Insulin resistance, lipotoxicity and endothelial ...

Biochimica et Biophysica Acta 1801 (2010) 320–326

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Review

Insulin resistance, lipotoxicity and endothelial dysfunction Helen Imrie ⁎, Afroze Abbas, Mark Kearney Division of Cardiovascular and Diabetes Research, Leeds Multidisciplinary Cardiovascular Research Centre, The LIGHT Laboratories, Clarendon Way, Leeds, LS29JT, UK

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Article history: Received 6 July 2009 Received in revised form 17 September 2009 Accepted 30 September 2009 Available online 8 October 2009 Keywords: Nitric oxide Reactive oxygen species Free fatty acid

a b s t r a c t The number of people with the insulin-resistant conditions of type 2 diabetes mellitus (T2DM) and obesity has reached epidemic proportions worldwide. Eighty percent of people with T2DM will die from the complications of cardiovascular atherosclerosis. Insulin resistance is characterised by endothelial dysfunction, which is a pivotal step in the initiation/progression of atherosclerosis. A hallmark of endothelial dysfunction is an unfavourable imbalance between the bioavailability of the antiatherosclerotic signalling molecule nitric oxide (NO) and proatherosclerotic reactive oxygen species. In this review we discuss the mechanisms linking insulin resistance to endothelial dysfunction, with a particular emphasis on a potential role for a toxic effect of free fatty acids on endothelial cell homeostasis. © 2009 Elsevier B.V. All rights reserved.

1. Background Obesity and type 2 diabetes are major risk factors for the development of cardiovascular atherosclerosis and its complications. The number of people with type 2 diabetes mellitus (T2DM) and obesity (a major risk factor for the development of T2DM) has reached epidemic proportions worldwide. Eighty percent of people with T2DM will die from the complications of cardiovascular atherosclerosis resulting in an increased risk of death equivalent to 15 years of aging [1]. A recent study showed that obesity per se is associated with an increased risk of first acute myocardial infarction (AMI) equivalent to over 10 years of aging [2]. We recently demonstrated that despite the use of contemporary secondary prevention therapies patients with: 1) impaired glucose tolerance, 2) de novo and 3) existing T2DM, sustaining an AMI have not benefited from the improvement in mortality seen in similar patients without T2DM [3-6]. Resistance to the action of insulin in its classical target tissues (liver, muscle, fat) is a hallmark and key pathophysiological feature of obesity and T2DM [7]. Whole body insulin resistance is associated with hyperinsulinaemia, hyperglycaemia, hypertension, hypercoagulability and dyslipidaemia, all of which predispose to atherosclerosis (for review see 7). Several studies support a role for systemic insulin resistance in the development of premature atherosclerosis independent of T2DM and obesity [8-10]. As a result a substantial proportion of patients presenting with T2DM already have established atherosclerosis [11]. In this review we will discuss mechanisms underlying the accelerated atherosclerosis characteristic of the insulin resistanceassociated conditions of T2DM and obesity, with a particular focus on the role of reduced nitric oxide (NO) bioavailability, reactive oxygen

⁎ Corresponding author. E-mail address: [email protected] (H. Imrie). 1388-1981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2009.09.025

species and their source and a potential lipotoxic role for elevated free fatty acids (FFA). 2. Atherosclerosis and nitric oxide bioavailability Atherosclerosis is a disease of arterial lipid deposition characterised by a portfolio of biological responses central to which is a macrophage dominated oxidative/inflammatory reaction [12]. Pathological studies have demonstrated a defined series of changes in the vessel wall during atherogenesis [13]. There is now compelling evidence that very early in the atherogenic process before the onset of morphological changes there are subtle alterations in endothelial cell phenotype, now frequently described as endothelial dysfunction, arguably the most crucial aspect of which is a decline in the bioavailability of NO. This occurs as a result of reduced biosynthesis of NO and/or increased degradation by reactive oxygen species (ROS). NO has vasorelaxant, [14] anti-inflammatory, [15] antiproliferative, [16] antioxidant, [17] and antiplatelet actions [18]. Consonant with this, longitudinal studies have shown that impaired NO-dependent vasodilatation can predict future cardiac events [19] and the development of coronary artery disease (CAD: [20]). 3. Endothelial dysfunction and nitric oxide The vascular endothelium, traditionally regarded as an inactive barrier between tissue and circulating blood, is now recognised as an active site controlling the release of vasoactive molecules with the essential role of maintaining vascular homeostasis (for review see 7). The diverse actions of endothelial cells include: regulation of vessel integrity, vascular growth and remodelling, tissue growth and metabolism, immune responses, blood fluidity, platelet and white cell aggregation and vascular permeability. In the healthy endothelium, vascular homeostasis is maintained by a critical balance

