Article

---

Introduction
Cardiovascular disease is the leading cause of mortality in noninsulin dependent diabetes mellitus patients (NIDDM). Although originally thought to be a metabolic problem, widespread systemic complications are now recognized. The cardiovascular complications include intermittent claudication, atherosclerosis, hypertension, retinopathy, nephropathy, and congestive heart failure. The vascular problems of NIDDM individuals are largely traceable to alterations in endothelial function. Diabetes results in significant impairment of endothelium-dependent vasodilation in response to acetylcholine or increases in flow. This impairment is likely the result of decreased effectiveness of nitric oxide (NO) mediated functions within the vasculature. There appears to be an interrelationship between NO metabolism and diabetes. Transgenic mice in which the eNOS enzyme has been knocked out exhibit insulin resistance and metabolic syndrome [1].
Exercise has been very useful in the management of diabetes. Although most human studies have focused more on skeletal muscle metabolism, exercise has also been shown to improve vascular as well as cardiac function in NIDDM patients. Whereas some of the physiological consequences of the diabetes on the vasculature are understood, less is known about the molecular mechanisms responsible. And even less is understood about the effects of exercise on the diabetic microvasculature. A clearer understanding of the molecular mechanisms that are influenced by diabetes and exercise will ultimately serve to improve health care management of diabetic individuals.
NO and Endothelial Function
In addition to regulating vascular tone, NO inhibits components of the atherogenic process including platelet aggregation, monocyte adhesion, and vascular smooth muscle migration [2-4]. Additionally, NO influences myocardial energy consumption [5-7]. Different mechanisms of endothelial dysfunction related to altered NO release have been proposed and include changes in the rate of NO breakdown, decreased synthesis of nitric oxide synthase (NOS), altered NOS sensitivity, and down regulation of the signal transduction pathways that either activate NOS or maintain its synthesis [8-11]. All of these proposals involve significant changes in the endothelial phenotype and central to most of them are changes in NOS function as the rate-limiting step in NO production [12-14].
NO synthesis is synthesized by the nitric oxide synthase enzyme. Three isoforms of the NOS enzyme are known; that are the products of three separate genes and include endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS). These isoforms are similar and have a sequence identity of about 50-60% at the protein level. All use L-arginine, O2, and NADPH to catalyze the synthesis of NADP, citrulline, and NO as well as superoxide. Biochemical structural studies have identified different regions that separate the oxygenase and reductase domains within the NOS molecule [15]. eNOS function is subject to several layers of regulation. The eNOS enzyme can function either as a dimer or as a monomer in the membrane. Dimerization is a requirement for NO synthesis but full catalytic activity requires the formation of a complex that incorporates calmodulin, FAD, tetrahydrobiopterin (BH4), and iron protoporphyrin IX (haem) [15]. Only eNOS undergoes palmitoylation and myristoylation and it is this latter modification which is required for insertion into the caveolae of endothelial cells [16, 17]. Localization of eNOS to the caveolae is important for rapid modulation of eNOS activity by different stimuli including shear stress. Regulation by phosphorylation is also significant in that eNOS has multiple phosphorylation sites. Phosphorylation of the serines 617, 635 and 1179 will activate eNOS, while phosphorylation of the Thr497 site appears to serve as an intrinsic switch to enhance coupling of eNOS in favor of NO production at the expense of superoxide [18]. All of these factors point towards a complex regulation of eNOS allowing it to respond to varying environments.
The composition of the eNOS complex is critical for the formation of NO as opposed to superoxide formation. Whereas the dimer complex produces NO, the monomer preferentially synthesizes superoxide [19]. BH4 preserves the eNOS dimer conformation and supports eNOS activity [20]. The mechanisms responsible for uncoupling remain unclear, however, increases in glucose or peroxynitrite have been shown to reduce the dimer:monomer ratio [19]. More recently, Chen reported that peroxynitrite promotes the oxidation of tetrahydrobiopterin leading to the destabilization of the eNOS dimer [21]. Increased oxidative stress is evident within the diabetic vasculature. Diabetes is thought to have a significant impact on dimerization and a decrease in the dimer:monomer eNOS ratio have been reported in tissues from diabetic animals [19]. Supplementation of tetrahydrobiopterin (BH4) using sepiapterin can restore NO mediated flow-dependent dilation to type I diabetic rat arterioles [22]. Acute infusion of BH4 restored acetylcholine induced vasodilation in type II diabetic subjects [23]. These findings are important because the shift could contribute to increased cellular oxidative stress, which could exacerbate the decline in NO bioavailability.
eNOS protein levels are altered in response to different stimuli. Decreases were found in different forms of pathological overload including hypertension or heart failure, whereas chronic exercise increased vasculature eNOS protein levels [24, 25]. Aging is associated with decreased eNOS protein in the endothelium of coronary microvessels [26]. Control of eNOS expression lies at the transcriptional level. Both shear stress and VEGF will induce eNOS expression [27-34]. Gender differences in arteriole function have been linked to estrogen, and are also thought to act at the level of transcription [35-37]. The 5’ flanking region of the eNOS gene contains several transcription factor binding sites, including SP1, AP1, and cAMP response elements [28, 38]. Of these a VEGF response element that is highly similar to a consensus AP1 binding site appears responsible for the initial increases in eNOS expression [39]. This element is separate and distinct from a shear stress response element within the eNOS gene. Shear stress has also been reported to increase eNOS-mRNA stability a separate mechanism for the up regulation of eNOS expression [40, 41]. The impact of diabetes on eNOS expression is unclear, but several reports indicate that elevated glucose will increase eNOS-mRNA [42-44]. Insulin has also been shown to increase eNOS expression in the vascular stroma of lean Zucker rats but not insulin-resistant Zucker fatty rats [45]. This suggests that insulin resistance negatively impacts on the vasculature. Because NO inhibits smooth muscle growth, it is also possible that the loss of insulin sensitivity may result in a proatherosclerotic state. Both increases and decreases in eNOS expression have been reported from different models of diabetes including streptozotocin-diabetes [46, 47], alloxan-diabetes [42], in fatty Zucker rats [45] and in GK rats [48]. However, these findings were from analysis of tissues and the larger conduit arteries, and less is known about the microvasculature, which is responsible for regulation of blood pressure and the distribution of blood flow. A decrease in eNOS would link lower NO bioavailablity to gene expression levels. An increase or no changes in eNOS would support the idea that diabetes may result in an inactivation of eNOS. Clarification of these issues will be important for understanding the molecular mechanisms responsible for vasculature dysfunction.
Hyperglycemia has been shown to act as a NO scavenger [49]. Although the mechanism is not entirely clear, an acute change in eNOS conformation may be involved. In normal individuals, a single oral glucose challenge can reduce NO-specific vasodilatation, an action that was blocked by supplementation with tetrahydrobiopterin (BH4) [50]. It known that shifts in BH4 concentration will directly influence the rate of NO or superoxide anion O2¾ production [51-55]. Using an inhibitor of tetrahydrobiopterin (2,4-diamino-6-hydroxypyrimidine), Yamashio demonstrated a reduction in endothelium-dependent vasodilatation [56]. Meninnger et.al. isolated endothelial cells from diabetes-prone and non-diabetes-prone rats, a model of IDDM [57]. They determined that tetrahydrobiopterin concentration, GTP-cyclohydrolase-1 enzyme activity, and GTP-cyclohydrolase-1 protein levels were all decreased in the endothelial cells of diabetic rats compared to age matched control rats. Similarly in the Goto-Kakizaki rat (a nonobese model of NIDDM), Biter etal demonstrated that tetrahydrobiopterin was significantly decreased while oxidized biopterin was increased [12]. In conjunction with this oxidant stress in the form of superoxide was significantly increased in the Goto-Kakizaki vasculature [12, 58]. BH4 preserves the eNOS dimer conformation which supports NO-dependent eNOS activity, and a decline in BH4 results in an increase in superoxide formation [20]. Supplementation with sepiapterin, a precursor of tetrahydrobiopterin, restored NO-dependent endothelial function to skeletal muscle microvessels from type 1 diabetic rats [22]. These findings suggest that diabetic-induced changes in eNOS function rather than a direct change in eNOS protein content are more important in the decline in NO bioavailability in the diabetic vasculature.
Exercise and NIDDM
There is considerable evidence to demonstrate the benefits of exercise in the management od diabetes including improved glycemic control, an increase in quality of life and a reduction of cardiovascular risk factors. Exercise with and without dietarychanges resulted in a significant reduction in glycosylatedhemoglobin (HbA1c), increased insulin sensitivity, improved blood lipidlevels, and lowered blood pressure [59-63]. Even low intensity forms of exercise such as walking will benefit NIDDM patients [59]. This is good news since diabetic-related complications such as obesity, peripheral neuropathies, and ischemic heart disease represent real limitations on the exercise capacity of many NIDDM patients.
