Participation of leptin in lipolysis

A decade ago the identification of functional leptin receptors (OB-R) in white adipose tissue suggested the involvement of leptin in the direct regulation of adipocyte metabolism at a peripheral level. In fact, leptin was shown to participate in lipid metabolism control through lipogenesis inhibition and lipolysis stimulation (Frühbeck, Reference Frühbeck2001). At a local level, leptin has the ability to repress acetyl-CoA carboxylase gene expression, fatty acid synthesis and lipid synthesis, which are biochemical reactions leading to lipid accumulation (Bai et al. Reference Bai, Zhang, Kim, Lee and Kim1996; Wang et al. Reference Wang, Lee and Unger1998). Additionally, leptin has been shown to exert an autocrine–paracrine lipolytic effect on white adipose tissue both in vitro and ex vivo (Frühbeck et al. Reference Frühbeck, Aguado and Martínez1997, Reference Frühbeck, Aguado, Gómez-Ambrosi and Martínez1998, Reference Frühbeck and Gómez-Ambrosi2001b; Wang et al. Reference Wang, Lee and Unger1998).

A functional relationship between leptin and NO has been established in several physiological processes (Frühbeck et al. Reference Frühbeck, Gómez-Ambrosi, Muruzábal and Burrell2001a; Frühbeck, Reference Frühbeck2006). Given the observed co-localisation of both factors in adipocytes and their involvement in lipolysis, the potential role of NO in the leptin-induced lipolytic effect has been investigated (Frühbeck & Gómez-Ambrosi, Reference Frühbeck and Gómez-Ambrosi2001b). A dose-dependent increase in both serum NO concentrations and basal adipose tissue lipolytic rate was observed 1 h after exogenous leptin administration, with simple linear regression analysis demonstrating that 27% of the variability taking place in lipolysis is attributable to the changes in NO concentrations. Inhibition of NO synthesis by using N ^ω-nitro-l-arginine methyl ester pretreatment was shown to be followed by a reduction in leptin-mediated lipolysis stimulation compared with leptin-treated control animals. In contrast, in adipocytes obtained from rats under acute ganglionic blockade by chlorisondamine administration it was found that the leptin-induced lipolytic effect is not different from the lipolytic rate achieved by leptin in control rats treated with saline (9 g NaCl/l; Fig. 3).

Fig. 3.

Lipolysis under nitric oxide synthase (NOS) inhibition and acute ganglionic blockade. Wistar rats under pharmacological pretreatment consisting of intravenous administration of vehicle (saline (9 g sodium chloride/l); control group), NOS inhibition (N ^ωnitro-l-arginine methyl ester (L-NAME); 30 mg/kg) or acute ganglionic blockade (chlorisondamine; 30 mg/kg) were further injected with either saline (□) or leptin (100 mg/kg; ////) to study the effect on basal lipolysis of fat cells. The lipolytic activity was measured as the amount of glycerol released by isolated adipocytes after 1 h. Results are expressed as the percentage of basal lipolysis of fat cells from saline-treated control animals and are mean values with their standard errors represented by vertical bars for eight animals per group (lipolytic experiments were performed in duplicate). Mean values were significantly different from those for the saline-treated animals within the same pharmacological pretreatment group: **P<0·01, ***P<0·001. Mean values were significantly different from those for the control leptin-treated animals: †P<0·05. Statistical analyses were performed using ANOVA and post hoc pair-wise comparisons.

It is well known that a rise in cAMP as a result of either adenylate cyclase activation or phosphodiesterase inhibition stimulates lipolysis. In order to gain further insight into the potential mechanisms involved, lipolysis has been stimulated in isolated adipocytes using a number of agents acting at different levels of the lipolytic pathway (Fig. 4): (1) at the β-adrenergic receptor (isoproterenol); (2) at adenylate cyclase (forskolin); (3) at phosphodiesterase E (isobutylmethylxanthine); (4) at protein kinase A (dibutyryl-cAMP). In order to investigate the modulation of leptin-induced lipolysis by NO, the effect of S-nitroso-N-acetyl-penicillamine (SNAP), a known NO donor, was determined in vitro in adipocytes isolated from control rats and incubated with leptin, isoproterenol and combinations of the different lipolytic agents. The effect of OB-R deficiency on lipolysis activation was examined by further studying the effect of leptin, SNAP and catecholamines in fat cells of obese fa/fa rats. Leptin administration was found to have no significant effect on the lipolysis rate of white adipocytes derived from fa/fa rats (Frühbeck & Gómez-Ambrosi, Reference Frühbeck and Gómez-Ambrosi2001b). The addition of isoproterenol or SNAP to the incubation medium, however, was found to result in a marked lipolytic response, thus suggesting that adipocyte preparations obtained from fa/fa rats respond to other known lipolytic agents. When leptin and SNAP were present simultaneously in the incubation medium of adipocytes isolated from Wistar rats an additive effect on in vitro lipolysis was observed compared with the effect elicited by the products acting individually. Only the NO donor SNAP was able to exert a marked inhibitory effect on isoproterenol-stimulated lipolysis. It was further shown that leptin does not interfere with catecholamine-mediated lipolysis. On the other hand, it was shown that NO is a potentially relevant autocrine–paracrine physiological signal, regulating lipolysis by facilitating leptin-induced lipolysis and, at the same time, being capable of inhibiting catecholamine-induced lipolysis (Frühbeck & Gómez-Ambrosi, Reference Frühbeck and Gómez-Ambrosi2001b).

Fig. 4.

Diagram of the site of action of diverse pharmacological agents at different levels of the lipolytic pathway. FSK, forskolin; AC, adenylate cyclase; Bt₂-cAMP, dibutyryl-cAMP; HSL, hormone-sensitive lipase; PTX, pertussis toxin; Gi, inhibitory G-coupled protein; PKA, protein kinase A; CPA, N⁶-cyclopentyladenosine; ADA, adenosine deaminase; Adenos, adenosine; A₁, adenosine receptor; PDE, phosphodiesterase E; P, phosphate; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; IBMX, isobutylmethylxanthine; −, inhibitor; +, promoter.

