Twenty-five years since the discovery of endothelium-derived relaxing ...

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REVIEW / SYNTHÈSE

Twenty-five years since the discovery of endothelium-derived relaxing factor (EDRF): does a dysfunctional endothelium contribute to the development of type 2 diabetes?1 Chris R. Triggle, Andrew Howarth, Zhong Jian Cheng, and Hong Ding

Abstract: Twenty-five years ago, the discovery of endothelium-derived relaxing factor opened a door that revealed a new and exciting role for the endothelium in the regulation of blood flow and led to the discovery that nitric oxide (NO) multi-tasked as a novel cell-signalling molecule. During the next 25 years, our understanding of both the importance of the endothelium as well as NO has greatly expanded. No longer simply a barrier between the blood and vascular smooth muscle, the endothelium is now recognized as a complex tissue with heterogeneous properties. The endothelium is the source of not only NO but also numerous vasoactive molecules and signalling pathways, some of which are still not fully characterized such as the putative endothelium-derived relaxing factor. Dysfunction of the endothelium is a key risk factor for the development of macro- and microvascular disease and, by coincidence, the discovery that NO was generated in the endothelium corresponds approximately in time with the increased incidence of type 2 diabetes. Primarily linked to dietary and lifestyle changes, we are now facing a global pandemic of type 2 diabetes. Characterized by insulin resistance and hyperglycaemia, type 2 diabetes is increasingly being diagnosed in adolescents as well as children. Is there a link between dietary-related hyperglycaemic insults to the endothelium, blood flow changes, and the development of insulin resistance? This review explores the evidence for and against this hypothesis. Key words: diabetes, endothelium, hyperglycaemia, insulin, nitric oxide, oxidative stress. Résumé : Il y a 25 ans, la découverte du facteur de relaxation dérivé de l’endothélium a ouvert une voie qui a révélé un rôle nouveau et excitant pour l’endothélium dans la régulation du débit sanguin et a mené à la découverte du rôle multi-fonctionnel du monoxyde d’azote (NO) comme nouvelle molécule de signalisation cellulaire. Au cours des 25 années suivantes, notre compréhension de l’importance de l’endothélium et du NO s’est considérablement améliorée. Longtemps considéré comme une simple barrière ente le sang et le muscle lisse vasculaire, l’endothélium est maintenant reconnu comme un tissu complexe doté de propriétés hétérogènes. L’endothélium est la source non seulement du NO, mais aussi de nombreuses molécules vasoactives et voies de signalisation dont certaines n’ont pas encore été totalement caractérisées, tel l’hypothétique facteur hyperpolarisant dérivé de l’endothélium. La dysfonction de l’endothélium est un facteur de risque important de maladies macro- et micro-vasculaires, et la découverte de la production du NO dans l’endothélium coïncide avec l’augmentation de l’incidence du diabète de type 2. À l’origine lié à des changements dans les habitudes alimentaires et le style de vie, le diabète de type 2 atteint maintenant des proportions endémiques. Caractérisé par l’insulinorésistance et l’hyperglycémie, il est de plus en plus diagnostiqué chez les adolescents et les enfants. Existe-il un lien entre les crises d’hyperglycémie liées à l’alimentation et l’endothélium, les modifications du débit sanguin et le développement de l’insulinorésistance? Cette synthèse examine les arguments confortant et réfutant cette hypothèse. Mots clés : diabète, endothélium, hyperglycémie, insuline, monoxyde d’azote, stress oxydatif. [Traduit par la Rédaction]

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Received 30 May 2005. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 30 August 2005. C.R. Triggle2 and H. Ding. School of Medical Sciences, Bundoora West Campus, RMIT University, Victoria 3083, Australia. A. Howarth and Z.J. Cheng. Smooth Muscle Research Group, University of Calgary, Calgary, AB, Canada. 1 2

This review is part of the Special Issue entitled A Tribute to Ed Daniel, and has undergone the review process. Corresponding author (e-mail: [email protected]).

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doi: 10.1139/Y05-069

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Introduction Our knowledge of vascular homeostasis was dramatically changed when Furchgott and Zawadski (1980) published their landmark paper that described the acetylcholinemediated release of an endothelium-derived relaxing factor (EDRF) from rabbit aortic rings. EDRF was later identified as nitric oxide (NO) (Palmer et al. 1987). The discovery and later identification of EDRF as the free radical gas NO has significantly enhanced our understanding of cell-to-cell signalling processes in the vasculature. More importantly, we now accept that NO is not just an endothelium-derived paracrine factor but a molecule that enjoys an ubiquitous role as, from an evolutionary perspective, one of the earliest signalling molecule present throughout the vertebrate and invertebrate animal kingdom as well as, it would appear, in the plant kingdom (Torreilles 2001). In 1992, NO was recognised as “Molecule of the Year” by the journal Science (Koshland 1992), and in 1998, the Nobel Prize in Medicine was awarded jointly to Robert Furchgott, Louis Ignarro, and Ferid Murad for their pioneering research on the biological functions of NO (SoRelle 1998). It can be argued that acetylcholine-mediated vasodilatation was well established in the literature long before the publication of the 1980 paper from Furchgott’s laboratory. For example, in the 3rd Edition of The Pharmacological Basis of Therapeutics (Goodman and Gilman 1965) the intravenous effects of acetylcholine are described as: “causes vasodilatation, especially of smaller blood vessels, and a fall in blood pressure”. However, what was missing in that earlier literature was an explanation for the apparent discrepancy between data obtained from in vivo studies, in which acetylcholine action was associated with vasodilatation and a fall in blood pressure, and in vitro studies with isolated blood vessels, in which, for many reports, acetylcholine produced a contractile response. The 1980 publication by Furchgott and Zawadski in explained these discrepant findings and, although not realised at that time, provided the first description of endothelial dysfunction. Specifically, how endothelial cell damage (removal of the endothelium was described in the case of the Furchgott and Zawadski paper) dramatically affects vascular function. Thus, endothelial dysfunction (ED) has come to be defined as a reduced endothelium-dependent vasodilator response to acetylcholine.3 Although the endothelium plays many additional functions on vascular homeostasis beyond the control of vascular tone, this reduced response to acetylcholine serves as an important indicator and assay of vascular dysfunction (Andrews et al. 2005; De Vriese et al. 2000; Pannirselvam et al. 2003; Triggle et al. 2003). In the past 25 years, other significant advancements have been made in the diagnosis and management of vascular diseases but it was the discovery of the importance of endogenously generated NO that tops the list (Olin et al. 2004). The endothelium The endothelium is a single layer of cells that line all blood vessels, weighs about 1 kg in an adult, and comprises 3

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approximately 10 trillion cells (Galley and Webster 2004). Although not truly an organ, the heterogeneous nature of endothelial cells (Andrews et al. 2005) and the endocrine-like function of the endothelium as the source of numerous vasoactive factors involved in the short- and long-term regulation of vascular tone function, regulation of leukocyte adhesion, vascular smooth muscle cell growth, and platelet aggregation, is now well recognised (Galley and Webster 2004). Whereas NO remains a key regulator of many of these processes (Rubio and Morales-Segura 2004), when we think about the regulation of vascular smooth muscle tone we must also consider the contribution of other endothelialderived factors. In addition to NO, prostacyclin (PGI2), its origin first described by the late Sir John Vane (who won the Nobel Prize in 1982 for his work on the mode of action of aspirin), is an important endothelium-derived vasodilator that also has important functions as an inhibitor of platelet aggregation (Moncada et al. 1976). The endothelium also produces contracting factors and, in 1988, the discovery of an extremely potent vasoconstrictor peptide, endothelin, was reported (Yanagisawa et al. 1988). Additional endotheliumderived vasoactive mediators and cellular processes remain undefined and, notably in the resistance vessels, another endothelial-vascular smooth muscle cell communication pathway is mediated by an as yet unidentified mediator and (or) transmission process that has been termed the endotheliumderived hyperpolarizing factor (EDHF) (Vanhoutte 2004). EDHF may be a true chemical mediator, and several candidate molecules have been identified (McGuire et al. 2001; Ahluwalia and Hobbs 2005) or, alternatively, it represents purely electronic conduction involving low-electrical resistance coupling involving myoendothelial gap junctions. One could also explain the EDHF phenomenon with the possibility of chemical mediation via gap junctions (Griffith 2004; McGuire et al. 2001; Sandow 2004). Heterogeneity exists within the vasculature not only in a vessel- and genderdependent fashion, but also in the nature of the mediators that are released by different endothelium-dependent vasodilators in any given blood vessel (McGuire et al. 2001). Such heterogeneity may provide the basis for the physiological fine-tuning of blood flow and also, in pathological states, dysfunction of these pathways may provide a basis for the development of vascular disease (Andrews et al. 2005). However, the role of EDHF in the pathophysiology of vascular disease remains largely unexplored. ED, which is associated with hypercholesterolemia, hypertension, diabetes, and smoking, is an early indicator of the development of cardiovascular disease and it plays an early and prominent role in the process that precedes the appearance of atherosclerotic plaque and the frank symptoms of peripheral vascular disease (Andrews et al. 1987; Cohen et al. 1988; Landmesser et al. 2004; Lüscher and Barton 1997). The appearance of ED is a key indicator of patients with a high risk for cardiovascular disease. We have already defined ED as the reduced endothelium-dependent vasodilator response to acetylcholine, but, in addition to a reduction in the vasodilator response to acetylcholine, ED may also reflect an enhanced contribution of endothelium-derived

