Modulation of Hormone-sensitive Lipase and Protein Kinase A-mediated Lipolysis by Perilipin A in an Adenoviral Reconstituted System

• Published in: The Journal of biological chemistry Vol. 277; no. 10; pp. 8267 - 8272
• Main Authors: Souza, Sandra C., Muliro, Kizito V., Liscum, Laura, Lien, Ping, Yamamoto, Mia T., Schaffer, Jean E., Dallal, Gerard E., Wang, Xinzhong, Kraemer, Fredric B., Obin, Martin, Greenberg, Andrew S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.03.2002; American Society for Biochemistry and Molecular Biology
• Subjects: 3T3 Cells; Adenoviridae - genetics; Alanine - chemistry; Animals; Bacterial Outer Membrane Proteins - metabolism; Blotting, Western; Carrier Proteins; Cell Line; Colforsin - pharmacology; Cyclic AMP-Dependent Protein Kinases - metabolism; Hydrolysis; Lipid Metabolism; Mice; Microscopy, Fluorescence; Mutagenesis, Site-Directed; Perilipin-1; Phosphoproteins - metabolism; Phosphorylation; Plasmids - metabolism; Protein Binding; Protein Conformation; Serine - chemistry; Sterol Esterase - metabolism; Triglycerides - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M108329200

Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin treatment induced Peri A hyperphosphorylation and abrogated the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri AΔ3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduced Peri A hyperphosphorylation and blocked constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells.