Several clinical studies have shown the beneficial cardiovascular effects of fibrates in patients with diabetes and insulin resistance. The ligands of peroxisome proliferator-activated receptor-α (PPAR-α) reduce ischemia-reperfusion injury in nondiabetic animals. We hypothesized that the activation of PPAR-α would exert cardioprotection in type 2 diabetic Goto-Kakizaki (GK) rats, involving mechanisms related to nitric oxide (NO) production via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. GK rats and age-matched Wistar rats (n ≥ 7) were given either 1) the PPAR-α agonist WY-14643 (WY), 2) dimethyl sulfoxide (DMSO), 3) WY and the NO synthase inhibitor NG-nitro-l-arginine (l-NNA), 4) l-NNA, 5) WY and the PI3K inhibitor wortmannin, or 6) wortmannin alone intravenously before a 35-min period of coronary artery occlusion followed by 2 h of reperfusion. Infarct size (IS), expression of endothelial NO synthase (eNOS), inducible NO synthase, and Akt as well as nitrite/nitrate were determined. The IS was 75 ± 3% and 72 ± 4% of the area at risk in the Wistar and GK DMSO groups, respectively. WY reduced IS to 56 ± 3% in Wistar (P < 0.05) and to 46 ± 5% in GK rats (P < 0.001). The addition of either l-NNA or wortmannin reversed the cardioprotective effect of WY in both Wistar (IS, 70 ± 5% and 65 ± 5%, respectively) and GK (IS, 66 ± 4% and 64 ± 4%, P < 0.05, respectively) rats. The expression of eNOS and eNOS Ser1177 in the ischemic myocardium from both strains was increased after WY. The expression of Akt, Akt Ser473, and Akt Thr308 was also increased in the ischemic myocardium from GK rats following WY. Myocardial nitrite/nitrate levels were reduced in GK rats (P < 0.05). The results suggest that PPAR-α activation protects the type 2 diabetic rat myocardium against ischemia-reperfusion injury via the activation of the PI3K/Akt and NO pathway.
- peroxisome proliferator-activated receptor-α ligand
- endothelial function
- phosphatidylinositol 3-kinase
type 2 diabetes is a metabolic disorder characterized by insulin resistance, hyperglycemia, and dyslipidemia. Thus, along with hypoglycemic and insulin-sensitizing therapy, patients with type 2 diabetes may benefit from treatment with lipid-lowering agents, such as fibrates. Indeed, several clinical studies have demonstrated the beneficial cardiovascular effects of fibrates in patients with type 2 diabetes (9, 20, 32, 37). These effects are thought to be primarily attributed to their ability to lower triglycerides and low-density lipoprotein cholesterol and to raise high-density lipoprotein cholesterol (34). However, several clinical trials have shown that the reduction in coronary events was greater than would have been expected, based on the reduction in low-density lipoprotein cholesterol (20, 32), suggesting additional mechanisms beyond the improvement of dyslipidemia by fibrates.
Fibrates are ligands of the peroxisome proliferator-activated receptor-α (PPAR-α) transcription factor, which is expressed in a number of tissues, including the myocardium and the vessel wall (1, 16, 21). PPAR-α regulates the metabolism of lipoprotein and fatty acids, as well as the transcription of several factors involved in inflammatory response and oxidative stress (14, 15, 26, 27). Moreover, the ligands of PPAR-α, including fibrates, have been shown to reduce myocardial ischemia-reperfusion injury in nondiabetic animals in vivo (40, 41). The mechanisms suggested to be involved in the cardioprotective effect of PPAR-α activation include anti-inflammatory effects via the inhibition of proinflammatory genes regulating the production of cytokines, adhesion molecules, endothelin-1, and matrix metalloproteinase-2 and -9 (7, 27, 41). We recently demonstrated that WY-14643 (WY), a selective agonist of PPAR-α, exerts cardioprotection in a rat model of ischemia-reperfusion injury via mechanisms related to the production of nitric oxide (NO) and endothelin-1 (5). Moreover, PPAR-α ligands were reported to increase NO production via a mechanism involving the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in endothelial cells (29, 39).