Vascular responsiveness in diabetics is compromised at the onset of exercise and during submaximal exercise compared to healthy individuals suggestive of microvascular pathology [64-67]. Not all exercise programs are equally efficacious in that adaptations have been observed in response to endurance training protocols but not after strength or speed training programs [68, 69]. The influence of chronic exercise extends across several levels including induction of angiogenesis and shifts in vasculature sensitivity [70-73]. The endothelial cells are an important target for these adaptations. Increased NO production is an early adaptation and has been observed as soon as one week after the start of the training program [71, 74]. There are several potential mechanisms that may be responsible for this increase in NO release. Exercise increases the sensitivity to endothelium-dependent relaxation by acetylcholine, but not the endothelium-independent response to sodium nitroprusside indicating that the impact lies more at the endothelium rather than smooth muscle [13, 75]. At the onset of exercise, blood flow to the working muscles increases dramatically and in proportion to the intensity of exercise. Blood flow to other organ systems such as the kidney, G-I tract, and liver is compromised by exercise and all decrease as exercise intensity and sympathetic outflow increases. Earlier studies observed that training increased eNOS protein [24, 25, 76]. Training effects appear limited to the vasculature of the working muscles since no effect was observed in the mesenteric arterioles (a nonworking vascular bed) [13, 77-79]. From this it has been suggested that flow-induced increases shear stress may be responsible for inducing increases in eNOS expression [27-30]. There is ample evidence from both in vivo and in vitro studies to support this concept [13, 77, 79, 80]. However, studies of diabetic animals have yielded somewhat different results. In OLETF rats (an obese model of NIDDM) exercise improved endothelium dependent vasodilatation was observed in the mesenteric arteries [81]. Similarly, in a human study, exercise and diet modification improved flow-mediated dilation (FMD) of the brachial artery during an exercise protocol recruiting the lower limbs [82]. These findings are very important as they suggest that mechanisms other than localized stimuli (such as increased shear stress or localized VEGF release) are important for diabetic individuals.
eNOS function is subject to complex regulation. Exercise-induced increases in vascular eNOS protein has been shown by several investigations [24, 25, 76, 83]. Chronic exercise increases the sensitivity to insulin-stimulated phosphorylation of eNOS. [84]. Zhang et al. [84] reported that the shift in eNOS phosphorylation (ser1179) status appears mediated through the Akt signaling pathway and significantly enhances myocardial contractility. However, shifts in phosphorylation status do not fully explain the findings improved myocardial contractility. To date no one has reported exercise-induced alterations in eNOS-Thr497 phosphorylation status. Further, Fulton et al demonstrated that phosphorylation of ser1179 was not required for correct intracellular localization [85]. Myristoylation is required for initial targeting of eNOS to the plasma membrane, while palmitoylation is thought to serve to stabilize eNOS in the membrane [86-88]. To date no one has examined the issue of exercise-induced changes in myristoylation or palmitoylation of eNOS.
Chronic exercise improves eNOS dimerization in the diabetic heart [83]. Increased dimerization of the eNOS protein increases coupling of the enzyme and shifts its enzymatic activity towards improved NO generation, at the expense of superoxide production. Any reduction in oxidant stress should serve to promote eNOS dimerization. Plasma levels of angiotensin II (Ang II) are elevated in diabetics with poor glucose control and in untreated diabetic animal models [89-93]. Chronically elevated Ang II levels are associated with increased expression of NADPH oxidase components and oxidative stress [94]. Conversely, normalization of Ang II levels can be achieved with improved glucose handling, ACE inhibition, as well as by exercise [89, 90, 94, 95]. Exercise training has long been known to improve glycemic control in diabetics and it may serve a multi-faceted role to lower angiotensin II impact and more locally to decrease hyperglycemic scavenging of NO [96, 97]. Exercise has also been shown to decrease NADPH oxidase activity [83]. Evidence that oxidant stress reduces tetrahydrobiopterin is significant in that preservation to tetrahydrobiopterin may restore its function. In aged skeletal muscle but not young muscle, chronic exercise elevates tetrahydrobiopterin levels [98]. In conjunction with this supplementation improved vasodilatation in aged sedentary individuals but not those that were exercise trained suggesting that training restores tetrahydrobiopterin levels [99]. These several factors; improved glycemic control, reduction of angiotenisin II activity, reduction of oxidant stress will all promote increased eNOS dimerization leading to better coupling of the eNOS enzyme [83].
Although increases in eNOS protein have been reported by several investigations, our own results observed a paradox in that exercise training of diabetic animals lead to an increase in eNOS protein, but a decrease in eNOS-mRNA. Others have reported that in comparison to controls within the diabetic heart eNOS protein was decreased while eNOS-mRNA was elevated in the vasculature or the heart[12, 42, 83]. And similar results have also been observed in cultured endothelial cells chronically exposed to elevated media glucose [44]. In cultured endothelial cells, exogenous H2O2 increased eNOS transcription and eNOS-mRNA stability [100]. While diabetic-induced oxidative stress depresses eNOS function it also serves to activate compensatory mechanisms. Exercise training reverses this. Both eNOS-driven and NADPH oxidase derived ROS are lowered by exercise training and this should serve to diminish eNOS transcription [83]. Exercise-induced improvements in glycemic control are well known and should have the effect to improve NO bioavailability [96, 97]. In conjunction with this, increased NO bioavailability is also a negative feedback mechanism to decrease eNOS transcription [101, 102]. As a result, the decreases in eNOS-mRNA following exercise training were possibly the result of the reduced drive for eNOS transcription.