H. Imrie et al. / Biochimica et Biophysica Acta 1801 (2010) 320–326

between secretion of vasodilators, of which NO is key, and vasoconstrictors (endothelin-1 (ET-1), angiotensin II and reactive oxygen species (ROS)). Subsequent diffusion of endothelial cellderived NO to vascular smooth muscle cells induces vasorelaxation. An imbalance between these secreted vasoactive molecules results in endothelial dysfunction characterised by a decrease in the synthesis of NO or increased ROS production. Decreased NO bioavailability as discussed above promotes a range of proatherosclerotic pathogenic events including vasoconstriction, leukocyte adherence, platelet activation, mitogenesis, oxidation, thrombosis, impaired coagulation and vascular inflammation.

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sclerotic endothelial cell phenotype as evidenced by impaired insulin, acetylcholine and calcium ionophore mediated NO release and an increase in endothelial cell NADPH oxidase-derived ROS. Kahn and colleagues have described an insulin-resistant model of endothelium targeted deficiency of insulin receptors (VENIRKO) [37]. Although endothelial cell function and NO bioavailability were not addressed in the study downregulation of total endothelial (e) NOS mRNA expression was observed. This is in contrast to data from our model but can potentially be explained by fundamental differences in the conditional knockout vs. mutant receptor overexpression transgenic approach or may be an age-related effect as VENIRKO mice when studied were substantially older than the ESMIRO mice in our study.

4. Insulin resistance and nitric oxide bioavailability A potentially important pathophysiological reciprocal relationship between insulin sensitivity and NO bioavailability has emerged over the last 10 years (for review see [21]). We have used complementary studies in humans and gene modified mice to explore the mechanisms underpinning this relationship in more detail. 4.1. Studies in humans In insulin-resistant young Asian men and healthy insulin sensitive white European men we examined basal NO production and brachial artery flow mediated vasodilatation (FMD: [22]). Asian men had reduced forearm resistance vessel vasoconstriction in response to the nitric oxide synthase (NOS) inhibitor L-NMMA (indicative of reduced basal NO) and a lower FMD than white Europeans. In healthy subjects [23], those with the metabolic syndrome [24], and severely obese subjects [25] we have demonstrated a strong inverse correlation between insulin resistance and FMD. A range of other studies performed by our group and others have provided compelling evidence supporting a strong association between insulin resistance and NO bioavailability in humans ([26-31] and [32] for review). 4.2. Studies in gene modified mice While there is a strong association between insulin resistance and reduced NO bioavailability definitive evidence for a cause and effect relationship has been lacking. To dissect the temporospatial relationship between global insulin resistance and NO bioavailability we, and others, have used a number of murine models. In a diet-induced model of obesity we demonstrated that insulin-mediated NO release is blunted early in the development of obesity [33]. Moreover, we demonstrated that this was associated with increased endothelial cell-derived ROS. In a non-obese model of whole body insulin resistance (mice with haploinsufficiency of the insulin receptor: IRKO) we demonstrated reduced basal and insulin-mediated NO release [34]. In this model of mild insulin resistance acetylcholine (endothelium dependent see below for details) mediated vasodilatation was preserved. As IRKO mice reached adulthood however, they developed blunting of acetylcholine-mediated vasorelaxation in aortae at least in part due to increased endothelial cell NADPH oxidase-derived ROS [35] While these studies demonstrate that insulin resistance at a whole body level influences vascular insulin sensitivity and both models are consistent with the paradigm of insulin resistance in the vasculature being a progressive disorder associated with vascular oxidative stress, they do not clearly establish whether endothelium specific insulin resistance per se impacts on endothelial function. To address this question and circumvent the problem of activation of different compensatory mechanisms seen in models of whole body insulin resistance we generated a mouse with endothelial cell specific expression of a dominant negative mutant human insulin receptor (ESMIRO) [36]. This allowed us to demonstrate for the first time that insulin resistance restricted to the endothelium leads to a proathero-