Since myocardial infarction is a common complication in patients with diabetes, it is important to evaluate the possibility to limit the extent of myocardial infarct size in models of diabetes. Importantly, it has been reported that protection against ischemia-reperfusion injury is less pronounced in animal models of diabetes (13, 38). Furthermore, in consideration of the finding that endothelial dysfunction, characterized by reduced bioavailability of NO, is common in diabetes, it may be anticipated that compounds like PPAR-α ligands, acting via the NO pathway, would be less efficient in models of diabetes. Therefore, it is important to investigate whether the activation of PPAR-α protects against ischemia-reperfusion injury in the Goto-Kakizaki (GK) rat, a model of type 2 diabetes with documented endothelial dysfunction (2, 6). This inbred strain of Wistar rat origin is nonobese, and hyperglycemia develops early in life in association with an impaired insulin secretion. Insulin resistance in skeletal muscle and liver is rather mild and, to a large extent, secondary to glucotoxicity (30).
Thus the primary aim of the present study was to determine whether the myocardium of type 2 diabetic GK rats can be protected by WY, a ligand of PPAR-α. The secondary aim was to determine whether the protection involves the PI3K/Akt and NO signaling pathway.
MATERIALS AND METHODS
Animal preparation and experimental protocol.
The study was approved by the Regional Ethical Committee for Laboratory Animal Experiments and conforms to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).
Male GK rats of the Stockholm GK colony at 12 wk of age were used in the study. As controls, age-matched male Wistar rats (B&K Universal, Sollentuna, Sweden) were used. The rats were anesthetized with pentobarbital sodium (50 mg/kg ip, followed by continuous infusion of 5 mg·kg−1·h−1 iv; Apoteksbolaget, Stockholm, Sweden), tracheotomized, intubated, and ventilated with air by a rodent ventilator (50 strokes/min, 8–10 ml/kg tidal volume). The rectal temperature was maintained at 38.0 ± 0.5°C by a heated operation table. The right carotid artery was cannulated and connected to a pressure transducer (Statham P23Db) for measurement of mean arterial pressure (MAP), which was continuously recorded on a Grass polygraph (model 7D, Grass Instruments, Quincy, MA). Heart rate (HR) was determined from the arterial pressure curve. The left jugular vein was cannulated for the administration of drugs. The heart was exposed via a left thoracotomy. A ligature was placed around the left coronary artery. After completion of the surgical preparation, the rats were allowed to stabilize for 15 min before drug administration. Both GK and Wistar rats were randomly divided into the following treatment groups (n ≥ 7): 1) 10% dimethyl sulfoxide (DMSO); 2) the PPAR-α agonist 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY, 1 mg/kg); 3) the combination of WY and the nonselective NO synthase (NOS) inhibitor NG-nitro-l-arginine (WY + l-NNA, 2 mg/kg); 4) l-NNA alone; 5) the combination of WY and a specific inhibitor of PI3K wortmannin (15 μg/kg), or 6) wortmannin. All treatments were given as intravenous injections 30 min before coronary artery occlusion. The dose of WY, l-NNA, and wortmannin (Sigma, St. Louis, MO) and the concentration of DMSO were based on previous studies of myocardial ischemia-reperfusion injury (5, 28, 36).
Thirty minutes after the drug administration, the coronary artery was occluded by tightening the ligature. This was associated with a decrease in MAP and an appearance of a cyanotic color of the myocardial area at risk. Reperfusion was initiated 35 min following the induction of ischemia by the removal of the snare and was maintained for 2 h. The reperfusion was associated with hyperemia and the disappearance of the cyanotic color of the myocardium.
Determination of infarct size.
After 2 h of reperfusion, the left coronary artery was reoccluded and 1.5 ml of 2% Evans blue was injected into the right atrium via the left jugular vein to outline the ischemic myocardium (area at risk). The rats were euthanized with an overdose of anesthetics, and the heart was rapidly excised. The atria and the right ventricle were removed. The left ventricle was cut into 1–1.5-mm-thick slices perpendicular to the heart base-apex axis. The slices were scanned from both sides for area-at-risk measurements and weighed. One slice from each heart was saved for further tissue analysis. The residuary slices were put into 0.8% triphenyltetrazolium chloride for 15 min at 37°C to distinguish the viable myocardium from the necrotic (10). After an overnight incubation in 4% formaldehyde, the slices were again scanned from both sides, and the extent of myocardial necrosis and the area at risk was determined by planimetry of computer images (Photoshop 6.0, Adobe Systems, San Jose, CA).