Although it might be argued that acetylcholine is not a physiological mediator of endothelium-dependent relaxation (but see Welsh and Segal 1997), acetylcholine and, in some instances in the clinical setting, methacholine are commonly employed as the probe in both clinical and experimental studies for defining endothelial function and dysfunction. © 2005 NRC Canada

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contracting factors such as endothelin-1, thromboxane, reactive oxygen species (ROS), as well as yet to be identified endothelium-derived contracting factors (Pannirselvam et al. 2005; Saifeddine et al. 1998; Vanhoutte et al. 2005). Changes in endothelial cell function are now recognised as key indicators of the health of the vasculature, and ED may reflect a premature aging or senescence of the endothelial cells that is commonly seen in cardiovascular disease. Vascular disease may be linked to accelerated endothelial cell death and reduced regeneration of endothelial cells as many of the changes reported in endothelial cell function in cardiovascular disease are also seen in senescent cells (Ding and Triggle 2005; Minamino et al. 2004). We previously stated, “a man is only as old as his endothelium,” (Ding and Triggle 2005), reflecting the view that a healthy endothelium is essential for the maintenance of the appropriate balance of profibrinolytic and prothrombotic activity (Landmesser et al. 2004). A blunted endothelium-dependent dilation response is now recognized as a key predictive indicator for the future development of atherosclerosis in man (Bisoendial et al. 2002). Endothelial cells do not normally have a high turnover rate, but cardiovascular disease may induce the premature aging and senescence of the endothelium and a reduced capacity for repair of the endothelium by progenitor cells may be the basis for poor cardiovascular health (Minamino et al. 2004; Voghel et al. 2004). A link between telomere shortening, cellular senescence (Karlseder et al. 2002), and human vascular disease has also been reported (Minamino and Komuro 2002). In the Zucker diabetic rat, a model of insulin resistance and obesity, endothelial cells undergo premature senescence (Brodsky et al. 2004). Overall, these observations support the view that any damage to the endothelial cell layer will lead to an increased risk for the development of vascular disease. Diabetes In recent years the incidence of type 2 diabetes (characterized initially by insulin resistance, whereas type 1 is characterized by insulin deficiency) has grown to epidemic proportions and, according to the World Health Organization, there are now 150 million diagnosed type 2 diabetics in the world with current estimates indicating that the total will exceed 300 million people by 2025. However, many more diabetics probably remain undiagnosed, notably in third world countries. When we think about past epidemics, we always relate to those associated with infectious diseases. The Spanish Flu or La Grippe influenza pandemic of 1918– 1919 was a global disaster that killed over 20 million people worldwide and was termed a pandemic because it affected the entire world. We are now seeing a diabetes pandemic that may have even more serious affects on human mortality than did the 1918–1919 influenza pandemic. Why has there been such an enormous increase in the incidence of type 2 diabetes? Whereas definitive evidence has yet to be produced, it is thought that increases in caloric intake, especially carbohydrates, as well as decreases in the level of physical activity in the population at large is to blame. These diet and life style changes are also manifested in the alarming increase in the number of overweight adults and children and have resulted in an emergence of diabetes in populations in which it was once almost entirely absent

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(Diamond 2003; Zimmet et al. 2001). The metabolic syndrome, or insulin resistance syndrome, is present in almost one-half of older individuals and is associated with dyslipidemia (especially low high-density lipidoprotein cholesterol, increased triglycerides, and apolipoprotein B levels), hypertension, insulin resistance, glucose intolerance, hyperglycemia, and visceral adiposity (as well as lipid deposition in other nonadipose sites) (Eckel et al. 2005). Diabetes is a vascular disease It is the vascular sequellae of diabetes that represents the major clinical problem associated with this disease. Diabetes leads to a 2- to 4-fold increase in the incidence of coronary artery disease and a 10-fold increase in peripheral vascular disease. Blindness, end-stage renal failure, and neuropathies all result from microvascular disease within the tissues. These macro- and microvascular complications result in diabetic patients who have a mortality rate 3–4 times that of the general population (Haffner et al. 1998; Laakso 1999). Patients suffering from type 2 diabetes have a high risk of ED, which occurs from early stage in diabetes and is a hallmark of diabetic vascular disease; this subject has been extensively reviewed (Cosentino and Lüscher 1998; De Vriese et al. 2000; Pannirselvam et al. 2003). Hyperglycaemia, insulin resistance, dyslipidemia, hypertension, and advanced glycation end products have all been suggested to contribute to the development of ED and vascular pathologies (Cosentino and Lüscher 1998; Creager et al. 2003; De Vriese et al. 2000; Pannirselvam et al. 2003) with the majority view that ED and vascular disease occur secondarily to the development of insulin resistance and the resultant decrease in the disposal of glucose and hyperglycaemia. A role for acute hyperglycaemia in the initiation of endothelial dysfunction? The prevailing view has been that vascular disease develops secondarily to chronic hyperglycaemia and that the formation of advanced glycation end products (AGE) play an important role in the initiation of this pathophysiology. There are many competing theories that link elevated plasma glucose to the development of diabetic complications, most notably those related to vascular disease (Baynes and Thorpe 1999). However, the importance of postprandial glucose elevations is receiving increased attention as a result of a number of studies that indicate that postprandial hyperglycaemia and the effects of acute glycaemia are linked to an increased risk of cardiovascular disease (Ceriello 2005; Yamagishi et al. 2005). In high carbohydrate meals the glycaemic potential (or glycaemic index; see below) is of particular relevance to both the prevention and management of coronary disease (Brand-Miller 2004). Data from a number of clinical studies have recently been summarized by Ceriello (2005) and indicate that postprandial hyperglycaemia is a direct and independent risk factor for the development of cardiovascular disease. As borne out by the UK Prospective Diabetes Study (UKPDS), the importance of glycaemic control in the development of vascular disease may be greater for microangiopathy than for macroangiopathy (Laakso 1999), but nonetheless, a number of clinical and basic research studies have shown that a short-term exposure to high glucose, such as may be seen postprandial, rapidly suppresses © 2005 NRC Canada

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endothelium-dependent vasodilatation. Of particular interest is that, following a glucose load, such as occurs for a glucose tolerance test, ED is more severe in patients with impaired glucose tolerance (IGT) than in control subjects, but worse in patients with frank diabetes (Kawano et al. 1999). Acute hyperglycaemia uncouples eNOS The speed of onset of ED following an oral glucose load suggests that glucose may directly lower NO bioavailability and, indeed, this is indicated by the work of Brodsky et al. (2001) who demonstrated, using electrospray ionization tandem mass spectrometry, that glucose covalently binds to NO. ED can also be linked to a glucose-mediated increase in oxidative stress that might reflect a dysfunctional, or uncoupled, eNOS. ED could be reversed in subjects by the provision of the 6R isoform of the critical cofactor for eNOS dimerization and activation, 6R tetrahydrobiopterin (6RBH4), but not with the 6SBH4 isomer (Ihlemann et al. 2003). Interestingly, both 6RBH4 and 6SBH4 possess antioxidant activity; however, 6RBH4 has close to 100 times the affinity for eNOS activation (Klatt et al. 1994; Ihlemann et al. 2003). Furthermore, comparable studies have been performed in animals in which transient hyperglycaemia has been reported to induce ED that is linked to a reduced availability of BH4 (Bagi et al. 2004), and the over-expression of the ratelimiting enzyme for BH4 formation, GTP cyclohydrolase I (GTPCH), also protects against postprandial ED (Alp et al. 2004). Adenovirus-mediated gene transfer of GTPCH has also been shown to rescue eNOS activity in human aortic endothelial cells exposed to high glucose (Cai et al. 2005). Cai et al. (2005) reported that although high glucose increased total eNOS protein levels 1.5-fold, the eNOS was present principally in the monomeric form; however, GTPCH gene transfer increased cellular biopterin levels, augmented the eNOS dimer:monomer ratio 2.6-fold, increased NO production, but decreased superoxide production. These data strongly suggest that acute increases in plasma glucose initiate ED by the uncoupling of eNOS and this leads to an increase in superoxide production and a cascade of cellular events culminating in impaired vascular disease (Alp and Channon 2004). Support for this hypothesis is provided by Cosentino et al. (2001) who reported that in the GTPCHdeficient mouse (hph-1), which is deficient in BH4, endothelium-dependent relaxations to acetylcholine in the aortae were mediated by superoxide, but not NO. How does oxidative stress affect endothelial cell function? It has been shown that an elevation of ROS increases sodium content in calf pulmonary artery endothelial cells, possibly via activation of nonselective cation channels and endothelial cell depolarization, and, despite an overall increase in cytosolic free calcium, this leads to the inhibition of agonist-stimulated influx of external calcium (Koliwad et al. 1996). Further, Matsuzaki et al. (2005) reported that, in both mouse and rat aortic endothelial cells, depolarization enhances ROS formation from membrane NADPH oxidase. Thus, any stimulus that enhances plasma ROS formation, as would occur with a sudden reduction in blood flow, may also enhance endothelial cell ROS formation and depolarize endothelial cells. The effects of ROS may be indirect via the