Summary
Although diabetes was originally thought to be a metabolic problem, widespread systemic complications related to vascular dysfunction are now recognized. Exercise has been highly useful in the management of diabetes. Both human and animal studies have found significant evidence to indicate that exercise improves cardiovascular function in the diabetic. Central to these improvements is increased NO bioavailability. Nitric oxide synthase is the rate limiting step in NO bioavailability, and it is eNOS function that has the largest impact on cardiovascular function Diabetes disrupts the complex regulation of eNOS function on several levels. Although it remains unclear if enzyme localization is influenced by chronic exercise; changes in protein content, phosphorylation status, and enzme conformation are sensitive to exercise training. Several mechanisms are likely to contribute to exercise-induced improvements in eNOS function in diabetics and many orginate from exercise-induced improvements in glycemic control. This one change brings about a host of intracellular changes centered on decreasing oxidant stress that leads to improved eNOS function.

References

---

1.Shankar RR, Wu Y, Shen HQ, Zhu JS, Baron AD: Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance. Diabetes 2000, 49(5):684-687.
2.Bath PM, Hassall DG, Gladwin AM, Palmer RM, Martin JF: Nitric oxide and prostacyclin. Divergence of inhibitory effects on monocyte chemotaxis and adhesion to endothelium in vitro. Arterioscler Thromb 1991, 11(2):254-260.
3.Garg UC, Hassid A: Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 1989, 83(5):1774-1777.
4.Radomski MW, Palmer RM, Moncada S: The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmacol 1987, 92(3):639-646.
5.Recchia FA, McConnell PI, Loke KE, Xu X, Ochoa M, Hintze TH: Nitric oxide controls cardiac substrate utilization in the conscious dog. Cardiovasc Res 1999, 44(2):325-332.
6.Recchia FA, Osorio JC, Chandler MP, Xu X, Panchal AR, Lopaschuk GD, Hintze TH, Stanley WC: Reduced synthesis of NO causes marked alterations in myocardial substrate metabolism in conscious dogs. Am J Physiol Endocrinol Metab 2002, 282(1):E197-206.
7.Loke KE, McConnell PI, Tuzman JM, Shesely EG, Smith CJ, Stackpole CJ, Thompson CI, Kaley G, Wolin MS, Hintze TH: Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption. Circ Res 1999, 84(7):840-845.
8.Lang MG, Noll G, Luscher TF: Effect of aging and hypertension on contractility of resistance arteries:  modulation by endothelial factors. Amer J Physiol 1995, 269:H837-H844.
9.Kung CF, Luscher TF: Different Mechanisms of Endothelial dysfunction with Aging and Hypertension in Rat Aorta. Hyper 1995, 25:194-200.
10.Tschundi MR, Barton M, Bersinger NA, Moreau P, Cosentino F, Noll G, Malinski T, Luscher TF: Effect of Age on Kinetics of Nitric Oxide Release in Rat Aorta and Pulmonary Artery. J Clin Invest 1996, 98(4):899-905.
11.Flavahan NA, Shimokawa H, Vanhoutte PM: Pertussis toxin inhibits endothelium-dependent relaxations to certain agonists in porcine coronary arteries. J Physiol (Lond) 1989, 408:549-560.
12.Bitar MS, Wahid S, Mustafa S, Al-Saleh E, Dhaunsi GS, Al-Mulla F: Nitric oxide dynamics and endothelial dysfunction in type II model of genetic diabetes. Eur J Pharmacol 2005, 511(1):53-64.
13.Sun D, Huang A, Koller A, Kaley G: Short-term daily exercise activity enhances endothelial NO synthesis in skeletal muscle arterioles of rats. J Appl Physiol 1994, 76(5):2241-2247.
14.Sun D, Huang A, Koller A, Kaley G: Enhanced NO-mediated dilations in skeletal muscle arterioles of chronically exercised rats. Microvasc Res 2002, 64(3):491-496.
15.Alderton WK, Cooper CE, Knowles RG: Nitric oxide synthases: structure, function and inhibition. Biochem J 2001, 357(Pt 3):593-615.
16.Prabhakar P, Thatte HS, Goetz RM, Cho MR, Golan DE, Michel T: Receptor-regulated translocation of endothelial nitric-oxide synthase. J Biol Chem 1998, 273(42):27383-27388.
17.Michel T: Targeting and translocation of endothelial nitric oxide synthase. Braz J Med Biol Res 1999, 32(11):1361-1366.
18.Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA, Jr., Sessa WC: Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem 2003, 278(45):44719-44726.
19.Zou MH, Shi C, Cohen RA: Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest 2002, 109(6):817-826.
20.Cai S, Alp NJ, McDonald D, Smith I, Kay J, Canevari L, Heales S, Channon KM: GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation. Cardiovasc Res 2002, 55(4):838-849.
21.Chen W, Druhan LJ, Chen CA, Hemann C, Chen YR, Berka V, Tsai AL, Zweier JL: Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase: transition from reversible to irreversible enzyme inhibition. Biochemistry 2010, 49(14):3129-3137.
22.Bagi Z, Koller A: Lack of nitric oxide mediation of flow-dependent arteriolar dilation in type I diabetes is restored by sepiapterin. J Vasc Res 2003, 40(1):47-57.