Adenosine A₁ receptors have been shown to be markedly expressed in adipocytes and influence fat cell metabolism via the regulation of adenylate cyclase, and therefore participate in lipolysis control via the inhibitory GTP-binding proteins (Honnor et al. Reference Honnor, Dhillon and Londos1985a,b). The adenosine deaminase gene had been previously shown to be linked to increased adiposity (LaNoue & Martin, Reference LaNoue and Martin1994; Bottini & Gloria-Bottini, Reference Bottini and Gloria-Bottini1999; Rankinen et al. Reference Rankinen, Zuberi, Chagnon, Weisnagel, Walts, Pérusse and Bouchard2006). The adenosinergic system reportedly increases leptin secretion by directly activating adenosine A₁ in white adipose tissue (Rice et al. Reference Rice, Fain and Rivkees2000). Thus, the involvement of leptin in the transmembrane adenosinergic signalling system of adipocytes has been shown to be feasible from both a genetic and a biochemical and functional point of view. The results of these studies (Frühbeck et al. Reference Frühbeck, Gómez-Ambrosi and Salvador2001b) suggest a molecular mechanism that causes or maintains an increased adipose tissue mass, based on an altered functional regulation of lipolysis as a result of defective leptin-induced stimulation opposing the adenosine-mediated tonic inhibition. In fact, it has been shown (Frühbeck et al. Reference Frühbeck, Gómez-Ambrosi and Salvador2001b) that the lipolytic effect of leptin is located at the adenylate cyclase-inhibitory G proteins step, which provides an explanation for the defective stimulation of adipocyte adenylate cyclase and the blunted lipolysis observed in leptin-deficient rodents and in rodents lacking OB-R, as well as in morbidly-obese human subjects (Greenberg et al. Reference Greenberg, Taylor and Londos1987; Vannucci et al. Reference Vannucci, Klim, Martin and LaNoue1989; Martin et al. Reference Martin, Klim, Vannucci, Dixon, Landis and LaNoue1990).

Aquaporin-7

Recently, AQP7 has emerged as a new piece in the complex puzzle of obesity and insulin resistance. Over eons, mammalian evolution has selected for genes that allow for survival during famine or drought periods. The primary role of fat cells is to store triacylglycerols during periods of energy excess and to mobilise this reserve when expenditure exceeds intake. Among the complex mechanisms underlying energy balance control, AQP7 has emerged as a novel pathway critical to the modulation of lipid metabolism (Frühbeck Reference Frühbeck2005b; Frühbeck et al. Reference Frühbeck, Catalán, Gómez-Ambrosi and Rodríguez2006). AQP are a family of integral membrane proteins favouring water movement across cell membranes (King et al. Reference King, Kozono and Agre2004). AQP transport water, in some cases together with small molecules such as glycerol and other small solutes. The identification of AQP has allowed the recognition of the relevance of water channels for homeostatic control, and their true contribution to human health and disease has started to unfold (Verkman, Reference Verkman2005). AQP7 belongs to the subcategory termed aquaglyceroporins, which comprise channels known to permeabilise glycerol as well as water (King et al. Reference King, Kozono and Agre2004; Hara-Chikuma & Verkman, Reference Hara-Chikuma and Verkman2006). Originally named AQPap, this water–glycerol pore was cloned from human adipose tissue (Kuriyama et al. Reference Kuriyama, Kawamoto, Ishida, Ohno, Mita, Matsuzawa, Nishizawa, Matsubara and Okubo1997; Kishida et al. Reference Kishida, Shimomura, Kondo, Kuriyama, Makino and Nishizawa2001; Kondo et al. Reference Kondo, Shimomura, Kishida, Kuriyama, Makino and Nishizawa2002). AQP7 deficiency in adipocytes has been associated with adult-onset obesity in mice; thus, providing evidence that AQP7 functions as a glycerol channel in vivo whereby adipocyte glycerol permeability exerts a key role in the regulation of fat accumulation (Maeda et al. Reference Maeda, Funahashi, Nagasawa, Kishida, Kuriyama, Nakamura, Kihara, Shimomura and Matsuzawa2004; Hara-Chikuma et al. Reference Hara-Chikuma, Sohara, Rai, Ikawa, Okabe, Sasaki, Uchida and Verkman2005; Hibuse et al. Reference Hibuse, Maeda, Funahashi, Yamamoto, Nagasawa and Mizunoya2005; Wintour & Henry Reference Wintour and Henry2006). Fat cells of AQP7-knock-out mice exhibit an increase in glycerol kinase activity, which stimulates triacylglycerol synthesis, ultimately leading to adipocyte hypertrophy and subsequent development of obesity. The control exerted by AQP7 over the efflux of glycerol from fat cells further highlights its role in aged mice as a determinant of whole-body glucose homeostasis and insulin sensitivity (Hibuse et al. Reference Hibuse, Maeda, Funahashi, Yamamoto, Nagasawa and Mizunoya2005). Interestingly, the coordinated regulation of fat-specific AQP7 and liver-specific AQP9 may be key to determining glucose metabolism in physiology and insulin resistance (Kuriyama et al. Reference Kuriyama, Shimomura, Kishida, Kondo, Furuyama and Nishizawa2002). Furthermore, the expression of AQP7 in fat cells has been reported to be sensitive to nutritional and neuroendocrine factors (Fig. 5) such as fasting, insulin, TNFα, steroids, adrenergic agonists and PPARγ (Kishida et al. Reference Kishida, Shimomura, Kondo, Kuriyama, Makino and Nishizawa2001; Kondo et al. Reference Kondo, Shimomura, Kishida, Kuriyama, Makino and Nishizawa2002; Kuriyama et al. Reference Kuriyama, Shimomura, Kishida, Kondo, Furuyama and Nishizawa2002; MacDougald & Burant, Reference MacDougald and Burant2005).