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effects of peroxynitrite (ONOO–), formed nonenzymatically when superoxide anion interacts with NO. Authentic ONOO– has been shown to inhibit large conductance calciumactivated potassium channel currents (BKCa) in human coronary arterioles (Liu et al. 2002); an action that might also explain the effects of elevated glucose to inhibit voltagegated K+ channels in both rat and human coronary arteries (Li et al. 2003, 2004; Liu et al. 2001). A component of NO endothelial cell generation appears to be regulated by small- and intermediate-conductance endothelial cell calcium-activated K+ channels (SKCa and IKCa, respectively) and a combination of the SKCa and IKCa inhibitors, apamin and charybdotoxin, inhibits the soluble guanylyl cyclase-sensitive component (as reflected by an inhibition of acetylcholine-mediated relaxation by the guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a) quinoxalin-1-one) of acetylcholine-mediated relaxation in mouse small mesenteric arteries (Ding et al. 2000). A hypothesis may therefore be advanced whereby glucose-induced increases in ROS, perhaps again via ONOO– formation, inhibits SKCa and IKCa, and depolarizes endothelial cells and further reduces NO generation, thus contributing to ED. Hyperglycaemia-induced superoxide production, probably of mitochondrial origin, and the activation of the oxidative stress transcription factor activator protein-1 (AP-1), has also been associated with an early increase followed by a decrease in eNOS protein and RNA expression in human aortic endothelial cells (Srinivasan et al. 2004). Hyperglycaemia also downregulates connexin 43, an important component of myo-endothelial junctions (Sandow 2004), and this may be a contributing factor to ED (Sato et al. 2002). It can thus be seen that several glucose-activated cellular pathways may collectively lead to the elevated formation of superoxide anions, formation of ONOO–, the reduced bioavailability of NO, and the rapid onset of ED (Fig. 1). Several reports also indicate that brief exposure of 3 h of bovine aorta endothelial cells (BAECs) to elevated glucose (23 mmol/L) results in dramatic changes in agonist-induced and cellular calcium homeostasis that could be linked to increases in oxidative stress, PKC, and the regulation of calcium entry (Kimura et al. 1998a, 1998b, 2001). These data support the above hypothesis that acute increases in plasma glucose will rapidly affect endothelium-dependent vasodilatation via changes in calcium-dependent signalling. A number of studies have also reported that high glucose, via PKC activation, reduces connexin 43 expression (Kuroki et al. 1998) and changes connexin 43 phosphorylation levels; these changes may also contribute to the development of ED, but seem to require exposure times of days rather than hours. Acute hyperglycaemia can also enhance blood flow Not all reports support the view that a local increase in plasma glucose immediately attenuates endotheliumdependent vasodilatation. For example, high local levels of glucose have a vasodilator effect on resistance vessels in skeletal muscle of the forearm that is not modified by local hyperinsulinaemia (van Veen et al. 1999). Enhanced blood flow that results from an increase in plasma glucose may be in part due to a glucose-mediated activation of endothelial eNOS (Taubert et al. 2004) and in part by, possibly via an © 2005 NRC Canada

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Fig. 1. Endothelial cell dysfunction. Binding of insulin to its receptor-insulin receptor substrate (IRS) complex activates the lipid phosphatidylinositol-3-kinase (PI3K) and generates phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 is a second messenger essential for the translocation of Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinases (not shown). Protein phosphatase 2A (PP2A) is responsible for dephosphorylation of Akt. Activation of Akt enhances eNOS activity via Akt-mediated phosphorylation of serine1179 of eNOS. Hyperglycaemia, via protein glycosylation, may decrease eNOS activitation through the IRS-PI3 kinase/Akt pathway. Activation of the muscarinic receptor by acetylcholine (ACh), via calcium mobilization and increases in intracellular calcium (Ca) as well as extracellular calcium entry via store-operated channels (TRPC), activate eNOS with subsequent generation of NO and L-citrulline from the amino acid L-arginine. In the presence of hyperglycaemia-induced oxidative stress and elevated superoxide anion the level of tetrahydrobiopterin (BH4) a key cofactor for eNOS function, falls, resulting in an uncoupled eNOS. An uncoupled eNOS produces superoxide rather than NO. Superoxide can also be generated from cyclooxygenase, cytochrome p450, NADPH oxidase, mitochondria, as well as xanthine oxidase. Elevation of superoxide levels results, via a nonenzymatic interaction with NO, in the formation of peroxynitrite, ONOO–, which will nitrosylate a number of proteins including endothelial and smooth muscle KV and KCa channels, potentially leading to endothelial and smooth muscle cell depolarization. Endothelial cell depolarization will reduce the driving force for calcium entry via small and intermediate conductance calcium-activated potassium channels (SKCa and IKCa, respectively). Endothelial cell depolarization will thus reduce calcium entry and reduce eNOS activation. The SKCa and IKCa channel blockers, apamin and TRAM-34/charybdotoxin (ChTX), also depolarize endothelial cells and will reduce calcium entry. In addition, hyperglycaemia, via O-linked glycosylation modification of signalling proteins, modifying insulin-receptor-IRS-PI3K-Akt activation of eNOS as well as connexin and myo-endothelial gap junction communication. Solid lined arrows represent activation and dotted lined arrows represent inhibitory events.

osmotic action, activation of platelet eNOS (Massucco et al. 2005). Cipolla et al. (1997) studied the effects of increasing extracellular glucose (up to 44 mmol/L) on basal tone and myogenic activity in isolated rat cerebral arteries and reported an NO-mediated vasodilator action of glucose that was inhibited by endothelium removal or by the NOS inhibitor, L-NNA with indomethacin having a marginal inhibitory action. The acute increases in glucose impaired cerebrovascular reactivity to changes in transmural pressure and denuded vessels constricted in the presence of elevated glucose. Hoffman et al. (1999) studied the effects of acute hyperglycaemia on sympathetic activity and vascular function in

8 young normal control subjects (28 ± 3 years). Muscle sympathetic nerve activity and forearm vascular resistance measurements indicated that acute hyperglycaemia caused sympathoexcitation and peripheral vasodilatation. Studies with mannitol, however, suggested that the vascular actions of glucose may, in part, be mediated by increased osmolar load. Oomen et al. (2002) investigated the effects of acute hyperglycaemia and hyperinsulinaemia, both separately and in combination, on skin microvascular flow, capillary permeability, capillary recruitment, and ED in subjects with type 1 diabetes mellitus. Hyperglycaemia and hyperinsulinaemia alone or in combination increased flow measured with laser Doppler, but without capillary recruitment or evident © 2005 NRC Canada

