INTRODUCTION

Insulin resistance, impaired glucose tolerance, and overt diabetes, are associated with an increased risk of cardiovascular disease.¹ All these conditions are accompanied by the presence of oxidative stress. Oxidative stress has been proposed as the pathogenic mechanism linking insulin resistance with β -cell dysfunction and endothelium-dependent alterations, eventually leading to overt diabetes and cardiovascular disease.²

When caloric intake exceeds the energy expenditure, the substrate-induced increase in citric acid cycle activity generates an excess of mitochondrial NADH (mNADH) and reactive oxygen species (ROS). To protect themselves against harmful effects of ROS, cells may reduce the formation of ROS and/or enhance ROS removal. Prevention of ROS formation is accomplished by preventing the build-up of mNADH by inhibiting insulin-stimulated nutrient uptake and preventing the entrance of energetic substrates (pyruvate, fatty acids) into the mitochondria.³

Acetyl-CoA, derived either from glucose through pyruvate or from beta-oxidation of fatty acids, combines with oxaloacetate to form citrate, which enters the citric acid cycle and is converted to isocitrate. NAD⁺-dependent isocitrate dehydrogenase generates NADH. When excessive NADH cannot be dissipated by oxidative phosphorylation (or other mechanisms), the mitochondrial proton gradient increases and single electrons are transferred to oxygen, leading to the formation of free radicals, particularly superoxide anion.

It has been suggested that interrupting the overproduction of superoxide by the mitochondrial electron transport chain would normalize the pathways involved in the development of the oxidative stress.⁴

The implications of oxidative stress in the development of atherosclerosis and type 2 diabetes have been recently reviewed elsewhere.^2,4 This article will be focused on the role of some causative triggers of oxidative stress such as iron and cytokines.

THE ROLE OF IRON IN OXIDATIVE STRESS, INSULIN RESISTANCE, AND ATHEROSCLEROSIS

Iron is intimately linked to oxidative stress. Iron participates, through the Fenton reaction, in the formation of highly toxic free radicals, such as hydroxide and the superoxide anion, which are capable of inducing lipid peroxidation. In order for iron to act as a prooxidant agent it must be in its free form. Iron can be released from ferritin by the action of reducing agents that convert Fe³⁺ into Fe²⁺.⁵ Glycation of transferrin decreases its ability to bind ferrous iron⁶ and, by increasing the pool of free iron, stimulates ferritin synthesis. Glycated holotransferrin is additionally known to facilitate the production of free oxygen radicals, such as hydroxide, that further amplify the oxidative effects of iron.⁷

The fraction of non-used and highly toxic iron is stored as ferritin molecules in order to be neutralized. Apoferritin, the protein fraction of ferritin, is spatially folded to create a central groove that holds oxidized iron molecules [Fe³⁺]. Apoferritin is a high-molecular weight (450 kDa), multimeric protein (24 subunits of heavy and light chains) that exhibits exquisite high capacity for iron storage (4500 moles of iron per mole of ferritin). Synthesis of apoferritin is induced, at both the transcriptional and posttranscriptional levels, by the presence of free iron. The increase in Fe²⁺ downregulates the affinity of iron-regulatory element binding protein (IRE-BP) for its IRE binding site in the 5' region of ferritin mRNA leading to increased ferritin translation.

The heavy chain in the apoferritin molecule exerts ferroxidase activity, catalyzing the oxidation of Fe²⁺ into Fe³⁺, which prevents iron-induced cyclic red-ox reactions that would spread and amplify the oxidative damage. This activity occurs under aerobic conditions, allowing the storage of intracellular iron. When concentrations of antioxidants are low, the reducing potential and anaerobiosis progressively increase, facilitating a rapid release of iron from ferritin. Additionally, the ferroxidase activity in the heavy chain is downregulated in this setting, decreasing the incorporation of iron into ferritin. The overall result of oxidative reactions is an increase in the availability of free iron from the ferritin molecule as well as from other molecules undergoing degradation, such as the heme group. These events, in turn, can enhance and amplify the process of generation of free radicals, causing cellular and tissue damage. The oxidative stress also downregulates the affinity of IRE for IRE-BP. Thus, ferritin can act both as a source or iron, which induces oxidative stress, and as a mechanism that protects against iron toxicity.⁶

Hyperferritininemia is present in 6.6% of unselected patients with type 2 diabetes.⁸ Serum concentrations of ferritin are usually increased in poorly controlled type-1 and type-2 diabetic subjects and ferritin has been shown to predict HbA_1C independently of glucose,⁹ probably reflecting increased oxidative stress. Short-term improvement in glycemic control is followed by variable decreases in serum ferritin concentration.

