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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

FFA uptake by differentiated adipocytes.(A) The uptake of BODIPY-C12 FA by preadipocytes and adipocytes were observed under a fluorescent microscope. Scale = 50 μm. (B) Time-dependent BODIPY-C12 FA uptake by adipocytes. Intracellular emission of BODIPY-C12 FA was monitored every 20 s up to 3,000 s in adipocytes and preadipocytes in the presence and absence of insulin (100 nM). Insulin-treated (+) and untreated (-). (C) The rate of FFA uptake was assessed with the adipocytes cultured in the presence and absence of 25 mM glucose. Blue circle (no insulin, no glucose), red square (with insulin, no glucose), green triangle (no insulin, with glucose), purple reverse triangle (with insulin, with glucose). Inset shows FFA uptake with an extended assay time. At 6,000 s, insulin-dependent FFA uptake shows the saturation of FFA uptake-dependent fluorescent signals. *P < 0.05.
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pone.0120289.g001: FFA uptake by differentiated adipocytes.(A) The uptake of BODIPY-C12 FA by preadipocytes and adipocytes were observed under a fluorescent microscope. Scale = 50 μm. (B) Time-dependent BODIPY-C12 FA uptake by adipocytes. Intracellular emission of BODIPY-C12 FA was monitored every 20 s up to 3,000 s in adipocytes and preadipocytes in the presence and absence of insulin (100 nM). Insulin-treated (+) and untreated (-). (C) The rate of FFA uptake was assessed with the adipocytes cultured in the presence and absence of 25 mM glucose. Blue circle (no insulin, no glucose), red square (with insulin, no glucose), green triangle (no insulin, with glucose), purple reverse triangle (with insulin, with glucose). Inset shows FFA uptake with an extended assay time. At 6,000 s, insulin-dependent FFA uptake shows the saturation of FFA uptake-dependent fluorescent signals. *P < 0.05.

Mentions: To assess the FFA uptake by adipocytes, we examined the cellular uptake of the fluorescently-labeled medium-chain FFA dodecanoic acid (BODIPY-C12-FA) [24,28]. The BODIPY-C12-FA was quenched in culture media as indicated by the lack of fluorescent emission from the extracellular space; however, once C12-FFAs were incorporated into the cytoplasm of adipocytes, the quenchers were dissociated and intracellular fluorescent emission was observed [10]. While undifferentiated 3T3-L1 preadipocytes did not display any detectable fluorescent emission, differentiated adipocytes displayed a time-dependent and robust accumulation of BODIPY-C12-FA in the form of lipid droplets within adipocytes (Fig. 1A, 1B), which was consistent with previously reported observations [12,24].


The exocyst complex regulates free fatty acid uptake by adipocytes.

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

FFA uptake by differentiated adipocytes.(A) The uptake of BODIPY-C12 FA by preadipocytes and adipocytes were observed under a fluorescent microscope. Scale = 50 μm. (B) Time-dependent BODIPY-C12 FA uptake by adipocytes. Intracellular emission of BODIPY-C12 FA was monitored every 20 s up to 3,000 s in adipocytes and preadipocytes in the presence and absence of insulin (100 nM). Insulin-treated (+) and untreated (-). (C) The rate of FFA uptake was assessed with the adipocytes cultured in the presence and absence of 25 mM glucose. Blue circle (no insulin, no glucose), red square (with insulin, no glucose), green triangle (no insulin, with glucose), purple reverse triangle (with insulin, with glucose). Inset shows FFA uptake with an extended assay time. At 6,000 s, insulin-dependent FFA uptake shows the saturation of FFA uptake-dependent fluorescent signals. *P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120289.g001: FFA uptake by differentiated adipocytes.(A) The uptake of BODIPY-C12 FA by preadipocytes and adipocytes were observed under a fluorescent microscope. Scale = 50 μm. (B) Time-dependent BODIPY-C12 FA uptake by adipocytes. Intracellular emission of BODIPY-C12 FA was monitored every 20 s up to 3,000 s in adipocytes and preadipocytes in the presence and absence of insulin (100 nM). Insulin-treated (+) and untreated (-). (C) The rate of FFA uptake was assessed with the adipocytes cultured in the presence and absence of 25 mM glucose. Blue circle (no insulin, no glucose), red square (with insulin, no glucose), green triangle (no insulin, with glucose), purple reverse triangle (with insulin, with glucose). Inset shows FFA uptake with an extended assay time. At 6,000 s, insulin-dependent FFA uptake shows the saturation of FFA uptake-dependent fluorescent signals. *P < 0.05.
Mentions: To assess the FFA uptake by adipocytes, we examined the cellular uptake of the fluorescently-labeled medium-chain FFA dodecanoic acid (BODIPY-C12-FA) [24,28]. The BODIPY-C12-FA was quenched in culture media as indicated by the lack of fluorescent emission from the extracellular space; however, once C12-FFAs were incorporated into the cytoplasm of adipocytes, the quenchers were dissociated and intracellular fluorescent emission was observed [10]. While undifferentiated 3T3-L1 preadipocytes did not display any detectable fluorescent emission, differentiated adipocytes displayed a time-dependent and robust accumulation of BODIPY-C12-FA in the form of lipid droplets within adipocytes (Fig. 1A, 1B), which was consistent with previously reported observations [12,24].

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