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changes in microvascular permeability or endothelial markers. Other studies of subjects with type 1 diabetes have reported both normal endothelial function, but reduced vascular response to NO (sodium nitroprusside-mediated vasodilatation) (Calver et al. 1992), normal endothelial and vascular function (Smits et al. 1993), and, based on the ability of NG-monomethyl-L-arginine (L-NMMA) to reduce blood flow in healthy control subjects vs. type 1 diabetic patients, reduced endothelial function (Elliott et al. 1993). Effects of high glucose on endothelial function in vitro vs. in vivo Numerous studies with isolated vascular preparations have, however, demonstrated that raising extracellular Dglucose, but not L-glucose, results in an almost immediate (within 30 min) reduction in endothelium-dependent, but not endothelium-independent relaxation. The data are comparable regardless of whether large vessels such as the aorta (Tesfamariam and Cohen 1992), or small mesenteric (Bohlen et al. 2002; Bohlen and Lash 1993, Jin and Bohlen 1997; Taylor and Poston 1994), renal (Gomes et al. 2004), skeletal muscle (Lash et al. 1999), or cerebral preparations (Mayhan and Patel 1995; Sercombe et al. 2004) are studied. The threshold concentration for glucose-induced damage, as has been pointed out by Bohlen (2004), may be reduced by insulin resistance (for example), as vessels from Zucker rats were more susceptible than intestinal arterioles from control rats (Bohlen and Nase 2002). The rapidity of glucoseinduced ED and the higher susceptibility in insulin resistance suggests that ED will occur under any conditions wherein endothelial cell exposures to abnormally high fluctuations in glucose concentrations are unusually prolonged. Substantive evidence obtained from studies with endothelial cell cultures, whole blood vessels and clinical studies indicates that ED most likely results from a glucose-mediated increase in oxidative stress (Kawano et al. 1999; Nishikawa 2000a, 2000b), linked to a rapid uncoupling of eNOS and a shift to the monomeric form of the NOS (Cai et al. 2005). Thus, the susceptibility of the endothelial cell to injury will also be dependent on the level and viability of antioxidant defence mechanisms and this will include not only the standard cell antioxidants such as glutathione but also eNOSgenerated NO. Increased production of hydrogen peroxide in endothelial cells will also increase PKC activity (Taher et al. 1993) and this will lead to a decrease in eNOS activity via PKC-mediated phosphorylation of eNOS specifically at Thr497 (Matsubara et al. 2003). However, as already noted, in vivo the contribution of both endothelial and platelet eNOS activation by glucose must be considered (Massucco et al. 2005; Taubert et al. 2004). The intracellular effects of elevating glucose in vascular tissue have also been extensively studied. Kashiwagi et al. (1996) investigated the association between intracellular glucose metabolism and oxygen radical scavenging function via the glutathione redox cycle in human umbilical vein endothelial cells (HUVECs) exposed to 33 vs. 5.5 mmol/L glucose conditions and reported a near 50% reduction of the pentose phosphate pathway NADPH supply to the glutathione redox cycle. Although these results link glucose metabolism to oxidative stress and a reduction in the level of endogenous antioxidants, the glucose exposure was over a

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5–7 day time period. Hyperglycaemia, in tissues such as the endothelium where uptake of glucose is insulin independent, will rapidly saturate the hexokinase-mediated phosphorylation of glucose into glucose 6-phosphate leading to overflow of the products of the polyol pathway and the initiation of various metabolic imbalances that may contribute to ED (Yabe-Nishimura 1998). When intracellular glucose is elevated, the aldose reductase-catalyzed reduction represents an ever-increasing route for glucose utilization, leading to the accumulation of osmotically active sorbitol (Bhatnagar and Srivastava 1992) that can contribute to tissue damage (Costantino et al. 1999). Nonetheless, the role of aldose reductase and the use of aldose reductase inhibitors in determining hyperglycemic changes in vascular tissue remains poorly understood (Srivastava et al. 2005). Although the source of the glucose-induced oxidative stress following acute exposure has not been determined with absolute certainty, work by Brownlee and colleagues suggests that, at least in bovine aortic endothelial cells, the mitochondria rapidly produce superoxide when exposed to high glucose (Nishikawa 2000a, 2000b). Numerous other cellular sources of superoxide exist with NADPH oxidase, xanthine oxidase, cytochrome P450, cyclooxygenase (COX), as well as nitric oxide synthase (NOS), as potential sources (Ellis and Triggle 2003). However, the contribution of an uncoupled eNOS, the oxidation of BH4, must be emphasised as an important and early trigger, as several studies indicate that correcting BH4 deficiency prevents or reverses ED in both clinical and animal studies (Alp and Channon 2004; Bagi et al. 2004; Ihlemann et al. 2003; Pannirselvam et al. 2002). BH4 also increases insulin sensitivity in patients with type 2 diabetes (Nystrom et al. 2004). Indeed, in pro-oxidant states where an uncoupled eNOS is favoured, the glucose-mediated activation of endothelial and platelet eNOS described by Taubert et al. (2004) and Massucco et al. (2005), respectively, will lead to the generation of superoxide rather than NO. Thus, a scenario can be envisaged in which postprandial glucose produces reversible ED in normal subjects but the progressive development of ED in vulnerable individuals. Libby and Plutzky (2002) have pointed out that it may well be the time-dependent accumulation of hyperglycaemia-induced damage that is important and this will determine which therapies and over what time period interventions will prove effective. An analogy to make is that of chronic traumatic encephalopathy, a serious health problem in boxing that leads to the “punch drunk” syndrome, or dementia pugilistica (McCrory 2002). Chronic traumatic encephalopathy results from the cumulative long term neurological consequences of repetitive concussive and subconcussive blows to the head and one can readily see how high glucose may have the same effects on the oxidant defense mechanisms in the endothelium. What is unclear is what determines the susceptibility of an individual to postprandial glucose? Likely multiple factors are involved and it also can be anticipated that there is considerable heterogeneity among individuals, but clearly this is an area of research that requires urgent attention. The association between hyperglycaemia, ED, and neovascularization and wound healing may, to some extent, depend on the numbers and viability of endothelial progenitor cells that are assumed to be of bone marrow origin (Asahara et al. 1997). Endothelial progenitor cell numbers and angio© 2005 NRC Canada

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genic activity is reduced in patients with type 1 diabetes, thus providing another potential contribution to glucoseinduced endothelial damage (Loomans et al. 2004). Glucose transport into endothelial cells Endothelial cells express the ubiquitous but noninsulinregulated glucose transporter GLUT-1 (except in retinal vessels where GLUT-3 is also expressed; Knott et al. 1996) as well as the sodium/glucose cotransporter-1 (SGLT-1) (Nishizaki et al. 1995). In addition, GLUT-2–5 have also been shown to be expressed in endothelial cells, with GLUT3–5 primarily in brain endothelial cells, suggesting considerable vessel heterogeneity (Gaudreault et al. 2004). Gaudreault et al. (2004) report that, after 8 weeks of streptozotocin (STZ)-induced hyperglycaemia in rats, there was a downregulation of GLUT-1 and GLUT-3–5, but GLUT-2 levels were increased in the endothelium of the septal coronary artery. Gaudreault et al. (2004) argue that the abluminal upregulation of this low affinity/high capacity transporter may be a compensatory, but counterproductive, attempt to protect endothelial cells by facilitating the transfer of glucose from the blood to the vascular wall. Reducing GLUT-2 expression may therefore be beneficial in diabetes. Chronic exposure of endothelial cells to high glucose will has also been shown to reduce GLUT-1 expression, and a 36-h exposure of BAECs and smooth-muscle cells to 23 mmol/L glucose led to a down-regulation of the rate of glucose transport as well as GLUT-1 mRNA and protein (Alpert et al. 2002). The same group, however, failed to show changes in BAECs after a 24-h exposure to high glucose (Kaiser et al. 1993). Taubert et al. (2004) demonstrated that, in a study with passage 2 porcine aortic endothelial cells, 20 mmol/L Dglucose, but not L-glucose, induced an increase in intracellular calcium and a transient release of NO that was inhibited by either the sodium/calcium exchange (NCX) inhibitor, dichlorobenzamil, or the SGLT-1 inhibitor, phlorizin. D-glucose also augmented the vasodilator action of insulin and enhanced insulin-induced relaxation of porcine artery ring preparations as well as insulin-mediated NO response in porcine aortic endothelial cells; interestingly prolonged exposure to 25 mmol/L D-glucose resulted in a progressive loss of NO release and after 2 h complete inhibition (Taubert et al. 2004). The glucose-induced increase in intracellular calcium was linked to an increase in intracellular sodium secondary to the activation of SGLT, and the inhibition of the calcium signal with the NCX inhibitor, dichlorobenzamil, supports this conclusion (Taubert et al. 2004). In the continued presence of elevated glucose and SGLT activation may lead to a reduction in NCX activity and NOS activation (Hattori et al. 2000). One explanation, suggested by Taubert et al. (2004), is that the initial effects of elevated glucose on endothelial function serve, via an increase in NO generation, to increase tissue perfusion and enhance the action of insulin, thus rapidly lowering plasma glucose levels and minimizing the potential for glucose toxicity. This hypothesis is in keeping with those studies that indicate that raising glucose does produce an initial increase in NO generation and subsequent vasodilator action. However, a delicate balance exists regarding NO generation and oxidative stress such that ED can rapidly develop as reflected by the

