Adiponectin circulating levels: A new emerging biomarker of cardiovascular risk

Pharmacological Research 56 (2007) 459–467

Review

Adiponectin circulating levels: A new emerging biomarker of cardiovascular risk D. Giannessi ∗ , M. Maltinti, S. Del Ry CNR Institute of Clinical Physiology, Laboratory of Cardiovascular Biochemistry, Pisa, Italy Accepted 17 September 2007

Abstract Fat is now considered as an endocrine organ that produces a lot of molecules having biological activity, called adipocytokines. Among these, adiponectin, a 247 aminoacid protein produced abundantly and specifically by adipose tissue, besides its effects on glucose metabolism, plays important protective function against cardiovascular diseases. Circulating levels lower than those of healthy control subjects have found to be associated to conditions such as obesity, diabetes and cardiovascular diseases. In animal experimental models, administration of adiponectin has been shown to have beneficial effects against the development of obesity-related vascular diseases, including atherosclerosis. In humans, circulating levels can be raised by life style modification (weight loss or exercise training) or pharmacological treatments. Adiponectin is present in the human plasma in different isoforms: a large multimeric structure of high molecular weight and in a trimer and examer form, whereas the monomeric form is found only in the adipose tissue. The biological activities of the different multimers are not yet fully known, although the different isoforms appear to have different functional importance following the different diseases. This paper reports the main biological features of adiponectin in order to highlight its possible role as diagnostic/prognostic marker in cardiovascular diseases. Particular attention is paid to practical considerations relative to the analytical determination of this protein in humans. © 2007 Elsevier Ltd. All rights reserved. Keywords: Adiponectin; Adipocytokines; Multimers; Immunoassay; Cardiovascular diseases

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adiponectin—structure and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adiponectin measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Circulating levels of adiponectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnostic/prognostic value of adiponectin in cardiovascular diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adiponectin as a therapeutic target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction In the last years many biological markers [1] have been proposed for screening, diagnosing, prognostication, prediction of disease recurrence as well as for therapeutic monitoring

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Corresponding author at: CNR Institute of Clinical Physiology, Laboratory of Cardiovascular Biochemistry, Research Area-Via G. Moruzzi, 1 Pisa, Italy. E-mail address: [email protected] (D. Giannessi). 1043-6618/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2007.09.014

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of cardiovascular diseases, which represent the first cause of morbidity and mortality in the industrialized countries [2]. Among the biomarkers that could help the clinicians to better identify high-risk subjects and to effectively prognosticate and treat the patient with cardiovascular diseases, an increasing importance has been given to adiponectin, a protein produced by adipocytes [3–6] and present at high concentration in human peripheral circulation [4]. In fact, adipose tissue is now studied as an endocrine organ [7] because, in response to specific extracellular stimuli or to variations in the metabolic conditions, it

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releases many biologically active substances, called adipocytokines or adipokines. The adipocytokine family, besides the classical cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-␣, produced probably by inflammatory cells embebbed in the fat tissue, includes other molecules with high biological activity such as ghrelin, leptin and adiponectin. These proteins, besides the well-known metabolic actions, have other different biological effects. In particular, adiponectin, due to its anti-inflammatory and anti-atherogenic properties, appears to have important protective effects at cardiovascular level [8]. In these last years, a progressive increase in the knowledge of the biological actions of adiponectin has been observed, which, in turn, led to recognize the importance of its functions until to suppose a cardioprotective role [9]. In fact, a plenty of papers have been published on the issue “adiponectin and heart”; considered as a whole, these studies indicate that this effector has a diagnostic–prognostic value in cardiovascular diseases and that it can be also considered a therapeutic target [10–12]. Some features of adiponectin make this protein a suitable marker of cardiovascular risk [11]. Among these, the most important for treatment and prevention is represented by the possibility of modulating its circulating levels by pharmacological treatments, life style changes or diet. Moreover, adiponectin is present at high levels in peripheral circulation, so that it can be measured by using little amounts of sample, and shows poor intra-individual variability and lacks circadian rhythm. For these reasons and for its possible role at cardiovascular level, the measure of circulating levels of adiponectin constitutes a new interesting parameter for the bio-humoral characterization of patients with cardiovascular diseases [10]. This paper reports an overview of the recent literature about the role of adiponectin in cardiovascular diseases in order to evaluate a possible clinical use of this effector as a new cardiovascular biomarker [1]. Moreover, special attention is addressed to the problems related to the analytical determination of this

