The body’s most abundant energy reserves are stored as triacylglycerol (TAG) in the lipid droplets of adipocytes. Perilipin A is the major lipid droplet-associated protein in adipocytes and has a critical role in the regulation of TAG storage and lipolysis. When the body is in the fed state, adipocytes store excess calories as TAG in perilipin-coated lipid droplets. Perilipins assist TAG storage by forming a surface barrier on lipid droplets to shield stored TAG from abundant cytosolic lipases. When energy is required by the body, catecholamines bind to ß-adrenergic receptors on the adipocyte plasma membrane, initiating a signaling cascade that elevates cAMP levels and activates cAMP-dependent protein kinase (PKA). The PKA-mediated phosphorylation of several proteins promotes lipolysis and the consequent release of fatty acids to provide energy to the body.
The adipocyte lipid droplet is a largely uncharacterized organelle. Few lipid droplet-associated proteins have been identified, yet it is clear that these proteins play major roles in the control of whole body energy metabolism. Over the past 2 years, we have conducted a proteomic project to identify additional proteins on lipid droplets isolated from 3T3-L1 adipocytes incubated under either basal or lipolytically-stimulated conditions. It was a very challenging task due to the hydrophobic nature of the lipid droplet-associated proteins. To overcome the experimental difficulty, SDS-PAGE and capillary reversed-phase HPLC-MS/MS were used. We have successfully identified more than 50 proteins, including CGI-58, a member of the lipase subfamily of a/ß-hydrolase-fold enzymes, on adipocyte lipid droplets. Our data allowed us to analyze the protein profile differences of lipid droplet isolated from 3T3-L1 adipocytes under the basal and lipolytically-stimulated conditions. The associations of several identified proteins to the lipid droplet were confirmed by cytofluorescence microscopy and Western blot. This study resulted a substantial information that can help us in understanding how lipid droplet-associated proteins changes in response to energy storage and consumption in our body.
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