Molecular Mechanisms of Active Lipolysis in 3T3-L1 Adipocytes

Abstract

Lipid droplets (LDs) of mammalian cells are coated with lipid-associated proteins (LAP) such as perilipin (PLIN), hormone sensitive lipase (HSL), and adipose triglyceride lipase (ATGL), which play important roles in regulating fat storage and mobilization, by interacting with each other. By using coherent anti-Stokes Raman Scattering (CARS) microscopy which visualizes LDs in living cells, we observed numerous micro-LDs (mLDs) emerging after lipolytic stimulation. However, the interplay of LAP and the physiological significance of mLDs are not fully understood. PURPOSE: In this study, we examined the localization, trafficking, and interactions of key proteins involved in lipolysis (i.e. LAP) as well as the physiological significance of mLDs during lipolysis in differentiated 3T3-L1 adipocytes. METHODS: The localization and trafficking of LAP during 2 hrs of cAMP-dependent PKA activation were examined by immunostaining. In addition, dynamic morphological changes of LDs and fatty acid (FA) turnover during lipolysis were obtained using multiplex CARS microspectroscopy, which is able to distinguish between deuterium-labeled FA and non-labeled FA. Lipolytic activity and the protein-protein interaction were biochemically analyzed. RESULTS: In the basal state, the localization of HSL was cytoplasmic, while that of PLIN was on large LDs. Upon lipolytic stimulation, HSL being redistributed to the surface of large LDs where being co-localized with PLIN. Furthermore, HSL and PLIN were co-localized at particulate structures, which are supposed to be mLDs. We found that these structures are derived from endoplasmic reticulum (ER) where nascent LDs are formed via triacylglycerol (TAG) synthesis, and perform active lipolysis. On the other hand, lipolytic stimulation did not alter the cytoplasmic distribution of ATGL. Following subcellular fractionation, we observed that ATGL and its co-activator CGI-58 were co-fractionated upon lipolytic stimulation in membrane fraction where organelles are enriched. Furthermore, immunoprecipitation analysis demonstrated that a small but significant amount of ATGL was co-precipitated with Myc-tagged CGI-58 in membrane fraction, in response to lipolytic stimulation. CONCLUSIONS: These results suggest that mLDs have important functions to enhance the efficiency of lipolysis, by increasing the total LD surface area where PLIN and HSL can interact. It is also possible that active lipolysis occurs by means of interplays of CGI-58 and ATGL, in organelles other than LDs.