5. Potential mechanisms linking reduced nitric oxide bioavailability and insulin resistance NO production is catalysed by 3 isoforms of nitric oxide synthase (NOS) [38]. Of these, endothelial (e)NOS (NOS 3) and neuronal (n) NOS (NOS 1) are constitutively expressed and synthesise small amounts of NO basally with an increase upon stimulation. Inducible (i)NOS (NOS 2) is transcriptionally regulated and absent from most cells. Its expression is dependent upon stimulation by inflammatory cytokines with a capacity to produce up to 1000× more NO than eNOS. Under physiological conditions NO is synthesised in endothelial cells from L-arginine in a reaction that requires oxygen, NADPH and the essential co-factors tetrahydrobiopterin (BH4), FAD and FMN. NO production and bioavailability are regulated/dysregulated at several levels. 5.1. Expression of eNOS Although eNOS is constitutively expressed, in vivo and in vitro studies have established that eNOS expression is subject to regulation by several factors, including insulin [39], shear stress [40] and cytokines [41]. Consistent with a role for insulin to upregulate eNOS transcription, we have demonstrated that non-diabetic mice with hyperinsulinaemia within the physiological range have increased eNOS mRNA in aortic tissue and functional evidence of increased NO production [42]. 5.2. Abnormalities of agonist-mediated NO release The activation of eNOS occurs through a number of signalling pathways. Classical activation of eNOS (e.g. by acetylcholine) involves a rise in intracellular Ca2+ and binding of Ca2+/calmodulin to the enzyme. More recently, a Ca2+-independent regulatory pathway for eNOS has been described [43]. Shear stress and agonists such as insulin have been shown to increase endothelial NO production via activation of PI3-K and protein kinase-B (PKB/AKT), which phosphorylates eNOS. In a series of in vitro studies Quon et al. dissected the pathway by which insulin stimulates NO release from endothelial cells [44,45]. These studies demonstrated important roles for eNOS, PI3-K, AKT and the insulin receptor (IR) in insulin-mediated NO release. Consistent with these findings we have demonstrated that insulin has a significant vasodepressor action in healthy young [46] and elderly humans [47]. In lean and obese mice we have shown using a number of bioassays that insulin-mediated eNOS activation is endothelium, eNOS, PI3K and insulin receptor dependent [48,49]. More recently, the notion of disrupted insulin signalling, via impaired PI3K/Akt/eNOS phosphorylation, as the major determinant of decreased NO and endothelial dysfunction has been challenged. In a mouse model of diet-induced obesity Symons et al. reported abolition of insulinstimulated eNOS phosphorylation yet preserved upstream Akt signalling [50]. The suggestion that the defect may lie at the level of eNOS dysfunction comes from evidence of diminution of eNOS dimers in arteries of obese/diabetic mice [51]. An associated increase in levels