Fig. 5.

Schematic representation of the nutritional and neuroendocrine factors regulating aquaporin-7 (AQP7) expression in adipocytes.

The potential contribution of AQP7 to human obesity development has not been completely elucidated. The AQP7 gene, which resides in chromosome 9p13, has not been previously identified as a candidate gene for obesity (Ishibashi et al. Reference Ishibashi, Yamauchi, Kageyama, Saito-Ohara, Ikeuchi, Marumo and Sasaki1998; Kishida et al. Reference Kishida, Shimomura, Kondo, Kuriyama, Makino and Nishizawa2001; Rankinen et al. Reference Rankinen, Zuberi, Chagnon, Weisnagel, Walts, Pérusse and Bouchard2006). The loss of function associated with mutation of the AQP7 gene has been identified only in a single human subject who showed no signs of development of obesity or diabetes (Kondo et al. Reference Kondo, Shimomura, Kishida, Kuriyama, Makino and Nishizawa2002). The only observed alteration associated with the homozygous mutation is an impaired increase in plasma glycerol in response to exercise.

The extent, as well as the true influence, of aquaglyceroporins in human energy balance remains to be fully established. A microarray analysis (Sjöholm et al. Reference Sjöholm, Palming, Olofsson, Gummesson, Svensson, Lystig, Jennische, Brandberg, Torgerson, Carlsson and Carlsson2005) has shown increased AQP7 expression levels in the adipose tissue of obese women compared with obese men, while no differences were observed between the subcutaneous and omental depots. Elucidating the exact contribution of AQP7 and its role in the regulation of glycerol permeability, together with the potential impact on the current worldwide ‘diabesity’ epidemic, will provide a fertile area of research.

Caveolins

Caveolae are 50–100 nm flask-shaped cell-surface plasma-membrane invaginations implicated in a wide range of cellular functions such as endocytosis, cholesterol homeostasis and cell signalling control (Cohen et al. Reference Cohen, Hnasko, Schubert and Lisanti2004). The discovery of caveolin (Rothberg et al. Reference Rothberg, Heuser, Donzell, Ying, Glenney and Anderson1992; now termed caveolin-1) as a protein marker and main component of caveolae has provided more insight into the roles of these organelles. Caveolins are integral membrane proteins that serve as the structural elements of caveolae. They function as scaffolding as well as being capable of recruiting numerous signalling molecules to the plasma membrane lipid rafts and regulating their activity (Stan, Reference Stan2005; Parton et al. Reference Parton, Hanzal-Bayer and Hancock2006). Interestingly, caveolins are cholesterol-binding proteins with a unique hairpin topology that allows both the amino and carboxy terminals to face the cytoplasm (Fig. 6). Three different caveolin isoforms have been identified so far, exhibiting a unique tissue distribution pattern. While caveolin-3 is expressed in skeletal, cardiac and smooth muscle cells (Tang et al. Reference Tang, Scherer, Okamoto, Song, Chu, Kohtz, Nishimoto, Lodish and Lisanti1996), caveolin-1 is expressed in the majority of cells, excluding those of the muscular lineage, with expression being particularly high in adipocytes, fibroblasts and epithelial and endothelial cells (Parton et al. Reference Parton, Joggerst and Simons1994; Scherer et al. Reference Scherer, Lewis, Volonte, Engelman, Galbiati, Couet, Kohtz, van Donselaar, Peters and Lisanti1997). Expression of caveolin-2 most closely follows the distribution pattern of caveolin-1, although it has been recently shown that caveolin-2 is also found in cardiac myocytes (Rybin et al. Reference Rybin, Grabham, Elouardighi and Steinberg2003).

Fig. 6.

Location of caveolin in the plasma membrane invaginations (or caveolae) and potential underlying mechanisms that lead ultimately to insulin resistance via insulin receptor (Ins-R) signalling defects.

The study of the phenotypes of caveolin-knock-out mice has considerably enhanced the understanding of the functional contribution of these proteins in diverse tissues and cellular processes (Le Lay & Kurzchalia, Reference Le Lay and Kurzchalia2005). An in vivo role for caveolin-1 in the development of obesity and insulin resistance through a direct participation in lipid homeostasis has been clearly established (Razani et al. Reference Razani, Combs, Wang, Frank, Park, Russell, Li, Tang, Jelicks, Scherer and Lisanti2002; Cohen et al. Reference Cohen, Combs, Scherer and Lisanti2003a,Reference Cohen, Hnasko, Schubert and Lisantib). Despite an evident hyperphagia, caveolin-1-knock-out mice show a lean phenotype with overt resistance to diet-induced obesity resulting from an impaired adipocyte functioning as a consequence of altered lipid handling. Adipocytes of caveolin-1-null mice lack caveolae, a derangement that with increasing age translates into a systemic inability for lipid accumulation, resulting in a near complete ablation of fat depots accompanied by histological alterations, including reduced fat cell number and diameter. The difference in weight of caveolin-1-knock-out mice when compared with wild-type mice becomes even more striking when the rodents are challenged with a high-fat diet. Caveolin-1-deficient mice show normal circulating glucose, insulin and cholesterol concentrations at the same time as exhibiting increased triacylglycerol and NEFA levels. The impaired triacylglycerol clearance takes place in the setting of normal total and hepatic lipoprotein lipase activity. It has further been shown that caveolin-1 is essential for adequate non-shivering thermogenesis in brown adipose tissue (Cohen et al. Reference Cohen, Schubert, Brasaemle, Scherer and Lisanti2005). Since caveolin-3 presents a high amino acid sequence homology to caveolin-1 (Song et al. Reference Song, Scherer, Tang, Okamoto, Li, Chu, Chafel, Chu, Kohtz and Lisanti1996), a role in energy balance and glucose homeostasis for this muscle-specific isoform could be expected. In contrast to the effect of caveolin-1 deficiency, in caveolin-3-knock-out mice there is an increased body weight at the expense of an elevated adiposity, accompanied by a normal food intake (Capozza et al. Reference Capozza, Combs, Cohen, Cho, Park and Schubert2005).