Oxidative stress also influences both glucose and iron metabolism. Oxidative stress induces both insulin resistance--by decreasing internalization of insulin¹⁰-- and increased ferritin synthesis.

Cytokines may also cause simultaneously an increase in transferrin receptors on the cell surface, favoring tissue deposition of iron¹¹ and insulin resistance.¹²

The evidences of the iron-insulin resistance-type 2 diabetes relationship are explained in the table 1.

RECENT DEVELOPMENTS

Visfatin [also known as pre-B-cell colony-enhancing factor],¹³ is a novel adipokine that is predominantly secreted by visceral adipose tissue.¹⁴ As serum ferritin,^9,15 plasma visfatin has been found to be increased in human type 2 diabetes.¹⁶ As stated above, iron participates, through the Fenton reaction, in the formation of highly toxic free radicals, such as hydroxil radicals (OH·, from Fe and H₂O₂), which are capable of inducing lipid peroxidation.¹⁷ Other free radicals such as O₂· are formed by NADPH oxidase and mitochondrial electron transfer. In this sense, visfatin is a a nicotinamide phosphoribosyltransferase, a cytosolic enzyme involved in NAD biosynthesis.¹⁸ The maximum level of visfatin mRNA was found in the liver tissue, and the next highest amount was found in muscle tissue.¹³ These tissues are classically insulin sensitive but are also characterized to be central in iron metabolism. High expression of visfatin was also found in bone marrow, the source of circulating iron.¹³ Given this coincidence of visfatin expression in iron-rich tissues, one recent study investigated the possible interaction of visfatin with iron metabolism.¹⁹

Serum visfatin was found to be associated with different parameters of iron metabolism such as serum prohepcidin and serum soluble transferrin receptor (sTfR). These associations differed according to obesity status and glucose tolerance status. In nonobese subjects, serum sTfR concentration and prohepcidin contributed independently to visfatin variance after controlling for age and BMI. Insulin sensitivity, however, was the only factor contributing to serum visfatin when it was accounted for. In obese subjects, only sTfR contributed to visfatin variance when BMI, age, prohepcidin, insulin sensitivity and glucose tolerance status were controlled for. These findings suggest that insulin sensitivity would be the main factor influencing serum visfatin concentration across the range of insulin action observed in nonobese subjects. When insulin resistance develops (obese subjects), sTfR would turn into one of the factors influencing serum visfatin concentration.¹⁹

Serum visfatin correlated negatively with serum sTfR concentration. Subjects with altered glucose tolerance and the lowest sTfR concentration showed the highest circulating visfatin. This suggests that increased iron stores would lead to increasing visfatin synthesis. Factors related to hyperglycemia or oxidative stress can mediate in part the association between visfatin and parameters of iron metabolism (sTfR, prohepcidin) in subjects with altered glucose tolerance.¹⁹

Circulating visfatin thus appears to be linked to parameters of iron metabolism, mainly in subjects with altered glucose tolerance.¹⁹

In summary, a scenario can be envisioned in which the physiological action of insulin leads to increased uptake of different nutrients and iron. Any factor causing hyperinsulinemia (weight gain, aging, repeated usual-life infections, periodontitis) amplifies this process, determining increased deposition of iron, which in the long term worsens insulin resistance.