23.Heitzer T, Krohn K, Albers S, Meinertz T: Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with Type II diabetes mellitus. Diabetologia 2000, 43(11):1435-1438.
24.Woodman CR, Muller JM, Laughlin MH, Price EM: Induction of nitric oxide synthase mRNA in coronary resistance arteries isolated from exercise-trained pigs. Am J Physiol 1997, 273(6 Pt 2):H2575-2579.
25.Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH: Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res 1994, 74(2):349-353.
26.Csiszar A, Ungvari Z, Edwards JG, Kaminski P, Wolin MS, Koller A, Kaley G: Aging-induced phenotypic changes and oxidative stress impair coronary arteriolar function. Circ Res 2002, 90(11):1159-1166.
27.Nishida K, Harrison DG, Navas JP, Fisher AA, Dockery SP, Uematsu M, Nerem RM, Alexander RW, Murphy TJ: Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest 1992, 90(5):2092-2096.
28.Malek AM, Jiang L, Lee I, Sessa WC, Izumo S, Alper SL: Induction of nitric oxide synthase mRNA by shear stress requires intracellular calcium and G-protein signals and is modulated by PI 3 kinase [published erratum appears in Biochem Biophys Res Commun 1999 Mar 5;256(1):255]. Biochem Biophys Res Commun 1999, 254(1):231-242.
29.Ziegler T, Silacci P, Harrison V, Hayoz D: Nitric oxide synthase expression in endothelial cells exposed to mechanical forces. Hyper 1998, 32:351-355.
30.Xiao Z, Xhang Z, S.L. D: Shear Stress Induction of the Endothelial Nitric Oxide Synthase Gene is Calcium-Dependent But Not Calcium-Activated. Jour of Cell Phys 1997, 171:205-211.
31.Lin C, Ho H, Chen K, Lin G, Nunes L, Lue T: Intracavernosal injection of vascular endothelial growth factor induces nitric oxide synthase isoforms. BJU Int 2002, 89:955-960.
32.Bouloumie A, Schini-Kerth VB, Busse R: Vascular endothelial growth factor up-regulates nitric oxide synthase expression in endothelial cells. Cardiovasc Res 1999, 41(3):773-780.
33.Kroll J, Waltenberger J: VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2 (KDR). Biochem Biophys Res Commun 1998, 252(3):743-746.
34.Shen B-Q, Lee DY, T.F. Z: Vascular Endothelial Growth Factor Governs Endothelial Nitric-oxide Synthase Expression via a KDR/Flk-1 Receptor and a Protein Kinase C Signaling Pathway. Jour of Bio Chem 1999, 274(46):33057-33063.
35.Huang A, Sun D, Koller A, Kaley G: 17beta-estradiol restores endothelial nitric oxide release to shear stress in arterioles of male hypertensive rats. Circulation 2000, 101(1):94-100.
36.Koller A, Huang A, Sun D, Kaley G: Differences in function of arterioles of female and male hyptertensive rats:  role of nitric oxide and estrogen. 20th European Conference on Microcirculation 1998:251-256.
37.Virdis A, Ghiadoni L, Pinto S, Lombardo M, Petraglia F, Gennazzani A, Buralli S, Taddei S, Salvetti A: Mechanisms Responsible for Endothelial Dysfunction Associated With Acute Estrogen Deprivation in Normotensive Women. Circulation 2000, 101(19):2258-2263.
38.Zhang R, Min W, Sessa WC: Functional analysis of the human endothelial nitric oxide synthase promoter. Sp1 and GATA factors are necessary for basal transcription in endothelial cells. J Biol Chem 1995, 270(25):15320-15326.
39.Koai E, Rincon Rios T, Edwards JG: Vascular Endothelial Growth Factor Increases Endothelial Nitric Oxide Synthase Transcription In HUVEC Cells. WebmedCentral Physiology 2010, 1(11):WMC001111.
40.Weber M, Hagedorn CH, Harrison DG, Searles CD: Laminar shear stress and 3' polyadenylation of eNOS mRNA. Circ Res 2005, 96(11):1161-1168.
41.Kosmidou I, Moore JP, Weber M, Searles CD: Statin treatment and 3' polyadenylation of eNOS mRNA. Arterioscler Thromb Vasc Biol 2007, 27(12):2642-2649.
42.Zhao G, Zhang X, Smith CJ, Xu X, Ochoa M, Greenhouse D, Vogel T, Curran C, Hintze TH: Reduced coronary NO production in conscious dogs after the development of alloxan-induced diabetes. Am J Physiol 1999, 277(1 Pt 2):H268-278.
43.Cai S, Khoo J, Mussa S, Alp NJ, Channon KM: Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerization. Diabetologia 2005, 48(9):1933-1940.
44.Ding QF, Hayashi T, Packiasamy AR, Miyazaki A, Fukatsu A, Shiraishi H, Nomura T, Iguchi A: The effect of high glucose on NO and O2- through endothelial GTPCH1 and NADPH oxidase. Life Sci 2004, 75(26):3185-3194.