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of peroxynitrite was attributed to this eNOS dimer disruption. It follows that vascular dysfunction may occur via an alternative mechanism to defective IR signalling, involving FFA accumulation and increased ROS production. 5.3. Inactivation of NO by reactive oxygen species Oxidative stress is thought to play a pivotal role in the pathophysiology of atherosclerosis [52] and insulin resistance [53]. ROS are thought to promote atherosclerosis through a number of different mechanisms including but not limited to; enhanced oxidation of lipoproteins, activation of pro-inflammatory genes, alteration of vascular smooth muscle cell phenotype, and possibly most importantly reduction of NO bioavailability. The principal focus of this review will be superoxide as this appears to be most important in the development of atherosclerosis. There are several mechanisms by which NO bioavailability may be reduced by superoxide: 1) by rapidly reacting with NO to produce the potent oxidant peroxynitrite (ONOO). 2) Superoxide may modify the NO synthase co-factor BH4 leading to dysfunctional eNOS. 3) Superoxide can modify endothelial NO production by oxidation of the zinc-thiollate centre of eNOS. 4) Superoxide can lead to post translational modification of eNOS by oxidation of methionine in calmodulin which may negatively affect eNOS activity. 5) Superoxide by simply combining with NO may decrease its bioavailability. 5.4. Sources of ROS Potential sources of ROS in the arterial wall include: 1) xanthine dehydrogenase, 2) the mitochondrial electron transfer chain, 3) cytochrome p450 based enzymes, 4) infiltrating inflammatory cells, 5) NOSs, which under normal conditions generate NO, can instead generate O.2− in the setting of deficiency of the essential co-factor BH4 (for review see [54]). 6) A major source of ROS to emerge over the last 10 years is NADPH oxidase [55] for review). NADPH oxidase was originally identified in phagocytes where it exists as a multisubunit complex consisting of a membrane bound cytochrome b558 (made up of subunits p22 phox and gp91phox), which is the major component responsible for enzyme stability and activity, and at least four cytosolic subunits (p47phox, p67phox, p40phox and Rac 1), which translocate to the membrane upon activation. It is now clear that the NADPH oxidases are a family of enzyme complexes with each member or isoform being distinguished by the membrane spanning catalytic NOX or Duox subunit that transfers electrons from NADPH to molecular oxygen termed NOX1-NOX5 where gp91phox is renamed NOX2. NOX1, NOX2 and NOX4 are the principal isoforms expressed in the cardiovascular system with NOX3 and NOX5 being predominantly expressed in the inner ear and lymphoid tissue respectively. The expression profiles of NOX1, NOX2 and NOX4 differ between cell types: NOX2 and NOX4 are expressed in endothelial cells whereas NOX1 is expressed principally in vascular smooth muscle cells [56]. Our group and others have shown that NOX4 expression far exceeds NOX2 expression in endothelial cells ([36] and for review see [57]). Although the expression levels of NOX4 (at least at an mRNA level) appear to be substantially higher than NOX2 this may not necessarily reflect the relative contributions of the different isoforms to ROS production in the endothelium, under basal or pathophysiological conditions. For example Selemidis et al. [58] showed that downregulation of NOX2 in human endothelial cells by 50% reduces O.-2 generation by 50% whereas a similar reduction in NOX4 expression reduced O.-2 by only 25%. A possible explanation for this is that NOX4 principally generates H2O2 whereas NOX2 generates superoxide. Transgenic mice with endothelium specific expression of NOX2 or NOX4 under direction of the Tie-2 promoter-enhancer provide evidence to support this hypothesis. Mice overexpressing human

NOX2 in the endothelium have significantly elevated basal superoxide [59] whereas mice overexpressing NOX4 in the endothelium have enhanced H2O2 production and relative hypotension compared to wild type littermates [60]. Data from studies in mice with whole body deletion of NOX2 support the hypothesis that NOX2 NADPH oxidase may be an important determinant of the increased ROS seen in insulin-resistant mice. NOX2 deficient mice have enhanced vasorelaxation to acetylcholine [61] and are protected against the oxidative stress associated with renovascular hypertension [62].