IRON DEPLETION AND ATHEROSCLEROSIS

The general effect of catalytic iron is to convert poorly reactive free radicals such as H₂O₂, into highly reactive ones (OH^- and O₂^-). Free radicals and other oxidation by-products are well known factors that impair the mechanisms of vasodilatation²⁰ and cause endothelial depletion of endogenous antioxidants, such as ascorbic acid.²¹ Iron chelation blocks oxidation of low-density lipoprotein and iron released from heme and ferritin favors oxidation of this lipoprotein.²² Thus, increased iron availability is, theoretically, expected to contribute to macrovascular disease because iron has an adverse effect on endothelium²³ and accelerates the development of atherosclerosis.²⁴ In fact, ferritin gene expression increases in the course of atherosclerotic plaque formation.²⁵

In subjects with hemochromatosis medium-size arteries are characterized by an eccentric hypertrophy and decreased distensibility that are partially reversible after iron depletion.²⁶ These findings seem to be linked to iron-induced fibrogenesis determining an increased total collagen content in arteries from these patients. There is also some evidence for iron-dependent growth of arterial wall tissue: iron chelation by deferoxamine inhibits vascular smooth muscle cell proliferation.²⁷

Long-term use of the modified iron chelator hydroxyethyl starch conjugated-deferoxamine prevented endothelial dysfunction associated with experimental diabetes mellitus.²⁸ In type 2 diabetic patients, coronary artery responses to cold stress testing improved substantially after deferoxamine administration.²⁹ Similarly, iron chelation was shown to ameliorate endothelial dysfunction of patients with coronary heart disease.³⁰

Improvement of nitroglycerine-induced vasodilatation was also observed following blood letting in type 2 diabetic patients. The improvement in vascular reactivity paralleled the decrease in serum transferrin saturation, total hemoglobin (markers of circulating iron) and blood glycated hemoglobin.³¹ These observations suggest that diabetic vascular dysfunction seems partially reversible and that the circulating compartment acts as a reservoir of transition metals that directly affects vascular function.³² Increased hemoglobin, an iron-enriched protein, is deleterious for endothelial function, as normal blood vessels exposed to total and glycated hemoglobin are known to experience impaired vascular relaxation (Figure 1).³³

Fig. 1. A high resolution external ultrasound of the brachial artery before (upper panel) and after (lower panel) ischemia-induced vasodilation.

The relationship between iron and atherosclerosis is, however, controversial.³⁴ Thus, some animal experimental data regarding the effect of manipulating iron stores on atherosclerosis, and human data showing improvement of vascular structure and function following iron depletion,^26,29,30,31 are both consistent with the theory that iron contributes to the development of vascular disease. However, the current epidemiological data associating iron stores with either atherosclerosis or coronary heart disease (reviewed in 34) do not fully support this hypothesis.

In summary, the impact of transition metals, in general, and iron, in particular, on human physiology has only begun to be elucidated in the last decades. Because iron is a first-line prooxidant, it contributes to regulate the clinical manifestations of numerous systemic diseases, including diabetes mellitus and atherosclerosis. Iron regulation of the cell oxidative stress can explain, at least in part, its close association with abnormalities in insulin sensitivity.

CYTOKINES, OXIDATIVE STRESS, AND CARDIOVASCULAR DISEASE

Different cytokines induce mitochondrial alterations and increased production of free radicals. Cytokines have also an important role in the endothelial injury induced by inflammation. Vascular endothelium is involved in the inflammatory response to atherosclerosis,^35-38 and changes in endothelium function could underlie the association between cardiovascular disease and inflammation.

In recent years, increasing evidence has shown how chronic inflammation is simultaneously linked to endothelial dysfunction, atherosclerosis and insulin resistance.^39,40 Endothelial dysfunction is one of the earliest abnormalities that can be detected in people at risk for cardiovascular events and it is linked to insulin resistance and type 2 diabetes.^36,37 Plasma concentrations of proinflammatory cytokines such as interleukin (IL)-18, IL-6 and tumor necrosis factor alpha (TNF-α ) and of several other inflammatory markers are increased in patients with ischemic heart disease.^39,41-43 Men who have been exposed to increased inflammation-sensitive plasma proteins have higher fatality in future coronary events even after adjustment for traditional risk factors.^41,44 Circulating cytokines are also elevated in type 2 diabetes, obesity and insulin resistance syndrome and play a central role in the pathogenesis of these disorders.³⁹

