title: The autophagic inhibitor 3-methyladenine potently stimulates PKA-dependent lipolysis in adipocytes.
abstract: BACKGROUND AND PURPOSE The class III PI3K inhibitor, 3-methyladenine (3-MA), is commonly used to selectively block autophagy. Recent findings suggest a strong relationship between autophagy and lipid turnover. Here, we explore the effect of 3-MA on adipocyte lipolysis. EXPERIMENTAL APPROACH Assays were performed in 3T3-L1 cells. Cells were treated with 3-MA and wortmannin, a pan PI3K and autophagy inhibitor. Pharmacological and genetic manipulation of endogenous autophagic and lipolytic pathways was used to ascertain the contribution of 3-MA to the observed effects on lipolysis. KEY RESULTS 3T3-L1 cells that were exposed to 3-MA showed a consistent increase in lipolysis, approximately 50% over basal levels. The effect of 3-MA was not secondary to autophagic inhibition as treatment of 3T3-L1 cells with wortmannin yielded no such changes. Dosing and time course experiments showed that 3-MA's ability to activate lipolysis was more sensitive than its inhibitory effect on autophagy. Knockdown of adipose triglyceride lipase (ATGL) negated the stimulatory effect of 3-MA by >90%, indicating that 3-MA enhanced ATGL-dependent hydrolysis of triacylglycerols. Additionally, the lipolytic effect of 3-MA was dependent on the activation of PKA and 3-MA induced a rapid and potent elevation of intracellular cAMP levels in adipocytes. CONCLUSIONS AND IMPLICATIONS Cumulatively, we show that 3-MA potently modulated a cellular mechanism and its underlying signalling pathways not associated with autophagy. Furthermore, we describe a novel stimulatory effect on a major signalling pathway. Our findings provide valuable information to studies employing 3-MA as a specific inhibitor for PI3K and autophagy.
authors: Department of Biochemistry & Molecular Biology, Mayo Clinic, Scottsdale, AZ, USA.
authors: Bradlee L
authors: BL
authors: Heckmann
authors: Xingyuan
authors: X
authors: Yang
authors: Xiaodong
authors: X
authors: Zhang
authors: Jun
authors: J
authors: Liu
mesh_terms: D041721:3T3-L1 Cells; D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D017667:Adipocytes / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D006868:Hydrolysis; D008049:Lipase / Q000378:metabolism; D008066:Lipolysis / Q000502:physiology*; D051379:Mice; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D002787:Sterol Esterase / Q000378:metabolism; D014280:Triglycerides / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034741:RNA, Small Interfering; D014280:Triglycerides; C025946:3-methyladenine; D002787:Sterol Esterase; D008049:Lipase; D000225:Adenine; D000077191:Wortmannin