5.5. Kindling radicals During normal physiology NOX2 and NOX4 dependent NADPH oxidases are thought to generate low levels of ROS in a highly regulated fashion for use in redox dependent signalling ([63] for review). Under these conditions eNOS and xanthine dehydrogenase perform their normal catalytic roles of NO production and purine metabolism respectively. When generation of ROS from NOX2 and possibly NOX4 NADPH oxidases exceeds their homeostatic threshold in endothelial cells antioxidant defences are unable to deal with the excess ROS. Superoxide then reacts with NO to form ONOO which through oxidation of BH4 and of critical cysteine residues on xanthine dehydrogenase, ONOO converts these enzymes into ROS generating systems (for review see 57). Moreover, NADPH oxidase-derived ROS can impact the major cellular source of ROS—the mitochondria, to enhance superoxide production from this organelle [64]. As a result of this complex interplay between sources of ROS a vicious cycle can ensue amplifying endothelial ROS production, reducing NO bioavailability and generating an increasingly proatherosclerotic environment. As discussed above the majority of ROS production in mammals is thought to come from mitochondrial electron transport. Studies from the laboratory of Clay Semenkovich [65] examined the role of uncoupling protein 1 (UCP-1) in vascular smooth muscle cells (VSMC) in ROS production and atherosclerosis. Using a mouse with inducible transgenic overexpression of UCP-1 specific to VSMC (using SM22α a VSMC specific promoter) they demonstrated that UCP-1 generated ROS from VSMC leads to elevated ROS, reduced NO bioavailability and accelerated atherosclerosis in mice on an ApoE deficient background. The role of mitochondrial dysfunction and ROS in diabetic related complications in the endothelium has been extensively examined in Michael Brownlees' laboratory. Brownlees' group demonstrated that free fatty acids at pathophysiological concentrations increased ROS production in macrovascular endothelial cells [66]. In contrast to the findings of Semenkovich overexpression of UCP-1 blunted free fatty acid induced ROS production. Suggesting that overexpression of UCP-1 in the endothelium may afford protection against diabetes related endothelial cell dysfunction. It is possible that there are cell specific effects of the UCPs a possibility that warrants future studies.

6. Oxidative stress, NADPH oxidase and insulin resistance In lean mice with whole body [35] and endothelium specific [36] insulin resistance we have shown the presence of reduced NO bioavailability and significant vascular oxidative stress, the principal source of which was endothelial cell NADPH oxidase (Fig. 1). NADPH oxidase has recently emerged as a major source of superoxide in obese humans [67], those with the metabolic syndrome [68], patients with type 2 diabetes [69] and as we demonstrated recently, humans with chronic heart failure [70]. In experimental models, and, consistent with findings in humans, NADPH oxidase has been shown to be a key source of ROS in adipose tissue of obese mice [71].


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Fig. 1. (A) Acetylcholine (Ach) relaxation responses in 6-month-old mice heterozygous for a knockout of the insulin receptor (IRKO) mice and wild type littermates (WT). (B) Restored Ach responses by superoxide dismutase mimetic MnTMPyP. (C) NADPH dependent superoxide production in IRKO and WT. (D) NOX2 and NOX4 expression in endothelial cells from IRKO and WT. (E) Ach relaxation responses in mice with endothelium specific expression of mutant insulin receptors which have a dominant negative effect (ESMIRO) mice and WT. (F) Restored Ach responses in ESMIRO by MnTMPyP. (G) NADPH dependent superoxide production in ESMIRO and WT. (H) NOX2 and NOX4 expression in endothelial cells from ESMIRO and WT. Data taken from [35] and [36].

7. Insulin resistance, free fatty acids and oxidative stress: a complex collection of interlocking vicious cycles

connection may promote the development of a vicious cycle of worsening whole body insulin resistance.

A number of studies have shown that discrete steps in triglyceride and FFA handling determine systemic patterns of metabolism, inflammation and atherosclerosis (for review see [72]). Insulin resistance at a whole body level leads to elevated concentrations of FFA and, as a result, elevated FFA in the circulation are common in obesity [28] and T2DM [73]. Increased FFAs are also intimately related to hypertriglyceridaemia and abnormal accumulation of triglyceride in skeletal muscle and liver. FFA themselves are also thought to promote insulin resistance; while the mechanisms are unclear this

8. Free fatty acids and atherogenic dyslipidaemia Obesity and T2DM are associated with atherogenic dyslipidaemia which manifests as an increase in plasma triglycerides, a reduction in high-density lipoproteins (HDL)–cholesterol concentrations and the presence of small, dense low-density lipoprotein (LDL) particles. A significant early event in the progression of atherosclerosis is the permeation of small LDL particles across the endothelial barrier and subsequent accumulation in the vascular wall [74]. Phospholipids