Interleukins

In particular, the release of IL-6, mainly from abdominal adipocyte sources, has been claimed to have a pivotal role in the relationship between oxidative stress and endothelial dysfunction.³ IL-6 is a mediator of the inflammatory response and it is linked to dyslipidemia, type 2 diabetes and risk of myocardial infarction.^39,45-47 IL-6 is secreted by a variety of different cell types including lymphoid and endothelial cells, fibroblasts, skeletal muscle and adipose tissue. Circulating IL-6 levels also correlate with obesity and insulin resistance, and may predict the development of type 2 diabetes.^47-50

It is difficult to ascertain whether cytokines induce endothelial injury directly or at least in part through insulin resistance-associated inflammation. Recently, an independent association between insulin resistance and vascular dysfunction has been described in 81 patients with type 2 diabetes.³⁷ Insulin resistance was accompanied by a decreased endothelium dependent vasodilation and increased low grade inflammation. In type 2 diabetes, a characteristic sensitivity of endothelium to insulin resistance has also been suggested.³⁷

In other study, a negative correlation between IL-6 levels and endothelium dependent vasodilation, which remained significant after adjusting for insulin sensitivity and other risk factors, was described in apparently healthy men.⁵¹ In this study the authors analyzed healthy men in order to minimize the confounding effect of other risk factors or treatments which could interfere in the interpretation of our results.⁵¹ The association between serum IL-6 levels and endothelium dependent vasodilation was especially significant in subjects with strictly normal fasting glucose.⁵¹ Other factors and cytokines in the context of hyperglycemia and insulin resistance may blunt the relationship between IL-6 and endothelial dysfunction when fasting glucose increases.

Endothelial and smooth muscle cells have been shown to produce IL-6.⁵² Inflammation may produce endothelial dysfunction by different mechanisms. Inflammation has the capacity to impair flow-mediated vasodilatation both by increasing vasoconstriction or reducing endothelium-derived vasodilators.⁵³ Cytokines may also induce vasoconstriction through different pathways such as induced synthesis of endothelin-1, decreased expression of endothelial nitric oxide (NO) synthase or by reducing the bioavailability of NO.⁴⁰

In the Framinghan offspring study, an inverse correlation between IL-6 concentrations and endothelial function was shown, this relationship was attenuated after adjusting for traditional risk factors.⁵⁴ The relationship between IL-6 and flow mediated vasodilation has also been described in subjects with acute coronary syndrome and hypercholesterolemia.^55,56 In these studies, despite the close relationship between metabolic syndrome and cardiovascular risk, the effect of insulin resistance on endothelium function was not assessed.

Pioglitazone has shown to improve vascular reactivity in the same manner that they reduce IL-6 levels, suggesting a role of this cytokine on the effect of insulin resistance on vascular function.⁵⁷

Another explanatory hypothesis is that a low grade inflammation (increased interleukin 6) could constitute a first hit, a first aggression that would exert a central role in the cluster inflammation/insulin resistance by inducing predominantly endothelial dysfunction. In a second, subsequent hit, the effect of insulin resistance over endothelium would turn to be more important per se, playing then the central role of the metabolic abnormalities associated with vascular dysfunction.⁵¹ Prospective studies analyzing simultaneously inflammation, insulin sensitivity and endothelial function will be necessary to better understand the underlying mechanism of these processes.

It should also be recognized that the chronic inflammatory response in the context of insulin resistance and endothelium dysfunction concern not only interleukin 6 but probably a myriad of other factors. In future studies, it will be interesting to sort out whether interleukin 6 is more important than other circulating cytokines and factors.