The relative importance of caveolins in insulin signalling in vivo has been investigated, as well as the clarification of the mechanisms that underlie potential tissue- or cell-specific signalling defects (Ishikawa et al. Reference Ishikawa, Otsu and Oshikawa2005). Caveolin-1-null mice exhibit a markedly decreased glucose uptake in response to an insulin tolerance test and develop postprandial hyperinsulinaemia when placed on a high-fat diet (Cohen et al. Reference Cohen, Razani, Wang, Combs, Williams, Scherer and Lisanti2003b). Since caveolin has been previously reported to operate as a positive regulator of insulin signalling (Yamamoto et al. Reference Yamamoto, Toya, Schwencke, Lisanti, Myers and Ishikawa1998), the involvement of caveolin-1 in this process has been examined. No changes in insulin receptor gene expression are evident in caveolin-1-deficient mice (Cohen et al. Reference Cohen, Razani, Wang, Combs, Williams, Scherer and Lisanti2003b). However, there is >90% reduction in insulin receptor protein levels in adipose tissue of these knock-out rodents, with ectopic expression of caveolin-1 being sufficient to restore expression to that of wild-type mice. The in vivo metabolic consequences of the genetic ablation of caveolin-3 in mice also affect glucose metabolism and lipid homeostasis, with caveolin-3-null mice displaying a marked postprandial hyperinsulinaemia and impaired glucose tolerance that progresses to whole-body insulin resistance with decreased insulin-stimulated whole-body glucose uptake. In particular, there is decreased glucose metabolic flux in skeletal muscle, as well as reduced insulin-mediated suppression of hepatic glucose production. Even in white adipose tissue, which does not express caveolin-3, an approximately 70% decrease in insulin-stimulated glucose uptake is observed that also suggests an insulin-resistant state for this tissue (Capozza et al. Reference Capozza, Combs, Cohen, Cho, Park and Schubert2005). Interestingly, caveolin-3-null mice have also been reported to develop whole-body insulin resistance associated with an impaired glucose tolerance (Oshikawa et al. Reference Oshikawa, Otsu, Toya, Tsunematsu, Hankins, Kawabe, Minamisawa, Umemura, Hagiwara and Ishikawa2004; Capozza et al. Reference Capozza, Combs, Cohen, Cho, Park and Schubert2005). Caveolae act as communication platforms, serving as a concentrating point for numerous signalling cascades. Effective insulin signalling in adipocytes is dependent on the localisation in caveolae of insulin-responsive elements. Caveolin-1 seems to play a role in the regulation of glucose homeostasis, both through a direct interaction with the insulin receptor and as an agent for GLUT4-mediated glucose uptake (Cohen et al. Reference Cohen, Combs, Scherer and Lisanti2003a). Caveolin-3 appears to attenuate the insulin-stimulated activation of insulin receptors and downstream molecules, such as insulin receptor substrate 1 and Akt, in skeletal muscles (Oshikawa et al. Reference Oshikawa, Otsu, Toya, Tsunematsu, Hankins, Kawabe, Minamisawa, Umemura, Hagiwara and Ishikawa2004; Capozza et al. Reference Capozza, Combs, Cohen, Cho, Park and Schubert2005).

Leptin

The role of leptin in the development of cardiovascular complications, beyond its effects on energy balance, only started to unfold some years after the identification of the hormone (Correia & Haynes, Reference Correia and Haynes2004; Matsuzawa, Reference Matsuzawa2005). Leptin has been shown to contribute to blood pressure homeostasis by inducing a pressor response attributable to sympathoactivation (Correia & Haynes, Reference Correia and Haynes2004) and a depressor response attributable to the vasodilation of conduit and resistance vessels (Frühbeck, Reference Frühbeck1999; Beltowski et al. Reference Beltowski, Wojcicka and Jamroz-Wisniewska2006; Beltowski, Reference Beltowski2006a). In relation to this role, the author's group was the first to identify NO as the key molecule responsible for the depressor response induced by leptin (Frühbeck, Reference Frühbeck1999). Subsequent studies have further shown that leptin acts on the endothelium, inducing the synthesis of NO via the activation of endothelial NO synthase, which induces an endothelium-dependent vasodilation (Kimura et al. Reference Kimura, Tsuda, Baba, Kawabe, Boh-oka, Ibata, Moriwaki, Hano and Nishio2000; Lembo et al. Reference Lembo, Vecchione, Fratta, Marino, Trimarco, d'Amati and Trimarco2000; Winters et al. Reference Winters, Mo, Brooks-Asplund, Kim, Shoukas, Li, Nyhan and Berkowitz2000; Beltowski et al. Reference Beltowski, Wójcicka and Borkowska2002; Vecchione et al. Reference Vecchione, Maffei, Colella, Aretini, Poulet, Frati, Gentile, Fratta, Trimarco, Trimarco and Lembo2002; Beltowski, Reference Beltowski2006a). Since OB-R are also expressed in the underlying smooth muscle layer, it is considered that VSMC also represent an important target for the vascular actions of leptin (Fortuño et al. Reference Fortuño, Rodríguez, Gómez-Ambrosi, Muñiz, Salvador, Díez and Frühbeck2002; Rodríguez et al. Reference Rodríguez, Frühbeck, Gómez-Ambrosi, Catalán, Sáinz, Díez, Zalba and Fortuño2006). It has been shown that leptin acts on VSMC, inhibiting the increase in cytosolic Ca induced by angiotensin II, thus blunting the contractile response caused by this potent vasoactive peptide. However, the intracellular mechanisms underlying this endothelium-independent vasodilation induced by leptin have not been completely elucidated. Given that the main signalling cascades activated by OB-R include the Janus kinase and signal transduction and activator of transcription cascades as well as the phosphoinositol-3 kinase/Akt pathways (Frühbeck, Reference Frühbeck2006; Peelman et al. Reference Peelman, Couturier, Dam, Zabeau, Tavernier and Jockers2006), it is hypothesised that an up-regulation of inducible NO synthase underlies the inhibitory effect of leptin on the angiotensin II-induced response in VSMC via the Janus kinase/signal transduction and activator of transcription and phosphoinositol-3 kinase/Akt pathways, as illustrated in Fig. 7.

