title: Acylated and unacylated ghrelin attenuate isoproterenol-induced lipolysis in isolated rat visceral adipocytes through activation of phosphoinositide 3-kinase γ and phosphodiesterase 3B.
abstract: The acylated peptide ghrelin (AG) and its endogenous non-acylated isoform (UAG) protect cardiomyocytes, pancreatic β-cells, and preadipocytes from apoptosis, and induce preadipocytes differentiation into adipocytes. These events are mediated by AG and UAG binding to a still unidentified receptor, which determines the activation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) ERK1/2. AG and UAG also possess antilipolytic activity in vitro, but the underlying mechanism remains unknown. Thus, the objective of the current study was to characterize the molecular events involved in AG/UAG receptor signaling cascade. We treated rat primary visceral adipocytes with isoproterenol (ISO) and forskolin (FSK) to stimulate lipolysis, simultaneously incubating them with or without AG or UAG. Both peptides blocked ISO- and FSK-induced lipolysis. By direct measurement of cAMP intracellular content, we demonstrated that AG/UAG effect was associated to a reduction of ISO-induced cAMP accumulation. Moreover, the cAMP analog 8Br-cAMP abolished AG/UAG effect. As AG and UAG were ineffective against lipolysis induced by db-cAMP, another poorly hydrolyzable cAMP analog, phosphodiesterase (PDE) involvement was hypothesized. Indeed, cilostamide, a specific PDE3B inhibitor, blocked AG/UAG effect on ISO-induced lipolysis. Furthermore, the PI3K inhibitor wortmannin and AKT inhibitor 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4piperidinyl)-2H-benzimidazol-2-one trifluoroacetate also blocked AG/UAG action, suggesting a role in PDE3B activation. In particular, PI3K isoenzyme gamma (PI3Kγ) selective inhibition through the compound AS605240 prevented AG/UAG effect on ISO-stimulated lipolysis, hampering AKT phosphorylation on Ser(473). Taken together, these data demonstrate for the first time that AG/UAG attenuation of ISO-induced lipolysis involves PI3Kγ/AKT and PDE3B.
authors: Department of Anatomy, Pharmacology, and Forensic Medicine, Division of Medical Pharmacology, University of Torino, Via P. Giuria 13, 10125 Torino, Italy.
authors: Alessandra
authors: A
authors: Baragli
authors: Corrado
authors: C
authors: Ghè
authors: Elisa
authors: E
authors: Arnoletti
authors: Riccarda
authors: R
authors: Granata
authors: Ezio
authors: E
authors: Ghigo
authors: Giampiero
authors: G
authors: Muccioli
mesh_terms: D000215:Acylation; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D058544:Class Ib Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D054439:Ghrelin / Q000378:metabolism / Q000494:pharmacology*; D005990:Glycerol / Q000378:metabolism; D050152:Intra-Abdominal Fat / Q000166:cytology; D007545:Isoproterenol / Q000378:metabolism / Q000494:pharmacology*; D008066:Lipolysis / Q000187:drug effects*; D008297:Male; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D015363:Quinolones / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D045162:Thiazolidinediones / Q000494:pharmacology
chemical_list: C532657:1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one; C505144:5-quinoxalin-6-ylmethylenethiazolidine-2,4-dione; D000318:Adrenergic beta-Agonists; D001562:Benzimidazoles; D054439:Ghrelin; D010726:Phosphodiesterase Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015363:Quinolones; D011810:Quinoxalines; D045162:Thiazolidinediones; D005576:Colforsin; C021294:cilostamide; D000242:Cyclic AMP; D058544:Class Ib Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D007545:Isoproterenol; D005990:Glycerol