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Endocrine Reviews 24(3):278 –301 Copyright © 2003 by The Endocrine Society doi: 10.1210/er.2002-0010

Insulin Resistance and Chronic Cardiovascular Inflammatory Syndrome ´ MANUEL FERNA ´ NDEZ-REAL JOSE

AND

WIFREDO RICART

Section of Diabetes, Endocrinology and Nutrition, University Hospital of Girona “Dr. Josep Trueta,” 17007 Girona, Spain Insulin resistance is increasingly recognized as a chronic, low-level, inflammatory state. Hyperinsulinemia and insulin action were initially proposed as the common preceding factors of hypertension, low high-density lipoprotein cholesterol, hypertriglyceridemia, abdominal obesity, and altered glucose tolerance, linking all these abnormalities to the development of coronary heart disease. The similarities of insulin resistance with another inflammatory state, atherosclerosis, have been described only in the last few decades. Atherosclerosis and insulin resistance share similar pathophysiological mechanisms, mainly due to the actions of the two major proinflammatory cytokines, TNF-␣ and IL-6. Genetic predisposition

to increased transcription rates of these cytokines is associated with metabolic derangement and simultaneously with coronary heart disease. Dysregulation of the inflammatory axis predicts the development of insulin resistance and type 2 diabetes mellitus. The knowledge of how interactions between metabolic and inflammatory pathways occur will be useful in future therapeutic strategies. The effective administration of antiinflammatory agents in the treatment of insulin resistance and atherosclerosis is only the beginning of a promising approach in the management of these syndromes. (Endocrine Reviews 24: 278 –301, 2003)

I. Introduction II. Hypertension and Chronic Inflammation A. Hypertension and the inflammatory cascade B. Hypertension and TNF-␣ C. Hypertension, IL-6, and other cytokines III. Dyslipidemia as a Chronic Inflammatory Disease A. Dyslipidemia and TNF-␣ B. Lipid metabolism, IL-6 and other cytokines IV. Insulin Resistance and Inflammation A. Insulin action on adipose tissue and inflammation B. Insulin action on muscle and inflammation C. Insulin action on liver and inflammation D. General insulin action and inflammation V. Evidence Linking Proinflammatory Cytokines to Cardiovascular Disease through Metabolic Pathways A. TNF-␣ and cardiovascular disease B. IL-6 and cardiovascular disease VI. Inflammatory Markers Predict Type 2 Diabetes Mellitus and Its Complications VII. Antiinflammatory Agents Improve Insulin Resistance and Prevent Development of Type 2 Diabetes Mellitus VIII. Possible Interpretations, Unifying Hypothesis IX. Summary

preceding factor of hypertension, low high-density lipoprotein (HDL) cholesterol, hypertriglyceridemia, abdominal obesity, and altered glucose tolerance, linking all of these abnormalities to the development of CHD (1). Although different potential genes may lead to the insulin resistance syndrome (2), prospective cohort studies confirmed hyperinsulinemia, at first in univariate analysis and more recently in multivariate analysis, as a major underlying abnormality driving cardiovascular disease (3). In 1996, a link between a direct measure of insulin resistance itself and atherosclerosis was definitively demonstrated (4). Impressive evidence has accumulated over the past decade that the atherosclerotic process is regulated by inflammatory mechanisms (reviewed in Refs. 5 and 6). Insulin resistance has also been increasingly recognized as having an important role in inflammatory pathways (7–10). Initially, in the late 1980s, active chronic inflammatory disease was found to lead to peripheral insulin resistance (11). After achieving remission of the inflammatory process, normalization of the glucose handling and insulin sensitivity was observed. The data were interpreted as follows: “The linkage between inflammatory indices and glucose metabolism might reflect a special consequence of inflammation . . . ” (12). In 1993, circulating insulin was found to be associated with white blood cell count and C-reactive protein (CRP) in patients with angina pectoris (13). More recently, chronic subclinical inflammation has been proposed as a part of the insulin resistance syndrome (10). Epidemiological evidence that inflammatory markers predict the development of diabetes and glucose disorders is also beginning to accumulate (14 –16). In both insulin resistance and atherosclerosis, the acute-phase response is enhanced. The study of the factors that regulate the acute-phase response in apparently healthy subjects has yielded consistent results implicating cytokines and growth factors in the pathophysiology of insulin resis-

I. Introduction

I

N THE LAST few decades, a cluster of metabolic factors that can lead to coronary heart disease (CHD) have been identified. Hyperinsulinemia was proposed as the common

Abbreviations: ACE, Angiotensin converting enzyme; APP, acute-phase protein(s); BMI, body mass index; CBG, corticosteroid binding globulin; CHD, coronary heart disease; CRP, C-reactive protein; DHA, docosahexaenoic acid; FA, fatty acid(s); ICAM, intercellular adhesion molecule-1; IHD, ischemic heart disease; iNOS, inducible NOS; LDL, low-density lipoprotein; LPL, lipoprotein lipase; LPS, lipopolysaccharide; NO, nitric oxide; NOS, NO synthase; s, soluble; TNFR, TNF-␣ receptor; VLDL, very LDL; WHR, waist-to-hip ratio.

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Ferna´ ndez-Real and Ricart • Insulin Action, Inflammation, and Atherosclerosis

tance and atherosclerosis and in their complications. Two viewpoints exist on the initiation of this exaggerated acutephase response. The first holds that the acute-phase response is activated by ongoing intraarterial inflammation, in which arterial wall-resident macrophages secrete proinflammatory cytokines in response to multiple stimuli. According to the second view, extravascular stimuli induce a chronic, lowlevel activation of the acute-phase response. The low-level activators would include smoking, mucosal infections (bronchitis, gastritis, or periodontitis), aging, and obesity (17). The final result of both views would be the triggering of the inflammatory cascade leading to insulin resistance and atherosclerosis (Fig. 1). Recent reviews are focused on inflammatory markers in CHD and are circumscribed to the atherosclerotic process (5, 6). In this article, we will try to embrace a systemic approach in which the metabolic response to injury is physiologically linked to inflammatory pathways. Dysregulation of these homeostatic mechanisms ultimately leads to atherosclerosis. We will summarize evidence according to which insulin resistance is pivotal in triggering and/or modulating the inflammatory response to physical, chemical, and biological stimuli. First, we will describe the knowledge linking each component of the insulin resistance syndrome to alterations of the inflammatory cascade. This review will focus specifically on the two major cytokines, TNF-␣ and IL-6. It should be stated that cytokines act primarily as autocrine/paracrine factors, and circulating levels may have nothing to do with their pathophysiological roles. IL-6 is possibly the exception to this rule. It is thought that IL-6 is secreted by a specific tissue (adipose tissue, among others), circulates in the bloodstream, and acts on distant tissues (the definition of hormone). It should also been kept in mind that whether or not TNF-␣ or IL-6 is elevated in one condition vs. another may not be relevant to pathophysiology of insulin resistance. The majority of published studies do little to in-

FIG. 1. Possible pathways leading to chronic inflammation, resulting in atherosclerosis.

Endocrine Reviews, June 2003, 24(3):278 –301

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dicate which factors are primary and which are acquired in the evolution of the syndrome. From current available evidence, it is not still clear whether inflammatory parameters are markers or mediators of insulin resistance and/or cardiovascular disease, i.e., whether these parameters are elevated secondary to ongoing atherosclerosis or represent the direct cause of accelerated atherogenesis. Thus, any inference on causality should still be demonstrated with adequately designed studies. II. Hypertension and Chronic Inflammation A. Hypertension and the inflammatory cascade

The mechanism by which primary, essential, or idiopathic forms of hypertension occur in humans is multifactorial and is based on a chronic metabolic disturbance. A large percentage of human hypertensive patients are salt sensitive, referring to the dependence of hypertension on sodium intake, but the cause of the salt sensitivity is not known. Although several mechanisms may contribute to salt-sensitive hypertension, the nitric oxide (NO) system appears to play a major role (18). NO is an important messenger molecule that plays a critical role in a wide variety of physiological functions, including immune modulation, vascular relaxation, neuronal transmission, and cytotoxicity. There are at least three isotypes of NO synthase (NOS): endothelial cell NOS, the neuronal type NOS, and the so-called inducible NOS (iNOS). iNOS is implicated in host defense and is synthesized de novo in response to a variety of inflammatory stimuli. Studies in humans indicate that NO production is decreased during hypertension (19). Interestingly, a polymorphism within the promoter of the iNOS candidate gene, NOS2A, revealed both increased allele sharing among sibpairs and positive association of NOS2A to essential hypertension (20). These facts are linked to insulin resistance be-

