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The exocyst complex regulates free fatty acid uptake by adipocytes.

Inoue M, Akama T, Jiang Y, Chun TH - PLoS ONE (2015)

Bottom Line: Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Metabolism, Endocrinology & Diabetes (MEND), Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

The exocyst complex mediates insulin-induced FFA uptake in adipocytes.(A) Exo70 gene expression was suppressed (Exo70 KD) with siRNA and FFA uptake by adipocytes was assessed. Gray circle (control siRNA, no insulin), black circle (control siRNA, with 100 nM insulin), yellow triangle (Exo70 siRNA, no insulin), red triangle (Exo70 siRNA, 100 nM insulin). (B) Sec8 gene expression was silenced (Sec8 KD) and FFA uptake by adipocytes in the presence and absence of insulin was determined. Gray circle (control siRNA, no insulin), black circle (control siRNA, with 100 nM insulin), yellow triangle (Sec8 siRNA, no insulin), red triangle (Sec8 siRNA, 100 nM insulin). (C) The effect of nocodazole (Noc) treatment on FFA uptake of adipocytes. Mean ± SEM. N = 4. *P < 0.05.
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pone.0120289.g004: The exocyst complex mediates insulin-induced FFA uptake in adipocytes.(A) Exo70 gene expression was suppressed (Exo70 KD) with siRNA and FFA uptake by adipocytes was assessed. Gray circle (control siRNA, no insulin), black circle (control siRNA, with 100 nM insulin), yellow triangle (Exo70 siRNA, no insulin), red triangle (Exo70 siRNA, 100 nM insulin). (B) Sec8 gene expression was silenced (Sec8 KD) and FFA uptake by adipocytes in the presence and absence of insulin was determined. Gray circle (control siRNA, no insulin), black circle (control siRNA, with 100 nM insulin), yellow triangle (Sec8 siRNA, no insulin), red triangle (Sec8 siRNA, 100 nM insulin). (C) The effect of nocodazole (Noc) treatment on FFA uptake of adipocytes. Mean ± SEM. N = 4. *P < 0.05.

Mentions: We next aimed to determine the effect of Exo70 and Sec8 gene silencing on adipocyte FFA uptake. Gene silencing of Exo70 significantly reduced FFA uptake under both basal and insulin-treated conditions (AUC, baseline with control oligo, 3.6x107, with Exo70 siRNA, 3.1x107, P = 0.001 at t = 3,000 s; under insulin treatment, control oligo, 3.7x107; Exo70 siRNA, 3.3 x 107, P = 0.01 at t = 3,000 s) (Fig. 4A).


The exocyst complex regulates free fatty acid uptake by adipocytes.

Inoue M, Akama T, Jiang Y, Chun TH - PLoS ONE (2015)

The exocyst complex mediates insulin-induced FFA uptake in adipocytes.(A) Exo70 gene expression was suppressed (Exo70 KD) with siRNA and FFA uptake by adipocytes was assessed. Gray circle (control siRNA, no insulin), black circle (control siRNA, with 100 nM insulin), yellow triangle (Exo70 siRNA, no insulin), red triangle (Exo70 siRNA, 100 nM insulin). (B) Sec8 gene expression was silenced (Sec8 KD) and FFA uptake by adipocytes in the presence and absence of insulin was determined. Gray circle (control siRNA, no insulin), black circle (control siRNA, with 100 nM insulin), yellow triangle (Sec8 siRNA, no insulin), red triangle (Sec8 siRNA, 100 nM insulin). (C) The effect of nocodazole (Noc) treatment on FFA uptake of adipocytes. Mean ± SEM. N = 4. *P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359155&req=5

pone.0120289.g004: The exocyst complex mediates insulin-induced FFA uptake in adipocytes.(A) Exo70 gene expression was suppressed (Exo70 KD) with siRNA and FFA uptake by adipocytes was assessed. Gray circle (control siRNA, no insulin), black circle (control siRNA, with 100 nM insulin), yellow triangle (Exo70 siRNA, no insulin), red triangle (Exo70 siRNA, 100 nM insulin). (B) Sec8 gene expression was silenced (Sec8 KD) and FFA uptake by adipocytes in the presence and absence of insulin was determined. Gray circle (control siRNA, no insulin), black circle (control siRNA, with 100 nM insulin), yellow triangle (Sec8 siRNA, no insulin), red triangle (Sec8 siRNA, 100 nM insulin). (C) The effect of nocodazole (Noc) treatment on FFA uptake of adipocytes. Mean ± SEM. N = 4. *P < 0.05.
Mentions: We next aimed to determine the effect of Exo70 and Sec8 gene silencing on adipocyte FFA uptake. Gene silencing of Exo70 significantly reduced FFA uptake under both basal and insulin-treated conditions (AUC, baseline with control oligo, 3.6x107, with Exo70 siRNA, 3.1x107, P = 0.001 at t = 3,000 s; under insulin treatment, control oligo, 3.7x107; Exo70 siRNA, 3.3 x 107, P = 0.01 at t = 3,000 s) (Fig. 4A).

Bottom Line: Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Metabolism, Endocrinology & Diabetes (MEND), Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus