelin from bovine endothelial cells. Diabetes , 42, 351-358.

Hulman, S. & Falkner, B. (1994). The effect of excess dietary sucrose on growth, blood pressure, and metabolism in developing sprague-dawley rats. Pediatric Res. , 36, 95-101.

Jiang, F., Guo, Y., Salvemini, D. & Dusting, G.J. (2003). Superoxide dismutase mimetic M40403 improves endothelial function in apolipoprotein(E)-deficient mice. Br. J. Pharmacol ., 139 , 1127-1134.

Juan, C.C., Fang, V.S., Hsu, Y.P., Huang, Y.J., Hsia, D.B., Yu, P.C., Kwok, C.F. & Ho, L.T. (1998). Overexpression of vascular endothelin-1 and endothelin-A receptors in a fructose-induced hypertensive rat model. J. Hypertens. , 16, 1775-1782.

Juan, C.C., Shen, Y.-W., Chien, Y., Lin, Y.-J., Chang, S.-F. & Ho, L.T. (2004). Insulin infusion induces endothelin-1-dependent hypertension in rats. Am. J. Physiol. , 287, E948-E954.

Katakam, P.V., Pollock, J.S., Pollock, D.M., Ujhelyi, M.R. & Miller, A.W. (2001). Enhanced endothelin-1 response and receptor expression in small mesenteric arteries of insulin-resistant rats. Am. J. Physiol. Heart Circ. Physiol. , 280, H522-H527.

Katakam, P.V.G., Ujhelyi, M.R., Hoenig, M.E. & Winecoff-Miller, A. (1998). Endothelial dysfunction precedes hypertension in diet-induced insulin resistance. Am. J. Physiol. , 275, R788-R792.

Kaufman, L.N., Peterson, M.M. & DeGrange, L.M. (1993). Pioglitazone prevents diet induced hypertension in rats. Diabetes , 42, 47A.

Keaney, J.F., Jr., Gaziano, J.M., Xu, A., Frei, B., Curran-Celentano, J., Shwaery, G.T., Loscalzo, J. & Vita, J.A. (1994). Low-dose α-tocopherol improves and high-dose α-tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J. Clin. Invest ., 93 , 844-851.

Keller, K.B. & Lemberg, L. (2003). Obesity and the metabolic syndrome. Am. J. Crit. Care , 12, 167-170.

Kim, J.-Y., Nolte, L.A., Hansen, P.A., Han, D.-H., Kawanaka, K. & Holloszy, J.O. (1999). Insulin resistance of muscle glucose transport in male and female rats fed a high-sucrose diet. Am. J. Physiol. , 276, R665-R672.

Kitiyakara, C. & Wilcox, C.S. (1998). Antioxidants for hypertension. Curr. Opin. in Nephrol. Hypertens. , 7, 531-538.

Kohlmeier, M. (1986). Direct enzymatic measurement of glycerides in serum and lipoprotein fractions. Clin. Chem. , 32, 63-66.

Laakso, M., Edelman, S., Brechtel, G. & Baron, A.D. (1992). Impaired insulin mediated skeletal muscle blood flow in patients with NIDDM. Diabetes , 41, 1076-1083.

Laakso, M., Edelman, S.V., Brechtel, G. & Baron, A.D. (1990). Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man. J. Clin. Invest. , 85, 1844-1852.

Lee, D. H., Lee, J.U., Kang, D.G., Paek, Y.W., Chung, D.J. & Chung, M.Y. (2001). Increased cascular endothelin-1 gene expresseion with unaltered nitric oxide synthase levels in fructose-induced hypertensive rats. Metabolism , 50 , 74-78.

Liang, C.S., Doherty, J.U., Faillace, R., Maekawa, K., Arnold, S., Gavras, H. & Hood, W.B. (1982). Insulin infusion in conscious dogs: effects on systemic and coronary hemodynamics, regional blood flows, and plasma catecholamines. J. Clin. Invest. , 69, 1321-1336.

Mather, K.J., Lteif, A., Steinberg, H.O. & Baron, A.D. (2004). Interactions between endothelin and nitric oxide in the regulation of vascular tone in obesity and diabetes. Diabetes , 53, 2060-2066.

McIntyre, M., Bohr, D. & Dominiczak, A. (1999). Endothelial function in hypertension: the role of superoxyde anion. Hypertension , 34, 539-545.

McVeigh, G.E., Brennan, G.M., Johnston, G.D., McDermott, B.J., McGrath, L.T., Henry, W.R., Andrews, J.W. & Hayes, J.R. (1992). Impaired endothelium-dependent and independent vasodilation in patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia , 35, 771-776.

Meigs, J.B. (2003). Epidemiology of the insulin resistance syndrome. Curr. Diab. Rep. , 3, 73-79.

Neri, S., Signorelli, S.S., Torrisi, B., Pulvirenti, D., Mauceri, B., Abate, G., Ignaccolo, L., Bordonaro, F., Cilio, D., Calvagno, S. & Leotta, C. (2005). Effects of antioxidant supplementation on postprandial oxidative stress and endothelial dysfunction: a single-blind, 15-day clinical trial in patients with untreated type 2 diabetes, subjects with impaired glucose tolerance, and healthy controls. Clin. Ther ., 27 , 1764-1773.

Noronha, B.T., Li, J.M., Wheatcroft, S.B., Shah, A.M. & Kearney, M.T. (2005). Inducible nitric oxide synthase has divergent effects on vascular and metabolic function in obesity. Diabetes , 54, 1082-1089.

Onoue, H., Tsutsui, M., Smith, L., T., O.B. & Katusic, Z.S. (1999). Adventitial expression of recombinant endothelial nitric oxide synthase gene reverses vasoconstrictor effect of endothelin-1. J. Cereb. Blood Flow Metab. , 19, 1029-1037.

Perreault, M. & Marette, A. (2001). Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle. Nature Med. , 7, 1138-1143.

Piatti, P.M., Monti, L.D., Conti, M., Baruffaldi, L., Galli, L., Phan, C.V., Guazzini, B., Pontiroli, A.E. & Pozza, G. (1996). Hypertriglyceridemia and hyperinsulinemia are potent inducers of endothelin-1 release in humans. Diabetes , 45, 316-321.

Pitre, M., Nadeau, A. & Bachelard, H. (1996). Insulin sensitivity and hemodynamic responses to insulin in Wistar-Kyoto and spontaneously hypertensive rats. Am. J. Physiol. , 271, E658-E668.

Pitre, M., Santuré, M., Lévesque, M., Nadeau, A. & Bachelard, H. (2000). Implication du NO dans les effets vasculaires et métaboliques de l’insuline. Médecine Sciences , 16, 28.

Raij, L. & Baylis, C. (1995). Glomerular actions of nitric oxide. Kidney Int. , 48, 20-32.

Rattigan, S., Clark, M.G. & Barrett, E.J. (1999). Acute vasoconstriction-induced insulin resistance in rat muscle in vivo. Diabetes , 48, 564-569.

Reaven, G.M. (1991). Insulin resistance, hyperinsulinemia, hypertriglyceridemia, and hypertension. Parallels between human disease and rodent models. Diabetes Care , 14, 195-202.

Reaven, G.M. (1988). Role of insulin resistance in human disease. Diabetes , 37, 1595-1607.

Reaven, G.M. & Ho, H. (1991). Sugar-induced hypertension in Sprague-Dawley rats. Am. J. Hypertens. , 4, 610-614.

Reil, T.D., Barnard, R.J., Kashyap, V.S., Roberts, C.K. & Gelabert, H.A. (1999). Diet-induced changes in endothelial-dependent relaxation of the rat aorta. J. Surgical Res. , 85, 96-100.

Richard, V., Hogie, M., Clozel, M., Loffler, B.M. & Thuillez, C. (1995). In vivo evidence of an endothelin-induced vasopressor tone after inhibition of nitric oxide synthesis in rats. Circulation , 91, 771-775.

Richterich, R. & Dauwalder, H. (1971). Zur bertimmung der plasmaglucokonzentration mit der hexokinase-glucose-6-phosphat-dehydrogenase methode. Schweiz Med. Wochenschr , 101, 615-618.

Roberts, C.K., Barbard, R.J., Sindhu, R.K., Jurczak, M., Ehdaie, A. & Vaziri, N.D. (2005). A high-fat, refined-carbohydrate diet induces endothelial dysfunction, oxydant/antioxidant imbalance and depresses NOS protein expression. J. Appl. Physiol. , 98, 203-210.

Roberts, C.K., Vaziri, N.D., Liang, K.H. & Barnard, R.J. (2001). Reversibility of chronic experimental syndrome X by diet. Hypertension , 37, 1323-1328.

