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Papers In Press, published online ahead of print May 1, 2007
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* Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamigori, Hyogo 678-1297, Japan
Department of Applied Biological Science, Fukuyama University, Fukuyama 729-0292, Japan
Published, JLR Papers in Press, February 17, 2007.
1 To whom correspondence should be addressed. e-mail: osumi{at}sci.u-hyogo.ac.jp
A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes. We previously showed that Comparative Gene Identification-58 (CGI-58), a product of the causal gene of Chanarin-Dorfman syndrome, interacts with perilipin on LDs. In this study, we investigated the function of CGI-58 using RNA interference. Notably, CGI-58 knockdown caused an abnormal accumulation of LDs in both 3T3-L1 preadipocytes and Hepa1 hepatoma cells. CGI-58 knockdown did not influence the differentiation of 3T3-L1 adipocytes but reduced the activity of both basal and cAMP-dependent protein kinase-stimulated lipolysis. In vitro studies showed that CGI-58 itself does not have lipase/esterase activity, but it enhanced the activity of adipose triglyceride lipase. Upon lipolytic stimulation, endogenous CGI-58 was rapidly dispersed from LDs into the cytosol along with small particulate structures. This shift in localization depends on the phosphorylation of perilipin, because phosphorylated perilipin lost the ability to bind CGI-58. During lipolytic activation, LDs in adipocytes vesiculate into micro-LDs. Using coherent anti-Stokes Raman scattering microscopy, we pursued the formation of micro-LDs in single cells, which seemed to occur in cytoplasmic regions distant from the large central LDs. CGI-58 is not required for this process. Thus, CGI-58 facilitates lipolysis in cooperation with perilipin and other factors, including lipases.
Supplementary key words Comparative Gene Identification-58 • perilipin • coherent anti-Stokes Raman scattering microscopy
Abbreviations: ADRP, adipocyte differentiation-related protein; aP2, adipocyte lipid binding protein; ATGL, adipose triglyceride lipase; CARS, coherent anti-Stokes Raman scattering; CDS, Chanarin-Dorfman syndrome; CGI-58, Comparative Gene Identification-58; GFP, green fluorescent protein; GST, glutathione S-transferase; HSL, hormone-sensitive lipase; IBMX, 3-isobutyl-1-methylxanthine; LD, lipid droplet; PKA, cAMP-dependent protein kinase; PPAR, peroxisome proliferator-activated receptor; RNAi, RNA interference; shRNA, short hairpin RNA; TG, triglyceride; TPEF, two-photon excitation fluorescence
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