Perilipin A Increases Triacylglycerol Storage by Decreasing the Rate of Triacylglycerol Hydrolysis

Abstract

The perilipins are the most abundant proteins at the surfaces of lipid droplets in adipocytes and are also found in steroidogenic cells. To investigate perilipin function, perilipin A, the predominant isoform, was ectopically expressed in fibroblastic 3T3-L1 pre-adipocytes that normally lack the perilipins. In control cells, fluorescent staining of neutral lipids with Bodipy 493/ 503 showed a few minute and widely dispersed lipid droplets, while in cells stably expressing perilipin A, the lipid droplets were more numerous and tightly clustered in one or two regions of the cytoplasm. Immunofluorescence microscopy revealed that the ectopic perilipin A localized to the surfaces of the tiny clustered lipid droplets; subcellular fractionation of the cells using sucrose gradients confirmed that the perilipin A localized exclusively to lipid droplets. Cells expressing perilipin A stored 6 -30-fold more triacylglycerol than control cells due to reduced lipolysis of triacylglycerol stores. The lipolysis of stored triacylglycerol was 5 times slower in lipid-loaded cells expressing perilipin A than in lipid-loaded control cells, when triacylglycerol synthesis was blocked with 6 M triacsin C. This stabilization of triacylglycerol was not due to the suppression of triacylglycerol lipase activity by the expression of perilipin A. We conclude that perilipin A increases the triacylglycerol content of cells by forming a barrier that reduces the access of soluble lipases to stored lipids, thus inhibiting triacylglycerol hydrolysis. These studies suggest that perilipin A plays a major role in the regulation of triacylglycerol storage and lipolysis in adipocytes.Lipid droplets in adipocytes store the body's major energy reserves as triacylglycerols. These structures contain a large core of neutral lipid, primarily triacylglycerol, covered by a phospholipid monolayer. The intracellular mechanisms that control the storage and release of triacylglycerols are largely uncharacterized, yet are likely to be fundamental to understanding the regulation of energy metabolism in the body. Recent studies have shown that lipid droplets are covered with a proteinaceous coat; the functions and identities of the component proteins have not been fully elucidated. The first identified lipid droplet-specific proteins are the perilipins (1-7), a family of proteins coating the surfaces of lipid droplets of adipocytes and steroidogenic cells of adrenal cortex, testes, and ovaries, but lacking in other types of cells and in other cellular compartments. The perilipins are encoded by a single copy gene that gives rise to multiple mRNAs by alternative splicing mechanisms 1 ; these mRNAs are translated to yield the three described protein isoforms (2, 4). Perilipin A is the predominant isoform in both adipocytes and steroidogenic cells, perilipin B is found primarily in adipocytes, and perilipin C is unique to steroidogenic cells. Perilipin A is the most abundant protein on highly purified lipid droplets isolated from fully differentiated cultured 3T3-L1 adip...

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