Abstract
The exocyst is an octameric molecular complex that drives vesicle trafficking in adipocytes, a rate-limiting step in insulin-dependent glucose uptake. This study assessed the role of the exocyst complex in regulating free fatty acid (FFA) uptake by adipocytes. Upon differentiating into adipocytes, 3T3-L1 cells acquire the ability to incorporate extracellular FFAs in an insulin-dependent manner. A kinetic assay using fluoresceinated FFA (C12 dodecanoic acid) uptake allows the real-time monitoring of FFA internalization by adipocytes. The insulin-dependent uptake of C12 dodecanoic acid by 3T3-L1 adipocytes is mediated by Akt and phosphatidylinositol 3 (PI3)-kinase. Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes. Consistent with the roles played by Exo70 and Sec8 in FFA uptake, mCherry-tagged Exo70 and HA-tagged Sec8 partially colocalize with lipid droplets within adipocytes, suggesting their active roles in the development of lipid droplets. Tubulin polymerization was also found to regulate FFA uptake in collaboration with the exocyst complex. This study demonstrates a novel role played by the exocyst complex in the regulation of FFA uptake by adipocytes.
Highlights
Dietary lipids constitute approximately 40% of caloric intake in modern human diet [1]
The timedependent increase of fluorescent emission following the intracellular uptake of BODIPY-C12-FA was observed only in differentiated adipocytes but not in preadipocytes (Fig. 1B), suggesting that free fatty acid (FFA) uptake is a cellular process newly acquired during adipocyte differentiation
These results suggest that insulin-dependent FFA uptake in adipocyte is not significantly altered by the presence of extracellular glucose and that insulin directly regulates FFA uptake by adipocytes
Summary
Dietary lipids constitute approximately 40% of caloric intake in modern human diet [1]. Free fatty acids (FFAs) serve as important energy source for ATP synthesis and regulate intracellular signaling and transcription [2]. Circulating FFA levels are regulated by dietary FFA intake but by hormones and sympathetic tones [4]. Defining the molecular and cellular mechanisms that regulate FFA uptake should help us better understand the pathogenesis of obesity and insulin resistance. A cohort of receptors and transporters, e.g., CD36 and fatty acid transporters (FATP) 1–4, have been shown to regulate adipocyte FFA uptake [7,8,9,10,11,12]. The plasma membrane-mediated flip-flop mechanism of FFA translocation is suggested to regulate cellular FFA uptake [13,14]
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