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The vasoactive peptide adrenomedullin is secreted by adipocytes and inhibits lipolysis through NO-mediated ß-adrenergic agonist oxidation

Romain Harmancey^*, Jean-Michel Senard^*, Atul Pathak^*, Franck Desmoulin, Catherine Claparols, Philippe Rouet^* and Fatima Smih^*,1

^* INSERM U586, Insitut Louis Bugnard, CHU Rangueil, Université Paul Sabatier, TSA 50032, Toulouse, France;
UMR CNRS 5068, Université Paul Sabatier, Toulouse, France; and
Serv. Commun Spectrométrie de Masse FR 2599, Université Paul Sabatier, Toulouse, France

¹ Correspondence: Laboratoire de pharmacologie, Faculté de Médecine, 37 allées Jules Guesde, 31073 Toulouse, cedex 7, France. E-mail: fatima.smih{at}toulouse.inserm.fr

SPECIFIC AIMS

A dramatic increase in the prevalence of obesity and cardiovascular morbidity is anticipated in most developed countries. Adipocytes are known to secrete a number of adipokines, but many adipocyte secretions, as well as their functional importance, remain to be characterized. Our team is interested in the study of cardiac and vegetative adaptations to obesity since adiposity is one of the main risk factors for arterial hypertension and chronic heart disease. The links between adipose cells and the cardiovascular system are not thoroughly elucidated but certainly involve adipocyte secretions.

Adrenomedullin (AM) is a 52 amino acid peptide first isolated from a human phaeochromocytoma and is generally believed to be vasoactive and vasoprotective. Its most important known sources of secretion in the bloodstream are endothelial and vascular smooth muscle cells. Increased AM plasma levels have been reported in most experimental models of arterial hypertension and in hypertensive humans. These observations led us to consider AM as a counter-regulatory factor involved in the control of cardiovascular homeostasis. In addition to its vasodilating and natriuretic actions, AM has been shown to act at the cellular level, notably by decreasing cardiomyocyte diameter and inhibiting interstitial fibrosis in the heart. Moreover, we have recently shown that AM is able to up-regulate M2 muscarinic receptors in cardiomyocytes derived from the murine P19 cell line. Thus, AM could protect the heart from several complications implicated in obesity-linked cardiomorbidity, such as arterial hypertension, which inevitably leads to left ventricular hypertrophy, cardiac fibrosis, and finally heart failure.

Our first aim was to investigate secretions of the vasoactive peptide AM by white adipocytes using the 3T3-F442A murine preadipocyte cell line and isolated human adipocytes from abdominal dermolipectomies. Second, we looked for a possible paracrine/autocrine effect of AM at the level of lipolysis in adipocytes. Third, we investigated the mechanisms mediated by AM action on lipolysis in adipocytes.

PRINCIPAL FINDINGS

1. We show here for the first time by real-time PCR that isolated human adipocytes harbored higher levels of AM mRNA than known positive control sources, such as human right auricle

2. We demonstrate by radioimmunoassay that isolated human adipocytes secrete AM in conditioned media (0.26±0.1 fmol/10⁵ cells/h) and that once differentiated, the 3T3-F442A adipocyte cell line was also able to secrete AM (4.5±0.9 fmol/10⁵ cells/h at days 13–15 of differentiation)

3. Since AM is believed to act mainly as a paracrine/autocrine factor, we looked for AM receptors in our adipose cell models.
RT-PCR experiments indicated that both human adipocytes and differentiated 3T3-F442A adipocytes express mRNA encoding CL receptor and the 3 RAMP isoforms, suggesting the presence of functional AM₁ and AM₂ receptors formed by CL receptor/RAMP2 and CL receptor/RAMP3 combinations, respectively.

4. The physiological function of this feature was investigated at the level of lipolysis regulation since lipid storage is the main metabolic function of adipose tissue
Using fully differentiated 3T3-F442A adipocytes, we first found that AM alone is devoid of lipolytic function. We further demonstrated that AM inhibits ß-adrenergic (isoproterenol) stimulated lipolysis. This peptide is able to shift the concentration-response curve for isoproterenol by significantly decreasing its potency according to the change in pD2 value (8.6±0.2 vs. 9.8±0.1 with isoproterenol alone, P<0.001) (Fig. 1 ). This effect was shown to be mediated by a nitric oxide (NO) -dependent mechanism. The effect of AM is independent of NO-sensitive, soluble guanylate cyclase and is prevented by forskolin-induced direct stimulation of adenylate cyclase. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that AM-induced NO modifies isoproterenol through an extracellular oxidative reaction resulting in its aminochrome—namely, isoprenochrome (Fig. 2 ).

