The intracellular localization of the exocyst complex is known to be regulated through tubulin polymerization . C12 dodecanoic acid by 3T3-L1 adipocytes is usually 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 functions 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 functions 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. Introduction Dietary lipids constitute approximately 40% of caloric intake in modern human diet . Free fatty acids (FFAs) not only serve as important energy source for ATP synthesis but also regulate intracellular signaling and transcription . FFAs in blood circulation are rapidly incorporated into adipocytes, hepatocytes, and cardiac myocytes . Circulating FFA levels are regulated not only by dietary FFA intake but by hormones and sympathetic tones . Dysregulated FFA handling may contribute to impaired glucose metabolism found in obese and diabetic subjects [5,6]. Therefore, defining the molecular and cellular mechanisms that regulate FFA uptake should Beloranib 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) 1C4, have been shown to regulate adipocyte FFA uptake [7C12]. The plasma membrane-mediated flip-flop mechanism of FFA translocation is also suggested to regulate cellular FFA uptake [13,14]. However, the role of intracellular vesicle trafficking in the regulation of FFA uptake has not been examined to this date. The exocyst is usually a large protein complex composed of Sec3 Beloranib (Exoc1), Sec5 (Exoc2), Sec6 (Exoc3), Sec8 (Exoc4), Sec10 (Exoc5), Sec15 (Exoc6), Exo70 (Exoc7), and Exo84 (Exoc8). The exocyst complex was initially discovered in yeast as a molecular machinery that regulates the exocytosis of secretory vesicles . In mammalian cells, Beloranib the exocyst complex promotes the translocation of glucose transporter type 4 (GLUT4) from your intracellular compartment to the plasma membrane [16C18]. Diverse biological functions of the exocyst complex have been explained in different cell types including insulin secretion from pancreatic beta-cells [19,20], the trafficking of neurotransmitter receptors in synaptic terminals , and the membrane-localization of a matrix metalloproteinase (MMP) in malignancy cells . In adipocytes, however, the metabolic role played by the exocyst complex beyond insulin-dependent glucose uptake has not been fully explored. In this study, we have recognized a new role for the exocyst complex in the regulation of FFA uptake by adipocytes. Our findings may shed new light around the molecular mechanism underlying FFA handling in health and diseases. Materials and Methods Cell culture and adipocyte differentiation The 3T3-L1 cells (ATCC, CL-173) were managed in DMEM, 25 mM glucose (Gibco) with 10% new born calf serum (NCS, Hyclone) in a 5% CO2 incubator at 37C. The adipocyte differentiation of 3T3-L1 cells was induced by changing media to DMEM, 25 mM glucose with 10% fetal bovine serum (Hyclone) made up of a differentiation mix (100 nM insulin, 0.25 M dexamethasone, and 0.5 mM 3-isobutyl-1-methyxanthine, all from Sigma-Aldrich). Three Beloranib days after the induction of adipogenesis, 3T3-L1 adipocytes were cultured in an optical 96-well plates with DMEM supplemented with 25 mM glucose, 100 nM insulin, and 10% FBS. Free fatty acid uptake assay Lipid uptake assay was performed using QBT Fatty Acid Transporter Assay Kit (Molecular Devices) according to the manufacturers training . About 50,000 cells/well/100 L 3T3-L1 adipocytes were plated onto an optical 96 well plate (Fischer Scientific) and centrifuged at 1000 rpm for 5 min. After overnight incubation at 37C with 5% CO2, media were changed to serum-free DMEM of high-glucose (25 mM) or low-glucose concentration (5.5 mM), and incubated for additional 1 hour. Cells were stimulated with 10 nM insulin for 30min in 1x assay buffer (1x Hanks balanced salt answer with 20 mM HEPES and 0.2% fatty acid-free BSA) before the assay, then the fluorescent emission from each well was measured immediately after adding QBT Fatty Acid Uptake answer . The unquenched emission of intracellular BODIPY-dodecanoic acid was measured in a Victor II Multilevel Plate Reader (PerkinElmer) or Synergy Neo Multi-Mode Reader (Bio-Tek) in real time up to 3,000 seconds (ex = 480nm and em = 515nm). Inhibitors A phosphoinositide-3-kinase (PI-3K) inhibitor (Wortmannin), MEK inhibitor (U0126), mTOR inhibitor (rapamycin), Akt1/2 kinase inhibitor (Akt1/2I), and nocodazole were obtained from Sigma-Aldrich (St. Louis, MO). Stealth RNAi transfection MPSL1 into adipocytes 3T3-L1 adipocytes were transfected with stealth RNA interference (RNAi) duplexes (Invitrogen) using electroporation as.