Fig. 7.

Diagram illustrating the nitric oxide-dependent intracellular pathways activated by leptin in arteries participating in blood pressure regulation. OB-R, leptin receptor; PI3K, phosphoinositol-3 kinase; JAK, Janus kinase; STAT, signal transduction and activator of transcription; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; Ang, angiotensin; ACE, angiotensin-converting enzyme; AT₁-R, angiotensin receptor type 1; [Ca²⁺]_i, cystolic calcium; , increase.

Leptin has been postulated to be one of the potential links between adiposity and inflammation (Berg & Scherer, Reference Berg and Scherer2005; Fantuzzi, Reference Fantuzzi2005; Härle & Straub, Reference Härle and Straub2006). Although the increase in the expression of leptin mRNA in white adipose tissue and the elevation in circulating concentrations triggered by inflammatory stimuli in experimental animals have not been consistently observed in human subjects (Fantuzzi & Faggioni, Reference Fantuzzi and Faggioni2000; Gómez-Ambrosi et al. Reference Gómez-Ambrosi, Catalán, Diez-Caballero, Martínez-Cruz, Gil, García-Foncillas, Cienfuegos, Salvador, Mato and Frühbeck2004a), in general a proinflammatory role has been attributed to leptin. In this sense, the effects of leptin on inflammation and immunity need to be considered in a more broad and complex setting. In endothelial cells leptin has been shown to up regulate endothelin-1 and NO synthase, and stimulate the expression of adhesion molecules and monocyte chemoattractant protein-1, at the same time as inducing oxidative stress (Lau et al. Reference Lau, Dhillon, Yan, Szmitko and Verma2005; Beltowski, Reference Beltowski2006b). Leptin has also been observed to stimulate angiogenesis, platelet aggregation and atherothrombosis, and to have a direct effect on macrophages, promoting their accumulation of cholesterol at the same time as leading to an increased release of monocyte colony-stimulating factor. Leptin operates as a chemoattractant devoid of secretagogue properties but capable of inhibiting neutrophil chemotaxis to classical neutrophilic chemoattractants (Montecucco et al. Reference Montecucco, Bianchi, Gnerre, Bertolotto, Dallegri and Ottonello2006). This effect is reportedly dependent on the activation of intracellular kinases and, in particular, via p38 mitogen-activated protein kinase and Src kinase (Montecucco et al. Reference Montecucco, Bianchi, Gnerre, Bertolotto, Dallegri and Ottonello2006).

Fibrinogen, C-reactive protein and von Willebrand factor represent well-known markers of inflammation and endothelial dysfunction, which are increased in obese patients (Hansson, Reference Hansson2005). A positive association has been observed between these markers and body fat, which in the case of fibrinogen and von Willebrand factor is higher than the correlation observed with BMI (Gómez-Ambrosi et al. Reference Gómez-Ambrosi, Salvador, Páramo, Orbe, de Irala, Diez-Caballero, Gil, Cienfuegos and Frühbeck2002; Gómez-Ambrosi et al. Reference Gómez-Ambrosi, Salvador, Silva, Pastor, Rotellar, Gil, Cienfuegos and Frühbeck2006b). Despite its wide use, BMI does not provide an accurate measure of body composition. In this context more precise indicators need to be incorporated into the clinical diagnosis of obesity in order to better estimate the cardiovascular-related risk (Frühbeck, Reference Frühbeck2004b). Although these markers are significantly correlated with leptin concentrations (P<0·001 for C-reactive protein; Fig. 8), the statistical significance is lost after adjusting for body fat, suggesting that they are not regulated by leptin itself. Recent studies (Gómez-Ambrosi et al. Reference Gómez-Ambrosi, Salvador, Silva, Rotellar, Gil, Cienfuegos and Frühbeck2005, Reference Gómez-Ambrosi, Salvador, Silva, Pastor, Rotellar, Gil, Cienfuegos and Frühbeck2006b) have provided evidence of direct associations between adipose tissue stores and circulating concentrations of fibrinogen, C-reactive protein and von Willebrand factor that are not dependent on the influence of leptin.

Fig. 8.

Scatter diagram showing the highly significant positive correlation (n 553; r 0·144; P<0·001) between the circulating concentrations of log leptin and log C-reactive protein (CRP). (●), Men; (△), women.

Resistin

The seminal observation of the induction of insulin resistance in mice by a novel adipokine led to it being termed resistin (Steppan et al. Reference Steppan, Bailey, Bhat, Brown, Banerjee, Wright, Patel, Ahima and Lazar2001). Increased circulating resistin concentrations have been observed in genetically-obese rodents (ob/ob and db/db mice) as well as in diet-induced obesity. Immunoneutralisation of resistin ameliorates hyperglycaemia and insulin resistance in these obese mice, while recombinant resistin administration results in impaired glucose tolerance and insulin action in normal mice (Steppan et al. Reference Steppan, Bailey, Bhat, Brown, Banerjee, Wright, Patel, Ahima and Lazar2001; Steppan & Lazar, Reference Steppan and Lazar2004). Glucose, growth hormone (GH), glucocorticoids, insulin, β₃-adrenoreceptor stimulation and thiazolidinediones reportedly increase the expression of resistin (Koerner et al. Reference Koerner, Kratzsch and Kiess2005). However, insulin, TNFα, adrenaline, β-adrenergic receptor stimulation and thiazolidinediones have also been observed to decrease resistin gene expression (Steppan & Lazar, Reference Steppan and Lazar2004; Koerner et al. Reference Koerner, Kratzsch and Kiess2005). Moreover, the real contribution of resistin to human pathophysiology still remains controversial (Arner, Reference Arner2005a). Although resistin transcripts have been determined in adipose tissue of obese patients, no correlation between resistin mRNA levels and body weight, adiposity or insulin resistance has been observed (Gómez-Ambrosi & Frühbeck, Reference Frühbeck, Gibney, Elia, Ljungqvist and Dowsett2005a). The stromovascular fraction of adipose tissue and peripheral blood monocytes have been shown to express resistin. However, resistin mRNA is undetectable in human adipocytes of lean, insulin-resistant and obese patients and patients with diabetes (Koerner et al. Reference Koerner, Kratzsch and Kiess2005).

