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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

Akt- and PI3 kinase-dependent FFA uptake of adipocytes.(A) Adipocytes were pretreated with kinase inhibitors, then stimulated with 100 nM insulin for 30 minutes before FFA uptake assay. Blue, basal FFA uptake without insulin; red, with insulin; purple and black, pretreated with Wortmaninn 0.4 μM and 4 μM; navy blue, AktI 10 μM; brown, LY294002 10 μM. N = 5 each. * P <0.01 (B) Dose-dependent suppression of insulin-dependent FFA uptake by Akt inhibitor. FFA uptake induced by 100 nM insulin with the subtraction of basal FFA uptake. N = 3 each.
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pone.0120289.g002: Akt- and PI3 kinase-dependent FFA uptake of adipocytes.(A) Adipocytes were pretreated with kinase inhibitors, then stimulated with 100 nM insulin for 30 minutes before FFA uptake assay. Blue, basal FFA uptake without insulin; red, with insulin; purple and black, pretreated with Wortmaninn 0.4 μM and 4 μM; navy blue, AktI 10 μM; brown, LY294002 10 μM. N = 5 each. * P <0.01 (B) Dose-dependent suppression of insulin-dependent FFA uptake by Akt inhibitor. FFA uptake induced by 100 nM insulin with the subtraction of basal FFA uptake. N = 3 each.

Mentions: We explored the signaling pathways that are required for adipocyte FFA uptake using the kinetic assay of FFA uptake. While adipocytes were able to incorporate FFA without insulin, treatment with 100 nM insulin for 30 minutes significantly increased FFA uptake (AUC, control, 1.90 x 107; insulin treated, 2.96 x 107, P = 3.0 x 10-5 at t = 3,000 s, Fig. 2A). The inhibition of PI3-kinase activity with Wortmannin (0.4 μM) caused a substantial reduction in basal FFA uptake (AUC, 1.45 x 107, P = 0.0008 compared to control without insulin at t = 3,000 s) as well as in insulin-stimulated FFA uptake (AUC, 2.3 x 107, P = 0.0006 compared to control with insulin at t = 3,000 s) (Fig. 2A).


The exocyst complex regulates free fatty acid uptake by adipocytes.

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

Akt- and PI3 kinase-dependent FFA uptake of adipocytes.(A) Adipocytes were pretreated with kinase inhibitors, then stimulated with 100 nM insulin for 30 minutes before FFA uptake assay. Blue, basal FFA uptake without insulin; red, with insulin; purple and black, pretreated with Wortmaninn 0.4 μM and 4 μM; navy blue, AktI 10 μM; brown, LY294002 10 μM. N = 5 each. * P <0.01 (B) Dose-dependent suppression of insulin-dependent FFA uptake by Akt inhibitor. FFA uptake induced by 100 nM insulin with the subtraction of basal FFA uptake. N = 3 each.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120289.g002: Akt- and PI3 kinase-dependent FFA uptake of adipocytes.(A) Adipocytes were pretreated with kinase inhibitors, then stimulated with 100 nM insulin for 30 minutes before FFA uptake assay. Blue, basal FFA uptake without insulin; red, with insulin; purple and black, pretreated with Wortmaninn 0.4 μM and 4 μM; navy blue, AktI 10 μM; brown, LY294002 10 μM. N = 5 each. * P <0.01 (B) Dose-dependent suppression of insulin-dependent FFA uptake by Akt inhibitor. FFA uptake induced by 100 nM insulin with the subtraction of basal FFA uptake. N = 3 each.
Mentions: We explored the signaling pathways that are required for adipocyte FFA uptake using the kinetic assay of FFA uptake. While adipocytes were able to incorporate FFA without insulin, treatment with 100 nM insulin for 30 minutes significantly increased FFA uptake (AUC, control, 1.90 x 107; insulin treated, 2.96 x 107, P = 3.0 x 10-5 at t = 3,000 s, Fig. 2A). The inhibition of PI3-kinase activity with Wortmannin (0.4 μM) caused a substantial reduction in basal FFA uptake (AUC, 1.45 x 107, P = 0.0008 compared to control without insulin at t = 3,000 s) as well as in insulin-stimulated FFA uptake (AUC, 2.3 x 107, P = 0.0006 compared to control with insulin at t = 3,000 s) (Fig. 2A).

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