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time-dependent loss of the response to NO in the Taubert et al. (2004) study. Role of diet in the development of endothelial dysfunction It is well-established that dietary intake can affect cardiovascular health, and Western-style diets are reported to result in a considerable amount of the day spent in the postprandial state (Sies et al. 2005). In general, a diet that is characterized by higher intake of fruits, vegetables, legumes, whole grains, fish, and poultry will have a beneficial effect on the incidence of cardiovascular disease and the endothelium (LopezGarcia and Hu 2004). Conversely, a Western-style diet that is characterized by higher intake of red and processed meats, high sugar desserts, french (or “freedom”) fries, and refined carbohydrates, is associated with an impairment of endothelial function (Kromhout 2001). Data from animal studies suggest that this may be in part due to a combination of ROSmediated NO inactivation and changes in NOS protein expression (Roberts et al. 2003). Many studies have also indicated that Western-style diets lead to elevated blood pressure in human (Appel 2000) and animals (Yoshioka et al. 2000). In addition, a high fat diet combined with refined carbohydrate intake results in hypertension and insulin resistance (Barnard et al. 1998) that, in a study with rats fed such a diet for 7 months, leads to ED, enhanced oxidative stress, and decreased eNOS and Akt protein levels (Roberts et al. 2005). Attention should also be focused on the glycaemic index of food. The glycaemic index is calculated as the area under the glucose curve after the test food is eaten, divided by the corresponding area after the control food is eaten, and then multiplied by 100% to represent a percentage of the control food. Each unit of the glycaemic load represents the equivalent blood glucose-raising effect of 1 g of pure glucose or white bread (Liu and Willett 2002). High-glycaemic foods result in a higher and more rapid increase in blood glucose levels compared with the effects of low-glycaemic index foods. It has been argued that the adoption of the low-fat, high-carbohydrate diet of the mid 1980s, and the resultant attraction to low fat foods, may have contributed to an inappropriate swing to the consumption of high glycaemic, but low fat, foods and hence the current pandemic of obesity and type 2 diabetes (Weinberg 2004). Oral glucose loading, which is effectively what many of these so called low-fat carbohydrate foods produce, is well-known to result in a burst of oxidative stress and ED (Kawano et al. 1999; Title et al. 2000). Advanced glycation end products The persistent presence of hyperglycaemia will lead, via a nonenzymatic process, to protein glycation and the posttranslational modification of proteins. This process requires the formation of, over a period of hours, a Schiff base formed from the reaction between a free amino group on a protein and a carbonyl group from the sugar. Over a period of a few days, the labile Schiff base ultimately rearranges to a stable, essentially irreversible, ketoamine or Amadori product. Amadori products with time and via dicarbonyl intermediates, such as 3-deoxyglucosones, give rise AGEs. AGEs build up in tissues during prolonged hyperglycaemia and, via protein crosslinkling as well as binding to DNA, © 2005 NRC Canada

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can affect numerous cell events, and thus have been implicated in the development of diabetic complications (Ahmed 2005). The effects of protein glycation may be self-limited by high turnover and short half-life of many, but not all, cell proteins, and therefore, unless high glucose levels remain elevated for at least several days, AGE accumulation is normally negligible. Short periods of hyperglycaemia, as may occur in impaired glucose tolerance, may however be sufficient to increase the concentrations of aoxoaldehydes, such as glyoxal, methylglyoxal, and 3-deoxyglucosone, to initiate earlier glycation events (Thornalley et al. 1999). A 3-h exposure of rat mesenteric artery smooth muscle cells to methylglyoxal, a metabolite of glucose that causes nonenzymatic glycation of proteins and forms AGEs, elevated oxidative stress, and a 24-h exposure resulted in a 2-fold elevation of AGEs (Wu 2005). AGEs are also found in certain foods, and glycation was first recognized by the brownish appearance of food proteins as they age (i.e., the Maillard reaction), thus adding to the potential dietary contribution of diabetes, and their cellular accumulation likely contributes to the aging process as well as other pathologies such as Alzheimer’s. The cellular effects of AGEs primarily result from binding to a specific receptor appropriately termed receptor for AGE (RAGE). RAGE is thus an obvious target for drug development to prevent vascular disease in diabetic and nondiabetic subjects as well as for the treatment of Alzheimer’s (Hudson and Schmidt 2004). The dicarbonyl scavenger aminoguanidine (pimagedine) inhibits AGEs from forming and has been used in vivo and in vitro in animal models to slow the progression of diabetic vascular complications (Brownlee et al. 1986), but it did not slow the progression of nephropathy in humans (Thornalley 2003). In a study of cremaster arterioles from the STZ-diabetic rat, Hill and Ege (1994) reported that, under passive conditions, arterioles from STZ-diabetic animals were stiffer and less distensible compared with similar arterioles from control animals. Under active conditions, in the presence of extracellular calcium, arterioles from the diabetic group showed impaired myogenic reactivity. Chronic treatment with aminoguanidine prevented the diabetesinduced changes in the active and passive properties of the isolated arterioles, but did not improve the impaired endothelium-dependent vasodilator responses to acetylcholine. The altered stiffness and myogenic properties observed in the cremaster vessels from the diabetic rats may reflect the effects of AGE accumulation in the extracellular matrix affecting vessel rigidity. Aminoguanidine also inhibits NOS with reported selectivity towards inducible nitric oxide synthase (Southan and Szabo 1996), and this may confound an action on AGE accumulation in contributing to vascular and endothelial dysfunction; however, Hill and Ege (1994) demonstrated that methylguanidine, a NOS inhibitor, but not a RAGE antagonist, was ineffective in reversing both endothelial and myogenic dysfunction. Other compounds used to block the effects of AGE include pyridoxamine, ophenylenediamine, and cross-link breakers to remove already formed AGEs such as ALT-711. Several other drugs in use for cardiovascular disease and the treatment of diabetic complications have been shown to have an effect on AGE accumulation such as ramipril, the angiotensin-converting enzyme inhibitor, and metformin, the glucose-lowering drug

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(Hudson and Schmidt 2004; Cooper 2004). Forbes et al. (2004) have recently reported that treatment with either aminoguanidine or the AGE cross-link breaker, ALT-711, is associated with a small, but significant reduction of plaque area in the STZ-induced diabetic apoE-deficient mouse; an argument in favour of AGE in the development of plaque in the macro vasculature, but not necessarily supporting a role for AGE in the initiation of plaque formation. The Metabolic Syndrome The association between dyslipidemia, insulin resistance, glucose intolerance, hyperglycaemia, hypertension, and visceral adiposity was first described by Eskil Kylin in 1923 (Kylin 1923) and, many years later, was named “The Metabolic Syndrome”, also known as Syndrome X or the Insulin Resistance Syndrome (Eckel et al. 2005). It has been stated that this syndrome is present in approaching 50% of older individuals (Alexander 2003) and is a predictor of the future development of type 2 diabetes (Lorenzo et al. 2003). The incidence of the metabolic syndrome is increasing among younger individuals including adolescents, and, with the usual ethnic or racial differences, reflects the global improvements in the standard of living with increases in caloric intake, notably high glucose and high fat foods, and decreases in physical activity. Studies of obesity in men also indicate a correlation between oxidative stress and the development of insulin resistance (Urakawa et al. 2003). The metabolic syndrome can be referred to as a prediabetic state and is also clearly associated with an increased risk of cardiovascular disease (Isomaa et al. 2001). At what stage ED can be observed in subjects diagnosed with the metabolic syndrome has not been clearly determined, but of particular importance to the emergence of diabetes pandemic is the evidence that the maternal environment can affect vascular function in offspring. Epidemiological studies in humans and experimental studies in primarily rats indicate strong inverse relationships between birth mass and the risk of developing type 2 diabetes and the metabolic syndrome later in life (Barker et al. 1993; Hales and Ozanne 2002). The association between the intra-uterine environment and future cardiovascular disease has been extensively investigated (Barker 1993) and the evidence critically reviewed (Armitage et al. 2004). Furthermore, catch up growth following early growth restriction can also be detrimental (Hales and Ozanne 2003), but the cellular basis for this relationship remains unknown. Developmental programming of vascular disease Diabetes begets diabetes (Jovanovic 2004). Controlled animal studies, particularly in rodents with the same genetic background, provide an opportunity to determine the cellular processes that may contribute to the developmental programming influences on the cardiovascular system. Studies have shown that offspring of rats that were fed a high fat (lard) diet during pregnancy, designed mimic a Western style high fat diet, developed ED in the femoral artery but no blood lipid or glucose abnormalities (Ghosh et al. 2001). ED and enhanced contractile responses to vasoconstrictors have also been reported in blood vessels 15 days post-partum in the dams fed the high fat diet (Koukkou et al. 1998). Furthermore the nondiabetic offspring from rats with streptozotocin-induced diabetes that were also fed saturated fat during pregnancy dem© 2005 NRC Canada

