Cardiovascular Research 47 (2000) 457464 www.elsevier.com / locate / cardiores www.elsevier.

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Review

Antioxidants, diabetes and endothelial dysfunction

D.W. Laight*, M.J. Carrier, E.E. Anggard
The William Harvey Research Institute, St. Bartholomew s and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1 6 BQ , UK Received 3 January 2000; accepted 8 February 2000

Abstract While a damaged endothelium is recognised to be a key accessory to diabetic macroangiopathy, awareness is developing that impairments concerning endothelium- and nitric oxide (NO)-dependent microvascular function, may contribute to several other corollaries of diabetes, such as hypertension, dyslipidaemia and in vivo insulin resistance. There are now several reports describing elevations in specic oxidant stress markers in both insulin resistance syndrome (IRS) and diabetes, together with determinations of reduced total antioxidant defence and depletions in individual antioxidants. Such a pro-oxidant environment in diabetes may disrupt endothelial function through the inactivation of NO, resulting in the attenuation of a fundamental anti-atherogenic and euglycaemic vascular inuence. Indeed, experimental and clinical data suggest that the supplementation of insulin resistant or diabetic states with antioxidants such as vitamin E, normalises oxidant stress and improves both endothelium-dependent vasodilation and insulin sensitivity. However, the promising potential efcacy of antioxidant therapy in cardiovascular disease and diabetes, in either a primary or secondary preventative role, awaits denitive clinical demonstration. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Blood ow; Diabetes; Endothelial function; Free radicals; Nitric oxide
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1. Introduction Insulin resistance syndrome (IRS) or metabolic syndrome X [1,2], is characterised by a group of metabolic and haemostatic abnormalities, most of which represent independent risk factors for the development of type II diabetes (see Fig. 1). These include impaired glucose tolerance, hyperinsulinaemia, hypertension, dyslipidaemia, a pro-thrombotic / hypo-brinolytic state, oxidant stress and endothelial dysfunction [13]. This cluster also generates an increased risk of macroangiopathy in both diabetes and the prediabetic state represented by IRS, resulting principally from atherosclerotic and thrombotic pathologies [4]. Such macroangiopathy is often present at the diagnosis of type II diabetes [5] and the associated coronary artery,

cerebrovascular and peripheral vascular disease are leading causes of diabetic morbidity and mortality. Given the central pathogenic role of the dysfunctional endothelium in the atherosclerosis of large and mediumsized arteries [6,7], it is increasingly clear that endothelial cells are the ultimate target of pro-atherogenic mediators in IRS and diabetes such as oxidant stress [79]. Furthermore, a more disseminated endothelial dysfunction possibly mediated by reactive oxygen species (ROS), for example in resistance and microvascular beds, may even underlie the parallel development of some of the principal facets of IRS such as hypertension, insulin resistance, dyslipidaemia and altered haemostasis [10]. This notion is lent credence by ndings that in common with atherosclerosis, endotheliopathy is an early abnormality and precedes the development of type II diabetes [11]. The pathological role of the endothelium as both a target and mediator of diabetic macroangiopathy, has been the

*Corresponding author. Tel.: 144-171-982-6037; fax: 144-171-9826016. E-mail address: d.w.laight@mds.qmw.ac.uk (D.W. Laight).

Time for primary review 25 days.

0008-6363 / 00 / $ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0008-6363( 00 )00054-7

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insulin resistance in the development of essential hypertension. However, the role of this phenomenon in the maintenance of hypertension is less clear since, while the endothelial NO pathway is thought to be defective in this condition [15,24], insulin may still be able to potentiate endothelium-dependent vasodilation by what appears to be a compensatory, hyperpolarising mechanism in established essential hypertension [15]. Of course, endothelium-derived endothelin-1 and angiotensin II, two powerful vasoconstrictor and pro-mitogenic agents, are also clearly implicated in the progression of endothelial dysfunction and hypertension in diabetes [2530].

3. Endothelial dysfunction and in vivo insulin resistance

3.1. Blood ow and insulin-mediated glucose disposal

Fig. 1. Hypothetical scheme linking oxidant stress, endothelial dysfunction and insulin resistance in the setting of type II diabetes.

subject of a recent review by us [12]. The present review examines how endothelial dysfunction associated with oxidant stress, may help generate a number of risk factors for diabetic macroangiopathy, including vascular and metabolic insulin resistance, before considering evidence of an altered antioxidant status in diabetes and the potential of antioxidant therapy.