The ischemic and nonischemic myocardium of animals pretreated with DMSO and WY was collected and frozen at −80°C for subsequent evaluation of the expression of inducible NOS (iNOS), total endothelial NOS (eNOS), phosphorylated eNOS Ser1177, total Akt, phosphorylated Akt Ser473, and Akt Thr308 by immunoblotting. Frozen samples were homogenized in ice-cold lysis buffer containing 20 mM Tris (pH 7.8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% Triton X-100, 10% (wt/vol) glycerol, 10 mM NaF, 1 mM ethylenediaminetetraacetic acid, 5 mM Na pyrophosphate, 0.5 mM Na3VO4, 1 μg/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 mM benzamidine. The homogenates were centrifuged at 5,000 g for 20 min at 4°C, and the concentration of protein in the supernatant in each aliquot was determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). Protein extracts (50 μg/lane) were loaded onto a 10% SDS gel and separated by electrophoresis. Extracts from two separate groups were loaded on one gel, and the amount of protein was accordingly compared pairwise. Proteins were transferred to nitrocellulose membranes (Hybond-C pure, Amersham Biosciences, Little Chalfont, UK), and Ponceau solution was used to visualize protein loading.
The membranes were incubated overnight at 4°C with phosphospecific antibodies against eNOS Ser1177 (1:1,000, BD Biosciences Pharmingen), Akt Ser473, or Akt Thr308 (1:1,000, Cell Signaling Technology, Beverly, MA). After being washed in PBS-Tween, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG for eNOS Ser1177 (1:10,000, BD Biosciences Pharmingen) and donkey anti-rabbit IgG for Akt Ser473 and Akt Thr308 (1:2,500, Affinity Bioreagents, Golden, CO) for 1 h at room temperature, followed by additional washing. Proteins were visualized by enhanced chemiluminescence with ECL advanced Western blotting detection kit (Amersham Biosciences) and quantified using densitometry and Quantity One 4.5.1 software (Bio-Rad, Hercules, CA).
After immunoblotting with phosphospecific antibodies, the membranes were stripped and immunoblotted for either total eNOS (1:1,000, Affinity Bioreagents), iNOS (1:2,000, Becton Dickinson, Franklin Lakes, NJ), or Akt proteins (1:1,000, Cell Signaling Technology). β-Actin (1:5,000, Sigma-Aldrich) was used as a loading control.
Measurements of plasma glucose and insulin concentrations.
Basal blood samples were drawn from the tail vein of nonfasting anesthetized animals. Plasma glucose was measured by a glucose analyzer with plasma conversation (HemoCue, Ängelholm, Sweden). At the termination of the experiment, the arterial blood samples were collected from the carotid artery in plastic tubes containing Na citrate and centrifuged at 5,000 g for 15 min at 4°C, and the plasma was separated and stored at −80°C. Blood glucose concentrations were analyzed with a blood glucose meter YSI 2300 (VWR International, West Chester, PA). Plasma insulin concentrations were determined by radioimmunoassay (Pharmacia Insulin RIA kit; Pharmacia-Upjohn Diagnostics, Uppsala, Sweden) with rat insulin (Novo Nordisk, Bagsværd, Denmark) as a standard.
Myocardial nitrite/nitrate measurements.
Additional GK and age-matched Wistar rats (n = 5) were euthanized with an overdose of anesthetics, and the heart was rapidly excised. The levels of the NO metabolites nitrite and nitrate (NOx) were measured in heart tissue homogenates, using a chemiluminescence method described in detail elsewhere (24). Briefly, NOx levels were determined by chemiluminescence after reductive cleavage and a subsequent determination of the NO released into the gas phase. A rapid-response chemiluminescence NO system (Aerocrine, Stockholm, Sweden) was used to detect the NO signals and to collect the data that were further manipulated with Origin for Windows, version 7.0 (Microcal, Northampton, MA), and reported as the area under the curve. NOx were reduced to NO with a solution of vanadium (III) chloride in 1 N hydrochloric acid (saturated solution) at 95°C. The calibration curve was obtained with freshly prepared nitrate standard solution in ultrapure water. NOx levels were expressed in micromoles and adjusted for tissue wet weight.