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onstrated femoral artery dysfunction in the form of blunted endothelium-dependent dilation and enhanced constrictor responses to norepinephrine and the thromboxane mimetic U46619 as compared with young offspring of high-fat-fed normal dams (Koukkou et al. 1998). Further characterization of the impact of developmental programming on endothelial function has been pursued by Poston, Taylor and colleagues (Armitage et al. 2005). Following a similar protocol, ED, primarily owing to a reduced in the contribution of EDHF, was observed in small mesenteric arteries, but not in femoral arteries (where EDHF is less prominent), from both male and female offspring of female Sprague–Dawley rats that were fed a diet rich in lard (25.7% fat) 10 days before and throughout pregnancy and lactation (Taylor et al. 2004). Interestingly, in another study from this same group and using the same protocol, telemetric recording indicated that only female offspring developed hypertension (Khan et al. 2003). These latter data are particularly interesting as the dissociation between blood pressure and ED indicates that these 2 parameters are not always linked, whereas maternal diet seems to clearly impact on endothelial function, and ED may relate to changes in the regulation or contribution of EDHF. A refinement in this view may be needed based on the report from Khan et al. (2005) in which, again following the same high fat diet exposure in utero, 6 month old female offspring presented with raised blood pressure, elevated plasma insulin, and ED in small mesenteric arteries. However, comparable changes, although with heightened ED, were observed when exposure to the high fat diet was confined to 10 days by cross suckling pups derived from normal chow fed rats to dams that were on a high-fat diet. The effects of the high fat diet and the suckling phenomena have been attributed to changes in mitochondrial DNA and altered mitochondrial gene expression (Taylor et al. 2005). The importance of the background strain of the dam for the effects of suckling was reported some years ago for the spontaneously hypertensive rat where it was shown that offspring from the mating of spontaneously hypertensive rat parents had significantly lower blood pressures when they were suckled by a normotensive control dam (Cierpial and McCarty 1987). What is clear from all of these studies is that endothelial function can be radically affected by maternal as well as early postnatal dietary influences. Insulin resistance and fatty acid metabolism Although specific cellular mechanisms that are responsible for the development of insulin resistance in type 2 diabetes remain unclear, data from multiple studies link the accumulation of triglycerides in skeletal muscle to the development of both skeletal muscle and whole body insulin resistance (Pan et al. 1997; Krssak and Roden 2004; Kelley et al. 2002). Skeletal muscle accounts for greater than 80% of insulin-stimulated glucose uptake and subsequent storage of glycogen, but this is dramatically reduced in diabetics (Shulman et al. 1990). Infusions of lipids to induce increases in fatty acid plasma levels also reduces insulin-stimulated glucose disposal in rodents and human subjects (Boden and Shulman 2002; Boden 2001). The measurement of skeletal muscle triglyceride content from human subjects demonstrates a strong relationship between intracellular triglycer-

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ide content and insulin resistance (Pan et al. 1997; Perseghin et al. 1999). The fetal environment may also contribute to insulin resistance as it has been shown that the children of diabetics had a 50% reduction in the rate of insulinstimulated whole-body glucose metabolism indicating that glucose transport and (or) glycogen synthesis via reduced hexokinase activity is impaired at an early stage in the development of diabetes (Rothman et al. 1995). The classic view in which fatty acids induce insulin resistance was proposed by Randle et al. (1963) whereby free fatty acids inhibit pyruvate dehydrogenase and produce a feedback inhibition of further glucose uptake into skeletal muscle. Fatty acids have been shown to compete with glucose for oxidative processes and so Randle et al. (1963) argued that an increase in intramitochondrial acetyl coenzyme A (CoA) : CoA and NADH:NAD+ ratios would reduce pyruvate dehydrogenase activity and increase citrate accumulation. Elevated citrate, in turn, inhibits phosphofructokinase activity and a subsequent increase in glucose-6-phosphate inhibits hexokinase II activity. As a result of hexokinase II inhibition, intracellular glucose increases and reduces GLUT4-mediated transport of additional glucose. How relevant these mechanisms are for the regulation of glucose transport into endothelial cells is an unexplored area (Fig. 2). The subsequent inhibitory action on the glucose transporter, GLUT-4, leads to glucose intolerance and hyperglycaemia (McGarry 2002). Alternatively it has been argued that the accumulation of intramuscular fatty acids interferes with a very early step in the regulation of the GLUT-4 transporter that is then followed by a reduction in both the rate of muscle glycogen synthesis and glucose oxidation (Roden et al. 1996). The levels of intramuscular triglycerides are probably merely markers for more metabolically active lipid intermediates that inhibit insulin-signalling pathways. Diacylglycerol and ceramides are 2 products of fatty acyl-CoA that have been shown to accumulate in insulin-resistant muscle and liver and to inhibit early steps in insulin signalling via reducing PI3 kinase activation and hence a reduced insulin-stimulated glucose transport (Turinsky et al. 1990). Diacylglycerol activates protein kinase C (PKC), possibly the δ isoform (although it is also known that the β isoform plays an important role in diabetes-related vascular disease and selective inhibition improves endothelial function; Beckman et al. 2002; Ishii et al. 1998), which is also activated by oxidative stress (Talior et al. 2003), and this leads to serine phosphorylation of the insulin receptor substrate-1 (Cortright et al. 2000). Ceramide, a second messenger in the sphingomyelin signaling pathway, can inhibit insulin-induced Akt phosphorylation and activation and since ceramide is also elevated in the muscle of obese, insulin-resistant humans (Adams et al. 2004; Straczkowski et al. 2004) and obese Zucker rats (Turinsky et al. 1990), its accumulation may play a key role in disrupting downstream insulin signalling (Chavez et al. 2003) (Fig. 3). Convincing as these data may seem, not all of the published data supports the lipocentric viewpoint. Thus, peroxisome proliferator-activated receptor-γ (PPARγ) agonists are therapeutically employed as insulin sensitizers, and despite improving insulin sensitivity and lowering plasma glucose as a class, these agents increase lipid disposition (Berger et al. 2005). Lessard et al. (2004) reported that, in obese Zucker rats, the PPARγ agonist rosiglitazone enhanced glucose © 2005 NRC Canada

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Fig. 2. Fatty acid mediated inhibition of glucose metabolism. Increases in fatty acid levels in the cell enhance mitochondrial acetyl CoA/CoA and NADH/NAD ratios and has an inhibitory (-ve) action on pyruvate dehydrogenase (PDH). PDH inhibition leads to an increase in citrate, which, in turn, inhibits (-ve) phosphofructokinase (PFK). The inhibition of PFK leads to a build up of the substrate for PFK– glucose-6-phosphate (G6P) and a negative feed back (-ve) on hexokinase II (HK) with the result that intracellular glucose levels rise and inhibit the additional transport of glucose into the cell by the GLU4 transporter.

Fig. 3. Protein kinase C (PKC)-mediated inhibition of glucose transport. Fatty acid levels build up in the cell and are metabolized to produce diacylglycerol and ceramides leading to the activation of PKC and the serine-threonine kinase cascade that, via the phosphorylation of serine/threonine residues on the insulin receptor substrates (IRS-1 and IRS-2), reduces (-ve) the ability of the insulin receptor to activate the PI3 kinase pathway. PI3 kinase activation (+ve) is essential for the phosphorylation, translocation of the GLUT4 to the sarcolemmal membrane.

tolerance but increased skeletal muscle diacylglycerol and ceramide levels by 65% and 100%, respectively. These data indicate that rosiglitazone improves glucose tolerance by

cellular mechanisms distinct from reducing fatty acid accumulation in skeletal muscle and are in agreement with the reported pleiotropic actions of the PPARγ agonists (Berger © 2005 NRC Canada

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at al. 2005) and argue against a purely lipocentric basis of insulin resistance. The PPARγ agonist, troglitazone, through both PPARγ-dependent and -independent mechanisms, has been shown to increase endothelial cell NO generation in BAECs (Cho et al. 2004). The troglitazone-mediated increase in the expression of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, and subsequent eNOS-Ser1179 phosphorylation was inhibited by a PPARγ antagonist, whereas eNOS-Ser116 dephosphorylation was PPARγ-independent. Thus, NO-mediated vasodilatation, enhanced blood flow, and subsequent improved glucose disposal may explain the decrease in insulin resistance noted with PPARγ agonists. Of interest as well is the observation that treatment of the db/db type 2 diabetic mouse with a PPARγ agonist normalized metabolic abnormalities, endothelial function, and the expression of the sarcolemmal membrane associated protein, SLMAP, leading to the conclusion that dysregulation of the SLMAP gene may be linked to both metabolic and endothelial dysfunction in type 2 diabetes (Ding et al. 2005). The voltage-gated potassium channel Kv1.3 may also be involved in the regulation of peripheral insulin sensitivity as it has been shown that an inhibition of Kv1.3 activity facilitates the translocation of the GLUT-4 to the plasma membrane of skeletal muscle and adipose tissue (Xu et al. 2004). Further, Kv1.3 deficient mice demonstrate a resistance to weight gain, insulin resistance, and hyperglycaemia when provided a high-fat diet (Xu et al. 2004). The signalling pathways that link the KV1.3 channel to the regulation of peripheral insulin sensitivity remain to be elucidated. Insulin-mediated vasodilatation Although a strong case can be made that insulin resistance results from the reduced ability of insulin to activate specific glucose transporters (GLUT-4), with the resultant reduction in glucose disposal, particularly in skeletal muscle, leading to the development of hyperglycaemia (Shulman 2000), insulin also has direct effects on the endothelium. For instance, elevated plasma insulin may play a protective role and reduce hyperglycaemia-induced ED in the Zucker rat model of obesity-induced insulin resistance (Bohlen and Lash 1995) despite a pro-oxidant state that may counteract such actions (Laight et al. 2000). Of particular interest is that Goldstein et al. (2005) reported that, via the Nox4 subunit of NADPH oxidase, insulin-stimulated generation of cellular ROS results in the oxidative inhibition of proteintyrosine phosphatase 1B, a protein-tyrosine phosphatase known to be a major regulator of the insulin signaling cascade, and hence a facilitation of insulin action. On the other hand, insulin-mediated generation of superoxide may exacerbate endothelial function (McCarty 2002). The contribution of a defective insulin-mediated vasodilatation response as the basis for the development of insulin resistance and the subsequent reduction of glucose delivery to skeletal muscle has been rejected on the basis of indirect 13 C NMR studies (Shulman 2000). Nonetheless, there is substantial evidence that it is the action of insulin in the microvasculature that is important for mediating perfusiondependent increases in glucose metabolism in skeletal muscle (Laakso et al. 1990, 1992; Clerk et al. 2004). Baron’s pioneering work (Laakso et al. 1990, 1992) indicates that