2. Endothelial dysfunction, vascular insensitivity to insulin and hypertension

2.1. Vasodilator activity of insulin

Insulin per se elicits nitric oxide (NO)-dependent vasodilation in human skeletal muscle [13,14], possibly by enhancing agonist-stimulated endothelial vasodilator function [15,16]. This vasodilator action of insulin can be shown to be blunted in insulin resistant states such as obesity, hypertension and type II diabetes [1719]. Moreover, the recent report by Tack et al. [20] that impaired insulin-mediated endothelium-dependent vasodilation in obesity may be independent of frank endothelial vasodilator dysfunction, raises the fascinating possibility of a discrete vascular insensitivity to insulin or vascular insulin resistance at the level of the endothelium [21]. Such a defect in the putative tonic modulation of endothelial vasodilator function in resistance vessels by insulin, has already been suggested to contribute to hypertension in a genetic model of insulin resistance, the obese Zucker rat [22]. Studies in insulin resistant, hypertensive patients and spontaneously hypertensive rats by Lembo et al. [23] support a causative or permissive role of such vascular

Endothelial vasodilator dysfunction at the level of metabolically important arterioles such as those of skeletal muscle, may not only precede but also play a role in the development of IRS [10,31]. This is because a decit in endothelium-dependent vasodilation described in insulin resistant states including type II diabetes [19,3234], may impair the postprandial increase in blood ow in insulinsensitive tissues such as skeletal muscle, now considered to be a signicant determinant of glucose disposal, at least in insulin-sensitive individuals [17,3537]. This could explain the relationship between insulin sensitivity and NO-mediated, endothelial vasodilator function reported in normal subjects [3739], although such a link is not a universal nding [40]. Furthermore, as mentioned above, insulin is itself an endothelium-dependent vasodilator and this vascular effect in insulin-sensitive tissue is diminished in insulin resistant states [1719]. Indeed, impaired insulin-stimulated NO-mediated vasodilation in skeletal muscle has been accorded a major role in the pathogenesis of the in vivo insulin resistance associated with type I diabetes [41]. Furthermore, a defective insulin-mediated, endothelium-dependent regulation of blood ow in adipose tissue has itself been suggested to pose a signicant cardiovascular risk, since this may contribute to postprandial hyperlipidaemia [42].

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3.2. Role of endothelial transcytosis of insulin

Another aspect of endothelial dysfunction to consider in insulin resistant individuals is the delay in the endothelial transport of insulin to the interstitial space [35,43] which, although probably insufcient to account for poor insulin action under steady-state conditions [44], may nonetheless impose a rate-limiting step in glucose disposal in response to rapidly changing blood-borne insulin levels [45].

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3.3. Signicance of endothelial dysfunction to in vivo insulin resistance

Defects in both metabolically relevant endothelial vasoactive and transport function coupled with a reported reduction in skeletal muscle capillarisation in insulin resistant individuals [46], may all contribute to in vivo insulin resistance in man [47] and thus conceivably promote hyperglycaemia and hyperinsulinaemia and generate an enhanced cardiovascular risk. However, despite the identication of defects in endothelium-dependent vasodilation and the adverse implications for glycaemic control in insulin resistant states, reports in type II diabetic patients so far indicate that simply normalising skeletal muscle blood ow does not overcome established in vivo insulin resistance [39,48,49]. While this may not seem surprising in view of the fact that insulin resistance predominantly concerns a cellular defect in insulin-stimulated glucose uptake [50], it is important to distinguish between nutritive and non-nutritive microvascular ow in such studies; since only a rise in the former, which indeed may occur independently of a change in total blood ow, can be expected to enhance glucose and insulin delivery and therefore facilitate glucose disposal in insulin-sensitive tissue such as skeletal muscle [35].

vulnerability to an environmental pro-oxidative insult in established insulin resistance. Although the cellular source(s) of ROS is still unknown in such studies on the relationship between oxidant stress and insulin resistance, it is clear that endothelium-derived ROS potentially make a key contribution to poor insulin action at metabolically relevant sites (see above). Indeed, part of this contribution could concern a ROS-mediated reduction in trans-endothelial insulin transport [63]. Furthermore, the rapid inactivation of the endothelium-derived nitrovasodilator NO by vascular superoxide anion [6466], may account for the impaired endothelium-dependent vasodilation observed in type II diabetic patients [6769] and therefore contribute to a haemodynamic component of in vivo insulin resistance as well as more directly promoting atherogenesis [12]. It is interesting to note in this regard that our novel pro-oxidant model of type II diabetes in the obese Zucker rat [62], is also associated with evidence of a lesion in the NO signalling pathway in vivo (unpublished observation).
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4.2. ROS and dyslipidaemia

In addition to the inactivation of NO, a ROS-mediated loss of glycosaminoglycan may lead to reductions in capillary endothelium-bound lipoprotein lipase action and thereby help to account for the prole of plasma hypertriglyceridaemia and reduced high density lipoprotein levels in IRS and type II diabetes [10]. Furthermore, in addition to encouraging platelet aggregation by limiting the release of prostacyclin from the endothelium, dyslipidaemia and in particular elevated levels of very low density lipoprotein, contributes to the generation of a pro-thrombotic environment via an increase in circulating levels of the anti-brinolytic factor plasminogen activator inhibitor 1, derived from the endothelium [3]. In addition, the ability of the endothelium to oxidatively modify low density lipoprotein particles [12], which are small and dense in diabetes and particularly vulnerable to oxidation [70,71], creates the environment for enhanced foam cell formation in diabetic atherogenesis [72].

