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

Abstract

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 β-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 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%). Download figure Download PowerPoint 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. Download figure Download PowerPoint CONCLUSIONS AND SIGNIFICANCE Adipose tissue has long...