It has been established that resistin belongs to the RELM and FIZZ family of secreted proteins that have a tissue-specific pattern of expression and common signalling characteristics (Gómez-Ambrosi & Frühbeck, Reference Frühbeck2001). Indeed, resistin is identical to FIZZ3, while the amino acid sequence of RELMα is the same as that of FIZZ1, which had been previously shown to be overexpressed in allergic inflammation (Holcomb et al. Reference Holcomb, Kabakoff, Chan, Baker, Gurney and Henzel2000). Given that the pattern of expression and physiological functions described for these molecules resemble that of other well-known proinflammatory cytokines, and based on the superimposable expression pattern of FIZZ1/RELMα in inflammatory regions as well as cells, resistin has been proposed to be a critical mediator of the insulin resistance associated with sepsis and possibly other inflammatory settings (Gómez-Ambrosi & Frühbeck, Reference Frühbeck2001; Lehrke et al. Reference Lehrke, Reilly, Millington, Iqbal, Rader and Lazar2004).

Resistin has been shown to stimulate key processes in early atherosclerotic lesion formation, increasing the expression of monocyte chemoattractant protein-1 and cell adhesion molecules (vascular cell adhesion molecule-1 and intercellular adhesion molecule-1) in endothelial cells (Verma et al. Reference Verma, Li, Wang, Fedak, Li, Weisel and Mickle2003). In addition, resistin-treated cells have been reported to express lower TNFα receptor-associated factor, a potent inhibitor of CD40 ligand-mediated endothelial cell activation (Lau et al. Reference Lau, Dhillon, Yan, Szmitko and Verma2005). Interestingly, the resistin-induced up-regulation of adhesion molecules is antagonised by adiponectin (Kawanami et al. Reference Kawanami, Maemura, Takeda, Harada, Nojiri, Imai, Manabe, Utsunomiya and Nagai2004). The involvement of resistin in endothelial dysfunction in patients who are insulin resistant has been attributed to its direct effect on endothelial cells by promoting the release of endothelin-1 (Gómez-Ambrosi & Frühbeck, Reference Frühbeck, Gibney, Elia, Ljungqvist and Dowsett2005a). It seems plausible that the proliferative effect of resistin on VSMC underlies the increased incidence of restenosis common among patients with diabetes (Gómez-Ambrosi & Frühbeck, Reference Frühbeck2005b).

Adiponectin

In pathological conditions such as obesity and the characteristic insulin resistance accompanying both the prediabetic state and overt type 2 diabetes mellitus, a clear hypoadiponectinaemia has been observed (Frühbeck & Salvador, Reference Frühbeck and Salvador2004; Arner, Reference Arner2005a; Berg & Scherer, Reference Berg and Scherer2005; Koerner et al. Reference Koerner, Kratzsch and Kiess2005). Adiponectin (also termed Acrp30, AdipoQ, apM1 or GBP28) operates completely differently from other known adipokines, since it has been shown to improve insulin sensitivity, inhibit vascular inflammation and exhibit a cardio-protective effect. White adipose tissue adiponectin expression has been observed to be increased in lean individuals and women, exhibiting an association with lower extents of insulin resistance and TNFα expression (Yamauchi et al. Reference Yamauchi, Kamon, Ito, Tsuchida, Yokomizo and Kita2003). Glucocorticoids, TNFα and IL-6 inhibit the gene expression of adiponectin in human visceral adipose tissue, while insulin, insulin-like growth factor 1 and PPARγ agonists increase its expression. To date three putative adiponectin receptors have been cloned. Adiponectin receptor 1 has been shown to be abundantly expressed in skeletal muscle and adiponectin receptor 2 is predominantly present in liver, while a third receptor has been identified in endothelial cells and smooth muscle (Kadowaki & Yamauchi, Reference Kadowaki and Yamauchi2005).

The cardio-protective characteristics of adiponectin have been attributed to its involvement in the prevention of atherosclerotic plaque formation through the inhibition of monocyte adhesion to endothelial cells, by decreasing NFκB signalling via a cAMP-dependent pathway (Ouchi et al. Reference Ouchi, Kihara, Arita, Okamoto, Maeda and Kuriyama2000; Kawanami et al. Reference Kawanami, Maemura, Takeda, Harada, Nojiri, Imai, Manabe, Utsunomiya and Nagai2004). Interestingly, in rodent models of atherosclerosis, such as ob/ob and apoE-deficient mice, adiponectin reportedly exerts a protective effect on the development of both atherosclerosis and type 2 diabetes mellitus (Frühbeck, Reference Frühbeck2004a; Lau et al. Reference Lau, Dhillon, Yan, Szmitko and Verma2005). The findings in animals have a certain clinical parallel in human subjects, evidenced by a negative correlation between adiponectinaemia and markers of inflammation (Pischon et al. Reference Pischon, Girman, Hotamisligil, Rifai, Hu and Rimm2004; Xydakis et al. Reference Xydakis, Case, Jones, Hoogeveen, Liu, O'Brian, Nelson and Ballantyne2004). Circulating adiponectin levels have been observed to be inversely correlated with insulin resistance and C-reactive protein concentrations (Ouchi et al. Reference Ouchi, Kihara, Funahashi, Nakamura, Nishida and Kumada2003). In line with the cardio-protective properties of the adipokine, patients with CHD present with hypoadiponectinaemia compared with age- and BMI-adjusted controls, and a lower risk of myocardial infarction in men has been reported to be associated with high plasma adiponectin concentrations (Wellen & Hotamisligil, Reference Wellen and Hotamisligil2003). Furthermore, adiponectin has also been observed to regulate vascular inflammation via a direct effect on endothelial cells, as well as by decreasing VSMC proliferation and migration via a reduction in the effects of certain growth factors such as platelet-derived growth factor and heparin-binding epidermal growth factor (Frühbeck, Reference Frühbeck2004a). The detrimental effects of hypoadiponectinaemia in obesity, CVD and type 2 diabetes mellitus may be further related to an anti-inflammatory activity of the adipokine on macrophages. An inhibition of the endothelial inflammatory response by decreasing VSMC proliferation and vascular cell adhesion molecule-1 expression seems to underlie the anti-atherogenic effects of adiponectin (Lau et al. Reference Lau, Dhillon, Yan, Szmitko and Verma2005).