45.Kuboki K, MD, Jiang ZY, MD, PhD, Takahara N, MD, PhD, Ha SWM, PhD, Igarashi M, MD, Ph.D., Yamauchi T, MD,PhD, Feener EP, PhD, Herbert TP, MD, PhD, Rhodes CJ, PhD, King GL, MD: Regulation of Endothelial Constitutive Nitric Oxide Synthase Gene Expression in Endothelial Cells and In Vivo;  A Specific Vascular Action of Insulin. Circ 1999, 101:676-681.
46.Bojunga J, Dresar-Mayert B, Usadel KH, Kusterer K, Zeuzem S: Antioxidative treatment reverses imbalances of nitric oxide synthase isoform expression and attenuates tissue-cGMP activation in diabetic rats. Biochem Biophys Res Commun 2004, 316(3):771-780.
47.De Vriese AS, Stoenoiu MS, Elger M, Devuyst O, Vanholder R, Kriz W, Lameire NH: Diabetes-induced microvascular dysfunction in the hydronephrotic kidney: role of nitric oxide. Kidney Int 2001, 60(1):202-210.
48.Kobayashi T, Matsumoto T, Ooishi K, Kamata K: Differential expression of alpha2D-adrenoceptor and eNOS in aortas from early and later stages of diabetes in Goto-Kakizaki rats. Am J Physiol Heart Circ Physiol 2004, 287(1):H135-148.
49.Brodsky SV, Yamamoto T, Tada T, Kim B, Chen J, Kajiya F, Goligorsky MS: Endothelial dysfunction in ischemic acute renal failure: rescue by transplanted endothelial cells. Am J Physiol Renal Physiol 2002, 282(6):F1140-1149.
50.Ihlemann N, Rask-Madsen C, Perner A, Dominguez H, Hermann T, Kober L, Torp-Pedersen C: Tetrahydrobiopterin restores endothelial dysfunction induced by an oral glucose challenge in healthy subjects. Am J Physiol Heart Circ Physiol 2003, 285(2):H875-882.
51.Mayer B, Werner ER: In search of a function for tetrahydrobiopterin in the biosynthesis of nitric oxide. Naunyn Schmiedebergs Arch Pharmacol 1995, 351(5):453-463.
52.Mayer B, Klatt P, Werner ER, Schmidt K: Kinetics and mechanism of tetrahydrobiopterin-induced oxidation of nitric oxide. J Biol Chem 1995, 270(2):655-659.
53.Scott-Burden T: Regulation of nitric oxide production by tetrahydrobiopterin. Circulation 1995, 91(1):248-250.
54.Tiefenbacher CP: Tetrahydrobiopterin: a critical cofactor for eNOS and a strategy in the treatment of endothelial dysfunction? Am J Physiol Heart Circ Physiol 2001, 280(6):H2484-2488.
55.Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS, Karoui H, Tordo P, Pritchard KA, Jr.: Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A 1998, 95(16):9220-9225.
56.Yamashiro S, Kuniyoshi Y, Arakaki K, Miyagi K, Koja K: The effect of insufficiency of tetrahydrobiopterin on endothelial function and vasoactivity. Jpn J Thorac Cardiovasc Surg 2002, 50(11):472-477.
57.Meininger CJ, Marinos RS, Hatakeyama K, Martinez-Zaguilan R, Rojas JD, Kelly KA, Wu G: Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency. Biochem J 2000, 349(Pt 1):353-356.
58.Gupte R, Labinskyy N, Csiszar A, Ungvari Z, Edwards JG: Role of NAD(P)H oxidase in superoxide generation and endothelial dysfunction in diabetic Goto-Kakizaki (GK) rats. PLOS One 2010, 5(7):311800.
59.Walker K, Jones J, Piers L, O'Dea K, Putt R: Effects of regular walking on cardiovascular risk factors and body composition in normoglycemic women and women with type 2 diabetes. Diabetes Care 1999, 22:555-561.
60.Yamanouchi K, Shinozaki T, Chikada K: Daily walking combined with diet therapy is a useful means for obese NIDDM patients not only to reduce body weight but also to improve insulin sensitivity. Diabetes Care 1995, 18:775-778.
61.Lehmann R, Vokac A, Niedermann K, Agosti K, Spinas G: Loss of abdominal fat and improvement of the cardiovascular risk profile by regular moderate exercise training in patients with NIDDM. Diabetologia 1995, 38:1313-1319.
62.Boule NG, Kenny GP, Haddad E, Wells GA, Sigal RJ: Meta-analysis of the effect of structured exercise training on cardiorespiratory fitness in Type 2 diabetes mellitus. Diabetologia 2003.
63.Boule NG, Haddad E, Kenny GP, Wells GA, Sigal RJ: Effects of exercise on glycemic control and body mass in type 2 diabetes mellitus: a meta-analysis of controlled clinical trials. Jama 2001, 286(10):1218-1227.