adipokine, present in the human circulation in almost three multimeric forms [13]. 2. Adiponectin—structure and function Adiponectin, known also as Acrp30 [3], apM1 [4], AdipoQ [5] and GBP28 [6], is a protein of 247 aminoacids; it was isolated in 1995 from adipose tissue by four research groups independently by using different approaches [3–6]. It is constituted by four domains and presents a multimeric structure [14,15]. As shown in Fig. 1, adiponectin is formed by a 20residue amino-terminal signal sequence, a variable region, a collagenous domain and a carboxy-terminal globular domain. As demonstrated by X-ray crystallography of the globular domain, adiponectin presents a high structural homology with TNF-␣, suggesting an evolutionary link between TNF-␣ family and this adipokine [16]. By interactions at the collagenous domains, adiponectin is transformed within the adipocytes into multimeric forms [3,14,15], including the low molecular weight trimeric form (LMW, 75–90 kDa), made of three monomers strongly bounded at globular domain level, the middle molecular weight exameric form (MMW) and the high molecular weight form (HMW, about 500 kDa), constituted by eight or more monomers (Fig. 1). The trimer is formed by the association of the c-terminal globular domains with the triple helix formation at the collagenous domain level. The exameric form is obtained through disulphide bond formation at the cysteine (Cys)39 residue. HMW multimers are formed by noncovalent higher-order interactions. Both disulphide bond and the presence of cysteine at position 39 are necessary for multimerization; in fact, the replacement of cysteine with serine results in the lack of high-order structure formation. Similarly, heatdenaturated adiponectin is present only in the monomeric form, indicating that disulphide bridge is necessary for multimerization.

Fig. 1. Adiponectin structure (top); adiponectin multimeric forms (bottom).

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The different multimers have been identified by different methods, namely gel filtration chromatography and electrophoresis on acrilamide gel in the presence of sodium dodecyl sulphate (SDS-PAGE) and Western blotting analysis. The monomeric form, 28–30 kD molecular weight, is not found in peripheral circulation, but only in the adipose tissue. Four multimers of adiponectin can be found in circulation: the three multimeric forms produced by adipocytes together with a further form consisting of the trimer bound to the serum albumin (Alb-LMW). Fragments from adiponectin proteolysis, including the globular domains, can also be found in the plasma [9]. Both biological activity and transduction mode are different for the different multimeric forms of adiponectin. In fact, it has been recently observed that the HMW form is an active form, and its ratio with total adiponectin appears to be closely correlated with insulin sensitivity [17,18]. Moreover, it has been suggested that the percentage of each form with respect to the total adiponectin could vary as a function of the different physiopathological conditions [19]. These observations indicate the importance, from the clinician point of view, of measuring the concentrations of each multimer besides the total adiponectin level. Adiponectin synthesis and release are controlled by different mechanisms. It has been demonstrated that insulin stimulates adiponectin gene expression and its release by cultured adipocytes [3], and both insulin and insulin-like growth factor1 increase the adiponectin production in adipocytes isolated by visceral adipose tissue [20]. Moreover, the lipolysis in the human adipose cells appears to be hormone-dependent, catecholamines and natriuretic peptides, besides insulin, exerting important effects [21]. As to the action mode of adiponectin, two main specific receptors are known: adipoR1, mainly found in skeletric muscle, and adipoR2, mainly expressed in the liver, as well as in human monocytes and macrophages. They mediate the activation of adenosine monophosphate-activated protein kinase (AMPK), the oxidation of fatty acid, and glucose uptake. Specific receptors are expressed also in the cardiac tissue and particularly in cardiac myocytes, whose growth and intra-cellular interactions are modulated by adiponectin [9]. In eukaryotes only, T-cadherin, an extra-cellular protein present in endothelial and smooth muscle cells, can function as a further receptor for adiponectin MMW and HMW forms [22]. 3. Biological actions Adiponectin plays an important role in the regulation of insulin function and energetic homeostasis [16,23]. Circulating levels and adipose tissue gene expression are lower in the obese and in type 2 diabetes subjects with respect to healthy controls [24–26] and negatively correlate with the body mass index (BMI), the plasma levels of glucose, insulin, triglycerides and the insulin-resistance [25]. Moreover, it has been observed that adiponectin reduces glucose levels in different animal models of obesity/diabetes mellitus and this hypoglycemic effect is not associated to stimulation of insulin secretion, whose levels are reduced in parallel with those of glucose, but to an increased insulin sensitivity [27]. Although the mode of action