All values are presented as means ± SE. Differences in infarct size between the groups were calculated using one-way ANOVA, followed by the Tukey-Kramer multiple-comparisons test. Changes in hemodynamics were analyzed using ANOVA for repeated measurements, and differences in myocardial NOx and protein expression between groups were determined using an unpaired t-test. P < 0.05 was considered statistically significant.
Body weight and plasma glucose and insulin.
Body weight, basal and terminal plasma glucose, and terminal plasma insulin of GK and Wistar rats are shown in Table 1. GK rats had significantly lower body weight than age-matched Wistar rats. Both basal and terminal plasma glucose was significantly higher in GK rats compared with Wistar rats (Table 1). Terminal plasma glucose levels in both strains were twofold those of basal glucose values. There were no differences in plasma insulin between GK and Wistar rats. Treatment with WY did not significantly influence plasma glucose or insulin levels (Table 1).
Myocardial NOx levels.
Myocardial NOx levels were 60% lower (P < 0.05) in GK rats compared with age-matched Wistar rats (Fig. 1).
MAP, HR, and rate-pressure product (RPP) in Wistar and GK rats are presented in Tables 2 and 3, respectively. When compared with the vehicle group (DMSO), the group given WY + l-NNA had lower blood pressure before treatment as well as during ischemia and reperfusion in Wistar rats (Table 2). Accordingly, RPP was lower in this group. In GK rats, HR was higher in the l-NNA group compared with the DMSO group at 30 and 60 min of reperfusion (Table 3). However, the changes in HR did not significantly affect RPP, an indicator of myocardial oxygen demand.
The area at risk and infarct size are shown in Fig. 2. The area at risk was slightly larger in the Wistar WY + l-NNA group compared with the Wistar DMSO group (Fig. 2A). Apart from this, there were no significant differences in the area at risk in any group compared with the vehicle groups. The infarct size of the control groups (DMSO) was similar in the two strains (75 ± 3% and 72 ± 4% in Wistar and GK, respectively) (Fig. 2B). WY induced a significant reduction in infarct size both in Wistar (to 56 ± 3%, P < 0.05) and GK rats (to 46 ± 5%, P < 0.001) compared with the respective DMSO group. There was no significant difference regarding the degree of protection induced by WY in Wistar and GK rats. An administration of l-NNA reversed the cardioprotective effect of WY in Wistar rats (infarct size, 70 ± 5%; not significant) and GK rats (infarct size, 66 ± 4%, P < 0.05). l-NNA did not affect infarct size when given alone in either strain of rats (Fig. 2B). An administration of wortmannin also reversed the cardioprotective effect of WY in Wistar rats (infarct size, 65 ± 5%; not significant) and in GK rats (64 ± 4%, P < 0.05). Wortmannin did not affect infarct size per se (Fig. 2B).
eNOS, iNOS, and Akt expression and phosphorylation.
Total eNOS and iNOS expression, phosphorylation of eNOS at Ser1177, total Akt expression, and phosphorylation of Akt at Ser473 and Thr308 were determined in ischemic and nonischemic myocardium from the DMSO and WY groups in both Wistar and GK rats. There were no differences in the expression of total and phosphorylated eNOS in the nonischemic myocardium between the groups. WY increased the phosphorylation of eNOS at Ser1177 and the expression of total eNOS in the ischemic myocardium in both Wistar and GK rats (Fig. 3). No iNOS expression was detected in either the ischemic or nonischemic myocardium in any of the strains studied (data not shown).
There were no significant differences in the expression of total Akt in Wistar rats and the phosphorylation of Akt at Ser473 and Thr308 in the nonischemic myocardium of either rat strain. The expression of total Akt was significantly higher in the nonischemic myocardium of GK rats treated with WY than in that of GK DMSO rats (P < 0.05). WY significantly increased the phosphorylation of Akt at Ser473 and Thr308 and the expression of total Akt in the ischemic myocardium in GK rats (Fig. 3). There was a similar, but nonsignificant, trend regarding the change in phosphorylation of Akt and total Akt expression in Wistar rats (Fig. 3). The expression of all proteins in the ischemic myocardium was significantly decreased compared with the nonischemic myocardium in all groups of both rat strains (data not shown). No difference in myocardial expression of β-actin was found between the DMSO- and WY-treated groups.
