Regulation Of  Neutral Lipid Metabolism in Adipocytes and other cells.

Londos, Constantine

Abstract

All cells package neutral lipids in discrete storage droplets that are characterized by unique surface proteins. In adipose cells, the enormous droplets contain triacylglycerols, the primary bodily energy stores, whereas in steroidogenic cells, much smaller droplets contain cholesteryl esters, the precursors for steroid hormone synthesis. In the general cell population, available fatty acids are captured in even smaller droplets and are used eventually as energy sources or for membrane remodeling. The adipocyte remains our primary model system, and we focus on the processes whereby hormones regulate both the packaging and hydrolysis of stored neutral lipids. One important goal has been to dissect the molecular events subsequent to stimulation of adipose cells by lipolytic and antilipolytic hormones, such as epinephrine and insulin, respectively. Stimulation by catecholamines involves activation of adenylyl cyclase, elevation of cAMP, and activation protein kinase A. Hormone-sensitive lipase (HSL), an important enzyme of lipolysis, is phosphorylated by protein kinase A. Although the structure and PKA phosphorylation sites of HSL are known, there is little information on the process whereby cytoplasmic HSL gains access to its substrate, the triacylglycerols housed within the lipid storage droplets. We have found that PKA-phosphorylated HSL rapidly translocates and adheres to the surface of lipid droplets, but nothing is known of its cytoplasmic location in unstimulated cells, the translocation process, or the target locus on the droplet surface. It is this translocation and not HSL activation that accounts for the strong lipolytic enhancement following PKA activation. An important component in the lipolytic equation is the lipid droplet. Previously thought to be merely an amorphous accumulation of neutral lipids, we have identified a family of proteins, termed initially as perilipins, that are found exclusively at lipid droplet surfaces of adipocytes and steroidogenic cells. Perilipin is a single copy gene that gives rise, by alternative splicing, to three isoforms, A, B, and C. The perilipin gene is expressed most strongly in adipose cells where the A and B isoforms coat the triacylglycerol-containing droplets. The gene is expressed also in steroidogenic cells where perilipins A and C coat the cholesteryl estercontaining droplets. Like adipocytes, steroidogenic cells use a cAMP stimulated process and an HSL-like enzyme to release cholesterol, which serves as a substrate for steroid hormone synthesis. Perilipins are polyphosphorylated by PKA and, thus, their occurrence in only those cells in which lipolysis is mediated by increased cAMP points to a role for perilipin in the process of lipid breakdown. This hypothesis has been confirmed by the demonstration that the HSL translocation is dependent upon PKA-mediated phosphorylation of perilipin A. We have found that a related protein, adipose differentiation-related protein (ADFP), also termed adipophilin, coats the lipid droplet in most other types of cells. However, expression of perilipin in fibroblastic cells leads to the disappearance of ADRP, after which the lipid droplets acquire a coating of perillipin. The nonphosphorylated perilipin exerts a protective effect and suppresses lipolysis in such cells. Upon activation of PKA and subsequent phosphorylation of perilipin, a robust lipolysis ensues, which is due solely to perilipin phosphorylation, since the fibroblastic cells contain no PKAmediated lipases. A further manifestation of the protective effect of nonphosphorylated perilipin is the normal deposition of fat reserves in adipose tissue. We found that the perilipin null mouse had a 70% decrease adipose tissue, but are of normal weight and have similar caloric intake as wild type mice. Oddly, despite their greatly diminished adipose tissue, these animals have elevated plasma leptin values. The perilipin null animal also provided important clues on perilipin function. As expected, the adipocytes from these animals exhibit elevated basal lipolysis. Surprisingly, their adipocytes were also refractory to lipolytic stimulation, and we subsequently found that perilipin was required to elicit the PKA-mediated translocation of HSL from the cytosol to the surface of the lipid droplet. We have also shown that HSL translocation also requires phosphorylation of the enzyme at one of its Cterminal PKA sites. Thus, stimulated lipolysis is a concerted reaction requiring PKA phosphorylation of both perilipin and HSL. Recent evidence indicates that, in addition to HSL, a second lipase termed Adipocyte Triglyceride Lipase (ATGL) is also operative in adipocyte lipolysis and may be even more important than HSL in animal lipid metabolism. Although ATGL is not a PKA substrate, its access to lipid droplets is also blocked by perilipin and like HSL, PKA phosphorylation of perilipin facilitates access of ATGL to lipid droplets. We attempted to analyze the phenotype of a mouse in which the perilipin was rendered non-functional by introducing into the perilipin null mouse a perilipin transgene in which the crtical PKA sites had been mutated. In the course of these studies, we found that in the perilipin null mouse the pre-adipocytes are unable to differentiate into mature fat cells. This failure probably accounts, at least in part, to the 70% decrease in adipose tissue in the null mouse. In addition to the perilipins and ADFP, we have identified a number of related genes in Drosophila melanogaster and Dictyostilium discoidium, plus additional mammalian genes. When fused to GFP all of the proteins encoded by these genes target to lipid droplets when expressed in mammalian CHO fibroblasts. In addition to their sequence homologies, similarities in gene structures indicate that the mammalian genes derive from an ancient gene family, and it is likely that all of these proteins will be found to have a role in lipid metabolism. Indeed, recent publications from other groups show that these drosophila proteins play a role in lipid metabolism. There are now five lipid dropletassociated proteins in mammalian cells, perilipin, ADFP, TIP47, S312, and the newest member, LSDP5, which is expressed primarily in tissue that actively oxidize fatty acids. Collectively, these proteins comprise the PAT protein family. Like ADFP, TIP47 is widely expressed and is especially abundant in skeletal muscle cells. We have found that ADFP, TIP47, and LSDP5 serve to protect lipids from hydrolysis by lipases, but do not appear necessary for lipid droplet biogenesis. This profusion of seemingly unrelated names, has prompted a revised nomenclaure for the PAT proteins, in which perilipin, ADRP, TIP47, S3-12, and LSDP5 have been renamed, respectively, Perlipins (PLPN) 1, 2, 3, 4,and 5. Most recently, we have found that both perilipin and ADFP are regulated posttranslationally by stabilization upon binding to lipid droplets. Absent such stabilization, these proteins are ubiquinated and degraded by proteosomal action. Most recently, we have found that down-regulation of PAT proteins in AML12 liver cells leads to increased lipolyisis of lipid droplets, leading to the release of fatty acids to the cell interior. The cells become insulin resistant, which does not occur if the operative lipase ATGL is also downregulated. It is concluded that the primary function of the PAT proteins is to sequester fatty acids as TAG and thus prevent insulin resistance. More recently we have explored the possibility that triacylglycerols and cholesteryl esters my be stored in separate droplet rather than in a single droplet, and found that droplets loaded with labeled CE or FA target to distinctly different droplets. We are attempting to confirm this segregation of lipids with the use of proteomics to identify proteins that are unique to these droplet