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cause insulin stimulation of glucose uptake in skeletal muscles and adipose tissues in vivo seems to be NO dependent (21). Moreover, iNOS has recently been shown to be crucial for the development of insulin resistance (22). NO antagonizes the effects of angiotensin II on vascular tone and growth and also down-regulates the synthesis of angiotensin converting enzyme (ACE) and angiotensin II type 1 receptors (23). The role that inflammation plays in atherosclerosis is amplified by the renin angiotensin system via the effects on adhesion molecules, growth factors, and chemoattractant molecules, which modulate the migration of inflammatory cells into the subendothelial space. Clinical and basic research has increased our knowledge of the actions of the vasoactive hormone angiotensin II, showing that it has multifunctional properties beyond its hemodynamic effects. A new aspect of this peptide is coming into focus: its potential role as a proinflammatory modulator. Angiotensin II is important in stimulating the production of reactive oxygen species and the activation of ancient inflammatory mechanisms through its angiotensin II type 1 receptor (24). The relationships between these events and insulin resistance seem to play a role in humans, given the protective role of ACE inhibitors on the development of type 2 diabetes (see Section VII). Immunopathogenic mechanisms are increasingly recognized to be involved in the pathogenesis of hypertensive disease (25, 26). Abnormalities in immune system function, in both humoral and cellular immunity, and inflammatory mediators have been claimed to be responsible for the onset of hypertension (25, 26). A common finding in patients with hypertension is an elevated level of serum Ig, which is found in 20 – 40% of hypertensive subjects (27–29). Patients with borderline or established essential hypertension also display a delayed-type hypersensitivity to vascular antigens (30). The complement system, which plays an important role in the overall responsiveness of the innate system and in the initiation and regulation of inflammation, also seems to be involved in blood pressure regulation. A substantial part of patients with essential hypertension express the C3F protein (31), which binds more avidly to mononuclear cells than the other allele, C3S. Expression of the C3F protein more than doubles the risk of developing hypertension and increases the risk of ischemic heart disease (IHD) by more than 10-fold in hypertensive patients (32). Plasma concentrations of the third complement component (C3) have been found to be associated with blood pressure (33, 34) in parallel to insulin resistance (35). Evidence for immune dysregulation in animal hypertensive models is extensive (reviewed in Ref. 25), with the more prominent observation being that IL-2 significantly attenuated the elevated arterial pressure in the spontaneously hypertensive rat (36). It remains to be established whether immune alterations are primary, concomitant, or secondary to the hypertensive process. B. Hypertension and TNF-␣

The available information on the effects of TNF-␣ in experimental models suggests that it is involved in the patho-

physiology of hypertension. TNF-␣ stimulates the production of endothelin-1 (37) and angiotensinogen (38) in vitro. In the spontaneously hypertensive rat model, TNF-␣ synthesis and secretion are increased in response to lipopolysaccharide (LPS) stimulation compared with those in nonhypertensive control rats, and fat angiotensinogen mRNA increased after LPS in the former but not the latter (39). In humans, the TNF-␣ gene locus seems to be involved in insulin resistance-associated hypertension (40). However, in this study, a multivariant analysis of this gene locus effect on obesity and hypertension was not performed, and an obesityindependent effect of this gene on blood pressure is still obscure. A positive correlation has been found between serum TNF-␣ concentration and both systolic blood pressure and insulin resistance in subjects with a wide range of adiposity (41). Up-regulation of TNF-␣ secretion has also been observed in peripheral blood monocytes from hypertensive patients (42). TNF-␣ also determines endothelial dysfunction linked to insulin resistance (43). TNF-␣ signals through at least two known cell surface receptors (TNFRs), TNFR1 (p60) and TNFR2 (p80; Refs. 44 and 45). The soluble fractions of these receptors, sTNFR1 and sTNFR2, result from a proteolytic cleavage of the cell surface forms (46, 47) when TNF-␣ binds to its receptors. Measurements of the sTNFR concentrations in healthy individuals at different time lapses showed that the levels in the same subject were quite stable over time (48) and have been validated as sensitive indicators of TNF-␣ system activation (49). The ratio of soluble TNF-␣ receptors (sTNFR2/sTNFR1) correlated positively with systolic and diastolic blood pressure (P ⬍ 0.01) in a recent study (50). This ratio was significantly greater in type 2 diabetic patients than in type 1 diabetic patients and was greater in both than in control nondiabetic subjects. Interestingly, shedding of TNFR1 and TNFR2 was found to be associated with insulin resistance and vascular dysfunction in type 2 diabetic patients. After exercise-induced lowering of blood pressure, a concomitant decrease in the sTNFR2/sTNFR1 ratio was observed. It was concluded that insulin resistance and blood pressure are linked to altered shedding of TNFRs in type 2 diabetes mellitus (50). C. Hypertension, IL-6, and other cytokines

IL-6 is a multifunctional cytokine produced by many different cell types, including immune cells, endothelial cells, fibroblasts, myocytes, and adipose tissue, mediating inflammatory as well as stress-induced responses. In recent studies, blood pressure was a significant and independent predictor of circulating IL-6 concentrations in women but not in men (51, 52), but not all studies are concordant (53). A polymorphism in the promoter of the IL-6 gene has also been found to show divergent associations with blood pressure (54, 55). IL-6 stimulates the central nervous system and the sympathetic nervous system, which may result in hypertension (56, 57). The administration of IL-6 led to increased heart rate in healthy women and increased norepinephrine levels and heart rate in women with fibromyalgia (58). However, other mechanisms cannot be excluded. IL-6 might increase in concert with the modification of the redox state of the vascular


wall in chronic hypertension, as occurs in some hypertensive animal models (59), and in this vessel wall IL-6 also can lead to increased collagen (60). IL-6 is a well-characterized acute inducer of fibrinogen, and fibrinogen is a major determinant of blood viscosity (61). Finally, IL-6 might result in hypertension via effects on angiotensinogen expression (62), leading to higher concentration of angiotensin II, which is a potent vasoconstrictor. Interestingly, a cytokine-like molecule increasingly recognized to regulate several inflammatory pathways acting on a receptor of the IL-6 family (leptin; reviewed in Ref. 63) seems also to be associated with hypertension. The leptin signal, via central leptin receptors, is believed to interact with the central sympathetic nervous system (64). Infusion of leptin leads to increases in blood pressure (65). Transgenic mice overexpressing leptin had elevated blood pressure, normalized by ␣-adrenergic blockade (66). Recent findings implicate the leptin receptor gene locus with blood pressure regulation in men (67). Other cytokines might also play a role. The secretion of IL-1-␤ was significantly increased in peripheral blood monocytes from hypertensive patients vs. healthy individuals after stimulation with LPS (42). Similar findings were observed after stimulation with angiotensin II (42). Up-regulation of this cytokine was also seen at the RNA level in hypertensive


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patients. TGF-␤1 protein might also play a role in blood pressure regulation in humans (reviewed in Ref. 68). The potential link between hypertension and inflammatory mechanisms is summarized in Fig. 2. III. Dyslipidemia as a Chronic Inflammatory Disease

Lipid metabolic pathways and cytokines seem to share different pathophysiological mechanisms. During the acutephase response to infection and inflammation, cytokines induce tissue and plasma events that lead to changes in lipoprotein. The induced hyperlipidemia may represent a nonspecific immune response that can decrease the toxicity of a variety of harmful biological and chemical agents and serve to redistribute nutrients to cells important in host defense (69). We review the parallelism that exists between injury-induced and insulin resistance-induced changes in lipid metabolism. A. Dyslipidemia and TNF-␣

1. TNF-␣ and triglycerides. TNF-␣ has important effects on whole-body lipid metabolism. The mechanisms and dynamics of cytokine-induced hypertriglyceridemia have been reviewed elsewhere (69 –71). TNF-␣ acutely raises serum tri-

FIG. 2. Proposed pathophysiology of hypertension through inflammatory mechanisms (see Section II). CNS, Central nervous system; SNS, sympathetic nervous system.

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glyceride levels in vivo by stimulating very low-density lipoprotein (VLDL) production (72). Hypertriglyceridemia is well described in patients with frequent infections and chronic secretion of cytokines such as those with AIDS (73) or in patients with cystic fibrosis (74). In AIDS, other cytokines like interferon-␣ also contribute to hypertriglyceridemia (75). In apparently healthy people, a positive association between plasma concentration of the soluble fraction of TNFR-2 (sTNFR2, a surrogate of previous TNF-␣ effects) and total triglycerides has been described, in parallel to a negative one with HDL cholesterol (76). Because plasma sTNFR2 is thought to reflect insulin resistance (77, 78), the possible contribution of the latter to increased plasma triglyceride levels should not be ignored. Plasma TNF-␣ correlated positively with VLDL triglycerides in healthy men and postinfarction patients (79, 80) and negatively with HDL cholesterol in the latter (80). However, difficulties in measuring TNF-␣ in plasma should be recognized, where it is normally in a very low concentration (in the range of picograms per milliliter) and usually below the linear range of the assay. 2. TNF-␣ and cholesterol. The influence of cytokines on total and LDL cholesterol metabolism has been less well studied and seems species specific. TNF-␣ administration to cynomolgus monkeys resulted in hypocholesterolemia (81), and, in humans with chronic infections such as AIDS or with cystic fibrosis, decreased total, LDL, and HDL cholesterol levels are usually found (73, 74). In contrast to this evidence, TNF-␣ has been found to produce a 25% increase in serum cholesterol levels and a 2.3-fold increase in hepatic hydro3-methyl-glutaryl coenzyme A reductase activity in C57BL/6 mice (82). TNF-␣ is also capable of inducing sterol regulatory element binding protein-1 maturation, a key transcription factor in cholesterol biosynthesis, in a time- and dosedependent manner in human hepatocytes (83). The intensity, duration, and timing of TNF-␣ hypersecretion might contribute to explaining these opposite actions of TNF-␣ on cholesterol metabolism. In pathological conditions such as chronic infection, moderate to heavily increased TNF-␣ concentrations may activate pathways of cholesterol metabolism such as increased LDL-receptor expression leading to increased lipoprotein clearance, increased conversion of newly synthesized cholesterol into bile acids, or enhanced esterification and storage of cholesterol (reviewed in Refs. 84 and 85). In other chronic low-level inflammatory diseases, such as those caused by intracellular pathogens, the reverse might also be true. Chlamydia species have been found to induce production of TNF-␣, inhibiting the action of lipoprotein lipase (LPL), leading to accumulation of serum triglycerides and a decrease in serum HDL cholesterol (reviewed in 86). LPS, a bacterial component, binds to both HDL cholesterol and LDL cholesterol, which buffers its toxic capacity. However, in the long term, when this mechanism is overwhelmed, LPS makes LDL immunogenic or toxic to endothelial cells. In a vicious cycle, the accumulation of cholesteryl esters in macrophages exposed to LDL-immune complexes is again associated with increased synthesis and release of TNF-␣ (85), and activated macrophages are better

