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This study investigated the role of adenosine monophosphate–activated protein kinase (AMPK) in the regulation of lipolysis in visceral (VC) and subcutaneous (SC) rat adipocytes and the molecular mechanisms involved in this process. VC (epididymal and retroperitoneal) and SC (inguinal) adipocytes were isolated from male Wistar rats (160–180 g). Adipocytes were incubated either in the absence or in the presence of the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, 0–500 µmol/l). AMPK and acetyl-CoA carboxylase (ACC) phosphorylation, basal and epinephrine-stimulated (100 nmol/l) glycerol release, and hormone-sensitive lipase (HSL) phosphorylation and activity were determined. AICAR-induced (500 µmol/l) AMPK activation inhibited basal glycerol release by ∼42, 41, and 44% in epididymal, retroperitoneal, and inguinal adipocytes, respectively. Epinephrine-stimulated glycerol release was almost completely prevented by AICAR treatment in adipocytes from all fat depots. The AMPK inhibitor compound C (20 µmol/l) prevented AICAR-induced phosphorylation of AMPK and significantly increased basal (∼1.3-, 1.4-, and 1.7-fold) and epinephrine-stimulated (∼1.3-, 1.2-, 1.4-fold) glycerol release in epididymal, retroperitoneal, and inguinal adipocytes, respectively. AICAR increased phosphorylation of HSLSer565 and inhibited epinephrine-induced phosphorylation of HSLSer563 and HSLSer660. This was also accompanied by a 73% reduction in epinephrine-stimulated HSL activity. Compound C prevented the phosphorylation of HSLSer565 induced by AICAR and partially prevented the inhibitory effect of this drug on basal and epinephrine-stimulated lipolysis in adipocytes in VC and SC fat depots. In summary, despite different fat depots eliciting distinct rates of lipolysis, acute AICAR-induced AMPK activation suppressed HSL phosphorylation/activation and exerted similar antilipolytic effects on both VC and SC adipocytes.