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within a physiological concentration range, insulin increased total skeletal muscle blood flow in healthy human subjects in a fashion that paralleled the actions of insulin to increase glucose disposal. Laakso et al. (1990) concluded that in vivo insulin resistance is due to a defect in insulin action at the tissue level and (or) a defect in insulin’s haemodynamic action to increase blood flow to insulin sensitive tissues. Furthermore Bohlen and coworkers (Bohlen and Lash 1993; Bohlen and Nase 2002; Jin and Bohlen 1997) have described that NO-mediated vasodilatation is reduced within 15–30 min exposure to D-, but not L-, glucose in mesenteric, skeletal muscle, and cerebral microvascular beds (Bohlen 2004). Other evidence that supports a rapid effect of raised glucose on endothelial function has already been described. Clerk et al. (2004) argue that a vasodilator action of insulin in the smaller (< 150 µm diameter) precapillary arterioles that would be particularly important for the regulation of total blood flow and glucose disposition. These conclusions are supported by data from Bonadonna et al. (1998). In the latter study, forearm blood flow was measured in 10 subjects using physiological insulin concentrations (approximately 400 pmol/L) and an additional 10 at supraphysiological insulin concentrations (approximately 5600 pmol/L). Forearm blood flow was enhanced only by supraphysiological hyperinsulinaemia and this was accompanied by tissue recruitment; it was argued that this is relevant as a determinant of forearm (muscle) glucose uptake. In addition, the studies by Vincent et al. (2002) demonstrating that within physiological concentration ranges, insulin induces capillary recruitment, which precedes glucose uptake by rat skeletal muscle, and that this effect preceeds increases in total blood flow by 60–90 min. Finally, exercise training, which is well known to significantly reduce the risk of developing insulin resistance by improving glucose tolerance and insulin action (Hawley 2004), increases glucose transport into muscle by a process that is blocked by inhibition of nitric oxide synthase (Roberts et al. 1997). Insulin is well known to have direct effects on the endothelium and, for instance, stimulates NO release from endothelial cells (Montagnani et al. 2001; Zeng and Quon 1996; Zeng 2000) and inhibition of eNOS reduced glucose uptake and insulin-mediated capillary recruitment (Baron et al. 1995). It has been argued that endothelium-derived NO mediates insulin-induced stimulation of the perfusion of skeletal muscle in humans (Scherrer et al. 1994). Insulin increases eNOS protein and mRNA in native porcine coronary artery endothelial cells (Fisslthaler et al. 2003). The eNOS knockout mouse is insulin resistant The eNOS knockout mouse also demonstrates insulin resistance with fasting hyperinsulinaemia, hyperlipidemia, and a 40% lower insulin-stimulated glucose uptake than control mice (Duplain et al. 2001). Furthermore insulin resistance in the eNOS –/– mouse could be specifically associated with impaired NO generation and, in equally hypertensive 1-kidney/ 1-clip mice and renovascular hypertensive mice, insulinstimulated glucose uptake was normal (Duplain et al. 2001). Shankar et al. (2000) studied eNOS and nNOS –/– mice and reported that eNOS –/– mice were insulin resistant at the level of the liver and peripheral tissues, whereas the nNOS –/– mice were insulin resistant only in the latter. These data © 2005 NRC Canada

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indicate that although NO plays a role in modulating insulin sensitivity and carbohydrate metabolism it is eNOSgenerated NO, rather than nNOS that has the dominant role in modulating insulin sensitivity and carbohydrate metabolism. Cook et al. (2003) also have reported that eNOS –/– mice were hypertensive, insulin resistant, dyslipidaemic, had elevated plasma levels of leptin, uric acid, and fibrinogen, and that they developed glucose intolerance when challenged with a metabolic stress. Thus, a single gene defect is associated with a clustering of cardiovascular risk factors in young mice that are very similar to those seen in humans with the metabolic syndrome. eNOS, however, is also expressed in skeletal muscle and a role for skeletal musclederived NO in the regulation of insulin action has been argued (Kapur et al. 1997). Role of platelet eNOS in insulin-mediated vasodilatation Insulin-induced vasodilatation that is seen in vivo results may primarily result from the activation of platelet eNOS rather than endothelial cell eNOS (Randriamboavonjy et al. 2004). For example, Randriamboavonjy et al. (2004) demonstrated that although insulin failed to relax endotheliumintact porcine coronary artery ring preparations, the supernatant from insulin-stimulated human platelets induced a complete relaxation. Relaxation was attributed to the release of ATP and other vasoactive factors from platelet granules. Furthermore, platelet-mediated relaxation was not detected when platelets from eNOS –/– used were substituted in the protocol. Controversy exists as to whether the acute administration of physiologically relevant concentrations of insulin leads to a rapid activation of eNOS in isolated vascular preparations (Laight et al. 1998) and the data from the study by Randriamboavonjy et al. (2004) may provide the missing link to explain insulin-mediated vasodilatation. Importance of the PI3 kinase-Akt signalling pathway Zeng and Quon (1996) demonstrated that the inhibitor of phosphatidylinositol 3-kinase, wortmannin, inhibited insulinstimulated production of NO by approximately 50% in HUVECs. Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport thus supporting the view that NO is an effector of insulin signalling pathways in the vasculature. Zeng et al. (2000) have also reported that insulin receptor tyrosine kinase activity, PI3K, and Akt contribute to the insulin-stimulated production of NO in HUVECs. Montagnani et al. (2001) studied NO release from single NIH-3T3IR cells over-expressing eNOS, as well as HUVECs and BAECs, and concluded that insulin regulates eNOS activity via a calcium-independent mechanism that requires phosphorylation by Akt of eNOS serine 1179. There is substantial evidence suggesting defective Akt (aka protein kinase B) signalling in the development of insulin resistance and that changes in Akt signalling in the endothelium may be a contributing factor (Zdychova and Komers 2005). In a study of middle-aged men, it was found that exercise also improves fasting and postprandial vascular function thus indicating how diet and lifestyle interact to determine risk for cardiovascular disease (Gill, et al. 2004). It is therefore clear that numerous factors interact to determine how diet, life-

