title: Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulated lipolysis in adipocytes.
abstract: The regulation of lipolysis in adipocytes involves coordinated actions of many lipid droplet (LD)-associated proteins such as perilipin, hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and its activator protein, CGI-58. Here, we describe the cellular origin and physiological significance of micro LDs (mLDs) that emerge in the cytoplasm during active lipolysis, as well as the roles of key lipolytic proteins on mLDs in differentiated 3T3-L1 adipocytes. Multiplex coherent anti-Stokes Raman scattering (CARS) microscopy demonstrated that mLDs receive the fatty acid (FA) moiety of triglyceride from pre-existing LDs during lipolysis. However, when FA re-esterification was blocked, mLDs did not emerge. Time-lapse imaging of GFP-tagged LD-associated proteins and immunocytochemical analyses showed that particulate structures carrying LD-associated proteins emerged throughout the cells upon lipolytic stimulation, but not when FA re-esterification was blocked. Overall lipolysis, as estimated by glycerol release, was significantly lowered by blocking re-esterification, whereas release of free FAs was enhanced. ATGL was co-immunoprecipitated with CGI-58 from the homogenates of lipolytically stimulated cells. Following CGI-58 knockdown or ATGL inhibition with bromoenol lactone, release of both glycerol and FA was significantly lowered. AICAR, an activator of AMP-activated protein kinase, significantly increased FA release, in accordance with increased expression of ATGL, even in the absence of CGI-58. These results suggest that, besides on the surface of pre-existing central LDs, LD-associated proteins are actively involved in lipolysis on mLDs that are formed by FA re-esterification. Regulation of mLDs and LD-associated proteins may be an attractive therapeutic target against lipid-associated metabolic diseases.
authors.affiliation: Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan. thashimo@fc.ritsumei.ac.jp
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hashimoto
authors.forename: Hiroki
authors.initials: H
authors.lastname: Segawa
authors.forename: Masanari
authors.initials: M
authors.lastname: Okuno
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kano
authors.forename: Hiro-o
authors.initials: HO
authors.lastname: Hamaguchi
authors.forename: Tokuko
authors.initials: T
authors.lastname: Haraguchi
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hiraoka
authors.forename: Shiho
authors.initials: S
authors.lastname: Hasui
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Fumiko
authors.initials: F
authors.lastname: Hirose
authors.forename: Takashi
authors.initials: T
authors.lastname: Osumi
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D018929:Cell Culture Techniques; D008049:Lipase / Q000378:metabolism*; D050356:Lipid Metabolism; D008066:Lipolysis; D059627:Liquid Phase Microextraction; D051379:Mice; D008856:Microscopy, Fluorescence; D002787:Sterol Esterase / Q000378:metabolism*
chemical_list: D002787:Sterol Esterase; D008049:Lipase