Lipase-selective Functional Domains of Perilipin A Differentially Regulate Constitutive and Protein Kinase A-stimulated Lipolysis

Abstract

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMPdependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), Hydrolysis of triacylglycerol (TAG) 1 (lipolysis) in adipocytes is a key event that supplies the primary source of energy, free fatty acids, for other tissues. In times of energy need such as fasting (1-3) and exercise (4, 5), adipocyte lipolysis is regulated by hormones such as catecholamines, which activate cAMP-dependent protein kinase (PKA) (6, 7). Activation of PKA results in a marked increase in lipolysis as compared with spontaneous lipolysis in the absence of hormones (basal or constitutive lipolysis). Thus, basal and PKA-stimulated lipolysis reflect the lipolytic response of adipocytes to the changing energy requirements of the body. In obesity, basal lipolysis is increased, and PKA-stimulated lipolysis is blunted (8). This dysregulation of adipocyte lipolysis is associated with the development of insulin resistance and type 2 diabetes (9).TAG breakdown (lipolysis) is mediated by lipases, which hydrolyze TAG sequestered in intracellular lipid droplets. Approximately 50% of the neutral triglyceride lipase activity in white adipose tissue is attributable to hormone-sensitive lipase (HSL) (10 -12). HSL-mediated lipolysis is under the tight control of PKA. In the absence of PKA activation, constitutive HSL activity has been thought to mediate basal lipolysis (13). When PKA is activated, it phosphorylates HSL (13, 14), resulting in enhanced hydrolytic activity (15), translocation of HSL from cytosol to the lipid droplet surface (16 -18), and enhanced TAG breakdown. HSL was previously considered the rate-limiting enzyme in adipocyte lipolysis. This view, however, has recently been challenged by the following observations: 1) HSL-deficient mice are not obese, suggesting significant activity of lipase(s) other than HSL (10,12,19); 2) adipocytes derived from epididymal fat pads or embryonic fibroblasts of HSL null mice retain 50% of basal TAG lipase activity and are responsive to PKA activation (10,11,12,19); and 3) cell lines lacking endogenous HSL, i.e. 3T3-L1 preadipocytes (21), NIH 3T3 fibroblasts (22), and Chinese hamster ovary cells (23), exhibit significant TAG hydrolysis under basal and PKA-stimulated conditions. Thus, both HSL and non-HSL(s) play important roles in basal and PKA-stimulated lipolysis.The hydrolytic actions of HSL and non-HSLs are regulated at the lipid drop...

The Amino and Carboxyl Termini of Perilipin A Facilitate the Storage of Triacylglycerols

“…These observations suggest that the unique carboxyl terminus of perilipin A serves a shielding function against cytosolic lipases. A very recent report (27) has reported similar findings that the final 216 amino acid peptide of the carboxyl terminus of perilipin A serves an important role in reducing levels of basal lipolysis by non-HSL lipases; however, that study suggests that the amino terminus of perilipin A lacks protective function. We note that study of the larger amino-terminal truncations described in the recent report may have missed the effects that we have uncovered.…”

Protein kinase A suppresses the differentiation of 3T3-L1 preadipocytes

“…They bring about lipid accumulation and the adipocyte phenotype. It has previously been shown that cAMP and PKA are implicated in the regulation of lipid accumulation or lipolysis [26][27][28][29][30][31]. To avoid interference from the lipolysis effect of cAMP and PKA, all specific reagents used in this study (such as forskolin, SQ22536, 8-Br-cAMP and H89) were only presented in cell culture in the first 2 days of the entire induction period of adipogenesis.…”

AMP-activated Protein Kinase Is Activated as a Consequence of Lipolysis in the Adipocyte

“…Ϫ/Ϫ MEF Adipocytes-Adenoviruses expressing Aequoria victoria green fluorescent protein (Ad-GFP) or wild-type perilipin A (Ad-WT Peri A) were generated and verified as described previously (13,14,32). Recombinant adenovirus was transduced into Peri Ϫ/Ϫ MEFs with Lipofectamine Plus TM (Invitrogen) on day 3 after induction of differentiation.…”