Roberts, C.K., Vaziri, N.D., Ni, Z. & Barnard, R.J. (2002). Correction of long-term diet-induced hypertension and nitrotyrosine accumulation by diet modification. Atherosclerosis , 163, 321-327.

Roberts, C.K., Vaziri, N.D., Wang, X.Q. & Barnard, R.J. (2000). Enhanced NO inactivation and hypertension induced by a high-fat, refined-carbohydrte diet. Hypertension , 36, 423-429.

Roberts, C.K., Viziri, N.D., Sindhu, R.K. & Barnard, R.J. (2003). A high-fat, refined-carbohydrate diet affects renal NO synthase protein expression and salt sensitivity. J. Appl. Physiol. , 94, 941-946.

Saengsirisuwan, V., Perez, F.R., Sloniger, J.A., Maier, T. & Henriksen, E.J. (2004). Interactions of exercise training and α-lipoic acid on insulin signaling in skeletal muscle of obese Zucker rats. Am. J. Physiol. Endocrinol. Metab . 287 , E529-E536.

Santuré, M., Pitre, M., Marette, A., Deshaies, Y., Lemieux, C., Larivière, R., Nadeau, A. & Bachelard, H. (2002). Induction of insulin resistance by high-sucrose feeding does not raise mean arterial blood pressure but impairs hemodynamic responses to insulin in rats. Br. J. Pharmacol , 137, 185-196.

Scherrer, U., Randin, D., Vollenweider, P., Vollenweider, L. & Nicod, P. (1994). Nitric oxide release accounts for insulin’s vascular effects in humans. J. Clin. Invest. , 94, 2511-2515.

Schnackenberg, C.G. & Wilcox, C.S. (2001). The SOD mimetic tempol restores vasodilation in afferent arterioles of experimental diabetes. Kidney Int. , 59, 1859-1864.

Seidell, J.C. (2000). Obesity, insulin resistance and diabetes-a worlwide epidemic. Br. J. Nutr. , 83, S5-S8.

Shankar, R., Zhu, J.S., Ladd, B., Henry, D., Shen, H.Q. & Baron, A.D. (1998). Central nervous system nitric oxide synthase activity regulates insulin secretion and insulin action. J. Clin. Invest. , 102, 1403-1412.

Shankar, R.R., Wu, Y., Shen, H.-Q., Zhu, J.-S. & Baron, A.D. (2000). Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance. Diabetes , 49, 1-4.

Singh, M.K., Krisan, A.D., Crain, A.M., Collins, D.E. & Yaspelkis, B.B. (2003). High-fat diet and leptin treatment alter skeletal muscle insulin-stimulated phosphatidylinositol 3-kinase activity and glucose transport. Metabolism , 52, 1196-1205.

Srinivasan, K., Patole, P.S., Kaul, C.L. & Ramarao, P. (2004). Reversal of glucose intolerance by pioglitazone in high fat diet-fed rats. Methods Find Exp. Clin. Pharmacol. , 26, 327-333.

Steinberg, H.O., Brechtel, G., Johnson, A., Fineberg, N. & Baron, A.D. (1994). Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release. J. Clin. Invest. , 94, 1172-1179.

Steinberg, H.O., Chaker, H., Leaming, R., Johnson, A., Brechtel, G. & Baron, A. (1996). Obesity/insulin resistance is associated with endothelial dysfunction. Implication for the syndrome of insulin resistance. J. Clin. Invest. , 97, 2601-2610.

Teachey, M.K., Taylor, Z.C., Maier, T., Saengsirisuwan, V., Sloniger, J.A., Jacob, S., Klatt, M.J., Ptock, A., Kraemer, K., Hasselwander, O. & Henriksen, E.J. (2003). Interactions of conjugated linoleic acid and lipoic acid on insulin action in the obese Zucker rat. Metabolism . 52 , 1167-1174.

Thirunavukkarasu, V., Nandhini, A.T.A. & Anurakha, C.V. (2004). Cardiac lipids and antioxidant status in high fructose rats and the effect of α-lipoic acid. Nutr. Metab. Cardiovasc. Dis ., 14 , 351-357.

Thirunavukkarasu, V. & Anuradha, C.V. (2004). Influence of α-lipoic acid on lipid peroxidation and antioxidant defence system in blood of insulin-resistant rats. Diabetes. Obes. Metab . 6 , 200-207.

Verma, S., Bhanot, S. & McNeill, J.H. (1995). Effect of chronic endothelin blockade in hyperinsulinemic hypertensive rats. Am. J. Physiol. , 269, H2017-H2021.

Vincent, M.A., Dawson, D., Clark, A.D.H., Lindner, J.R., Rattigan, S., Clark, M.G. & Barrett, E.J. (2002)Skeletal muscle microvascular recruitment by physiological hyperinsulinemia precedes increases in total blood flow. Diabetes, 51 , 42-4 8.

Vollenweider, P., Tappy, L., Randin, D., Schneiter, P., Jequier, E., Nicod, P. & Scherrer, U. (1993). Differential effects of hyperinsulinemia and carbohydrate metabolism on sympathetic nerve activity and muscle blood flow in humans. J. Clin. Invest. , 92, 147-154.

Vrana, A., Kazdova, L., Dobesova, Z., Kunes, J., Kren, V., Bila, V., Stobla, P. & Klimes, I. (1993). Triglyceridemia, glucoregulation, and blood pressure in various rat strains. Effects of dietary carbohydrates. Ann. NY Acad. Sci. , 683, 57-68.

Wolpert, H.A., Steen, S.N., Istfan, N.W. & Simonson, D.C. (1993). Insulin modulates circulating endothelin-1 levels in humans. Metab. Clin. Exp. , 42, 1027-1030.

Yki-Järvinen, H. & Utriainen, T. (1998). Insulin-induced vasodilation: physiology or pharmacology? Diabetologia , 41, 369-379.

Yoshioka, S., Uemura, K., Tamaya, N., Tamagawa, T., Miura, H., Iguchi, A., Nakamura, J. & Hotta, N. (2000). Dietary fat-induced increase in blood pressure and insulin resistance in rats. J. Hypertens. , 18, 1857-1864.

Youngren, J.F., Paik, J. & Barnard, R.J. (2001). Impaired insulin-receptor autophosphorylation is an early defect in fat-fed, insulin-resistant rats. J. Appl. Physiol. , 91, 2240-2247.

Zeng, G., Nystrom, F.H., Ravichandran, L.V., Cong, L.-N., Kirby, M., Mostowski, H. & Quon, M.J. (2000). Role for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation , 101, 1539-1545.

Zeng, G. & Quon, M. (1996). Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. J. Clin. Invest. , 98, 894-898.

Zollner, S., Haseloff, R.F. & al., e. (1997). Nitroxides increase the detectable amount of nitric oxide released from endothelial cells. J Biol Chem , 272, 23076-23080.

*Standard laboratory rat chow (Teklab Global 18% Protein Rodent Diet, 2018)

†Purified high fat high sucrose diet:

-Protein is casein (purified high nitrogen, ICN Biochemicals, Montréal, Canada) and 0.3% dl-methionine

     -Vitamine mixture (No. 40060, Teklad, Madison, WI)

     -Mineral mixture (AIN-76 mineral mix, ICN Biochemicals)

     -Fiber (cellulose, Alphacel, ICN Biochemicals)

Values are means ± SE; n is the number of rats. *p<0.05 compared with Chow. † p<0.05 compared with Chow-tempol. § p<0.05 compared with HFHS.

Values are means ± SE. These parameters were measured in the unfasting state. *p<0.05 when compared to the untreated chow-fed group. †p<0.05 when compared to the tempol-treated chow-fed group.

Values are mean ± SEM.; n, is the number of rats. (The groups represent those used to assess the haemodynamic effects of insulin intravenously infused during the euglycemic hyperinsulinemic clamp studies). bpm, beat per minute.

Values are mean ± SEM.; n, is the number of rats. GIR60-120 is the glucose infusion rate required to maintain euglycemia during steady-state (60-120 min) plasma insulin concentration. *p<0.05 versus chow-fed rats, Student’s t-test for unpaired data. §p<0.05 versus tempol-treated chow-fed rats, Student’s t-test for unpaired data. ‡p<0.05 versus HFHS-fed rats.

Figure 1. Cumulative dose-response curves to increasing concentrationsof phenylephrine (0,001-10 µM) in rat isolated aortic rings incubated: (A) in the absence (HFHS, n=17; HFHS-tempol, n=19; Chow, n=15; Chow-tempol, n=18) or (B) in the presence of L-NAME (100 µM) (HFHS, n=8; HFHS-tempol, n=8; Chow, n=8; Chow-tempol, n=7). Values are means with SE shown by vertical lines. aP<0,05 for HFHS-tempol + L-NAME vs HFHS+ L-NAME.

Figure 2. Cumulative dose-response curves to increasing concentrations of carbachol (0,01-100 µM) in phenylephrine pre-contracted aortic rings incubated in the absence (bottom curves) (HFHS, n=17; HFHS-tempol, n=19; Chow, n=15; Chow-tempol, n=18) or in the presence of L-NAME (100 µM) (top curves) (HFHS, n=8; HFHS-tempol, n=8; Chow, n=9; Chow-tempol, n=8). Relaxations are expressed as percentage changes from the initial pre-contraction induced by phenylephrine, and values are means ± SE. aP<0,05 for HFHS-fed rats vs Chow-fed rats. bP<0,05 for HFHS-tempol vs HFHS cP<0,05 for HFHS + L-NAME vs HFHS. dP<0,05 for Chow + L-NAME vs Chow. eP<0,05 for HFHS-tempol + L-NAME vs HFHS-tempol. fP<0,05 for Chow-tempol + L-NAME vs Chow-tempol.

Figure 3. Cumulative dose-response curves to increasing concentrations of SNP (0,1-100 nM) in phenylephrine pre-contracted aortic rings (HFHS, n=17; HFHS-tempol, n=19; Chow, n=15; Chow-tempol, n=18). Relaxations are expressed as percentage changes from the initial pre-contraction induced by phenylephrine, and values are means ± SE.

Figure 4. Effect of insulin (150 nM) and treatment with tempol on cumulative dose-response curves to increasing concentrationsof phenylephrine (0,001-10 µM) in rat isolated aortic rings incubated in the absence or presence of L-NAME in both untreated and tempol-treated HFHS-fed rats in A (HFHS, n=17; HFHS+Insulin, n=17; HFHS+Insulin+L-NAME, n=10; HFHS-tempol+Insulin n=19; HFHS-tempol+Insulin+L-NAME, n=8), or in both groups of Chow-fed rats in B (Chow, n=15; Chow+Insulin, n=15; Chow+Insulin+L-NAME, n=9; Chow-tempol+Insulin n=18; Chow-tempol+Insulin+L-NAME, n=8). The developed tensions are expressed in gram, and values are means±SE. aP<0,05 when compared to HFHS in A, or to Chow in B. bP<0,05 when compared to HFHS+Insulin in A, or to Chow+Insulin in B. cP<0,05 when compared to HFHS+Insulin+L-NAME in A, or to Chow+Insulin+L-NAME in B. dP<0,05 when compared to HFHS-tempol+Insulin in A, or to Chow-tempol+Insulin in B. eP<0,05 for HFHS-tempol + Insulin vs HFHS+Insulin. fP<0,05 when compared to HFHS+Insulin in A, or to Chow+Insulin in B. gP<0,05 for Chow+Insulin vs Chow. hP<0,05 for Chow-tempol+Insulin vs Chow.

Figure 5. Effects of HFHS diet and treatment with tempol on endothelial nitric oxide synthase (eNOS) content in thoracic aortas isolated from overnight fasted treated and untreated HFHS- and Chow-fed rats. (A) Representative immunoblots showing eNOS (top) and alpha-tubulin (bottom) immunoreactivity in aortas from two different animals for each experimental group are shown. (B) Densitometric band intensities of eNOS are summarized. The mean level of eNOS in chow-fed rats (n=8) was normalized to 100%, and the relative levels of eNOS in tempol-treated chow-fed rats (n=7), HFHS-fed rats (n=8), and tempol-treated HFHS-fed rats (n=8) were calculated. Data are expressed as means ± SE. * P < 0.05 when compared to both groups of Chow-fed rats.

Figure 6. Effects of HFHS diet and treatment with tempol on endothelial nitric oxide synthase (eNOS) content in gastrocnemius muscles isolated from overnight fasted treated and untreated HFHS- and Chow-fed rats. (A) Representative immunoblots showing eNOS immunoreactivity in gastrocnemius muscles from two different animals for each experimental group are shown. (B) Densitometric band intensities of eNOS are summarized for each group: chow-fed rats (n=8), tempol-treated chow-fed rats (n=7), HFHS-fed rats (n=8), and tempol-treated HFHS-fed rats (n=8). Data are expressed as means ± SE. *p< 0.05 represent a significant difference between HFHS-fed rats and chow-fed rats. Student’s t test for unpaired data.

