Exercise training enhanced myocardial endothelial nitric oxide synthase (eNOS) function in diabetic Goto-Kakizaki (GK) rats

Cardiovascular Diabetology Original investigation Exercise training enhanced myocardial endothelial nitric oxide synthase (eNOS) function in diabetic Goto-Kakizaki (GK) rats James Grijalva 0 Steven Hicks 0 Xiangmin Zhao 0 Sushma Medikayala 0 Pawel M Kaminski 0 Michael S Wolin 0 John G Edwards 0 0 Address: Department of Physiology, New York Medical College , Valhalla NY , USA Background: Different mechanisms of diabetic-induced NO dysfunction have been proposed and central to most of them are significant changes in eNOS function as the rate-limiting step in NO bioavailability. eNOS exists in both monomeric and dimeric conformations, with the dimeric form catalyzing the synthesis of nitric oxide, while the monomeric form catalyzes the synthesis of superoxide (O2-). Diabetic-induced shifts to decrease the dimer:monomer ratio is thought to contribute to the degradation of nitric oxide (NO) bioavailability. Exercise has long been useful in the management of diabetes. Although exercise-induced increases expression of eNOS has been reported, it is unclear if exercise may alter the functional coupling of eNOS. Methods: To investigate this question, Goto-Kakizaki rats (a model of type II diabetes) were randomly assigned to a 9-week running program (train) or sedentary (sed) groups. Results: Exercise training significantly (p < .05) increased plantaris muscle cytochrome oxidase, significantly improved glycosylated hemoglobin (sed: 7.33 0.56%; train: 6.1 0.18%), ad improved insulin sensitivity. Exercise increased both total eNOS expression and the dimer:monomer ratio in the left ventricle LV (sed: 11.7 3.2%; train: 41.4 4.7%). Functional analysis of eNOS indicated that exercise induced significant increases in nitric oxide (+28%) production and concomitant decreases in eNOS-dependent superoxide (-12%) production. This effect was observed in the absence of tetrahydrobiopterin (BH4), but not in the presence of exogenous BH4. Exercise training also significantly decreased NADPH-dependent O2- activity. Conclusion: Exercise-induced increased eNOS dimerization resulted in an increased coupling of the enzyme to facilitate production of NO at the expense of ROS generation. This shift that could serve to decrease diabetic-related oxidative stress, which should serve to lessen diabetic-related complications. - Background In the management of diabetes there is considerable evidence to demonstrate the benefits of exercise including improved glycemic control, an increased quality of life, and a reduction of cardiovascular risk factors. Exercise with and without dietary changes resulted in a significant reduction in glycosylated hemoglobin (HbA1c), increased insulin sensitivity, improved blood lipid levels, and lowered blood pressure [1,2]. Even low intensity forms of exercise such as walking will benefit NIDDM patients [1]. Exercise induces angiogenesis and altered vasculature reactivity in different vascular beds [3,4]. Exercise increases the sensitivity to endothelium-dependent relaxation by acetylcholine, but not the endothelium-independent response to sodium nitroprusside [3]. Chronic exercise increases NO production as early as one week after the start of training [4]. These changes are thought to be the result of increased eNOS protein [5,6]. Training effects may be limited to the vasculature of the working muscles; no effect was observed in mesenteric arterioles, suggesting that exercise-induced increases in stress may have be the responsible mechanism [7]. Several groups have reported that shear stress induces increases in eNOS expression [8,9]. However, studies in both diabetic patients and in diabetic animals have yielded different results; that vascular beds not participating in the response to exercise demonstrate significant improvements, suggesting that mechanisms other than localized stimuli are important [10,11]. Nitric oxide (NO) signaling regulates vascular tone, inhibits components of the atherogenic process, and influences myocardial energy consumption [12,13]. NO synthesis is governed by nitric oxide synthase (NOS). Three isoforms of NOS have been identified which are the products of three separate genes; endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS). These isoforms share about 5060% sequence identity and all use Larginine, O2, and NADPH to catalyze the synthesis of NADP, citrulline, and NO as well as superoxide. Structural domain studies of the NOS molecule have identified separate oxygenase and reductase domains [14]. Dimerization is a requirement for catalytic activity of eNOS, although the truly active form is a complex that includes calmodulin, FAD, tetrahydrobiopterin (BH4), and iron protoporphyrin IX (haem) [14]. The dimeric form catalyzes the rate-limiting step in the synthesis of nitric oxide, while the monomeric form catalyzes the synthesis of O2-, a highly reactive oxidant species (ROS) [15]. The products catalyzed by eNOS are subject to complex regulation that we are just now beginning to understand. NO is an autocrine factor that regulates myocardial functioning via multiple mechanisms [16]. More recently Zhang et.al demonstrated that exercise training was associated with increased myocardial eNOS levels and enhanced myocardial contractility [17]. Different mechanisms of diabetic-induced NO dysfunction have been proposed and central to most of them are significant changes in eNOS function as the rate-limiting step in NO bioavailability. Several studies have reported decreased eNOS activity/protein levels in diabetic patients or animal models of diabetes [18-20]. The composition of the eNOS complex is critical for the relative formation of NO or superoxide formation. The mechanisms responsible for eNOS dysfunction remain unclear, however, a decrease in the dimer to monomer eNOS ratio within the myocardium of diabetic animals has been reported [15]. Although exercise-induced increases in eNOS expression have been documented, it is unclear if exercise may also alter the functional coupling of eNOS. To investigate this question, Goto-Kakizaki rats, a model of NIDDM, were exercise trained, to test if chronic exercise could improve eNOS function and enhance NO bioavailability. Methods Training Protocol Twenty male GK rats were randomly assigned to exercise training (train) or sedentary (sed) groups. Rats were run on a motor driven treadmill set at a ten-degree incline. Animals were initially run at approximately 50% VO2max and the animals were run for up to 60 minutes and 5 days/ week for 9 weeks. While training, animals were closely monitored to ensure animal safety and training compliance. Experimental protocols had institutional approval and animals were maintained in accordance with APS's Guiding Principles in the Care and Use of Animals and the Guide for the Care and Use of Laboratory Animals. Glucose Tolerance Test Following an overnight fast, animals were injected with Nembutal (40 mg/kg i.p.). To perform the gl (...truncated)