Visfatin

Among the more recently identified adipokines, visfatin is mainly produced and secreted by visceral white adipose tissue. It has putative anti-diabetogenic properties through its binding to the insulin receptor and the exertion of an insulino-mimetic effect both in vitro and in vivo (Fukuhara et al. Reference Fukuhara, Matsuda, Nishizawa, Segawa, Tanaka and Kishimoto2005; Sethi & Vidal-Puig, Reference Sethi and Vidal-Puig2005). Originally identified as pre-B-cell colony-enhancing factor, visfatin is a cytokine that is abundantly present in the broncho-alveolar lavage fluid of animal models of acute lung injury as well as in the neutrophils of patients with sepsis (Fantuzzi, Reference Fantuzzi2005). In spite of the descriptive name, plasma concentrations of visfatin and visceral visfatin mRNA expression have been reported to correlate with measures of obesity but not with the visceral fat mass or the waist:hip ratio. Furthermore, no differences have been observed in visfatin mRNA expression between the visceral and subcutaneous fat depots (Berndt et al. Reference Berndt, Kloting, Kralisch, Kovacs, Fasshauer, Schon, Stumvoll and Bluher2005). IL-6 has been reported to exert an inhibitory effect on visfatin expression, which is in part mediated by the p44/42 mitogen-activated protein kinase (Kralisch et al. Reference Kralisch, Klein, Lossner, Bluher, Paschke, Stumvoll and Fasshauer2005). A 2-fold increase in circulating concentrations of visfatin has been observed in patients with type 2 diabetes mellitus (Chen et al. Reference Chen, Chung, Chang, Tsai, Huang, Shin and Lee2006). Nonetheless, after adjusting for BMI and waist:hip ratio the independent association between visfatin and diabetes disappears. The pathophysiological relevance of visfatin deserves further investigation in order to clarify the paradoxical effects of simultaneously favouring fat accretion and promoting insulin sensitivity (Arner, Reference Arner2006). It is still not clear whether visfatin actively participates in the feedback mechanisms regulating fat accretion in the intra-abdominal depot and its accompanying insulin resistance, or merely represents an epiphenomenon that might be useful as a surrogate marker of increased omental adipose tissue.

Vascular endothelial growth factor

Microarray technology has been applied to the analysis of gene expression profiles in adipose tissue obtained from lean and obese individuals in order to identify differential expression patterns and key genes involved in obesity. Consequently, attention has been focused on the changes observed in angiogenic factors (Gómez-Ambrosi et al. Reference Gómez-Ambrosi, Catalán, Diez-Caballero, Martínez-Cruz, Gil, García-Foncillas, Cienfuegos, Salvador, Mato and Frühbeck2004b). VEGF is known to promote angiogenesis, inducing migration and proliferation of vascular endothelial cells (Berg & Scherer, Reference Berg and Scherer2005). Although VEGF is encoded by a single gene, four isoforms are produced by alternative splicing, which have been implicated in both normal blood vessel development and in pathogenic neovascularisation and atherosclerosis. In obese patients serum concentrations of one of the isoforms have been observed to be dependent on the intra-abdominal fat depot (Miyazawa-Hoshimoto et al. Reference Miyazawa-Hoshimoto, Takahashi, Bujo, Hashimoto and Saito2003). VEGF mRNA expression has been identified in various cell types, including endothelial, epithelial and mesenchymal cells. Interestingly, one of these microarray studies (Gómez-Ambrosi et al. Reference Gómez-Ambrosi, Catalán, Diez-Caballero, Martínez-Cruz, Gil, García-Foncillas, Cienfuegos, Salvador, Mato and Frühbeck2004b) has provided evidence for increased expression of VEGF-B mRNA in omental adipose tissue of obese patients, in accordance with the need for enhanced vascularisation to support adipose mass enlargement. In particular, the mRNA of VEGF-B₁₆₇ and VEGF-B₁₈₆, the two known isoforms of VEGF-B, were shown to be up regulated 1·6-fold and 2·1-fold respectively. This finding suggests a potential link in the involvement of VEGF-B in angiogenesis and obesity-related endothelial dysfunction. This aspect needs to be explored further in relation to the implication that VEGF is involved in vascular inflammation and remodelling through increased subendothelial macrophage accumulation and intima media thickening in the context of atheroma initiation and restenosis episodes.