View larger version (21K): [in this window] [in a new window]   Figure 1. AM modulates isoproterenol-stimulated lipolysis in 3T3-F442A cells. 12-day differentiated 3T3-F442A cells were deprived overnight in serum-free DMEM containing 2% BSA and further incubated with various chemicals in fresh 2% BSA-supplemented medium. After 90 min, media were collected and secreted NEFA were quantified to determine the lipolysis rate. Effects of increasing concentrations of isoproterenol (___) or AM (- - -) ranging from 10–11 M to 10–6 M and of isoproterenol from 10–11 M to 10–6 M with addition of 100 nM AM (····). Results are expressed as % of basal lipolysis determined from nonstimulated cells. Data are mean ± SE of 3 independent experiments. B = basal lipolysis (100%).

View larger version (17K): [in this window] [in a new window]   Figure 2. Increased aminochrome formation monitoring in cells conditioned media. 12-day differentiated 3T3-F442A cells were subjected to the following treatments for 10 min: 10–8 M isoproterenol (A), 10–8 M isoproterenol + 100 nM AM (B), and 10–8 M isoproterenol + 0.5 mM PAPA-NONOate (C). Conditioned media were then recovered, instantly frozen in liquid nitrogen, and kept at –80°C. Each sample was rapidly thawed just before LC-MS/MS analysis. Region of interest of LC-MS/MS MRM chromatograms obtained from media samples are presented. The peak at 15.4 min corresponds to characteristic isoprenochrome ion transition 208 166 and is indicated (). Each graphic shows 1 of 3 representative experiments.

CONCLUSIONS AND SIGNIFICANCE

Adipose tissue has long been taken into account as a simple lipid storage tissue, but it can now be regarded as a multipotent endocrine organ. We showed it is able to produce significant amounts of AM. We demonstrated the ability of AM to modulate isoproterenol-dependent lipolysis upstream of adenylate cyclase activation. We presented clear evidence for an NO-dependent mechanism implying the oxidation of a ß-agonist, thereby decreasing its pharmacological activity. The mechanism we propose to explain the following results is described in Fig. 3 . These findings open two interesting fields of investigation. First, they support the possibility that AM could act as a paracrine/autocrine factor to regulate lipid mobilization and thus promote the observed decrease of catecholamine-induced lipolytic rates in subcutaneous adipose tissue of obese subjects. Second, it proposes adipose tissue may function in NO oxidation-mediated clearance of circulating hormones, such as catecholamines, suggesting that adipose tissue is able to inactivate circulating drugs. This last finding may help explain the resistance of obese patients to some drugs and the development of obesity-associated diseases.

View larger version (82K): [in this window] [in a new window]   Figure 3. Schematic diagram of the proposed mechanism for AM-mediated inhibition of isoproterenol-induced lipolysis. Left panel: in the absence of AM, most of the isoproterenol can interact with ß-AR on adipocytes. The lipolytic pathway is fully activated () by the cAMP-dependent phosphorylation of HSL. Phosphorylated HSL hydrolyzes triglycerides into diglycerides and/or monoglycerides, FFA, and glycerol. FFA and glycerol are released by adipocytes in extracellular media. Right panel: when AM is added with isoproterenol in adipocyte media, the peptide binds to its receptors (AM1 and/or AM2), which leads to NOS activation and NO production. Extracellular NO partially oxidates isoproterenol into isoprenochrome, resulting in a weaker activation of the lipolytic pathway. Since less isoproterenol is available for binding to the receptor, less cAMP is produced (). Less FFA and glycerol are released under these conditions. AC, adenylate cyclase; AM, adrenomedullin; ß-AR, ß-adrenergic receptors; Gs, Gs protein; FFA, free fatty acids; HSL, hormone-sensitive lipase; NO, nitric oxide; NOS, nitric oxide synthase; PKA, cAMP-dependent protein kinase; RAMP, receptor activity modifying protein.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2868fje; doi: 10.1096/fj.04-2868fje

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