Adipocytes store the body's major energy supply in the form of triacylglycerols (TAGs) packaged into perilipin- coated lipid droplets. Dysregulation of adipose TAG metabolism in obese individuals leads to release of excess fatty acids into circulation, which contributes to the development of health complications including peripheral insulin resistance and hepatic steatosis. The regulation of adipose TAG storage and hydrolysis is complex;our understanding of these processes is rudimentary and incomplete. The proposed studies investigate the molecular mechanisms by which perilipin controls and coordinates TAG metabolism and lipid droplet dynamics. The overarching hypothesis of these studies is that perilipin forms a scaffold at the surfaces of lipid droplets in adipocytes that serves as an organizing center for lipid metabolic enzymes and trafficking factors. Under basal conditions, when TAG storage predominates over a low level of basal lipolysis, the perilipin scaffold binds CGI- 58, a Coenzyme A-dependent lysophosphatidic acid acyltransferase and co-activator of adipose triglyceride lipase (ATGL). When cell surface ?-adrenergic receptors are stimulated, cellular cAMP levels increase and protein kinase A (PKA) is activated. Perilipin is phosphorylated by PKA on as many as 6 serine residues;phosphorylation of these sites promotes lipolysis through several different mechanisms. Phosphorylation of PKA sites 1, 2, and 3 in the amino terminus of perilipin promotes the docking of PKA-phosphorylated hormone- sensitive lipase (HSL) on lipid droplets through a protein-protein interaction with perilipin, and HSL gains access to TAG and diacylglycerol substrates. Phosphorylation of carboxyl terminal PKA sites 4, 5, and 6 facilitates lipolysis by as yet poorly understood mechanisms, which include the promotion of lipid droplet fragmentation into myriad lipid micro-droplets with increased surface area for lipase (ATGL) binding. The goals of the proposed study are to 1) investigate the role of serine 517 (within PKA site 6) in lipid droplet association of perilipin and control of lipolysis using techniques of cellular and molecular biology, 2) investigate the role of PKA-mediated phosphorylation of serine 492 (within PKA site 5) in lipid droplet remodeling in a cultured cell model and lipolysis in both cells and mice, and 3) investigate the mechanisms by which CGI-58 co-activates ATGL, and perilipin serves as a platform for regulation of TAG hydrolysis by ATGL. Mutated variants of peri- lipin and CGI-58 will be studied in cultured cells and a novel transgenic mouse model of adipose-selective expression of mutated perilipin on a perilipin null background. The information gained from these studies will contribute to a long-term goal of defining lipid droplet-associated factors that control TAG metabolism in adipocytes.

Public Health Relevance

Adipocytes (fat cells) in adipose tissue store the major energy reserves of the body as triacylglycerols in struc- tures called lipid droplets. Obese individuals have enlarged lipid droplets in adipocytes when compared to lean individuals. Lipid droplets are covered with perilipin, a protein which controls the metabolism of triacylglycerols, and hence, the release of fatty acids into the blood for use as a source of energy by various tissues of the body. Obesity is characterized by inappropriate and excessive release of fatty acids into circulation;these fatty acids are taken up by muscle and liver and contribute to the development of insulin resistance (which occurs in type II diabetes) and fatty liver. Our understanding of the mechanisms by which perilipin controls triacylglycerol (fat) metabolism in adipocytes is incomplete;the proposed research will increase understanding of these mechanisms, and identify potential new targets for therapeutic intervention to control release of fatty acids from adipose tissue.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054797-13
Application #
8470621
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Haft, Carol R
Project Start
2000-08-15
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
13
Fiscal Year
2013
Total Cost
$305,520
Indirect Cost
$107,285
Name
Rutgers University
Department
Nutrition
Type
Schools of Earth Sciences/Natur
DUNS #
001912864
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
Brasaemle, Dawn L; Wolins, Nathan E (2012) Packaging of fat: an evolving model of lipid droplet assembly and expansion. J Biol Chem 287:2273-9
Caviglia, Jorge M; Betters, Jenna L; Dapito, Dianne-Helerie et al. (2011) Adipose-selective overexpression of ABHD5/CGI-58 does not increase lipolysis or protect against diet-induced obesity. J Lipid Res 52:2032-42
Wang, Hong; Bell, Ming; Sreenivasan, Urmila et al. (2011) Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein. J Biol Chem 286:15707-15
Montero-Moran, Gabriela; Caviglia, Jorge M; McMahon, Derek et al. (2010) CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase. J Lipid Res 51:709-19
Kimmel, Alan R; Brasaemle, Dawn L; McAndrews-Hill, Monica et al. (2010) Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins. J Lipid Res 51:468-71
Wang, Hong; Hu, Liping; Dalen, Knut et al. (2009) Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins. J Biol Chem 284:32116-25
Caviglia, Jorge M; Sparks, Janet D; Toraskar, Nikhil et al. (2009) ABHD5/CGI-58 facilitates the assembly and secretion of apolipoprotein B lipoproteins by McA RH7777 rat hepatoma cells. Biochim Biophys Acta 1791:198-205
Brasaemle, Dawn L; Subramanian, Vidya; Garcia, Anne et al. (2009) Perilipin A and the control of triacylglycerol metabolism. Mol Cell Biochem 326:15-21
Brasaemle, Dawn L (2007) Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis. J Lipid Res 48:2547-59
Marcinkiewicz, Amy; Gauthier, Denise; Garcia, Anne et al. (2006) The phosphorylation of serine 492 of perilipin a directs lipid droplet fragmentation and dispersion. J Biol Chem 281:11901-9

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