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style, and genetics affect endothelial function, cardiovascular morbidity, and mortality; likely the exact determinants that link ED, vascular disease, and diabetes are also variable. Lessons from insulin-receptor knockout mice Vicent et al. (2003) generated a vascular endothelial cell insulin-receptor knockout mouse using the Cre-loxP system. Interestingly, in this study, the loss of the endothelial cell insulin receptor had no major consequences on vascular development. In addition, fed and fasting glucose levels, fasting free fatty acids, and blood insulin levels were reported as normal in 2-month-old offspring, although fasting triglyceride levels were reduced by approximately 20% (Vicent et al. 2003). These data could be interpreted as indicating that the endothelial insulin receptor is superfluous for the regulation of glucose homeostasis; however, glucose tolerance, but not lipid metabolism, is also normal in the muscle-specific (Bruning et al. 1998) and fat-specific insulin-receptor knock out mice, who both have low fat mass and normal glucose tolerance (Bluher et al. 2002). It is also important to realize that glucose tolerance is disrupted in the liver-specific insulin-receptor knockout mice (Michael et al. 2000); severe insulin resistance and glucose intolerance is apparent in the skeletal muscle GLUT-4 knock out mice from an early age (Zisman et al. 2000). A GLUT-4 knock out in adipose tissue preserves GLUT-4 in muscle, but these mice still develop insulin resistance in both the muscle and liver (Abel et al. 2001). Thus, down regulation of GLUT-4 and glucose transport selectively in adipose tissue can cause insulin resistance and thereby increase the risk of developing diabetes. Overall, these findings, notably the differences in severity of glucose intolerance between skeletal muscle insulin receptor and GLUT-4 knock outs, are supportive of a vascular role for insulin that is perhaps mediated by NO for promoting glucose uptake into muscle. Nonetheless caution is required in the interpretation of these data either for or against the vascular theory as a basis for the development of insulin resistance. Insulin-induced oxidative stress Although elevated insulin enhances eNOS activity, it may also enhance oxidative stress. For example, Midaoui and de Champlain (2005) demonstrated that elevated plasma glucose levels alone do not induce vascular oxidative stress and hypertension in a rat unless it is combined with high level of insulin. Hyperinsulinaemia may also be involved in the generation of oxidative stress in humans via an NAD(P)Hdependent mechanism that involves the activation of phosphatidylinositol 3-kinase, and extracellular signal-related kinase (ERK)-1- and ERK-2-dependent pathways (Ceolotto et al. 2004). On the other hand, insulin was reported to preserve endothelial function by alleviating oxidative stress in the basilar artery of the STZ-diabetic rat (Matsumoto et al. 2004). Additional studies are required to determine the effects of elevated insulin on endothelial function, particularly in the presence of hyperglycaemia. Glucose, fat, or both? Considerable diversity exists throughout the vasculature both for endothelial and vascular muscle function (Andrews et al. 2005). Taking the analogy to the etiology of hypertension, it is quite likely that no one mechanism is responsible © 2005 NRC Canada

Triggle et al.

for the vascular disease associated with type 2 diabetes. The close association between obesity and the incidence of type 2 diabetes indicates that there is considerable merit in the lipocentric viewpoint as the basis for the development of insulin resistance (Savage et al. 2005). However, does this viewpoint take into account an extensive literature, reflecting both animal and clinical data, which indicates that increases in plasma glucose have a very rapid effect on blood flow? Evidence indicates that glucose can produce both vasodilatation as well as reduce endothelium-dependent increases in blood flow. Should we thus take the glucocentric point of view? The implications of glucose-induced ED on insulin-dependent and -independent total blood flow to skeletal muscle, would be dramatic (Clerk et al. 2004). The human clinical data presented by Kawano et al. (1999) indicates a range of glucose-load-induced ED in nondiabetic subjects with normal endothelial function, those with impaired glucose tolerance to severe ED in those with frank type 2 diabetes. In vitro studies also point to several cellular mechanisms that are rapidly affected by high glucose and lead to ED. The rapid global increase in type 2 diabetes also closely parallels dietary changes that, in part, reflect an increase in the intake of refined carbohydrates with high glycaemic indices (Weinberg 2004). Abuse of the endothelium by repeated exposure to rapid increases in glucose will lead to ED and, predictably, reduced blood flow and glucose disposition. What mediates the rapid effects of glucose on endothelial function? Activation of PKC, possibly the β isoform (Iishi et al. 1998), is a likely mediator, although the time period for activation remains unclear. Furthermore, diabetic- and hyperglycemia-induced increases in PKC activity and diacylglycerol levels in the heart and aorta of STZ-diabetic rats are preventable by insulin treatment (Inoguchi et al. 1994) (Fig. 4). Rabbit aorta treated for 10 min with 4-phorbol 12myristate 13-acetate, a PKC activator, showed decreased relaxations to the endothelium-dependent vasodilator, acetylcholine, as did aorta following a much longer 6-h exposure to elevated glucose (22 and 44 mmol/L) (Tesfamariam et al. 1991). On the other hand, rat cerebral pial arterioles exposed to 25 mmol/L glucose for 30 min showed a dramatic loss of endothelium-dependent relaxation that was reversed by prior exposure to the PKC inhibitors calphostin C or chelerythrine (Mayhan and Patel 1995). Elevations of glucose, via the involvement of phospholipase D and PKC activation, also lead to vascular smooth muscle proliferation and hypertrophy (Yasunari et al. 1996). It has been reported that PKC phosphorylates eNOS at threonine 497 and this leads to a decrease in eNOS activity that is likely linked to a decrease in the affinity of eNOS for calmodulin (Matsubara et al. 2003). However, Salt et al. (2003) studied the effects of a 48-h exposure of human aortic endothelial cells to 25 mmol/L glucose on insulinstimulated generation of NO and phosphorylation of eNOS at Ser1177, and they reported a decrease in NO generation with no effect on eNOS phosphorylation or of PKC inhibition. A role for PKC-mediated changes in ED is implicated by the studies reported by Cosentino et al. (2003) on the effects of high glucose on human aortic endothelial cells that showed an increase in cyclooxygenase 2, COX-2, mRNA,

693 Fig. 4. Multiple mechanisms increase protein kinase C (PKC). PKC activation can arise via an elevation of diacylglycerol (DAG) in the cell via the production of phosphatidic acid (PA) from either the action of phospholipase D (PLD) on phosphatidylcholine (PC) or from lysophosphatidic acid (LYSPA) via the glycolytic intermediates dihydroxyacetone phosphate (DHAP).

and protein expression following an undefined exposure time to 22.2 mmol/L glucose as well as activation of PKC after 1-h exposure. Interestingly, eNOS expression was increased 2-fold, but a reduction in NO formation and an enhanced production of ROS resulted. Phorbol ester also caused an increase of COX-2 and eNOS expression similar to that elicited by glucose, and this was prevented by the PKC inhibitor calphostin C (Cosentino et al. 2003). Thus, high glucose, via PKC-regulated changes in cell signalling, induces oxidative stress and up-regulation of COX-2, resulting in, despite the increase in eNOS expression, reduced NO bioavailability, increased oxidative stress, and peroxynitrite formation. Federici et al. (2002) reported that insulindependent activation of eNOS is impaired by O-linked glycosylation of serine/threonine residues of insulinsignalling proteins following a 3 day exposure of human coronary endothelial cells to 20 mmol/L glucose (Fig. 1). These data suggest that high glucose specifically inhibits insulin-stimulated NO generation and down-regulates some aspects of insulin-mediated signalling, but, as it turns out, not as a result of reduced Akt-mediated eNOS phosphorylation at Ser1177. O-lined glycosylation of cell proteins may play a major role in mediating the effects of elevated glucose on cell signalling (Whelan & Hart, 2003). A short 6-min exposure to elevated glucose in healthy young males has been found to activate platelet eNOS via a PKCbeta mediated process that could be mimicked by mannitol, thus suggesting an osmotic mechanism leading to elevations of platelet cGMP and cAMP. These data could at least partially explain the rapid vasodilatation effects of elevated glucose that are reported in vivo (van Veen et al. 1999), but, at the same time, reflect the complexities in determining the effects of hyperglycaemia on vascular function (Fleming 2005). © 2005 NRC Canada

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What is next? If frequent episodes of acute hyperglycaemia are responsible for ED and contribute to the development of insulin resistance, then clearly the specific cellular pathways remain unclear and additional research is required. Both clinical and animal studies support the viewpoint that acute hyperglycaemia can induce ED, but what determines the vulnerability of the endothelium and to what extent is the damage reversible? Presumably normal postprandial periods of glucose elevation should not produce permanent dysfunction of the endothelium. The initial response to acute hyperglycaemia may be to enhance insulin-mediated vasodilatation, but that is transient and can be readily compromised possibly because glucose levels rise too fast or remain elevated for too long; how fast is too fast and how long is too long remain to be defined. Does elevated ROS indirectly affect the generation of NO by altering the activation of eNOS (perhaps via endothelial cell depolarization and reducing the driving force for calcium entry into the endothelial cell)? Although considerable evidence indicates that the bioavailability of endothelium-derived NO is reduced in diabetes, the role of EDHF remains obscure. A better understanding of the cellular basis for EDHF in the microvasculature as well as studies of the EDHF pathway in disease states are required. Furthermore, is an elevated insulin level protective and, if so, in the presence of elevated insulin as in insulin resistance, why is eNOS uncoupled? Does platelet eNOS become uncoupled in diabetes? Is PKC activation involved in mediating the immediate effects of acute hyperglycaemia on endothelial function and, if so, which isoforms are important and what are their substrates in the endothelium?

Acknowledgements Professor Ed E. Daniel was Professor and the Chair of the Department of Pharmacology at the University of Alberta during the period when Chris Triggle was a graduate student in his department. Chris will always be indebted to Ed for his introductions to the discipline of pharmacology, to smooth muscle research, and for the advice to always critically review the literature and never, necessarily, accept what is written until you have explored all other appropriate possibilities.

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