The present study, to our knowledge, is the first one demonstrating the cardioprotective effect of a PPAR-α ligand against myocardial ischemia-reperfusion injury in an animal model of type 2 diabetes mellitus with reduced myocardial levels of NO metabolites. Moreover, we demonstrate that the inhibition of either NO production by l-NNA or PI3K by wortmannin abrogates the cardioprotection induced by the PPAR-α ligand in GK rats. In addition, a treatment with the PPAR-α ligand increased Akt and eNOS signaling in the ischemic myocardium of GK rats. Collectively, these data suggest that the activation of PPAR-α protects the type 2 diabetic heart against ischemia-reperfusion injury via a mechanism that involves PI3K/Akt and NO.
Fibrates are a group of drugs widely used for the treatment of hypertriglyceridemia and hypercholesterolemia via the activation of PPAR-α (21), which is involved in the regulation of the transcription of fatty acids oxidation enzymes. This may be of particular importance in type 2 diabetes, which is characterized by dyslipidemia along with hyperglycemia and insulin resistance. Accordingly, several clinical studies have demonstrated that fibrates more effectively lowered coronary events in type 2 diabetic than nondiabetic patients (9, 20, 32, 37). Moreover, the Helsinki Heart Study and the Department of Veterans Affairs High-Density Lipoprotein Intervention Trial have reported that the effects of fibrates were greater than expected from their lipid-lowering action (20, 32). Thus there are observations suggesting that not only the lipid-lowering effect of fibrates can explain benefits of PPAR-α ligands in patients with atherosclerosis, coronary artery disease, and type 2 diabetes.
Previous data from experimental studies have demonstrated that a short-term administration of fibrates limits myocardial ischemia-reperfusion injury in nondiabetic animal models (40, 41). In the present study, we demonstrate that the PPAR-α ligand WY also protects from ischemia-reperfusion injury in a model of type 2 diabetes. The magnitude of cardioprotection achieved by the presently used dose of WY was comparable in diabetic and nondiabetic rats. In accordance with previous data achieved in nondiabetic rats (5), the cardioprotective effect of the PPAR-α ligand was blocked by the NOS inhibitor l-NNA, suggesting that it was dependent on the maintained bioavailability of NO in both strains of rats.
We also tested the hypothesis that the PI3K/Akt pathway is involved in the cardioprotection induced by WY via NO. PI3K/Akt, a pathway suggested to be impaired in type 2 diabetic GK rats (19, 35), is important for eNOS phosphorylation and, thereby, for efficient NO production (8, 11). The cardioprotective effect of WY was abolished by the selective PI3K inhibitor wortmannin, supporting the hypothesis that the PI3K/Akt pathway is involved in the cardioprotective effect induced by WY.
To determine the molecular signaling pathway involved in the cardioprotective effect of WY, the expression and phosphorylation of eNOS, iNOS, and Akt protein were analyzed in the ischemic and nonischemic myocardium. The activation of PPAR-α increased the phosphorylation of eNOS at Ser1177 and Akt at Ser473 and Thr308, as well as the total eNOS and Akt expression in the ischemic myocardium of GK rats. PI3K activates Akt by phosphorylation at Thr308, which is necessary for Akt activation, and by phosphorylation at Ser473, which is required for its maximal activity. These phosphorylations, in turn, activate eNOS by their phosphorylation at Ser1177. These observations are in accordance with the study by Goya et al. (12) demonstrating that a pretreatment with WY increased eNOS protein expression in bovine aortic endothelial cells. Previous studies have also demonstrated that PPAR-α activation with WY induces mRNA and protein expression of the PI3K regulatory subunit p85 in human skeletal muscle cells (4, 31). Thus the mechanism of the WY effect on the PI3K/Akt pathway involves its transcriptional upregulation via the activation of PPAR-α. In accordance with our findings, the PPAR-α ligand bezafibrate was shown to increase eNOS expression at the transcriptional, posttranscriptional, and translational level in endothelial cells that involves the PPAR-α-mediated activation of PI3K and the MAPK signaling pathways (39), suggesting that the effects of bezafibrate on eNOS in the endothelium are mediated by both genomic action through PPAR-α and a nongenomic manner through PI3K signaling pathways. iNOS expression was undetectable in the present study, suggesting that iNOS is not significantly upregulated during this brief ischemia-reperfusion protocol in line with a previous observation (5). Collectively, our results suggest that PPAR-α activation induces the cardioprotective effects during ischemia-reperfusion in GK rats via the activation of PI3K, the activation of Akt, which results in the phosphorylation of eNOS, and the maintained bioavailability of NO.