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Fig. 2. Adipocytokines and the regulation of vascular function and the development of cardiovascular diseases. Modified from Ref. [12].

of adiponectin on glucose metabolism is not fully understood, experimental evidences indicate that adiponectin reduces the liver production of glucose, whose muscle utilization appears increased [16,23]. Besides these effects on glycidic metabolism, adiponectin exerts important functions at vascular and cardiac level (Fig. 2), especially as to endothelial function and the protection from ischemia/reperfusion injury. Moreover, it exerts anti-inflammatory, anti-apoptotic and anti-hypertrophic effects [9]. As to the vascular function, adiponectin controls the monocyte adhesion to vascular endothelium [28] and, in humans, low adiponectin levels appear to be closely correlated to the severity of endothelial dysfunction [29]. Moreover, hypoadiponectinemia is associated to a reduced endothelium-dependent vasodilation in subjects with and without type 2 diabetes [30]. Adiponectin attenuates the inflammatory response induced by different stimuli by modulating signal transduction mechanisms in different cells and these anti-inflammatory properties of adiponectin could account for its beneficial effects on cardiovascular and metabolic disorders, including atherosclerosis and insulin-resistance [31]. As can be seen in Fig. 3, where a summary of the adiponectin-mediated regulation of inflammatory responses in different cell types is depicted, adiponectin negatively modulates C reactive protein (CRP) and TNF-␣ expression in adipose tissue while both TNF-␣ and IL-6 inhibit adiponectin expression. In particular, in endothelial cells IL-8, vascular cell adhesion molecule (VCAM)-1 and reactive oxygen species (ROS) production is negatively regulated by adiponectin through cAMP-PKA-dependent pathway, while an activation of endothelial nitric oxide synthase (eNOS) is achieved by AMPK activation in the same cell type. In cardiac cells adiponectin reduces TNF-␣ production by activation of cyclo-oxygenase2 (COX-2)-prostaglandin (PG)E2 signalling. In macrophages, adiponectin reduces nuclear factor (NF)-kB activation resulting in an inhibition of TNF-␣ and IL-6 production. Moreover, in human macrophages, adiponectin, enhancing the expression of

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Fig. 4. Adiponectin and cardioprotection. Modified from Ref. [9].

actions of adiponectin could, partly, explain why the administration of drugs inhibiting COX-2 has negative collateral effects at cardiovascular level [41,42]. Besides these cardiovascular functions, adiponectin has also further actions such as, for example, the regulation of proliferation and function of hemopoietic stem cells [43].

Fig. 3. Regulation of inflammatory responses in different cells types by adiponectin. Modified from Ref. [31].