able to form foam cells. In this context, it is interesting to note that increased plasma cholesterol per se [as observed in hypercholesterolemic rabbits (87), LDL-receptor knockout mice (88), or diet-induced (89)] is associated with increased plasma TNF-␣ concentration or enhanced endotoxin-stimulated TNF-␣ and IL-1 gene expression in aortae (89). In apparently healthy subjects, sTNFRs circulate in proportion to total and LDL cholesterol (76, 90). High cholesterol could lead to an increased activity of TNF-␣ axis, of which increased sTNFRs would be its reflection. This rise in soluble fraction of cytokine receptors parallels increases in other soluble factors, such as soluble endothelial leukocyte adhesion molecules and soluble intercellular adhesion molecule-1 (sICAM-1) observed in other situations of hypercholesterolemia (91). Damage to the endothelium could provide a link explaining simultaneously increased sTNFRs, soluble endothelial leukocyte adhesion molecules, and sICAM-1, because elevated cholesterol levels are associated with endothelial dysfunction (92). An alternative explanation is that high levels of sTNFRs block the hypocholesterolemic action of TNF-␣ by inhibiting its interaction with cell surface receptors, resulting in high cholesterol levels. Another possible mechanism is one related to the stimulatory effect of insulin on LDL-receptor activity through the enhancement of LDL-receptor mRNA expression (93). In this sense, increased LDL cholesterol might be the result of hampered insulin action (possibly induced by TNF-␣) on LDLreceptor activity. B. Lipid metabolism, IL-6, and other cytokines

Recent investigations have shown that locally produced cytokines possess important autocrine/paracrine properties that influence diverse functions of the adipose tissue in addition to possible effects on other tissues. In this sense, IL-6 has been hypothesized to be responsible for the lipid abnormalities occurring in subjects with the insulin resistance syndrome (7, 8). This hypothesis is based on the findings of increased blood concentrations of IL-6 and markers of the acute-phase response, including CRP and cortisol in parallel with dyslipidemia (decreased plasma HDL cholesterol and increased plasma triglyceride concentration) in patients with this syndrome (7, 8). IL-6 inhibits adipocyte LPL activity (94) and induces increases in hepatic triglyceride secretion in rats (95). In man, IL-6 infusion leads to increased free fatty acid concentration (96), and fasting triglycerides, VLDL triglycerides, and postglucose load free fatty acids are linked to serum IL-6 concentration (97). The link between inflammation, insulin resistance, and CHD might be mediated through different pathways, including fatty acid (FA) metabolism. Dietary FA appear to modulate the release of different cytokines (98). The production of IL-1 ␤, TNF-␣, IL-6, and granulocyte and macrophage colony-stimulating factor by peripheral mononuclear cells decreases after dietary polyunsaturated FA supplementation in women (99, 100). Docosahexaenoic acid (DHA) and eicosapentaenoic acid inhibited in vitro human endothelial cell production of IL-6 (101). DHA also reduced endothelial expression of IL-6 in response to different stimuli (102). In


contrast, the consumption of a diet high in hydrogenated fat increases production of IL-6 and TNF-␣ (103). Hence, the profile of dietary FA strongly influences cytokine production. In fact, the proportion of saturated and polyunsaturated ␻-3 FA in serum of healthy volunteers was associated with circulating IL-6 concentration (104). Other circulating cytokines such as plasma granulocyte and macrophage colonystimulating factor concentration appear to be linked to serum DHA and eicosapentaenoic acid levels in healthy volunteers (105). IV. Insulin Resistance and Inflammation

Defects in insulin action on the main insulin-sensitive tissues (adipose tissue, muscle, and liver) are proposed to lead to a worsening of the chronic, low-grade inflammatory state. Independent of the triggering agent and of the initial events, the relationship is bidirectional; any process linked to chronic inflammation will decrease insulin action, and insulin resistance will lead to worsening of inflammation in a vicious cycle. A. Insulin action on adipose tissue and inflammation

There exists increased evidence that generalized and abdominal obesity constitute low-grade inflammatory states. Adipose tissue, long being misconstrued as a mere tissue of fat storage, is progressively acknowledged to be an active participant in energy homeostasis. The term “adipocytokines” was recently coined to describe the adipose-derived bioactive factors that modulate the physiological function of the other tissues in our body (106). 1. Abdominal obesity and TNF-␣. TNF-␣ seems to play a key role in regulating adipose tissue metabolism (107–112). In obese humans (111–113) and numerous rodent models (107– 109) of obesity-diabetes syndromes, TNF-␣ is overexpressed in the adipose tissue, as compared with tissues from lean individuals. A decreased processing rate of transmembrane TNF-␣ in mature adipocytes combined with an increase in TNF-␣ production may be a potential mechanism resulting in elevated membrane-associated TNF-␣ in adipose tissue in obesity (114). The TNF-␣ gene locus seems to influence the distribution of body fat according to sex. Although this locus exerted the most significant effects on waist circumference and suprailiac skinfold in men, the most significant impact in women was on upper thigh circumference and thigh skinfold (40). In fact, the only element in the TNF-␣ cascade that is known to have gender-specific regional effects is LPL (40). The mRNA level and the enzyme activity of LPL are higher in abdominal than in thigh adipose cells in men and vice versa in women (115). Obese women express approximately 2-fold more TNFR2 mRNA in fat tissue and approximately 6-fold more sTNFR2 in circulation relative to lean control subjects (77). Interestingly, adipose tissue expression of TNFR2 strongly correlates with body mass index (BMI) and with measures of abdominal obesity [waist-to-hip ratio (WHR); Refs. 77 and 78]. However, there exists some discrepancy in the relationships


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among sTNFR1, sTNFR2, and adiposity measures depending on study design and inclusion or not of morbidly obese subjects (116 –119). In one study, plasma sTNFR2 concentration was described to cosegregate with measures of obesity but not with insulin resistance in twins (120). However, mechanistically speaking, circulating sTNFR2 concentration changes with systemic insulin action (see Section IV.B). Subcutaneous adipose tissue also produced sTNFR1 as shown by arteriovenous differences (121), and diet-induced weight loss led to significantly decreased sTNFR1 levels (122). Administration of LPS or the recombinant cytokines TNF-␣ and IL-1 has been reported to induce leptin expression and secretion by adipose tissue (123, 124). Recent works have suggested the existence of a TNF-␣-leptin axis, in which leptin and TNF-␣ would be in a mutual interrelationship. TNF-␣ stimulates leptin secretion in cultured adipocytes and obese mice, and, as a feedback loop, leptin administration to rats decreased TNF-␣ expression by 40% (123–126). In fact, TNF-␣ administration increases serum leptin levels in humans (127), and plasma sTNFR1 concentration circulates in proportion to leptin (128). However, the possible role of leptin resistance in these interactions is still confusing in humans. These facts are important in understanding pathophysiology of abdominal obesity because leptin production is dependent on the distribution of body fat (129). Moreover, although leptin was first described for its role in modulating food intake and energy expenditure, there is now substantial evidence that leptin is also involved in immune function (63), as evidenced by its effect to enhance cytokine production and phagocytosis by macrophages (130). In fact, increased leptin concentrations correlate with increased concentrations of inflammatory markers in morbidly obese individuals (131). However, the role of leptin in human insulin action or obesity-associated inflammation is probably very small (132). 2. Abdominal obesity and IL-6. IL-6 is secreted from adipose tissue during noninflammatory conditions in humans. Omental adipose tissue produces 3-fold more IL-6 than sc adipose tissue (133). Dynamic studies of IL-6 concentration in humans showed that IL-6 increases postprandially, in parallel to glucose and insulin levels in the interstitial fluid of sc adipose tissue (134). This finding suggests that IL-6 might modulate adipose glucose metabolism in the fed state. It has been calculated that one third of total circulating concentrations of IL-6 originate from adipose tissue (135). A positive association between different measures of obesity and plasma IL-6 levels has been described in men and postmenopausal women (51, 52, 133). Because venous drainage from omental tissue flows directly into the liver, the increased physiological WHR of men is expected to have more metabolic impact. Abdominal arterial IL-6 was also associated with BMI (133). In contrast, plasma IL-6 levels were higher in obese patients with sleep apnea but not in obese controls in comparison with normal weight controls (136). In another study, the relationship between BMI and serum IL-6 was only observed in postmenopausal women, and this relationship was lost among those women receiving hormone replacement (52). In fact, estrogens are well-known inhibitors of IL-6 secretion (137). Adipose tissue-derived estrogens in