Figure 7. A) Confocal microscopy photographs of the thoracic aortas from one rat in each experimental group. Endothelium-specific eNOS protein expression was revealed using an Alexa fluor 594 conjugated rabbit anti-green fluorescent protein (GFP) (in red). The aortas were counter-stained with Alexa Fluor 488 conjugated phalloidin (in green) that has a high-affinity for F-actin. B) Densitometric band intensities of eNOS are summarized. The mean level of eNOS in chow-fed rats(n=5) was normalized to 100%, and the relative levels of eNOS protein in tempol-treated chow-fed rats (n=5), untreated HFHS-fed rats (n=5), and tempol-treated HFHS-fed rats (n=5) were calculated. Data are means ± SE. * P <0.05 when compared to untreated chow-fed rats. § P <0.05 when compared to tempol-treated chow-fed rats. † P <0.05 when compared to tempol-treated HFHS-fed rats.

Figure 8. Effects of HFHS diet and treatment with tempol on nitrotyrosine formation in thoracic aortas isolated from overnight fasted treated and untreated HFHS- and Chow-fed rats. Densitometric band intensities of nitrotyrosine are summarized. The mean level of nitrotyrosine in chow-fed rats (n=12) was normalized to 100%, and relative levels of nitrotyrosine in tempol-treated chow-fed rats (n=8), HFHS-fed rats (n=8), and tempol-treated HFHS-fed rats (n=9) were calculated. Data are expressed as means ± SE. * P <0.05 when compared to untreated and tempol-treated chow-fed rats. † P <0.05 when compared to untreated HFHS-fed rats.

Figure 9. Immunoreactive endothelin 1 (ir-ET-1) concentration in mesenteric arterial bed isolated from overnight fasted untreated HFHS- (n=8) and Chow-fed rats (n=9) and tempol-treated HFHS (n=7) and Chow-fed rats (n=8). Values are means ± SE shown by vertical lines. *p<0.05 HFHS vs chow,†p<0.05 HFHS vs HFHS-Tempol, §p<0.05HFHS vs Chow-tempol.

Figure 10. Insulin dose-response curve for stimulation of glucose uptake in (A) soleus and (B) EDL muscles. Muscles were dissected out from untreated chow-fed rats (n=10) and HFHS-fed rats (n=12), and from tempol-treated Chow (n=11) and HSHF-fed rats (n=9). Values are means ± SE shown by vertical lines. * p <0.05 for the HFHS-fed rats versus the chow-fed rats, §p<0.05 for the tempol-treated chow-fed rats versus the HFHS-fed rats, †p<0.05 tempol-treated HFHS-fed rats vs HFHS-fed rats. Student's t-test for unpaired data.

Figure 11. Cardiovascular changes elicited by control intravenous infusion of saline-0.2% bovine serum albumin (BSA) (n=11) or euglycemic infusion of insulin at a rate of 4 mU kg-1 min-1 in conscious untreated chow-fed (n=9) and HFHS-fed (n=9) rats or in tempol-treated chow-fed (n=8) and HFHS-fed rats (n=6). Values are means ± SE, shown by vertical lines. MAP, mean blood pressure; HR, heart rate. Data were analyzed for statistical significance by an ANOVA followed by Fisher’s test. * p< 0.05 for chow, chow-tempol or HFHS-tempol vs vehicle, § p< 0.05 for HFHS vs chow, † p< 0.05 for HFHS vs chow-tempol, and ‡ p< 0.05 for HFHS-tempol vs HFHS

Figure 12 . Changes in regional vascular conductances elicited by control iv infusion of saline-0.2% BSA (n=11) or euglycemic infusion of insulin at a rate of 4 mU kg-1 min-1 in conscious untreated chow-fed (n=9) or HFHS-fed (n= 9) rats or in tempol-treated chow-fed (n=8) and HFHS-fed rats (n=6). These data were derived from the data shown in Fig. 11. Values are means ± SE, shown by vertical lines. Data were analyzed for statistical significance by an ANOVA followed by Fisher’s test. *p<0.05 for chow, chow-tempol or HFHS-tempol vs vehicle, §p<0.05 for HFHS vs chow, † p<0.05 for HFHS vs chow-tempol, and ‡p<0.05 for HFHS-tempol vs HFHS

© Mylene Badeau, 2006