PERILIPIN IS LOCATED ON THE SURFACE-LAYER OF INTRACELLULAR LIPID DROPLETS IN ADIPOCYTES

Immunocytochemistry was used to determine the intracellular location o f perilipins in adipocytes and the occurrence of these proteins in tis sues involved in triacylglycerol metabolism. Confocal microscopy and 3 -dimensional analysis of 3T3-L1 adipocytes showed that perilipin immun ofluorescence, present on the surfaces of all sized lipid droplets, ap peared unevenly dispersed on the surfaces of many large lipid droplets . Electron microscopy revealed that immunogold staining for perilipin was located directly on the surface layer apposed to and surrounding t he core triacylglycerol of intracellular lipid droplets of adipocytes in culture or from white and brown adipose tissue. Freeze-fracture ele ctron microscopy indicated that the hydrophobic face of this surface m onolayer contained particles identical in size and distribution to int ramembranous particles (IMPs), which are unique structural features of the hydrophobic faces of bilayered membranes. Also, freeze-fracture r eplicas revealed areas of continuity between the surface layer of lipi d droplets and the membrane leaflets of endoplasmic reticulum, suggest ing that the droplet monolayer surface is an area of endoplasmic retic ulum membrane leaflet modified by its unique content of perilipin. Mic roperoxisomes, identified by immunostaining for catalase, were found c losely associated with lipid droplets, but external to and not in cont act with the lipid droplet surface layer. Vimentin, identified by immu nofluorescence, was present around the periphery of most lipid droplet s in 3T3-L1 cells during early stages of adipocyte development but, in contrast to perilipins, vimentin was not around the periphery of many large lipid droplets in mature cells. Although perilipin was at the s urface of lipid droplets in adipocytes of lactating mammary gland, non e was found to be associated with the milk lipid droplets in alveolar epithelial cells, nor was the protein found on the surfaces of lipid d roplets in hepatocytes. Studies in mammary gland show that perilipin i mmunostaining will be a valuable tool for the identification of tissue adipocytes severely depleted of their triacylglycerol stores and thus without their characteristic spherical shape. Perilipin's singular lo cation on the surface monolayer of intracellular lipid droplets suppor ts an intimate role for the protein in the triacylglycerol metabolic f unctions of adipocytes.