Abstract
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.
Highlights
A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes
The vector itself or a vector containing a mutant target sequence in which four nucleotides deviated from the wild-type sequence was used. 3T3-L1 preadipocytes transduced with each recombinant virus were exposed to the medium containing the adipogenic hormone mixture on the day when the cells reached confluence and treated for 2 days. mRNA levels of Comparative Gene Identification-58 (CGI-58) and markers for adipocyte differentiation [peroxisome proliferator-activated receptor (PPAR)g and adipocyte lipid binding protein] were first examined by RT-PCR (Fig. 1A)
Because adipocyte differentiation-related protein (ADRP) is involved in the accumulation of lipids and its protein level closely parallels the total fat cell mass, we examined whether the population and size of LDs are altered in RNA interference (RNAi)-treated cells (Fig. 2). 3T3-L1 cells treated with CGI-58 RNAi were stained with Nile Red, which labels LDs, and observed by phase-contrast and fluorescence microscopy
Summary
A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes. They further showed that activation of PKA dispersed CGI-58green fluorescent protein (GFP) from the surface of LDs to the cytosol, suggesting the involvement of CGI-58 in the lipolytic process. A significant increase in the level of ADRP was observed in the CGI-58 RNAi-treated cells at each stage of differentiation compared with the vector and mismatch controls.
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