IL-10, increases the production of the tissue inhibitor of metalloproteinase (TIMP)-1 [32]. Finally, in human macrophages adiponectin inhibits the macrophage-to-foam cell transformation by suppression of the expression of class A scavenger receptor (SR-A) [33]. As to the peripheral circulation, an inverse correlation has been demonstrated between the levels of adiponectin and inflammation markers such as CRP [34], that is considered to be an independent predictor of risk for cardiovascular disease outcome and of development of metabolic syndrome [35,36]. As to ischemia/reperfusion injury, it has been recently shown, in an animal model, that adiponectin is an endogenous protective factor against ischemia/reperfusion injury, functioning by reducing the oxidative stress mediated by nitric oxide (NO) and inhibiting, among other things, the inducible NO synthase (iNOS) [37]. High levels of adiponectin resulted associated to a lower risk for myocardial infarction, independently of the values of CRP and from glycemic situation [38]. After acute myocardial infarction, adiponectin plasma concentration rapidly decreases and negatively correlates with CRP [39], suggesting that hypoadiponectinemia is correlated with an increase of inflammatory response following cardiac ischemia. Studies in animals knock-out for adiponectin confirmed these observations and showed that preventive administration of esogenous recombinant adiponectin reduces the size of infarcted area [9]. It has been also observed that adiponectin inhibits apoptosis in cardiac myocytes and fibroblasts that have undergone hypossia/reossigenation damage [40] through AMPK-dependent mechanism. The protective action of adiponectin from ischemia/reperfusion injury at cardiac level is also due to the activation of COX-2 in cardiac cells [40] (Fig. 4). The two signal transduction pathways are independent: AMPK pathway mainly mediates the anti-apoptotic function while COX-2 pathway the anti-inflammatory actions. As to the COX-2 signalling, the proposed mechanism agrees with the observation that the treatment with selective inhibitors of this enzymatic pathway increases the risk of cardiovascular complications, including myocardial infarction [41]. The interference with the protective

4. Adiponectin measurement Many immunometric systems are commercially available for the determination of the total content of adiponectin in the peripheral circulation, after a sample treatment that quantitatively transforms all the adiponectin multimers present in the biological sample into the monomeric or dimeric form, respectively. For this, before the immunometric assay, the biological sample is treated with SDS at high temperature [24] to obtain adiponectin in monomeric form or with SDS at pH 3.0–3.5 [13] to have the dimeric form, respectively. The dose–response curve is built by using, as calibrator, recombinant adiponectin [24] or dimeric adiponectin [13] obtained by conversion of purified HMW adiponectin. The analytical performances of the main commercially available immunoassay systems are similar and suitable to reliably determinate adiponectin content in hematic samples. As an example, Fig. 5 shows original data obtained in our laboratory relative to the assay of total adiponectin by using two different immunoassay kits, both evaluating the adiponectin monomeric form. On the contrary, the absolute levels of total adiponectin obtained with commercial systems that evaluate the dimeric form are different from those obtained with systems determining the monomeric one, although closely related. The final result depends also by the calibrator chosen for the dose–response curve, thus, care must be exercised in the comparison of the adiponectin total concentration values obtained by different analytical methods.

Fig. 5. Comparison of two immunometric assays (Elisa) for the measurement of total adiponectin in plasma EDTA samples (n = 38). The methods compared are the B-Bridge International (San Jos´e, CA, US) and the Linco (St. Charles, MO, US). The regression plot and the main analytical features are reported.

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Fig. 6. Scheme of the procedure for the determination of the adiponectin multimers.

