Abstract
Lipid droplets (LDs) of mammalian cells are coated with one or more members of the PAT protein family (e.g. Perilipin), which serve important functions in regulating fat storage and mobilization by interacting with hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). However, the interplay of lipid-associated proteins (LAP) that regulate lipolysis is not fully understood. PURPOSE: In this study, we examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis as well as dynamic morphological change affecting LDs during lipolytic activation in differentiated 3T3-L1 adipocytes. METHODS: Time-lapse images of 3T3-L1 cells stably expressing GFP- and/or RFP-fused LAP were acquired during 2 hrs of cAMP-dependent PKA activation. In addition, dynamic morphological changes of LDs during lipolytic activation were obtained using coherent anti-Stokes Raman Scattering (CARS) microscopy, which visualizes LDs in living cells. RESULTS: During the course of stimulated lipolysis, GFP-perilipin (PLIN) and RFP-CGI58, which interacts with PLIN on LDs, began to disperse from central large LDs into the cytoplasm. In addition, using CARS microscopy, we observed numerous micro-LDs (mLDs) coated with CGI58 emerging after lipolytic stimulation. Further we showed that mLDs might be derived from a large central LDs coated with PLIN and CGI58. GFP-HSL and GFP-ATGL were found in the cytoplasm in basal condition. Upon lipolytic stimulation, GFP-HSL was targeted to various sized LDs, where it colocalized with PLIN. On the other hand, lipolytic stimulation did not alter the location of GFP-ATGL. Following subcellular fractionation, we observed that PLIN and CGI58 were predominantly fractionated with LDs in lysates from both basal and 1 hr PKA-stimulated adipocytes, but CGI58 also detected in membrane fraction, where organelles are involved, after 1 hr lipolysis. ATGL were found in membrane fraction as well as cytosolic fraction of both basal and PKA-stimulated adipocytes. CONCLUSION: These results suggest that mLDs are important physiologically to enhance the efficiency of lipolysis by increasing the total LD surface area where PLIN and HSL can interact. As well, it is possible that active lipolysis occurs by means of interplays of CGI58 and ATGL even in the organelles.
Published Version
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