64.Bauer TA, Reusch JE, Levi M, Regensteiner JG: Skeletal muscle deoxygenation after the onset of moderate exercise suggests slowed microvascular blood flow kinetics in type 2 diabetes. Diabetes Care 2007, 30(11):2880-2885.
65.Lalande S, Gusso S, Hofman PL, Baldi JC: Reduced leg blood flow during submaximal exercise in type 2 diabetes. Med Sci Sports Exerc 2008, 40(4):612-617.
66.Kingwell BA, Formosa M, Muhlmann M, Bradley SJ, McConell GK: Nitric oxide synthase inhibition reduces glucose uptake during exercise in individuals with type 2 diabetes more than in control subjects. Diabetes 2002, 51(8):2572-2580.
67.Kingwell BA, Formosa M, Muhlmann M, Bradley SJ, McConell GK: Type 2 diabetic individuals have impaired leg blood flow responses to exercise: role of endothelium-dependent vasodilation. Diabetes Care 2003, 26(3):899-904.
68.Tesch PA, Thorsson A, Kaiser P: Muscle capillary supply and fiber type characteristics in weight and power lifters. J Appl Physiol 1984, 56(1):35-38.
69.Daub WD, Green HJ, Houston ME, Thomson JA, Fraser IG, Ranney DA: Cross-adaptive responses to different forms of leg training: skeletal muscle biochemistry and histochemistry. Can J Physiol Pharmacol 1982, 60(5):628-633.
70.Laughlin MH: Endothelium-mediated control of coronary vascular tone after chronic exercise training. Med Sci Sports Exerc 1995, 27(8):1135-1144.
71.Delp MD: Effects of exercise training on endothelium-dependent peripheral vascular responsiveness. Med Sci Sports Exerc 1995, 27(8):1152-1157.
72.Hudlicka O: Is physiological angiogenesis in skeletal muscle regulated by changes in microcirculation? Microcirculation 1998, 5(1):5-23.
73.Tomanek RJ: Exercise-induced coronary angiogenesis: a review. Med Sci Sports Exerc 1994, 26(10):1245-1251.
74.Shen W, Zhang X, Zhao G, Wolin MS, Sessa W, Hintze TH: Nitric oxide production and NO synthase gene expression contribute to vascular regulation during exercise. Med Sci Sports Exerc 1995, 27(8):1125-1134.
75.Delp MD, McAllister RM, Laughlin MH: Exercise training alters endothelium-dependent vasoreactivity of rat abdominal aorta. J Appl Physiol 1993, 75(3):1354-1363.
76.Johnson LR, Rush JW, Turk JR, Price EM, Laughlin MH: Short-term exercise training increases ACh-induced relaxation and eNOS protein in porcine pulmonary arteries. J Appl Physiol 2001, 90(3):1102-1110.
77.Sun D, Huang A, Koller A, Kaley G: Adaptation of flow-induced dilation of arterioles to daily exercise. Microvasc Res 1998, 56(1):54-61.
78.Kaley G, Sun D, Huang A, Koller A: Exercise training: nitric oxide dependent changes in microvascular function. 20th European Conference on Microcirculation 1998:257-262.
79.Koller A, Huang A, Sun D, Kaley G: Exercise training augments flow-dependent dilation in rat skeletal muscle arterioles. Role of endothelial nitric oxide and prostaglandins. Circ Res 1995, 76(4):544-550.
80.Sun D, Hunag A, Koller A, Kaley G: Decreased arteriolar sensitivity to shear stress in adult rats is reversed by chronic exercise activity. Microcirculation 2002, 9:00-00.
81.Minami A, Ishimura N, Harada N, Sakamoto S, Niwa Y, Nakaya Y: Exercise training improves acetylcholine-induced endothelium-dependent hyperpolarization in type 2 diabetic rats, Otsuka Long-Evans Tokushima fatty rats. Atherosclerosis 2002, 162(1):85-92.
82.Hamdy O, Ledbury S, Mullooly C, Jarema C, Porter S, Ovalle K, Moussa A, Caselli A, Caballero AE, Economides PA et al: Lifestyle modification improves endothelial function in obese subjects with the insulin resistance syndrome. Diabetes Care 2003, 26(7):2119-2125.
83.Grijalva J, Hicks S, Zhao X, Medikayala S, Kaminski PM, Wolin MS, Edwards JG: Exercise training enhanced myocardial endothelial nitric oxide synthase (eNOS) function in diabetic Goto-Kakizaki (GK) rats. Cardiovasc Diabetol 2008, 7:34.
84.Zhang QJ, Li QX, Zhang HF, Zhang KR, Guo WY, Wang HC, Zhou Z, Cheng HP, Ren J, Gao F: Swim training sensitizes myocardial response to insulin: role of Akt-dependent eNOS activation. Cardiovasc Res 2007, 75(2):369-380.
85.Fulton D, Fontana J, Sowa G, Gratton JP, Lin M, Li KX, Michell B, Kemp BE, Rodman D, Sessa WC: Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme. J Biol Chem 2002, 277(6):4277-4284.