4. Endothelium and oxidant stress

4.1. ROS and insulin action

Oxidant stress is associated with type II diabetes [51,52]and has recently been identied in our laboratory in the insulin resistant, obese Zucker rat [53]. While the origin(s) of this oxidant stress remains to be fully dened, it is clear that the generation of ROS including superoxide anion may be elevated in type II diabetes and even during impaired glucose tolerance [54], due in part to glucose auto-oxidation and non-enzymatic protein glycation [55]. ROS generation has also been associated with the interaction of advanced glycation endproducts (AGE) with specic receptors (RAGE), present on endothelium [56]. In addition, hyperglycaemia has been demonstrated to generate superoxide anion via the stimulation of endothelial cyclo-oxygenase activity in vitro [57]. Other potential endothelial sources of ROS include the activity of NADPH oxidase and uncoupled NO synthase activity [58,59]. Oxidant stress contributes to insulin resistance in man [60] and we have made a similar observation in the obese Zucker rat, based on the ability of a lipophilic antioxidant, vitamin E, to improve insulin action in vivo while reducing elevated plasma levels of a lipid peroxidation marker [61]. Moreover, we have very recently shown that a pro-oxidant challenge in this insulin resistant animal in vivo, provokes a further deterioration in insulin action leading to a type II diabetic state [62]. This is the rst demonstration of such a

5. Antioxidants and diabetes

5.1. Antioxidants and endothelial function in diabetes

There is a great deal of evidence to support a role for ROS in the impaired endothelium-dependent vasodilation observed in a variety of vascular preparations isolated from experimental diabetic animals [7380]. This dysfunction is thought to principally involve the reduced bioavailability of NO resulting from its rapid inactivation by superoxide radical [64,65]. Such a disruptive inuence of ROS in diabetes is supported by the demonstration of a comparable endothelial dysfunction in vitamin E-deprived rats [81,82].

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Similarly, defective endothelium-dependent vasodilation in type II diabetic subjects [3234,69] can be reversed by dietary ascorbic acid [67]. This is also the case in type I diabetics [83]. Interestingly, ascorbic acid has recently also been demonstrated to reduce blood pressure in hypertensives [84]. These benecial actions of ascorbic acid may feasibly result from an enhanced bioavailability of vascular NO but could also conceivably arise from a stimulation of endothelial NO synthesis [85]. A reduction in NO formation could conceivably arise in diabetes following hyperglycaemia-dependent pseudohypoxia, since this condition is associated with the depletion of NADPH and tetrahydrobiopterin, which act as co-factors for NO synthase activity [12,86,87]. Furthermore, another result of pseudohypoxia, diacylglycerol formation, may activate protein kinase C and lead to the stimulation of vascular oxidase systems [8688]. This, together with the production of superoxide anion by uncoupled NOS, may generate signicant sources of endothelial superoxide anion (see above). Reductions in vascular NO signalling mediated by ROS, may be accompanied in diabetes by a reduced synthesis of prostacyclin [88,89] coupled with an undiminished or even enhanced formation of vasoconstrictor agents, which is also a factor in restricted vasodilation [79,80]. In particular, hyperglycaemia leads to endothelium-derived prostaglandin G 2 and thromboxane A 2 formation [90] and in common with hyperinsulinaemia, stimulates endothelin-1 (ET-1) synthesis in endothelial cells [91]. Indeed, ET-1 and another potent vasoconstrictor and pro-mitogenic peptide, angiotensin II derived from the vascular renin angiotensin system [92,93], have been linked with the development of clinical diabetic macroangiopathy [25,94]. In this regard, the angiotensin-converting enzyme inhibitor ramipril, has very recently been demonstrated to signicantly reduce the risk of myocardial infarction, stroke and cardiovascular death in a major clinical trial involving diabetics [95]. In addition, these vasoconstrictor peptides may also play a local role in the aetiology of microangiopathy, especially in the kidney, by maintaining post-capillary resistance in the face of diabetic arteriolar vasodilation and so facilitating capillary hypertension [96]. Ironically, superoxide anion may help mediate this paradoxical arteriolar vasodilation and increase in microvascular blood ow, which is an early feature of diabetes attendant on hyperglycaemia, by enhancing the calcium signalling pathway involved in agonist-stimulated NO synthesis [97,98]. An enhanced endothelial uptake of L-arginine, the substrate for NO synthase, has also been implicated in an effect of hyperglycaemia to stimulate basal NO synthesis [88]. NO per se has important antioxidant activities in the vessel wall, including the direct scavenging of superoxide anion and the inhibition of lipid peroxidation [99101]. Indeed, the efciency of NO in its antioxidant role, may make basal endothelium-dependent, NO-mediated vasodi-

