Insulin resistance is an independent risk factor for coronary artery disease, but the mechanisms predisposing insulin-resistant individuals to atherothrombosis are poorly understood [1]. Coronary artery disease has been related to chronic, subclinical inflammation, as indicated by elevated circulating levels of inflammatory proteins [2].

Obesity, insulin resistance, and atherosclerosis are closely linked phenomena, all of them often associated with low-grade inflammation [3]. The primum movens of the interplay among these complex phenomena remains at present unclear.

Over the past decade, obesity has been associated with inflammation by demonstrating that tumor-necrosis-factor (TNF)-α is constitutively expressed by adipose tissue [4]. Obese adipose tissue is characterized by inflammation and progressive infiltration by macrophages as obesity develops [5]. A low-grade chronic inflammatory state, characterized by increased levels of inflammatory and/or acute-phase proteins, is present in patients with obesity and metabolic syndrome (MS) [6]. In a large cohort of women participating in the Women’s Health Study, a strong linear increase was detected in C-reactive protein (CRP) levels as the number of components of MS increased. In addition, baseline CRP levels add clinically relevant prognostic information concerning future vascular risk [7].

Adipose tissue, far from being an inert organ, has the capacity to secrete biologically active mediators [8]. They may influence cardiovascular risk through their involvement not only in body weight homeostasis, but also in inflammation, coagulation, fibrinolysis, endothelial dysfunction, insulin resistance, and atherosclerosis [9].

Adiponectin, an adipokine with proven anti-atherogenic and anti-inflammatory properties, which is inversely related to insulin resistance, has been recently shown to act as an endogenous antithrombotic factor, since its over-expression attenuated thrombus formation in mice [10]. Moreover, circulating adiponectin is reduced and independently related to reduced IL-10 levels in android obese women [11]. In addition, the increase in circulating leptin, in obese women, is associated with markers of haemostatic system [12].

The subclinical inflammatory state derived from adipose tissue results in insulin resistance, as suggested by a pioneering work by Hotamisligil [13]. Whereas the proinflammatory state induces insulin resistance, by impairing the insulin signal transduction, this resistance to the metabolic and vascular actions of insulin [14] promotes inflammation further through an increase in free fatty acid concentration and interference with the anti-inflammatory effect of insulin [7,15], thus paving the way for the metabolic perturbations eventually leading to atherothrombosis.

We demonstrated a significant association between insulin resistance and markers of inflammation and thrombin generation. Indeed, obese women with impaired insulin sensitivity had significantly higher levels of CRP, transforming growth factor (TGF)-β, PAI-1, FVIIa, and factor 1+2, compared to healthy controls and non-insulin resistant obese women [16]. Moreover, insulin resistance elicits the gene transcription of PAI-1, which exerts anti-fibrinolitic actions, thus promoting a pro-coagulant state [17]. PAI-1 levels are significantly higher in android obese women compared with gynoid obese or non-obese women. A direct correlation exists between PAI-1 levels and oxidative stress, as assessed by 8-iso-PGF2α urinary excretion rate, in obese women [18].

The abnormal metabolic state that accompanies insulin resistance renders arteries susceptible to atherosclerosis, by altering the functional properties of multiple cell types, including platelets [19].

We have previously provided evidence of persistent thromboxane (TX)A2-dependent platelet activation in association with visceral obesity [20]. In this setting we characterized a putative biochemical link between obesity or diabetes and platelet activation, by investigating in vivo formation of F2-isoprostanes, as reflected by urinary excretion of 8-iso-PGF2α, in vivo marker of oxidative stress. The cause-and-effect relationship between oxidative stress and platelet activation is demonstrated by the linear relationship between the excretion rates of 8-iso-PGF2α and the TXA2 metabolite 11-dehydro-TXB2, marker of in vivo platelet activation, and by the down-regulation of these metabolites following weight loss. These abnormalities leading to TXA2-dependent platelet activation appeared to be driven by inflammatory triggers that were, at least in part, down-regulated following a successful weight-loss program.

Although visceral obesity is much more strongly linked to insulin resistance, this relation is not present in all obese individuals and non-obese, non-diabetic healthy individuals may be insulin resistant [21]. In addition, the finding that human platelets have insulin receptors that participate in the regulation of platelet function led to the hypothesis that platelets are potential sites of insulin resistance, thus causing the lack in the physiological anti-aggregating action exerted by insulin [22].

These observations prompted us to test the hypothesis that insulin resistance per se contributes to increased platelet activation in obesity, independently of underlying inflammation.

            We performed a cross-sectional study in 40 obese women, with a body mass index (BMI) > 30 kg/m2 at the time of screening [23]. To avoid confounding by other determinants of platelet activation, women were excluded if they had a history or evidence of atherothrombotic diseases, diabetes mellitus, cigarette smoking, dyslipidemia, or arterial hypertension. Twenty healthy women (BMI £ 25 kg/m2) were also recruited as a control group.

            Consistent with our earlier findings, obese women had markedly higher urinary 11-dehydro-TXB2 excretion rate, when compared with non-obese controls. Plasma CD40 ligand (CD40L) and CRP levels were also significantly higher and insulin sensitivity index (SI) significantly lower in obese than in non-obese women. Only SI and waist-to-hip-ratio (WHR) independently predicted urinary 11-dehydro-TXB2. In addition, impaired SI identified women with significantly higher urinary excretion of 11-dehydro-TXB2. Thus, inflammatory proteins such as CRP and CD40L were elevated in individuals with insulin resistance; however, their relation to increased platelet activation seems to be largely explained by differences in WHR and SI.

To investigate the cause-effect relationship of associations characterized in the cross-sectional study, we examined the effects of a short-term weight loss program (12 weeks) on urinary 11-dehydro-TXB2 in 10 of the 20 obese women with impaired SI. Moreover, to avoid confounding by weight loss, which might influence per se insulin resistance independently of platelet function, we performed a single-blind, placebo-controlled 3-week study with pioglitazone, 30 mg daily, a peroxisome proliferator-activated receptor-g ligand that acts as an insulin sensitizing agent in 10 women with android obesity and impaired insulin sensitivity. Both successful weight loss, defined as a reduction of at least 5 kg of the initial body weight, and pioglitazone short-term treatment were associated with a concomitant improvement in SI and platelet activation.

Our results suggest that insulin resistance, per se, represents a major independent determinant of TXA2-dependent platelet activation in obesity, with subclinical inflammation as an underlying event largely explained by the association between insulin resistance and platelet activation. In addition, we show that a substantial reduction in TXA2-dependent platelet activation in this setting may be achieved by increasing insulin sensitivity and provide a rationale for performing randomized studies comparing the effects of caloric restriction and insulin sensitizing agents.