WY did not significantly change the expression and phosphorylation of Akt in Wistar rats on the other hand. However, there was a similar trend toward an upregulation of Akt, and wortmannin also inhibited the cardioprotective effect of WY in the nondiabetic Wistar rats, suggesting that the PI3K/Akt pathway is also involved in this strain. Furthermore, it cannot be excluded that other signaling pathways also activated by PPAR-α are involved.
The activation of the PI3K pathway, which is required for a variety of insulin-mediated effects, including the regulation of glucose uptake and glycogen synthesis in skeletal muscle (33), is altered in the muscle of patients with type 2 diabetes (3, 23). An impaired PI3K activity might be one of the causes of insulin resistance in type 2 diabetes (42). Insulin resistance and an impaired PI3K/Akt signaling pathway leading to the development of endothelial dysfunction in type 2 diabetic rats could be important in the myocardial damage during ischemia-reperfusion. Previous in vitro studies performed on isolated heart models of ischemia-reperfusion in GK rats have reported that hearts from GK rats develop smaller infarcts than their age-matched controls (22, 38). Furthermore, hearts from the diabetic rats were more resistant to the protection induced by ischemic preconditioning (38). In the present in vivo study, the data suggest that the ischemia-reperfusion induced similar myocardial injury in the diabetic GK rats and in control Wistar rats, despite the reduced myocardial levels of NOx, which are indicative of impaired NO bioavailability (17) and endothelial dysfunction. Furthermore, the degree of cardioprotection induced by PPAR-α activation in the two strains was comparable. The increased eNOS expression in aortas of GK rats compared with Wistar rats found by Bitar et al. (2) was suggested to be a compensatory mechanism to increased oxidative stress in the diabetic rats. Increased eNOS expression may compensate the reduced NO availability in GK rats and result in myocardial injury following ischemia-reperfusion that is comparable with that obtained in Wistar rats.
There are limitations with the present study that should be mentioned. First, it cannot be clearly established that NO production blocked by l-NNA is derived from eNOS activity. Unfortunately, no selective eNOS is available. However, iNOS expression was undetectable, suggesting a minor role for NO produced from iNOS. The expression of the neuronal isoform of NOS was not determined since this isoform has been demonstrated not to be of importance for myocardial ischemia-reperfusion injury (18). Second, the phosphorylation of eNOS was only determined at the end of the experiment, whereas a protective effect by the activation of eNOS is likely to be mediated earlier during ischemia or reperfusion. However, the primary aim of the present study was to evaluate the cardioprotective effect of WY and whether it involved NO availability. The complete inhibition of the protective effect by l-NNA clearly indicates the involvement of NO. The increased phosphorylation of eNOS, although determined at the end of reperfusion, supports this conclusion. Third, myocardial NOx levels were not determined in animals included in the ischemia-reperfusion protocol due to a limited amount of tissue available for analysis following the determination of infarct size and protein expression. Furthermore, NOx levels are dependent on several factors other than NO production from l-arginine and NOS activity (25). Our finding that the protective effect of WY was blocked by a NOS inhibitor gives support to a functional involvement of NO production by NOS.
In conclusion, pretreatment with PPAR-α agonist WY limited ischemia-reperfusion-induced myocardial infarct size in type 2 diabetic GK rats. The effect was abolished by the NOS inhibitor l-NNA and the PI3K inhibitor wortmannin. WY also enhanced the phosphorylation and expression of eNOS and Akt in the ischemic myocardium. The results suggest that cardioprotection in type 2 diabetic GK rats induced by the activation of PPAR-α is related to the maintained bioactivity of NO and mediated via the PI3K/Akt signaling pathway.
The study was supported by Swedish Research Council Grants 10857, 20653, and 3541 and the Swedish Heart-Lung Foundation, Swedish Diabetic Association, Novo Nordisk Foundation, and Vinnova (Chronic Inflammation-Diagnostics and Therapy).
We thank Elisabeth Norén-Krog, Marita Wallin, and Carina Nihlen for technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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