lator function especially vulnerable to inhibition during an oxidative insult. Conversely, the loss of basal endotheliumderived NO in this manner, may also serve to help preserve vasodilator responses to endothelial agonists and NO donors [102]. However, any lesion in the endogenous NO signalling pathway has major pathological implications for macroangiographic disease progression in diabetes, resulting from a dysregulation of vascular tone, proliferation, platelet aggregation, coagulation, brinolysis, leukocyte adhesion, vascular permeability and lipoprotein oxidation [12]. Many of these pro-atherogenic sequelae may be triggered by hyperglycaemia via the formation of AGE, which been shown to inactivate NO and generate endothelial intracellular oxidant stress [9,12,56].

5.2. Antioxidant status in diabetes

There are several lines of evidence to suggest that antioxidant defences may be lower in diabetes. These include reports of reduced plasma / serum total antioxidant status or free radical scavenging activity and increased plasma oxidisability in type II diabetics, together with demonstrations of reduced levels of specic antioxidants such as ascorbic acid and vitamin E [54,103109]. In addition, the activities of the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase, have been described as reduced in diabetics [34,109,110]. A diminution in the endothelial synthesis of NO has also been suggested in type II diabetics [69], which apart from detracting from vascular antioxidant defence (see above), would of course compound any defect in the antiatherogenic signalling role of NO [12].

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5.3. The potential of antioxidant therapy

Antioxidant therapy, achieved by supplementation with pharmaceutical preparations of antioxidant nutrients and / or non-nutrients [111113], may conceivably confer both cardiovascular and metabolic benets in diabetes. This notion is well grounded in the theory surrounding the role of oxidative stress in disease [114]. In addition, it is supported by evidence of reduced antioxidant defences in diabetes and also by experimental ndings that antioxidants improve endothelium-dependent vasodilation and insulin sensitivity (see above). Indeed, epidemiological data provide strong associations between the dietary intake of antioxidant nutrients and protection against cardiovascular disease [113,115117]. Furthermore, a recent report has suggested a role for low serum carotenoid status in the development of insulin resistance and diabetes [118]. However, despite some early indications from clinical trials that vitamin E could protect against coronary artery disease [116,117,119], including that associated with diabetes [120], more recent studies with vitamin E, vitamin C and beta-carotene supplements have failed to clearly demonstrate a retardation in cardiovascular disease progression [119,121]. The reasons for these disappointing

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results are unclear; but may simply highlight the need to intervene at a suitable moment in disease progression in patients at specic risk, using the most appropriate balance of harmonised and correctly targeted antioxidants at an optimal dose and for an adequate duration [122]. A more comprehensive evaluation of the cardiovascular benets of a range of naturally occurring antioxidants, including vitamin E, vitamin C, beta-carotene and selenium as potential nutriceuticals, awaits the outcome of a number of ongoing clinical trials [119]. However, a preliminary report from one of these, the Heart Outcomes Prevention Evaluation (HOPE) study, indicates that vitamin E supplementation did not reduce the incidence of cardiovascular endpoints after 4.5 years of use [95].

6. Conclusions A multifaceted endothelial dysfunction, involving the generation of oxidant stress, is conceivably central to the

principal manifestations of IRS, including insulin resistance and associated macrovascular disease (see Fig. 2). Indeed, the parallel development of atherosclerotic, diabetic macroangiopathy and the metabolic corollaries of insulin resistance [10], can be considered as manifestations of endothelial dysfunction at distinct vascular sites. This would then account for the commonality of vascular endothelial dysfunction and relative resistance to insulinmediated glucose uptake in metabolic disorders such as type II diabetes and cardiovascular disease such as atherosclerosis, essential hypertension, congestive heart failure and cardiac syndrome X (microvascular angina) [123]. Furthermore, it is apparent that antioxidant intervention in both experimental and clinical diabetes, can reverse endothelial dysfunction which may itself be related to an insufcient antioxidant defence [34]. However, while antioxidants are proving essential tools in the investigation of oxidant stress-related diabetic pathologies and despite the obvious potential merit of a replacement style therapy, the safety and efcacy of antioxidant supplementation in any future treatment, remains to be established.
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Fig. 2. Hypothesised central role for a multifaceted, disseminated endothelial dysfunction in the development of insulin resistance syndrome, macroangiopathy and type II diabetes.

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