More recently, two kinds of proteases have been found, able to selectively digest adiponectin multimers [13], proteinase K (from Tritirachium album) and protease A (from Aspergillus oryzae). Proteinase K digests selectively the low (LMW and Alb-LMW) and the middle (MMW) molecular weight form while the high molecular weight form remains unchanged. Thus HMW form can be measured by immunoassay (after transformation to dimer or monomer, following the method used). The use of protease A, that digests the LMW e Alb-LMW form, allows us to measure together the MMW and the HMW forms. As shown in Fig. 6, the measure of the amount of adiponectin, before and after treatment with proteinase K and protease A, respectively, makes possible to obtain the concentration of the different adiponectin isoforms present in the biological samples. In a group of healthy control subjects the levels of the three multimeric forms of adiponectin resulted positively related to total concentration of the protein [13]. 5. Circulating levels of adiponectin The mean value of the circulating levels of adiponectin in nonobese subjects is of the order of tens micrograms per millilitre (3–30 ␮g/ml) [12] and, contrary to what happens for most proteins released by adipose tissue, a significant reduction (>50%) is found in the obese subjects [24]. Although the adiponectin expression is activated during adipogenesis, the “paradoxical” lowering of plasma levels as the body mass increases, a feature of this adipokine, could be due to a negative feedback effect on its production during obesity development. As to the action mechanism, still not clear, it could be supposed that the reduction of adiponectin in obese subjects could be due, at least partly, to the increase of TNF-␣, known to reduce the adiponectin secretion [44] as well as that of other adipokines whose expression is increased in obesity. As to the negative correlation with the BMI [24], this association is stronger with visceral adiposity than with subcutaneous adiposity [45]. A gender difference in the circulating levels of adiponectin has been also observed: considering non-obese subjects, mean plasma levels in men are lower than those found in women (e.g. 7.7 ± 3.1 vs. 10.6 ± 7.3 ␮g/ml) [24]. This gender difference, probably due to an estrogen action [46], is observed also

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in obese subjects [24]. In healthy pre- and post-menopausal women, plasma adiponectin was higher in middle-aged and older post-menopausal women compared with middle-aged pre-menopausal women; moreover, in normal weight women adiponectin is independently related to age, leptin, insulinresistance and menstrual status [47]. Concentrations lower than those of healthy controls are found also in patients with essential hypertension [48] and in diabetic subjects [25]. Administration of the insulin-sensitizing thiazolidinedione (TZD) increases the circulating levels of adiponectin by about three times in diabetic subjects [49]; also the exercise training is able to increase the circulating levels of adiponectin [50]. Adiponectin levels are negatively related to triglycerides, with atherogenic index (total cholesterol/HDL-cholesterol) and with apolipoproteins, B and E, while a positive correlation is observed with HDL-cholesterol and with apo-A-1 [51]. These correlations, found in a group of non-diabetic women, are independent of BMI, fat mass and age. The results of another recent study [52], made in a group of 1153 young subjects and devoted to evaluate the relationship between adiponectin and markers of cardiovascular risk, underline the importance of including adiponectin in the assessment of the cardiovascular risk. In fact, adiponectin concentration negatively correlates with insulin-resistance, visceral fat and the relative metabolic syndrome. Moreover, low levels of adiponectin are found in subjects with a family history for coronary artery disease (CAD), hypertension and diabetes. As to the adiponectin multimers, the levels of the high molecular weight form (HMW) account for the 50% of the total adiponectin, while both MMW and LMW forms constitute about the 25% of total adiponectin in control subjects [13]. The concentrations of HMW and MMW multimers resulted significantly higher in the women with respect to men [13,53] while no important gender differences were observed for the trimeric form (LMW) [13]. Similarly to plasma total adiponectin, HMW adiponectin correlates directly with HDL-cholesterol and inversely with triglycerides, insulin sensitivity, creatinine clearance and inflammatory markers in patients with type 2 diabetes [53]. HMW adiponectin as well as its ratio to total adiponectin resulted lower in patients with CAD with respect to those without CAD (the relation was stronger in men than in women) indicating that the HMW form determination and its ratio on total adiponectin are useful for evaluating CAD in type 2 diabetic patients [53]. Moreover, HMW form of adiponectin has been recently shown to be an independent factor able to influence the endothelial function, estimated by flow-mediated dilatation of the brachial artery during reactive hyperaemia in healthy young men [54], suggesting that HMW adiponectin may be more useful as a marker of endothelial dysfunction than total adiponectin. 6. Diagnostic/prognostic value of adiponectin in cardiovascular diseases The cardio-metabolic syndrome, associated with an increase of the risk of cardiovascular diseases in the industrialized countries, affects about one individual out of four. The molecular