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postmenopausal women would not be sufficient to reduce IL-6 in a similar way as endogenous estrogens in premenopausal women (52). It should be stated that an important variable is smoking, which was a significant confounding factor in the relationship between measures of body fat and circulating IL-6 concentration (51). Recently, the IL-6174G/C polymorphism has been found to be associated with indices of obesity in men (138). Of the aforementioned, obesity is expected to result in increased secretion of IL-6, with its detrimental metabolic effects (see Section IV.D.2.b). In contrast, mice lacking the gene encoding IL-6 (IL-6⫺/⫺) developed mature-onset obesity and disturbed carbohydrate and lipid metabolism, which were reversible by IL-6 replacement (139). The antiobesity effect of IL-6 was mainly exerted at the level of the central nervous system, being inactive when administered peripherally (139). At first glance, this is puzzling. However, IL-6 acts as a terminator as well as a prompter of inflammation. It should again be remembered that cytokines act in cascade, and total depletion of IL-6 might be detrimental because other proinflammatory cytokines (TNF-␣) are not adequately down-regulated, as is the case in other knockout models (140). Unfortunately, other cytokines were not studied in that report (139). B. Insulin action on muscle and inflammation

1. Muscle and TNF-␣. Obesity is not the only condition in which circulating sTNFR2 concentration is proportional to insulin resistance. Plasma sTNFR2 concentration has been described as being linked to markers of the muscle compartment as fat-free mass and midarm muscle circumference (78, 141, 142). Circulating sTNFR2 concentration is also associated with insulin resistance in other diseases characterized by muscle disease (143). TNF-␣ is a strong inducer of TNFR2 expression in adipocytes and other cell types (144), and, in this context, the association between the muscle compartment and insulin resistance might be attributed to increased production by the muscle of sTNFR2, leading to stabilization of TNF-␣ homotrimers, resulting in insulin resistance at the level of the adipocyte. These facts are related to the thrifty genotype hypothesis. Neel (145) postulated that a thrifty genotype existed that had a selection advantage as hunter-gatherers fluctuated between feast and famine. The thrifty genotype in type 2 diabetes contributes to the insulin resistance seen in muscle (146). A selective insulin resistance in muscle would have the effect of blunting the hypoglycemia that occurs during fasting but would allow energy storage in fat and liver during feeding. Both of these features could allow huntergatherers to have survival advantages during periods of food shortage (147–149). Although mild exercise seems to produce health benefits (150), strenuous exercise, taken to the extreme or during prolonged fasting conditions, initiates an immune and vascular proinflammatory response. In fact, acute strenuous exercise is considered to be a model of the acute-phase response that occurs in parallel to insulin resistance (150 – 152), and the cytokine response to strenuous exercise [both plasma TNF-␣ and sTNFR2 concentrations increase during exercise (151)] is similar to that found in sepsis and trauma

(150, 152). As recently hypothesized (9), the induction of muscle insulin resistance would allow a food-seeking behavior and would prevent the wasting of glucose to nonvital organs, protecting the brain and the immunological system. In the absence of food shortage, regular physical exercise led to a consistent decrease in circulating sTNFR2 in obese women (141, 153) and in type 2 diabetic patients (50), in parallel to improved insulin sensitivity. 2. Muscle, IL-6, and other cytokines. Several cytokines can be detected in plasma during and after strenuous exercise. The increase in TNF-␣, sTNFR2, IL-1␤, IL-1 receptor antagonist, IL-8, and IL-10 is accompanied by a dramatic increase in IL-6 (reviewed in Ref. 154). After exercise, IL-6 is produced in larger amounts by the contracting muscle than any other cytokine examined. Both IL-6 mRNA [which increases more than 10-fold after exercise (155)] and IL-6 receptor mRNA (156) have been detected in muscle. It has been suggested that depleted glycogen content or an energy crisis in the contracting muscle, rather than muscle damage, may be one stimulus for the IL-6 release (154). Thus, the possibility exists that the elevated serum IL-6 is a consequence of an increased production and release of IL-6 from muscle in response to the impaired insulin sensitivity, allowing hunter-gatherers to have survival advantages during periods of food shortage, in a similar way to the events described above. C. Insulin action on liver and inflammation

1. Acute-phase proteins (APP) and insulin resistance. After trauma or infection, the human body mounts a highly complex acute-phase response as part of the homeostatic response to injury. In the acute phase, the acute-phase response is protective because it counteracts the effects of injury and improves survival. Long-term exposure to stressful stimuli (mucositis, aging, increased fat intake, periodontitis, etc.) may result in disease rather than repair. The liver is the target of systemic inflammatory mediators and is also the organ responsible for determining the level of essential metabolites provided to the organism during the critical stages of stress. Among the most important aspects of this response is the reprioritization of hepatic protein synthesis with the increased production of a number of plasma proteins (positive APP) and reduced production of a number of normal export proteins (negative APP). It should be stated that expression of acute-phase reactants at high levels has also been recently identified in adipose tissue of mice, and this was especially remarkable in the diabetic state (157). The induction of APP production is thought to be regulated at the transcriptional level, and at least two signaling pathways have been identified within the hepatocyte. One pathway activates transcription of class I acute-phase genes such as CRP, serum amyloid A, and complement C3, whereas the other pathway activates transcription of class II acute-phase genes such as ␣1-antitrypsin and fibrinogen (158). Although the concentrations of multiple components of the acute-phase response increase together, not all of them increase uniformly in all patients. These variations indicate that the components of the acute-phase response are individually regulated, and this may be caused in part by dif-


ferences in the pattern of production of specific cytokines (159, 160). These facts would explain increased susceptibility to increased inflammatory activity among healthy volunteers with genetically increased rates of some cytokines (161). The hormonal and cytokine networks acting on the hepatocyte may lend a degree of fine-tuning to the spectrum of APP in response to different stimuli (158). Insulin seems to be one of the main regulators of the cytokine-associated acute-phase reaction (162, 163). Liver cells respond to many of the factors via their cell surface receptors. According to one vision (158), the inflammatory mediators fall into four major categories: 1) IL-6 type cytokines, of which IL-6 is the major representative; 2) IL-1 type cytokines (including IL-1␣, IL-1␤, TNF-␣, and TNF-␤); 3) glucocorticoids; and 4) growth factors (including insulin). The cytokines would act as primary stimulators of APP gene expression, whereas the glucocorticoids and growth factors function more as modulators of cytokine action. An adequate balance between these opposite pathways will result in resolution of the acute-phase process (Fig. 3). Insulin attenuates IL-1- and IL-6-type cytokine stimulation of most APP genes in human hepatoma cells (162, 163). Thus, the lack of significant insulin action, as found in type 2 diabetes, or unsubstituted insulin deficiency, would not be able to block TNF-␣, IL-1, and IL-6 actions, leading to prolonged acute-phase reaction. The acute-phase response changes are small in insulin resistance in comparison with those found in infection or trauma, but the potential damage is greater because of the chronicity of the changes. Systemic inflammation, measured by increased serum acute-phase reactants, has been recognized to occur in type 2 diabetes since the early work by McMillan (164). Significantly higher serum concentration of CRP, fibrinogen, ␣1acid glycoprotein, amyloid A, sialic acid, and orosomucoid have been described in patients with type 2 diabetes mellitus (7, 164). Serum CRP concentration and the acute-phase reaction have been significantly associated with clinical and biochemical indexes of insulin resistance (51, 165–172). The

FIG. 3. Balance of proinflammatory and antiinflammatory agents regulating the acute-phase response. An adequate balance will lead to resolution of the process.