Serum amyloid A

Recognition of obesity as a chronic low-grade inflammatory state has driven recent productive research efforts. Adipose tissue is considered an extremely active immune organ that secretes numerous immunomodulatory factors, and it has emerged as an important source of inflammatory signals known to be related to comorbidity development (Trayhurn & Wood, Reference Trayhurn and Wood2004; Sjöholm & Nyström, Reference Sjöholm and Nyström2005). Thus, inflammation within white adipose tissue represents a crucial step, contributing to the appearance of many of the pathological features accompanying increased adiposity. The mounting evidence of the relevance of inflammation to vascular disease and insulin resistance has orientated plentiful research efforts towards molecules that modulate leucocyte migration from the bloodstream to the vessel wall. Serum amyloid A (SAA) is an acute-phase reactant protein secreted by diverse cell types, including adipocytes. It has been associated with systemic inflammation, as well as being linked to atherosclerosis and serving as a predictor for coronary disease and cardiovascular outcome (Lau et al. Reference Lau, Dhillon, Yan, Szmitko and Verma2005). Circulating SAA concentrations are increased in obese patients and those with diabetes (Berg & Scherer, Reference Berg and Scherer2005). Under physiological circumstances white adipose tissue is known to express low levels of SAA, which have been shown to be strongly up regulated in obesity (Clément et al. Reference Clément, Viguerie, Poitou, Carette, Pelloux and Curat2004). From a mechanistic perspective, the detrimental effects of augmented SAA levels appear to be related to the displacement of apoA₁ from HDL-cholesterol, increasing its binding to macrophages, therefore decreasing the availability of the cardio-protective HDL-cholesterol (Lau et al. Reference Lau, Dhillon, Yan, Szmitko and Verma2005). In addition, SAA functions as a chemoattractant, an inducer of remodelling metalloproteinases and a stimulator of T-cell cytokine production (Berg & Scherer, Reference Berg and Scherer2005).

A positive correlation between BMI and circulating SAA concentrations has been reported (Urieli-Shoval et al. Reference Urieli-Shoval, Linke and Matzner2000). However, whether serum SAA levels are increased in obese patients in relation to the body fat compartment was not directly addressed. In a recent study (Gómez-Ambrosi et al. Reference Gómez-Ambrosi, Salvador, Rotellar, Silva, Catalán, Rodríguez, Gil and Frühbeck2006a) obese patients were found to exhibit a 6-fold increase in circulating SAA concentrations compared with lean individuals. Furthermore, increased expression of SAA mRNA in the omental fat depot of obese patients was established. Interestingly, it was found that weight loss following bariatric surgery was able to reduce SAA concentrations, which may play a role, in part, in the beneficial effects that accompany weight reduction following bariatric surgery. It can be concluded that the elevated SAA levels in both serum and omental adipose tissue observed in obese patients may contribute to the obesity-associated CVD risk, which can be beneficially influenced by weight loss.

Ghrelin

Ghrelin is a potent GH-releasing peptide that was originally isolated from the stomach and subsequently identified as an endogenous ligand for the GH secretagogue receptor. It has been shown to be involved in the regulation of food intake by exerting an orexigenic effect (Kojima et al. Reference Kojima, Hosoda, Date, Nakazato, Matsuo and Kangawa1999; Inui et al. Reference Inui, Asakawa, Bowers, Mantovani, Laviano, Meguid and Fujimiya2004). Similarly to leptin, other sources of ghrelin production have been located, providing evidence for a physiological role of the hormone beyond energy balance (Ghigo et al. Reference Ghigo, Broglio, Arvat, Maccario, Papotti and Muccioli2005). The almost universal distribution of GH secretagogue receptors, including in adipose tissue and the cardiovascular system (Papotti et al. Reference Papotti, Ghe, Cassoni, Catapano, Deghenghi, Ghigo and Muccioli2000; Gnanapavan et al. Reference Gnanapavan, Kola, Bustin, Morris, McGee, Fairclough, Bhattacharya, Carpenter, Grossman and Korbonits2002), supports the plausibility of vascular actions of ghrelin (Cao et al. Reference Cao, Ong and Chen2006). In particular, GH secretagogue receptors are expressed in both blood vessels and cardiomyocytes, providing evidence for direct cardiovascular effects of ghrelin. Exogenous administration of ghrelin has been shown to exert beneficial haemodynamic effects via a vasodilatory effect, reducing mean arterial pressure and increasing cardiac index and stroke volume without elevating heart rate in human subjects and different rodent models (Cao et al. Reference Cao, Ong and Chen2006). Circulating ghrelin concentrations are reportedly decreased in obese individuals. Interestingly, ghrelin has been shown to induce vasorelaxation, acting via an endothelium-independent mechanism, which reverses the effect of endothelin-1 on isolated human arteries (Wiley & Davenport, Reference Wiley and Davenport2002), and at the same time exerts an effect on the endothelium by increasing endothelial NO bioavailability (Shimizu et al. Reference Shimizu, Nagaya, Teranishi, Imazu, Yamamoto, Shokawa, Kangawa, Kohno and Yoshizumi2003). Recent studies (Kawczynska-Drozdz et al. Reference Kawczynska-Drozdz, Olszanecki, Jawien, Brzozowski, Pawlik, Korbut and Guzik2006) have shown that ghrelin counteracts vascular oxidative stress through the inhibition of vascular superoxide production.

A direct cardio-protective effect of ghrelin has also been observed, with ghrelin operating as a trophic local factor to inhibit apoptosis of cardiomyocytes and endothelial cells in vitro through the activation of extracellular signal-regulated kinase-1/2 and Akt serine kinases (Baldanzi et al. Reference Baldanzi, Filigheddu, Cutrupi, Catapano, Bonissoni and Fubini2002). The signalling pathways underlying the vascular effects of ghrelin are being studied in more detail, extending to CD36, a multiligand scavenger receptor related to macrophage foam cell formation and the pathogenesis of atherosclerosis (Bodart et al. Reference Bodart, Febbraio, Demers, McNicoll, Pohankova, Perreault, Sejlitz, Escher, Silverstein, Lamontagne and Ong2002), as well as addressing the impact of the hormone on cell adhesion molecule expression (Skilton et al. Reference Skilton, Nakhla, Sieveking, Caterson and Celermajer2005). Taken together these findings highlight the involvement of ghrelin in the etiopathogenesis of hypertension as well as atherosclerosis.