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mechanisms that bound this condition with cardiovascular diseases are not fully understood. Among the different effectors considered, it has been supposed that adiponectin, showing anti-inflammatory and anti-atherogenic effects, could have a protective effect against the development of cardiovascular diseases. Recently, many clinical studies confirmed this hypothesis, suggesting for adiponectin an important role in cardiovascular diseases. The health professionals follow-up study [38] indicates that subjects with more elevated levels of adiponectin present a significant reduction of the risk of myocardial infarction, also after correction for HDL- and LDL-cholesterol and BMI. As to possible correlations between adiponectin levels and markers of cardiovascular risk, a study on 1174 patients with angiographically documented CAD showed (after correction for age and sex) a correlation with HDL-cholesterol, triglycerides and NT-proBNP, suggesting that dyslipidemia could be a link between adiponectin and atherosclerosis progression [55]. The correlation, observed in this study, between adiponectin and BNP has been also confirmed in patients with peripheral arteropathy, independently of other risk factors [56]. Moreover, in non-diabetic subjects, circulating levels of adiponectin resulted inversely related to CAD severity [57,58]. Low concentrations of adiponectin appear to be associated also with an early onset of coronary heart disease and with the presence of multiple atherosclerotic lesions on coronary arteries [59]. Finally, Frystyk and coworkers, that made a 10 years follow-up in a group of healthy old subjects, recently showed that elevated circulating concentrations of adiponectin are associated with a lower risk for CAD independently of other well-known risk factors [60]. Adiponectin appears to have a role in many other related clinical conditions: a negative correlation between adiponectin and carotid intima media thickness [61] and with the intra-stent ristenosis was observed [62]. As to the heart failure (HF), adiponectin plasma levels increase as a function of disease severity (NYHA class) and correlate with BNP and TNF-␣ [63]. High levels of this protein constitute a predictor of death independently of other risk markers [64]. Similar results were obtained by George and coworkers [65], that found a positive correlation with NT-proBNP and negative with CRP; moreover, in a follow-up of 2 years, adiponectin values higher than 75◦ percentile appear to be independent predictors of mortality/hospitalisation for HF. These findings have been confirmed in a study evaluating the total and HMW adiponectin in a large group of Japanese peoples followed for 5 years [66]. In their cohort of patients, both total and HMW adiponectin increase with the severity of NYHA functional class and both are mainly associated with BMI and cardiac natriuretic peptides. Moreover, total adiponectin appears more useful for assessing mortality risk that HMW form and high plasma total adiponectin levels are confirmed as an independent prognostic predictor, especially in HF patients with normal BMI. The less useful prognostic value of the active form, HMW, is not easily to explain, but it might to be hypothesized that high HMW adiponectin has a cardioprotective effect while an increased total adiponectin reflects the severity of HF [66]. The evaluation of the results of the epidemiological studies on this disease is often difficult owing to the many bio-humoral and functional factors