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relationship between increased CRP and decreased insulin action might be intrinsically due to insulin resistance itself (51, 162, 163). Elevated serum CRP concentrations have been demonstrated consistently in overweight and obese adults, even among young adults aged 17–39 yr (165). From 34 –39% of U.S. people with diabetes had elevated CRP levels, and this association was not completely explained by increases in BMI (166). Of note was that CRP and IL-6 decreased significantly after improvement of metabolic control in type 2 diabetic patients, indicating that the inflammatory pathways are modulated by insulin (173). CRP may not measure all of the relevant effectors of inflammation because the concentration of CRP may be subject to posttranscriptional regulation (159). In one study, CRP levels were associated with several metabolic parameters in men and women, but in a multiple linear regression analysis, CRP was associated independently with IL-6 only in men (51). Interestingly, both constitutive and IL-6-dependent acute-phase expression of the human CRP transgene require testosterone in transgenic mice, implying a potential mechanism for this gender dimorphism (174). Other acute-phase reactants, such as fibrinogen, plasminogen activator inhibitor-1, and amyloid A are associated with insulin resistance and predict development of type 2 diabetes (Ref. 167; also see Section IV). 2. Corticosteroid binding globulin (CBG). CBG is the major blood transport protein for cortisol, the major antiinflammatory hormone in humans. Scarce data in the literature have suggested that CBG is a negative acute-phase reactant (175, 176). CBG level was negatively associated with insulin secretion in obese and glucose-tolerant subjects who also had lower CBG levels than obese and glucose-intolerant subjects (177). It has been shown that both insulin secretion and insulin sensitivity independently contributed to CBG changes after glucose-induced insulin stimulation (178). The insulin response after a glucose challenge was linked to acute CBG changes in lean subjects.

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CBG has been recently found to be negatively associated with several indexes of insulin resistance such as BMI, WHR, and homeostasis model assessment, and with inflammatory parameters such as sTNFR1, sTNFR2, and IL-6 concentrations (179). A polymorphism of the IL-6 gene promoter, which is linked to increased IL-6 levels and to insulin resistance, was also associated with low CBG levels (161). In women, both decreased CBG and free cortisol independently contributed to homeostasis model assessment variance in a multiple linear regression analysis, suggesting that both mechanisms would be metabolically additive (179). Because CBG secretion has been shown to be negatively regulated by both insulin (180) and IL-6 (181, 182), it is tempting to propose that CBG concentration is an index of insulin resistance and inflammation. On the other hand, constitutive low CBG levels might also contribute to insulin resistance by increasing cortisol biodisposal to target cells, including muscular cells. Interestingly, physiological increments in plasma insulin concentrations have been described to affect synthesis of other hepatic proteins in normal humans (183). In addition, insulin is also able to promote albumin distribution to peripheral tissues by increasing the protein transcapillary escape rate (184). As shown for low serum albumin in the Atherosclerosis Risk in Communities Study (14), it has been suggested that low CBG, another liver protein, could be an interesting index for the development of type 2 diabetes as well as for incident cardiovascular disease as reported early in postmenopausal women (185). It remains to be established whether low circulating or tissue CBG concentration impacts on systemic insulin action and metabolism. D. General insulin action and inflammation

Aging is usually associated with increased insulin resistance (186). In parallel to age-related insulin resistance, the production of circulating APP (187), the secretion of cytokines from monocytes and macrophages (188), and the production of TNF-␣ (186) and sTNFR2 (189) are all increased with age. We will review the evidence according to which cytokines are involved in general insulin action. 1. TNF-␣ and insulin resistance a. TNF-␣ gene and insulin resistance. The relative importance of TNF-␣ in insulin action has been tested by inducing a targeted null mutation in the TNF-␣ gene. Mice with this mutation were spared from obesity-induced deficiencies in insulin-receptor signaling in fat and muscle tissues (190, 191). However, it should be recognized that insulin resistance is alleviated but not eliminated in these models. In humans, similar information can be obtained by comparing individuals with different transcription rates of the TNF-␣ gene. Some substitutions in the TNF-␣ promoter gene lead to different constitutive and inducible levels of transcription of the TNF-␣ gene than the wild-type allele (192, 193). Those subjects that are homozygotes for the absence of the restriction site (resulting from a guanine to adenine substitution) at position ⫺308 of the TNF-␣ promoter showed an increased percentage of fat mass and leptin levels and a decreased

insulin sensitivity index (194). The 308G/A TNF-␣ polymorphism has subsequently been linked to obesity in an epidemiological basis (195, 196) to body fat content (197), to insulin resistance (198, 199), and to fasting glucose (200). Secretion of TNF-␣ from adipose tissue also differed among nonobese subjects according to TNF-␣ ⫺863C/A polymorphism (193). Adipose tissue from subjects with the rare allele ⫺863/A secreted significantly less TNF-␣ than adipose tissue from nonobese subjects carrying the ⫺863C allele. This indicated that C to A substitution at position ⫺863 represented a functional polymorphism, which leads to decreased TNF-␣ gene expression and thereby less production and secretion of the cytokine (193). In parallel to decreased TNF-␣ secretion, fasting serum triglycerides were significantly lower, and insulin sensitivity was significantly higher in subjects with the rare allele ⫺863/A in a subsequent study (201). Thus, different adipocyte secretion rates of TNF-␣ according to ⫺863C/A TNF-␣ gene polymorphism are mirrored at the level of insulin action, at least in moderately obese subjects. Another substitution, the homozygous ⫺857T allele, tended to be higher in obese patients with diabetes than in lean subjects with normal glucose tolerance (202). Mutations within regulatory elements of the TNF-␣ gene were not associated with an increase in the prevalence of noninsulin-dependent diabetes mellitus (202, 203). However, in the latter reports, insulin resistance was not evaluated. In other studies, the inclusion of subjects with morbid obesity probably led to false-negative associations when evaluating TNF-␣ ⫺308G/A polymorphism and obesity phenotypes (204), in contrast to the studies that have not included them (40, 194) or that have evaluated a small proportion of these subjects (195, 197, 200). Some associations among obesity, obesity-associated phenotypes, and cytokines have been found to be most significant in nonmorbidly obese individuals (40). Enhanced activity of cytokines due to the development of obesity is, on one hand, predicted to contribute to the development of obesity-associated phenotypes but is, on the other hand, expected to limit the progression of obesity. These mechanistic relationships are probably lost in subjects with morbid obesity. Differences in study design have also contributed to disparity of results: insulin sensitivity or insulin secretion was not significantly different in healthy young relatives of type 2 diabetic patients with the A allele (205–207). Finally, interethnic differences, gene-gene and gene-environment interactions are important confounders of any association between a single polymorphism and disease (208 –210). Other components of the TNF-␣ axis seem to be genetically linked to insulin resistance. Mice lacking the TNFR-2 gene, TNFR2 (p75⫺/⫺), fed a high-fat diet, consistently gained less weight and displayed reduced insulin levels, as expression of improved insulin sensitivity, in comparison with wildtype mice that followed a similar diet (211). In humans, a mutation in the TNFR2 gene has been associated with increased BMI and leptin levels in parallel to insulin resistance in nondiabetic subjects and increased BMI and leptin concentration in diet-treated type 2 diabetic patients (212). Interestingly, other mutations in this locus have been associated with other components of the insulin resistance syndrome such as hypertension (213), dyslipidemia (214),


and cardiovascular disease (90), and with other degenerative diseases such as osteoporosis (215). This locus has also been associated with polycystic ovary syndrome, an entity with known defects in insulin action (216). The differences in insulin resistance seem to be restricted to the TNF-␣ gene because polymorphisms of the TNF-␤ gene, a closely associated gene, were not associated with this phenotype (217). It should be remembered that much of TNF-␣ is secreted, whereas most of TNF-␤ is on the membrane of the lymphocytes. Again, interethnic differences have also been described. Insulin resistance was significantly lower in variant TNF-␤ homozygotes vs. wild-type allele in Japanese subjects (209), whereas hyperinsulinemia, attributed to TNF-␤ gene polymorphism, was described in Caucasians with CHD (218). b. TNF-␣ action and insulin resistance. TNF-␣ blocks the action of insulin in cultured cells and whole animals (107– 109). The induction of insulin resistance is mediated through its ability to produce serine phosphorylation of insulin receptor substrate 1, decreasing the tyrosine kinase activity of the insulin receptor (109). Neutralization of TNF-␣ in obese fa/fa rats by iv administration of a sTNFR-IgG chimeric protein substantially improved insulin sensitivity and restored the tyrosine kinase activity in fat and muscle (107). It also reverted the insulin-induced phosphorylation of insulin receptor substrate 1 to levels observed in lean animals (109). In contrast, treatment of type 2 diabetic or obese human subjects with an antibody specific for TNF had no effect on insulin sensitivity (219, 220). However, one of these studies was performed using one single administration of antibody in adults with established diabetes (219). Moreover, this approach does not affect the autocrine and paracrine effect of TNF-␣ and is not directed against the primary endogenous stimulus for increased TNF-␣ secretion. Again, it should be remembered here that increased sTNFR2 levels circulate in association with insulin resistance in healthy volunteers (77, 78), in lean nondiabetic offspring of type 2 diabetic subjects (221), and in young obese subjects with normal and impaired glucose tolerance (142), but not in older subjects (222). 2. IL-6 and insulin resistance a. IL-6 gene and insulin resistance. Mice with a targeted null mutation in the IL-6 gene, made obese by a high-fat diet, became more insulin resistant compared with wild-type controls (139). However, as stated above, total depletion of IL-6 might be detrimental because other proinflammatory cytokines (TNF-␣) are not adequately down-regulated. This information is apparently at odds with what is observed in humans with different transcription rates of the IL-6 gene. Subjects with increased constitutive transcription rate of this cytokine, associated with ⫺174 C/G IL-6 promoter substitution, showed decreased insulin sensitivity (161), and this polymorphism was more frequently present among Pima Indians and Caucasians with type 2 diabetes (223). Again, divergent results have been described in other populations (224) in keeping with the observation that IL-6 promoter haplotypes (rather than simply single variant sites) influence IL-6 gene expression in vitro (225). Importantly, at