86.Robinson LJ, Michel T: Mutagenesis of palmitoylation sites in endothelial nitric oxide synthase identifies a novel motif for dual acylation and subcellular targeting. Proc Natl Acad Sci U S A 1995, 92(25):11776-11780.
87.Robinson LJ, Busconi L, Michel T: Agonist-modulated palmitoylation of endothelial nitric oxide synthase. J Biol Chem 1995, 270(3):995-998.
88.Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Ying Y, Anderson RG, Michel T: Acylation targets emdothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem 1996, 271(11):6518-6522.
89.Bluher M, Kratzsch J, Paschke R: Plasma levels of tumor necrosis factor-alpha, angiotensin II, growth hormone, and IGF-I are not elevated in insulin-resistant obese individuals with impaired glucose tolerance. Diabetes Care 2001, 24(2):328-334.
90.Ferriss JB, O'Hare JA, Kelleher CC, Sullivan PA, Cole MM, Ross HF, O'Sullivan DJ: Diabetic control and the renin-angiotensin system, catecholamines, and blood pressure. Hypertension 1985, 7(6 Pt 2):II58-63.
91.Lip PL, Chatterjee S, Caine GJ, Hope-Ross M, Gibson J, Blann AD, Lip GY: Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy: effects of laser photocoagulation and angiotensin receptor blockade. Br J Ophthalmol 2004, 88(12):1543-1546.
92.Taguchi K, Kobayashi T, Hayashi Y, Matsumoto T, Kamata K: Enalapril improves impairment of SERCA-derived relaxation and enhancement of tyrosine nitration in diabetic rat aorta. Eur J Pharmacol 2007, 556(1-3):121-128.
93.Hollenberg NK, Stevanovic R, Agarwal A, Lansang MC, Price DA, Laffel LM, Williams GH, Fisher ND: Plasma aldosterone concentration in the patient with diabetes mellitus. Kidney Int 2004, 65(4):1435-1439.
94.Hattori Y, Akimoto K, Gross SS, Hattori S, Kasai K: Angiotensin-II-induced oxidative stress elicits hypoadiponectinaemia in rats. Diabetologia 2005, 48(6):1066-1074.
95.Cornelissen VA, Fagard RH: Effects of endurance training on blood pressure, blood pressure-regulating mechanisms, and cardiovascular risk factors. Hypertension 2005, 46(4):667-675.
96.Sato Y, Nagasaki M, Nakai N, Fushimi T: Physical exercise improves glucose metabolism in lifestyle-related diseases. Exp Biol Med (Maywood) 2003, 228(10):1208-1212.
97.Stewart KJ: Exercise training and the cardiovascular consequences of type 2 diabetes and hypertension: plausible mechanisms for improving cardiovascular health. Jama 2002, 288(13):1622-1631.
98.Sindler AL, Delp MD, Reyes R, Wu G, Muller-Delp JM: Effects of ageing and exercise training on eNOS uncoupling in skeletal muscle resistance arterioles. J Physiol 2009, 587(Pt 15):3885-3897.
99.Eskurza I, Myerburgh LA, Kahn ZD, Seals DR: Tetrahydrobiopterin augments endothelium-dependent dilatation in sedentary but not in habitually exercising older adults. J Physiol 2005, 568(Pt 3):1057-1065.
100.Drummond GR, Cai H, Davis ME, Ramasamy S, Harrison DG: Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res 2000, 86(3):347-354.
101.Grumbach IM, Chen W, Mertens SA, Harrison DG: A negative feedback mechanism involving nitric oxide and nuclear factor kappa-B modulates endothelial nitric oxide synthase transcription. J Mol Cell Cardiol 2005, 39(4):595-603.
102.Vaziri ND, Wang XQ: cGMP-mediated negative-feedback regulation of endothelial nitric oxide synthase expression by nitric oxide. Hypertension 1999, 34(6):1237-1241.

Source(s) of Funding

---

Acknowledgements: The authors have no conflicts of interest to report.  Supported in part by NIH HD065551 and HL043023.

Competing Interests

---

The authors have no competing interests.

Disclaimer

---

This article has been downloaded from WebmedCentral. With our unique author driven post publication peer review, contents posted on this web portal do not undergo any prepublication peer or editorial review. It is completely the responsibility of the authors to ensure not only scientific and ethical standards of the manuscript but also its grammatical accuracy. Authors must ensure that they obtain all the necessary permissions before submitting any information that requires obtaining a consent or approval from a third party. Authors should also ensure not to submit any information which they do not have the copyright of or of which they have transferred the copyrights to a third party.
Contents on WebmedCentral are purely for biomedical researchers and scientists. They are not meant to cater to the needs of an individual patient. The web portal or any content(s) therein is neither designed to support, nor replace, the relationship that exists between a patient/site visitor and his/her physician. Your use of the WebmedCentral site and its contents is entirely at your own risk. We do not take any responsibility for any harm that you may suffer or inflict on a third person by following the contents of this website.