involved. Obesity, as an example, constitutes a risk factor for the development of HF. However, higher values of BMI are associated to a better prognosis in patients with chronic heart failure because wasting is strongly associated with an increase of death. For this, high adiponectin levels are predictors of death in patients with HF, a higher body mass (i.e. low adiponectin levels) improving the survival in overt HF. Thus, adiponectin appears to have a “paradox” behaviour during HF progression: in HF the increase of the adiponectin levels, associated to the decrease in body weight, have not the waited cardiovascular protective actions. High adiponectin levels act as indicators of disease severity, but its function as pathophysiological mediator is less relevant in this situation. This hypothesis could explain the lack of importance of the HMW form, which is the functionally more active multimer. These observations suggest the existence of a “functional adiponectin resistance” in HF as explain in a recent editorial on this issue [67]. In tune with these findings, it has been observed that mRNA and protein expressions of the receptor AdipoR1 are reduced in the left ventricle of infarcted mouse with respect to control hearts [68]. This confirms the possibility of an “adiponectin resistance” receptor-mediated induced by target end-organ damage [67]. There are no data on the adiponectin circulating levels in dilatative cardiomyopathy (DCM), although an increase of this protein has been observed in the cardiomyocytes from DCM patients or with acute myocardial infarction at autopsy [69]. Preliminary results relative to a group of DCM patients, with or without heart failure, obtained in our laboratory, indicate an increase of adiponectin circulating levels with respect to a healthy subject control group, in agreement with those of markers of inflammation and of cardiac and endothelial function, also in this clinical condition [70]. 7. Adiponectin as a therapeutic target Adiponectin and its receptors may be considered as targets of potential therapeutic agents for metabolic syndrome and related diseases. In fact, it has been suggested that hypoadiponectinemia, due to interaction between genetic factors and environmental factors such as obesity, would result in an increased insulin-resistance, that, in turn, could be associated to an increase in insulin secretion, as compensatory mechanism. This brings about as a consequence a down-regulation of adiponectin receptors, regulated also by insulin, that produces a reduction of the adiponectin-binding capacity. So, metabolism could enter into a “vicious circle” toward metabolic syndrome [71], a situation known to be associated with an increase of the risk of cardiovascular diseases. Moreover, studies in vitro and in animal models showed that direct administration of adiponectin causes an up-regulation of its concentration, and besides modulating the insulin sensitivity, can exert protective effects on the atherosclerosis development [72]. The direct administration may be difficult in the clinical practice, thus the up-regulation of adiponectin can be achieved by pharmacological treatments, capable of increasing peripheral levels of this protein such as the therapy with TZD drugs (rosiglitazone, pioglitazone, troglitazone), and by weight loss [72]. The action mode of the TZD

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in increasing the circulating levels of adiponectin is not known, however both a direct effect of TZD on the synthesis and/or secretion of adiponectin by adipocytes and an indirect effect by the reduction of the production of inflammatory cytokines [73,74] have been suggested. The results of a recently published meta-analysis, including 19 studies relative to the effect of TZD treatment on adiponectin levels, with special attention to avoid publication bias, clearly show an increase of endogenous serum adiponectin levels by intake of TZD and may indicate a potential new option to manage obesity-related diseases [75]. Also vanadium salts resulted capable of increasing the synthesis and release of adiponectin in 3T3-L1 adipocytes through a protein kinase B-dependent transduction pathway [76], probably exerting, by this action mode, their insulin sensitising effects. The medical use of trace of metal vanadium in the treatment of diabetes mellitus is known since the end of 18th century and the anti-diabetic effects of vanadium salts have been extensively described [77], which consist in vanadium mimicking the biological actions of insulin, such as decreasing circulating glucose levels in diabetic mice models and inducing glucose uptake in adipose and muscle cells [76]. These findings suggest that administration of vanadium compounds could be relevant in the light of adiponectin elevation as a new therapeutic strategy for obesity-related disorders. The weight loss is another possible way to increase plasma adiponectin: significant reductions of the body weight such as those obtained after gastric bypass or by important changes in the lifestyle (almost 14% reduction of BMI value) [72] resulted able to modify the circulating levels of adiponectin. Finally, it has been recently demonstrated that administration of eicosapentaenoic acid (fish-oil), well known in reducing the incidence of coronary heart disease, increases adiponectin secretion in rodent models of obesity and in human obese subjects [78], possibly through the improvement of the inflammatory changes in obese adipose tissue [79]. These data relative to fish-oil administration indicates that white adipose tissue has a specific role in regulating the improvement in systemic lipid homeostasis and atherosclerosis-mediated by dietary fish-oil [79]. Further therapeutic treatments, including the possibility to up-regulate the adiponectin receptors by specific receptor agonists [74], are at present under study. 8. Conclusions Adiponectin is a protein released specifically by adipose tissue, that exerts protective effects on the heart and vessels. In the heart, adiponectin functions by regulating the cardiac injury through its anti-inflammatory and anti-remodeling effects. Many studies need to identify what isoforms of adiponectin are associated to the cardioprotection and to clarify the signal transduction pathways receptor-mediated in different physiopathological conditions. The knowledge of the molecular and cellular bases of the biological actions of this protein will allow, among other things, to better understand how obesity influences the cardiac function and to address the development of new therapeutic approach for cardiovascular disease management.

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