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least two allelic polymorphisms in the IL-6 promoter region were found to cooperate in the regulation of IL-6 activity in vivo (225). b. IL-6 action and insulin resistance. Available data suggest that although TNF-␣ functions locally at the level of the adipocyte in a paracrine fashion, IL-6 circulates in plasma at high concentrations. In this sense, IL-6 may be more important systemically and perhaps represents a hormonal factor that induces muscle insulin resistance. In fact, IL-6 is named the endocrine cytokine (226). Although adipose cells contribute to one third of circulating IL-6 concentration (135), other sources are potentially important. In this sense, glucose stimulated IL-6 production by human peripheral blood monocytes has been demonstrated, but 33 mmol/liter glucose induced only a 1.56-fold increase in IL-6 compared with treatment with 11 mmol/liter glucose (227). Although this mechanism is not operative with normal fasting glucose levels, it could be of significance in patients with type 2 diabetes. IL-6 is a pleiotropic cytokine, and some of its metabolic actions have been evaluated after IL-6 administration. In in vitro studies, IL-6 induced a dose-dependent inhibition of the glucose-stimulated insulin release of rat pancreatic islets (228, 229). In vivo, administration of recombinant human IL-6 to normal subjects induced metabolic changes usually found in catabolic states, increasing plasma glucose levels in a dosedependent fashion without altering significantly plasma insulin or C-peptide concentrations (230). In another study in cancer patients, however, recombinant human IL-6 administration led to an increase in the metabolic clearance of glucose (96). To integrate these opposite actions, one has to consider that these metabolic effects of IL-6 have been studied mainly after exogenous treatment at relatively high doses. It is also important to keep in mind the metabolic milieu in which IL-6 is exerting its effects; cytokines act in cascade, and any single change in a given step could change the final result. Another way to evaluate IL action is to infer it from IL-6 concentration, but this approach cannot conclude which is the cause and which is the consequence. For instance, plasma IL-6 levels are elevated in type 2 diabetic patients, particularly in those with features of the insulin resistance syndrome (7, 8). One interpretation could be that type 2 diabetes mellitus and the insulin resistance syndrome lead to an ongoing acute-phase response through increased IL-6 derived from unsuppressed adipose or immune secretion acting on hepatocytes that are oversensitive (Fig. 4). In fact, this hypothesis is derived from the findings of increased blood concentrations of markers of the acute-phase response, including CRP, serum amyloid-A, ␣-1 acid glycoprotein, sialic acid, and cortisol in these conditions (7, 8). But the reverse could also be true. According to the opposite vision, increased IL-6 and markers of the acute-phase response are perhaps counteracting hyperglycemia and insulin resistance (Fig. 4, step 1). When proinflammation is enduring, chronic, or uncontrolled and when a challenge becomes overwhelming, the failure to reach the desirable effect results in worsening of hyperglycemia and insulin resistance (Fig. 4, step 2). Lack of exposure would result in proinflammation only when insulin resis-

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FIG. 4. Proposed mechanisms leading to insulin resistance-related inflammation. Type 2 diabetes mellitus and the insulin resistance syndrome lead to an ongoing acute-phase response through increased IL-6 derived from unsuppressed adipose or immune secretion acting on hepatocytes that are oversensitive (step 3). In fact, this hypothesis is derived from the findings of increased blood concentrations of markers of the acute-phase response, including CRP, serum amyloid-A, ␣-1 acid glycoprotein, sialic acid, and cortisol in these conditions. According to the opposite vision, increased IL-6 and markers of the acute-phase response are perhaps counteracting hyperglycemia and insulin resistance (step 1). When proinflammation is enduring, chronic, or uncontrolled, and when a challenge becomes overwhelming, the failure to reach the desirable effect results in worsening of hyperglycemia and insulin resistance (step 2). Lack of exposure would result in proinflammation only when insulin resistance is severe (step 3).

tance is severe (step 3). These possibilities are summarized in Fig. 4. According to recent studies, serum IL-6 is associated with insulin action in human subjects (51, 231–234). Insulin sensitivity has been evaluated using euglycemic clamp technique (231, 232), the minimal model approach (51, 233), or the fasting insulin resistance index (51, 234). In all of these reports, circulating IL-6 was associated with insulin resistance in men (51), in women with a mean BMI of 36 kg/m2 (233), in hyperandrogenic women (234), and in cancer patients (231). Circulating IL-6 levels in men were associated with insulin sensitivity even after controlling for BMI, absolute fat mass or percentage fat mass (51). Both insulin resistance and insulin secretion seemed to contribute to circulating IL-6 in Pima Indians (232). A threshold of visceral fat-derived IL-6 to impact on insulin action could be speculated. The influence of estrogens on IL-6 in premenopausal women could predominate on that exerted by insulin resistance. All of these findings are strengthened by the recent description of IL-6 receptors in human adipocytes (235) and by the dem-

onstration that IL-6 impairs insulin signaling in primary mouse hepatocytes and human hepatocarcinoma cell line, HepG2 (236). Plasma IL-6 and insulin sensitivity relationships seem to occur in parallel to increases in plasma nonesterified FA (233). A strong linear relationship between the secretions of IL-6 and TNF-␣ from the adipose tissue (r ⫽ 0.81; P ⬍ 0.0001) was also reported (233). Circulating IL-6 concentration has been described to predict the development of type 2 diabetes mellitus in women. The relative risk of future type 2 diabetes mellitus for women in the highest vs. lowest quintile of these inflammatory markers was 7.5 (95% confidence interval, 3.7– 15.4; Ref. 237). Acute infections determine insulin resistance, and even after clinical recovery, some impairment in carbohydrate metabolism persists (238). Both IL-6 action (229) and acute infections (238) are characterized by a defect in insulin-stimulated glucose use, despite normal carbohydrate oxidation. It cannot be excluded that chronic or subclinical infections have contributed simultaneously to increased IL-6 levels and


insulin resistance. Of note is that a higher peripheral white blood cell count has been associated with insulin resistance since the preliminary observations in 1992 (239). In particular, it was observed that peripheral white blood cell count correlated significantly with insulin-mediated glucose disposal during a euglycemic clamp (239). In subsequent studies, it was demonstrated that neutrophil and lymphocyte count correlated positively with several components of the insulin resistance syndrome and that plasma insulin concentration was specifically associated with the number of lymphocytes and monocytes (240). These associations were confirmed in healthy subjects. Given that IL-6 is involved in hematopoiesis (241), it has been suggested that these associations might in part be due to different IL-6 concentration. Those subjects with increased constitutive transcription rates of IL-6 would be prone, simultaneously, to insulin resistance and increased peripheral white blood cell count (161). Interestingly, increased platelet count, another component of hematopoiesis, occurs in parallel to white blood cell count in healthy subjects (242). 3. Other cytokines and insulin resistance. Recently, a low production capacity of IL-10, a centrally operating cytokine with strong antiinflammatory properties by antagonizing IL-6 and TNF-␣, was found to be associated with the metabolic syndrome and type 2 diabetes in old age (243). The production capacity of the antiinflammatory cytokine IL-10 was assessed in a whole-blood assay in which LPS was used as a stimulus. Serum concentrations of total cholesterol, LDL cholesterol, triglycerides, glucose, and hemoglobin A1c gradually decreased over strata representing higher IL-10 production capacity, whereas the concentration of HDL cholesterol gradually increased. The odds ratio for type 2 diabetes was 2.7 (95% confidence interval, 1.5– 4.9) when subjects with the lowest IL-10 production capacity were compared with those with the highest IL-10 production capacity (243).

FIG. 5. Integrated regulative pathways leading to decreased insulin action through modulation of the inflammatory cascade. Dotted line, Negative effect. NEFA, Nonesterified FA; IR, insulin resistance.


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The overall picture relating insulin resistance to inflammatory pathways could be summarized as shown in Fig. 5. V. Evidence Linking Proinflammatory Cytokines to Cardiovascular Disease through Metabolic Pathways

Risk factors for atherosclerosis, insulin resistance, and type 2 diabetes closely overlap, and the three disorders may share a mutual inflammatory and genetic basis. Some of the known cardiovascular risk factors [disorders of lipid metabolism (244), platelet function (245), and vascular function (246)] are genetically determined, but estimates of these known heritable risks explain less than half of the total inherited risk for IHD (247). The genetic basis of IHD may arise from a gene having a direct effect on the initiation of the disease process or a modifying effect on the development of the process after it is initiated. Given that abnormalities in immune system function and inflammatory mediators have been found to be interrelated with several classical cardiovascular risk factors reviewed here, such as hypertension, dyslipidemia, obesity, insulin resistance, and others like endothelial dysfunction and clotting activation (168, 169, 248), it is predicted that cardiovascular disease is the endpoint, common to metabolic and inflammatory pathways. A. TNF-␣ and cardiovascular disease

TNF-␣ decreases collagen synthesis and increases matrix metalloproteinase activity in vitro, perhaps leading to plaque rupture (249). Human atherosclerotic lesions have been found to contain TNF-␣ mRNA (250, 251). Early accumulation of monocytes and lymphocytes in the aortic intima with cytokine release may be essential in the development of the disease. The accumulation of cholesteryl esters in macrophages exposed to LDL is associated with increased synthesis and release of TNF-␣ (85).

