Perilipin Targets a Novel Pool of Lipid Droplets for Lipolytic Attack by Hormone-sensitive Lipase

Hsiao-Ping H Moore,Robert B Silver,Emilio P Mottillo,David A Bernlohr,James G Granneman

doi:10.1074/jbc.m506336200

Abstract

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.

Highlights

There is strong evidence that Plin is critically involved in regulating access of hormone-sensitive lipase (HSL) to stored triglyceride, several observations indicate that a simple barrier/translocation model is insufficient to explain lipolysis in vivo
The barrier/translocation model does not explicitly address the issue of whether all lipid storage droplet (LSD) surfaces are coated with Plin, the model implies that surfaces not coated by Plin should be subject to attack by HSL
Our results indicate that mature adipocytes contain at least two types of LSDs: a major or central LSD that is typically associated with unilocular fat cells, and novel peripheral LSDs that are interposed between the plasma membrane and the central LSD

Summary

EXPERIMENTAL PROCEDURES

Materials—Alexa-488-Bodipy was purchased from Molecular Probes (Eugene, OR). Rabbit anti-Plin and rabbit anti-HSL antibodies were kindly provided by Dr A. Fixed cells were first permeabilized with PBS containing 5% normal goat serum and 0.01% saponin (permeabilization buffer) for 1 h They were incubated for 1 h with primary antibodies against the first antigen (rabbit anti-HSL at 1:500 dilution, or rabbit anti-Plin at 1:1000 dilution). Cells cotransfected with ECFP-HSL and Plin-A-EYFP were treated under control and stimulated conditions for 30 min, fixed with 1% paraformaldehyde and imaged by spinning disc confocal microscopy, as detailed below. Under these conditions, no channel bleed-through was observed as assessed by cells stained with a single fluorescent probe. Quantitation of fluorescence intensities and colocalization were performed using the colocalization and line profile tools of Image-Pro Plus (Media Cybernetics, Inc., Silver Spring, MD)

RESULTS

DISCUSSION

An important concept that emerges from this work is that lipolysis