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.