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The associations of TNF-␣ gene polymorphism and myocardial infarction have been investigated in Northern Ireland and France (195). The TNFA2 allele, linked to increased transcription rate of the cytokine, was associated with parental history of myocardial infarction. The odds ratio for premature myocardial infarction in the presence of TNF-␣/308A was 1.76 (1.14 –2.70). In Northern Ireland, the prevalence of TNFA2 homozygosity tended to be higher in IHD patients than in controls (P ⫽ 0.06; Ref. 195). In another study, TNF-␣ gene polymorphism was not associated with clinical and angiographically assessed coronary stenosis (252). However, in the latter report, nearly 80% of IHD patients were male, in contrast to 43% of control subjects (252). The odds ratio for myocardial infarction tended to be higher (albeit not significantly) in TNF2 homozygotes in Brazil (253). These authors also described a tendency toward increased risk of myocardial infarction conferred by obesity. However, the specific effect of the homozygous state for the mutation could not be fully addressed in their investigation because of the small number of TNF2 homozygotes among Brazilian subjects (253). In another study, the TNF2 allele was associated with increased plasma homocysteine levels, a known potentiator of lipid-related oxidation, although it was not related to the prevalence of IHD (254). TNF2 homozygotes (n ⫽ 10) tended to have more fibrous lesions and calcification in their coronary arteries in an autopsy series (255). No association attributed to this polymorphism was found in angiographically examined patients and controls (256). Recently, a robust association between ⫺308 TNF-␣ gene polymorphism and IHD has been found, mainly attributed to women with type 2 diabetes (257). The latter would be even more susceptible by their accompanying charge of concomitant insulin resistance, hypertension, and dyslipidemia, amplifying the constitutively increased TNF-␣ transcription rate present in TNF2 carriers. Interestingly, this gender dimorphism also occurs in the association between several genetic polymorphisms and the development of obesity. For example, ␤3adrenoceptor, ␤2-adrenoceptor, LDL-receptor, and ⫺308 TNF-␣ gene polymorphisms have all been described to be linked to obesity in women but not in men (reviewed in Ref. 258). It could be hypothesized that women are prone to TNF-␣-induced metabolic derangements by their physiologically increased fat mass, ultimately leading to atherosclerosis. The gender-specific effect of the TNF-␣ gene could be the result of a gender difference in the regional expression of the gene itself or any other element involved in the cascade of events that leads from activation of the gene to its action in the target tissue. B. IL-6 and cardiovascular disease

IL-6 has been speculated to play a key role in the development of coronary disease through a number of metabolic, endothelial, and procoagulant mechanisms (reviewed in Refs. 259 and 260). Damage to the vessel wall results in endothelial cell disruption, resulting in exposure of the underlying vascular smooth muscle cells. Endothelial and smooth muscle cells produce IL-6 and IL-6 gene transcripts that are expressed in human atherosclerotic lesions (261, 262). Prospective studies of apparently healthy and high-risk

individuals indicate that increased IL-6 levels (54, 263, 264) and elevated CRP concentration (265, 266), a surrogate of IL-6 activity, predicted cardiovascular mortality (263) and future myocardial infarction (264). Substantive interrelationships among circulating IL-6, CRP, and traditional risk factors have been described in women (267). IL-6 has been demonstrated that is a strong independent marker of increased mortality in unstable coronary artery disease and identifies patients who benefit most from a strategy of early invasive management (268). Promoter polymorphisms regulating IL-6 gene expression have been found to be simultaneously associated with circulating levels of CRP (269), carotid artery atherosclerosis (270), and peripheral artery occlusive disease (271), but not with coronary artery disease (272), probably reflecting the heterogeneity of this disease and study designs. Recently, genetic variants of Toll-like receptor 4, which confers differences in the inflammatory response elicited by bacterial LPS, were associated with differences in circulating IL-6, acutephase reactants, and development of atherosclerosis (273).

VI. Inflammatory Markers Predict Type 2 Diabetes Mellitus and Its Complications

Inflammatory markers predict the development of type 2 diabetes, but evidence is less extensive compared with that for cardiovascular disease. The Atherosclerosis Risk in Communities (ARIC) study reported that higher fibrinogen, white cell count, and lower serum albumin predicted later type 2 diabetes. Those subjects with leukocyte counts in the highest quartile had a 50% increased risk of developing diabetes (14). A high leukocyte count also predicted a worsening of insulin action and the development of type 2 diabetes in Pima Indians (274). In the Insulin Resistance Atherosclerosis Study, subjects with diabetes at follow-up (5 yr) had higher baseline levels of fibrinogen, CRP, and plasminogen activator inhibitor-1, but the latter association was independent of insulin resistance (167). In the Cardiovascular Health Study, however, baseline fibrinogen, white blood cell and platelet counts, albumin, and factor VIIIc were not associated with development of diabetes, whereas elevated baseline CRP anticipated glucose progression even after adjustment for known baseline predictors of fasting glucose status (16). CRP was associated with progression in glucose status in those subjects with lower BMI. The later finding suggested to the authors that their results were not due to the production of proinflammatory cytokines by adipose cells (16). In contrast, the Atherosclerosis Risk in Communities (ARIC) study, sialic acid and orosomucoid remained significant predictors after adjustment for baseline BMI (14). A different prevalence of cytokine polymorphisms in obese and lean subjects cannot be excluded as significant confounders of these associations. More recently, different studies have confirmed that CRP and other inflammation-sensitive plasma proteins predict incident diabetes mellitus, metabolic syndrome (275–277), and, simultaneously, cardiovascular events (278). Gammaglobulin levels, a nonspecific marker of immune activation, have also been found to predict type 2 diabetes mellitus, whereas rheumatoid factor did not (15). Increased


serum ferritin, another acute-phase reactant, predicted the development of diabetes in epidemiological studies (279). However, in this case, a role for increased iron stores cannot be excluded (280). Recent information also suggests that inflammatory markers might predict not only diabetes mellitus but also diabeticassociated complications. The longitudinal development of increased urinary albumin excretion was significantly and independently determined by increased baseline CRP and fibrinogen in type 2 diabetics and nondiabetic individuals (281, 282). In fact, in early work by McMillan (164), progressive increases in plasma levels of CRP, ␣-1 glycoprotein, haptoglobin, and plasma viscosity correlated with increasing evidence of microangiopathy in type 2 diabetic subjects. Interestingly, TNF polymorphisms seem also to modulate the risk of diabetic retinopathy (283–285).

VII. Antiinflammatory Agents Improve Insulin Resistance and Prevent Development of Type 2 Diabetes Mellitus

Insulin is increasingly recognized as an antiinflammatory molecule (reviewed in Refs. 286 and 287). This general antiinflammatory activity has been advocated to explain the remarkable improvements in mortality and morbidity after low doses of insulin in patients with acute myocardial infarction (288) and in patients admitted to a surgical intensive care unit (289). In fact insulin, at physiologically relevant concentrations, causes a suppression of intranuclear nuclear factor-␬B (which induces the transcription of proinflammatory genes like TNF-␣ and IL-6), ICAM-1, and monocyte chemoattractant peptide in human aortic endothelial cells and in mononuclear cells in vivo. These effects have been related to the ability of insulin to induce the release of NO and to enhance the expression of constitutive NOS (Ref. 286; also see Section II.A). The insulin sensitizer thiazolidinediones decrease the plasma concentrations of TNF-␣, sICAM-1, monocyte chemoattractant peptide-1, and CRP, in parallel to improved insulin action (290 –292). These agents also cause an increase in the antiinflammatory cytokine IL-10 and decrease reactive oxygen species generation by mononuclear cells (286, 290). In the last few years, the adipocyte protein adiponectin has also been recognized to exhibit antiinflammatory actions. Interestingly, adiponectin is paradoxically decreased in obesity (293–295) in parallel with reduced insulin sensitivity (294, 295). Recent works show how adiponectin deficiency leads to increased TNF-␣ activity (140). Weight reduction leads to increases of its plasma levels (296), and adiponectin administration reverses insulin resistance associated with obesity (297). Salicylates have been described to improve insulin action in vitro and in vivo in animal models (298, 299). High doses of salicylate and inactivation of I␬B kinase-␤ prevent fatinduced insulin resistance in skeletal muscle by blocking fat-induced defects in insulin signaling and action. In humans, high-dose aspirin treatment resulted in an approximately 25% reduction in fasting plasma glucose, associated with an approximately 15% reduction in CRP and total cho-


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lesterol, an approximately 50% reduction in triglycerides, and an approximately 30% reduction in insulin clearance. Aspirin treatment also resulted in an approximately 20% reduction in basal rates of hepatic glucose production and an approximately 20% improvement in insulin-stimulated peripheral glucose uptake (300). On the other hand, genetic variability in insulin action inhibitor I␬B kinase-␤ does not play a major role in the development of type 2 diabetes in humans (301). The assignment to pravastatin therapy resulted in a 30% reduction in the hazard of becoming diabetic in the West of Scotland Coronary Prevention Study (302). Given the antiinflammatory effects of this statin [it has been shown to reduce circulating levels of IL-6 and TNF-␣ (303)], this might be the mechanism by which pravastatin favorably influences the development of diabetes. Similarly, a reduction in the incidence of type 2 diabetes in patients treated with ramipril (304, 305), an ACE inhibitor with presumed antiinflammatory effects, is consistent with type 2 diabetes being an inflammatory state. ␣-Lipoic acid, a naturally occurring compound, is able to stimulate glucose uptake in cytokine-treated cells that are insulin resistant (306). Oral treatment with ␣-lipoic acid also improved insulin sensitivity in patients with type 2 diabetes as demonstrated during isoglycemic glucose-clamp (307).

VIII. Possible Interpretations, Unifying Hypothesis

Humans live in close association with vast numbers of microorganisms that are present on the external and internal surfaces of our bodies. The ability to mount a prominent inflammatory response to bacterial pathogens confers an advantage in innate immune defense. Different nutrientsensing and metabolic pathways might have evolved in parallel with several mechanisms involved in our fight against infection (Fig. 6). Even first-line immune defenses seem to be associated with metabolic pathways. For instance, overweight seems to be a medium-term complication of tonsillectomy (308). Recent evidence shows that adipose cells play a role in the local immune defense during inflammatory processes. A link between the immune system and adipose tissue has been suggested on the basis of the adipocytokines. TNF-␣ has been hypothesized to arise by divergence from a primordial recognition molecule of the innate immune system (309). Adipocyte complement-related protein/AdipoQ/ adiponectin is a close homolog of the complement protein C1q, which is involved in the recognition of microbial surfaces. Interestingly, studies of the structure of C1q have revealed an unexpected homology to the TNF family, firmly establishing an evolutionary link between the C1q and TNF families (309). Other factors have been added recently to the list of primordial molecules implicated in the recognition of microbial surfaces that are simultaneously involved in immune system function and in the regulation of energy balance (310 –313). Soluble CD14, detectable at high concentrations constitutively present in the circulation, is believed to play a key role as intermediate in the neutralization of bacterial LPS under physiological conditions. Decreased sCD14 is associated with insulin resistance in healthy controls and

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FIG. 6. Glucose sensing is among the most conserved pathways in human evolution, given its vital importance for brain and immune system metabolism. These two systems are hypothesized to interact to obtain glucose, no matter the cost, from adipose, liver, and muscle tissues, even at the expense of pancreas exhaustion. Evolutively, the price to pay was very low. Those individuals with cytokine and metabolic genetic polymorphisms that implicated the best external/internal defense, preserving glucose for brain, immune system, placenta, and mammary gland, the classical insulin-independent tissues, survived and transmitted their genes.

in patients with type 2 diabetes (310). CRP is a member of the pentraxin family of oligomeric proteins involved with pattern recognition in innate immunity (311, 312). Myeloperoxidase, an enzyme principally associated with host defense mechanisms, has also been associated with CRP levels and cardiovascular risk (313). Myeloperoxidase seems to modulate vascular signaling and vasodilatory function of NO, linking the fight against infection with metabolic events (314). The association between several acute-phase reactants, insulin resistance, and IHD might be interpreted as the response of a body to chronic tissue infection, and decreased insulin action would be a byproduct of the inflammatory cascade triggered by physical, environmental, and infectious agents. Although it seems unlikely that one specific pathogen causes atherosclerosis, the infectious burden is increasingly claimed to be involved in the development of atherosclerosis (315) and possibly insulin resistance. The association between pathogen burden and extent of atherosclerosis was mainly driven by seropositivities to bacterial and viral infections. Family studies show that up to 60% of the variability in TNF-␣ production between individuals may be genetically determined (316). Similar proportions are probably shared by other cytokines. Wilson et al. (192) proposed that different cytokine genotypes may exist in the population as a result of the selective pressure of infectious diseases. It may be that specific cytokine genotypes are beneficial in the eradication of infectious diseases, but by creating a proinflammatory phenotype, they predispose to chronic inflammatory diseases or to a more severe form of inflammatory disease with a worse clinical outcome, irrespective of whether the initial

triggering event is an infectious agent, autoimmunity, or, indeed, any cause sufficient to stimulate an inflammatory response. Moreover, prenatal cytokine exposure may result in persisting metabolic and hormonal changes in the fetus by inducing gender-specific programming, leading to insulin resistance (317). Some ethnic groups such as American Indians display a high incidence of obesity and type 2 diabetes mellitus. In contrast, the frequency of type 2 diabetes is of the lowest in the world, despite increasing obesity among Europeans. It has been suggested that, because most diabetogenes are recessive, the greater the genetic heterogeneity of a population, the lower the chances of homozygosity, thus explaining this European paradox (318). In the presence of numerous infectious diseases, different alleles, protecting against individual pathogens, could make heterozygosity advantageous overall (319). South European history provides explanations of a much higher degree of interbreeding than almost any other area of the world (318). On the opposite side, American Indians, after centuries of relative isolation, have a dramatic history of recent epidemics of infectious disease after their first contact with Europeans. Those Pima Indians with the better defense against pathogens (high cytokine responder) eradicated this injury, but at the expense of developing insulin resistance nowadays. Interestingly, all subjects who were of full Pima Indian heritage had the IL-6-174 GG genotype, whereas in the American Indian subjects with nonPima admixture, type 2 diabetes was associated with IL-6 genotype (P ⫽ 0.001; Ref. 223). The G allele was also more frequently present among Caucasians with type 2 diabetes (223). This allele had been previously found to be associated with both increased serum IL-6 concentration and insulin


resistance (161). Hence, genetic diversity might have contributed, simultaneously, to defense against infection and to the lower prevalence of insulin resistance and type 2 diabetes mellitus among Europeans—and to its increased prevalence among Pima Indians (15). However, it should be stated that the genetic-environmental interactions are otherwise complex. For instance, Africans are one of the most genetically diverse populations [high cytokine responders? (according to Ref. 9)], and these peoples have increased prevalence of type 2 diabetes in a Westernized environment. On the other hand, glucose sensing is among the most conserved pathways in human evolution, given its vital importance for brain and immune system metabolism. Thus, it is not surprising that these two systems interact to obtain glucose, no matter the cost, from adipose, liver, and muscle tissues, even at the expense of pancreas exhaustion. Evolutively, the price to pay was very low; the priority was the defense against pathogens, and the mean survival was limited to 35– 40 yr. Those individuals with cytokine and metabolic genetic polymorphisms that implicated the best external/internal defense (preserving glucose for brain, immune system, placenta, and mammary gland, the classical insulin-independent tissues) survived and transmitted their genes (Fig. 6). Finally, it cannot be forgotten that our vision of chronic cardiovascular inflammatory disease is perhaps limited by the same tool we usually use for the measurement of serum/ plasma glucose concentration. Glucose is only one component of the metabolic network. A network consists of nodes connected by edges. Biologically speaking, substrates are the nodes, and the metabolic reactions are the connecting edges. Metabolic networks seem to belong to the class of scale-free networks that are highly inhomogeneous because they contain nodes that have a significantly higher number of connections than the average, acting like hubs. There are a few highly connected substrates that turned out to be the same across the species. Despite being robust and error-tolerant, scale-free networks are also sensitive to attacks on the highly connected nodes (320). The research in the area of the insulin resistance syndrome has been focused on glucose disorders, even after the demonstration that a serum glucose from 85 mg/dl was associated in a continuum manner with cardiovascular disease (321). That is, perhaps, one of the reasons why former therapeutic agents for insulin resistance were selected on the basis of their effects on serum glucose. We should take advantage of the glucose hub to treat other aspects of insulin resistance. The moment has arrived to look for earlier markers and hubs for cardiovascular disease, and insulin resistance-mediated inflammation is, perhaps, one of the keys.

IX. Summary

In the last few years, insulin action was increasingly recognized as an important effector mechanism of the inflammatory pathways. These interactions occur at all levels of the immune response. Given the crucial importance of insulin resistance and inflammation in cardiovascular disorders, the study of the interactions of these important pathophysiolog-


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ical mechanisms will shed light on new therapeutic strategies. Genetic research on factors involved in the inflammatory cascade will probably help to characterize individuals simultaneously prone to obesity, hypertension, dyslipidemia, insulin resistance, and cardiovascular disease. Acknowledgments Address all correspondence and requests for reprints to: J. M. Ferna´ ndez-Real, M.D., Ph.D., Unitat de Diabetes, Endocrinologia i Nutricio´ ., Hospital de Girona “Dr. Josep Trueta”, Carretera de Francia s/n, 17007 Girona, Spain. E-mail: [email protected]

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