Perilipin Promotes Hormone-sensitive Lipase-mediated Adipocyte Lipolysis via Phosphorylation-dependent and -independent Mechanisms

Hideaki Miyoshi,Sandra C Souza,Hui-Hong Zhang,Katherine J Strissel,Marcelo A Christoffolete,Julia Kovsan,Assaf Rudich,Fredric B Kraemer,Antonio C Bianco,Martin S Obin,Andrew S Greenberg

doi:10.1074/jbc.m601097200

Abstract

Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes. HSL-dependent lipolysis in response to catecholamines is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin A (Peri A), an essential lipid droplet (LD)-associated protein. It is believed that perilipin phosphorylation is essential for the translocation of HSL from the cytosol to the LD, a key event in stimulated lipolysis. Using adipocytes retrovirally engineered from murine embryonic fibroblasts of perilipin null mice (Peri-/- MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is LD-associated in the basal state and that PKA-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri Adelta1-6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri Adelta1-6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri Adelta1-6 failed to enhance PKA-stimulated lipolysis in either MEF adipocytes or differentiated brown adipocytes. Thus, the lipolytic action(s) of HSL at the LD surface requires PKA-dependent perilipin phosphorylation. In Peri-/- MEF adipocytes, PKA activation significantly enhanced the amount of HSL that could be cross-linked to and co-immunoprecipitated with ectopic Peri A. Notably, this enhanced cross-linking was blunted in Peri-/- MEF adipocytes expressing Peri Adelta1-6. This suggests that PKA-dependent perilipin phosphorylation facilitates (either direct or indirect) perilipin interaction with LD-associated HSL. These results redefine and expand our understanding of how perilipin regulates HSL-mediated lipolysis in adipocytes.

Highlights

Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes
Using adipocytes retrovirally engineered from murine embryonic fibroblasts of perilipin null mice (Peri؊/؊ MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is lipid droplet (LD)-associated in the basal state and that protein kinase A (PKA)-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri A⌬1– 6)
Characterization of PeriϪ/Ϫ MEF Adipocytes—Our experimental strategy was to develop a stable line of perilipin null adipocytes in which to examine the effects of ectopically expressed wild type or PKA sitedeficient perilipin on HSL translocation and HSL-mediated lipolysis

Summary

Perilipin Regulation of HSL Action

In vitro and in vivo studies demonstrate that reduced levels or absence of perilipin A results in increased basal lipolysis while substantially attenuating catecholamine (PKA)-stimulated lipolysis (20 –23). 2) PKA-dependent perilipin phosphorylation at six serine residues (consensus PKA sites) is required for stimulated lipolysis [14, 22, 26], presumably reflecting perilipin conformational changes that expose stored neutral lipid and facilitate HSL translocation to the LD [19, 22, 27]. This combination of abrogated barrier function and facilitation of HSL translocation is proposed as the mechanism by which PKA-dependent perilipin phosphorylation mediates catecholamine-stimulated lipolysis in adipocytes [19, 22, 23, 27]. These observations redefine the mechanism by which perilipin regulates HSL-mediated lipolysis in response to ␤-adrenergic stimuli

EXPERIMENTAL PROCEDURES

RESULTS

Cell fraction

DISCUSSION