THE ROLE OF TUMOR-NECROSIS FACTOR- α (TNF- α )

TNF-α is a proinflammatory cytokine that is also implicated in the pathogenesis of insulin resistance and endothelium dysfunction linked to this event.^39,58 Contradictory effects of TNF-α on endothelial function have been described in different studies.^59-61 Acute intra-brachial TNF-α infusion impairs endothelium-dependent vasodilatation, but TNF-α also enhances protective mechanism.^58-61

TNF-α mediates its multiple biological activities after binding to two different membrane receptors, TNFR1 and TNFR2, activating different signalling cascades, thus mediating distinct cellular responses.⁶² After binding to these receptors, a proteolytic cleavage of the extracellular parts elicits the soluble forms, named sTNFR1 and sTNFR2.⁶³ sTNFR1 and sTNFR2 concentrations are thought to reflect previous TNF-α effects.⁶³

Shedding of TNFR1 leads to increased sTNFR1, which antagonizes TNF-α .⁶⁴ Increased sTNFR1 expression reduced TNF-α bioactivity and protected the myocardium from infarction following ischemia and reperfusion.^65,66 sTNFR1 might have other protective roles through stimulating endothelial cell growth.⁶⁷ On the other hand, sTNFR2 levels have been linked to coronary artery disease,⁶⁸ insulin resistance,⁶⁹ and hypertension.⁷⁰

Insulin resistance states are also associated with impaired sTNFR1 shedding and increased sTNFR2 shedding.^70,71 Sustained up-regulation of human TNFR2 in transgenic mice leads to a chronic accumulation of cell surface and plasma receptor,⁷² providing them the capacity to be hyper-responders to circulating TNF-α .

These anti-atherosclerotic mechanisms induced by sTNFR1, and proatherosclerotic associated to sTNFR2 are in line with recent findings.⁷³ The relationship among soluble TNF-a receptors, insulin sensitivity and vascular reactivity in subjects with normal and impaired glucose tolerance was evaluated. A positive correlation between sTNFR1 levels and endothelium-dependent vasodilatation (r=0.291, P=.02) was found in subjects with normal glucose tolerance. In multiple regression analysis, serum sTNFR1 independently contributed to endothelium-dependent vasodilatation (EDVD) in these subjects, after adjusting for age, BMI, smoking status, systolic and diastolic blood pressure and insulin sensitivity (B=0.414, P=.002).⁷³ Circulating sTNFR2 was negatively associated with insulin sensitivity (r=-0.20, P=.04) and a trend was observed with EDVD (r=-0.190, P=.058). In glucose-intolerant subjects, serum sTNFR2 levels correlated negatively with EDVD (r=-0.366, P=.047). The relationship, however, was not significant after adjusting for confounding variables.⁷³ No association was found between endothelium-independent vasodilatation and circulating sTNFR1 or sTNFR2 levels.⁷³

This study thus provided divergent relationships between circulating sTNFR levels and endothelial function. While sTNFR1 was positively associated with EDVD, opposite relationships regarding sTNFR2 were observed, mainly in subjects with glucose intolerance.⁷³ The knowledge of how these interactions occur may have therapeutic implications. In this respect, it is perplexing that inactivation of one TNFR1 in mice increases atherosclerosis.⁷⁴ Inactivation of NF-κβ also increases vascular disease in mice.⁷⁵ Given these paradoxical observations, the possibility has been raised that the vasculature may attempt to limit the acquisition of cholesterol through processes related to insulin resistance.⁷⁶

CONCLUSIONS

In summary, the triggering agents leading to oxidative stress (iron and inflammatory cytokines), and the modulation of these processes are increasingly recognized. The knowledge of these interactions will undoubtedly have both preventive and therapeutic revenues.

ABBREVIATIONS

BMI: body mass index
EDVD: endothelium-dependent vasodilatation
IL: interleukin
IRE-BP: iron-regulatory element binding protein
Mnadh: mitochondrial NADH
NO: nitric oxide
ROS: reactive oxygen species
TNF-α : tumor necrosis factor alpha
TNFR1: tumor necrosis factor receptor I
TNFR2: tumor necrosis factor receptor II

Correspondence: Dr. J.M. Fernández-Real.
Department of Diabetes, Endocrinology and Nutrition.
Hospital Dr. Josep Trueta.
Ctra. França, s/n, 17007 Girona. Spain.
E-mail: uden.jmfernandezreal@htrueta.scs.es

References