Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulated lipolysis in adipocytes

Abstract

SummaryThe 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 reesterification. Regulation of mLDs and LD-associated proteins may be an attractive therapeutic target against lipid-associated metabolic diseases.

Lipid metabolism and exercise

“…A recent study demonstrated, also by CARS microscopy, that mLDs are formed de novo during lipolysis when cellular FAs are overloaded, and suggested that mLDs protect cells from the toxicity of excess FAs 13) . Recently, Hashimoto et al have established the cellular origin and physiological significance of mLDs as well as the functional interplay of LD-associated proteins during lipolysis in differentiated 3T3-L1 adipocytes 14) .…”

“…Hashimoto et al performed microscopy studies of the trafficking of key lipolytic proteins in differentiated 3T3-L1 adipocytes 14) . During lipolysis, GFP-perilipin was retained on the surface of large LDs, and also appeared on particulate structures in the cytoplasm (Fig.1A).…”

“…In order to elucidate the origin of the TAG contents in mLDs, Hashimoto et al employed multiplex CARS microscopy (CARS spectral imaging method, for details, see Okuno et al 15) ) 14) . For this purpose, Hashimoto et al pre-loaded 3T3-L1 adipocytes with deuterium-labeled palmitate (D-palmitate), and then subjected the cells to lipolysis in the absence of D-palmitate (see Fig.…”

“…For this purpose, Hashimoto et al pre-loaded 3T3-L1 adipocytes with deuterium-labeled palmitate (D-palmitate), and then subjected the cells to lipolysis in the absence of D-palmitate (see Fig. 2A) 14) . The deuterium in a CD bond is heavier than hydrogen, placing CD vibrational stretching frequencies into near 2100 cm -1 , which is an otherwise silent region of the biological Raman spectra.…”

“…Hashimoto et al examined the significance of mLDs as well as the functional interplay of LD-associated pro- ) JPFSM : Regulation of lipolysis teins in lipolysis 14) . The significance of mLDs might be explained by the increase in the surface area available for lipolysis.…”