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role in endothelial inflammation, insulin resistance and NO bioavailability [82]. In insulin-resistant murine models FFA-induced ROS production substantially inhibited prostacyclin synthase and eNOS activity in aortae [66]. The interlocking effects of FFA on insulin resistance and ROS may lead to a vicious cycle of lipotoxicity induced vascular dysfunction (see Fig. 2). 10. The endothelium and free fatty acid metabolism A further level of complexity is added by the recent report that supports a role for the endothelium in FFA handling. Elegant work from the Plutzky lab [83], showed that obese mice lacking endothelial cell PPAR-γ have markedly elevated FFA. Surprisingly these mice were not insulin-resistant and had decreased adiposity in response to a high-fat high calorie diet. The role of the endothelium per se in glucose and metabolic homeostasis remains an open question. 11. Conclusions

Fig. 2. Vicious cycle of lipotoxicity induced vascular dysfunction. The reciprocal effects of free fatty acids (ffa), insulin resistance, reactive oxygen species (ROS) and inflammation result in decreased nitric oxide (NO) production and ultimately endothelial dysfunction.

contained within LDLs are highly susceptible to oxidation by ROS to generate oxidized phospholipids (OxPLs), the formation of which triggers endothelial cell activation to facilitate monocyte binding and stimulate expression of inflammatory cytokines [75].

Insulin resistance associated with T2DM, obesity and atherosclerosis is a global health problem. Insulin resistance is characterised by a deleterious proatherosclerotic change in endothelial cell phenotype, central to which is reduced NO bioavailability. While an association between T2DM/obesity and reduced NO bioavailability is well established, the mechanisms underlying this relationship remain unclear and as a result therapeutic targets are limited. A portfolio of abnormalities of metabolic and vascular homeostasis accompanies T2DM and obesity and these are thought to conspire to lead to accelerated atherosclerosis and premature death. Elevated FFA are part of this portfolio of abnormalities and whether targeting FFA metabolism directly can impact on cardiovascular events warrants attention.

9. Free fatty acids, reactive oxygen species and NO bioavailability Acknowledgements It is well established that elevated FFA increase ROS with a range of studies showing elevated FFA may activate NADPH oxidase and the mitochondrial electron chain to generate superoxide (for review see [76]). As discussed, excessive ROS production from one source can lead to enhanced production from another. FFA may also have direct effects on NO biosynthesis (for review see 32). In human studies, short-term elevation of FFA (by infusion) significantly blunts NO-dependent basal leg blood flow and methacholine-induced vasodilation. With increased duration of FFA elevation, impaired insulin-mediated vasodilation develops in healthy individuals [66]. The observation that increased FFA levels impair basal and insulin-mediated endothelial NO production in a dose and time dependent manner has been further confirmed in vitro [77]. Incubation of bovine aortic endothelial cells (BAECs) with the saturated FFAs palmitate/oleic acid results in reduced basal and insulin-induced eNOS phosphorylation, blunted NO production [50], and an increase in ROS via increased vascular NADPH oxidase expression [78] and as discussed above mitochondrial uncoupling [79]. Activation of the transcription factor NFkB signalling pathway, via the serine kinase IKKB, has been demonstrated in human and bovine endothelial cells upon palmitate treatment and subsequent insulin stimulation. This regulator of the inflammatory pathway may disrupt insulin signalling via inhibition of IRS-1, Akt and eNOS phosphorylation and hence leading to decreased NO production, perhaps linking inflammation and endothelial dysfunction [80,81]. Further evidence from in vitro and in vivo studies implicates the inflammatory mediator toll-like receptor-4 (TLR4) signalling in IKKB activation and impaired endothelial insulin signalling. TLR4 siRNA treatment of HMECs incubated with palmitate protected against disruption of insulin signalling, supported by observations in TLR4 knockout mice. In a state of FFA excess TLR4 may have an important

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