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Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells.

Sebastián D, Guitart M, García-Martínez C, Mauvezin C, Orellana-Gavaldà JM, Serra D, Gómez-Foix AM, Hegardt FG, Asins G - J. Lipid Res. (2009)

Bottom Line: The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1.However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects.These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.

ABSTRACT
Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for beta-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

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Palmitoyl-CoA oxidation in mitochondrial fractions. Mitochondrial fractions that were obtained from cells infected with Ad-LacZ, Ad-FATP1, or Ad-CPT1A were incubated for 0.5 h with agitation in 400 μl of pregassed complete MKRH buffer and 50 μl of a 2.5 mM 5:1 palmitoyl-CoA-BSA complex containing 1 μCi/ml [1-14C]palmitoyl-CoA and palmitoyl-CoA, and oxidation to CO2 (A) and to ASPs (B) was measured. Data are the mean ± SE of three experiments performed in duplicate. *P < 0.01 versus Ad-LacZ; &P < 0.01 versus the overexpression of only one protein (CPT1A or FATP1).
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fig4: Palmitoyl-CoA oxidation in mitochondrial fractions. Mitochondrial fractions that were obtained from cells infected with Ad-LacZ, Ad-FATP1, or Ad-CPT1A were incubated for 0.5 h with agitation in 400 μl of pregassed complete MKRH buffer and 50 μl of a 2.5 mM 5:1 palmitoyl-CoA-BSA complex containing 1 μCi/ml [1-14C]palmitoyl-CoA and palmitoyl-CoA, and oxidation to CO2 (A) and to ASPs (B) was measured. Data are the mean ± SE of three experiments performed in duplicate. *P < 0.01 versus Ad-LacZ; &P < 0.01 versus the overexpression of only one protein (CPT1A or FATP1).

Mentions: To assess whether the role of FATP1 in the increase in mitochondrial fatty acid oxidation was due to its acyl-CoA synthetase activity, providing the substrate for CPT1, we measured palmitoyl-CoA oxidation to CO2 and ASPs in isolated mitochondria, where palmitoyl-CoA is oxidized in the absence of acyl-CoA synthetase activity. The overexpression of either CPT1 or FATP1 alone produced an increase in fatty acid oxidation to CO2 compared with LacZ control cells (2.20 ± 0.11 nmol palmitoyl-CoA/mg protein × h), which was additive when both proteins were overexpressed together (Fig. 4A). However, no significant difference was observed in palmitoyl-CoA oxidation to ASPs in FATP1-overexpressing cells compared with LacZ control cells (27.6 ± 0.37 nmol palmitoyl-CoA/mg protein × h; Fig. 4B).


Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells.

Sebastián D, Guitart M, García-Martínez C, Mauvezin C, Orellana-Gavaldà JM, Serra D, Gómez-Foix AM, Hegardt FG, Asins G - J. Lipid Res. (2009)

Palmitoyl-CoA oxidation in mitochondrial fractions. Mitochondrial fractions that were obtained from cells infected with Ad-LacZ, Ad-FATP1, or Ad-CPT1A were incubated for 0.5 h with agitation in 400 μl of pregassed complete MKRH buffer and 50 μl of a 2.5 mM 5:1 palmitoyl-CoA-BSA complex containing 1 μCi/ml [1-14C]palmitoyl-CoA and palmitoyl-CoA, and oxidation to CO2 (A) and to ASPs (B) was measured. Data are the mean ± SE of three experiments performed in duplicate. *P < 0.01 versus Ad-LacZ; &P < 0.01 versus the overexpression of only one protein (CPT1A or FATP1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724792&req=5

fig4: Palmitoyl-CoA oxidation in mitochondrial fractions. Mitochondrial fractions that were obtained from cells infected with Ad-LacZ, Ad-FATP1, or Ad-CPT1A were incubated for 0.5 h with agitation in 400 μl of pregassed complete MKRH buffer and 50 μl of a 2.5 mM 5:1 palmitoyl-CoA-BSA complex containing 1 μCi/ml [1-14C]palmitoyl-CoA and palmitoyl-CoA, and oxidation to CO2 (A) and to ASPs (B) was measured. Data are the mean ± SE of three experiments performed in duplicate. *P < 0.01 versus Ad-LacZ; &P < 0.01 versus the overexpression of only one protein (CPT1A or FATP1).
Mentions: To assess whether the role of FATP1 in the increase in mitochondrial fatty acid oxidation was due to its acyl-CoA synthetase activity, providing the substrate for CPT1, we measured palmitoyl-CoA oxidation to CO2 and ASPs in isolated mitochondria, where palmitoyl-CoA is oxidized in the absence of acyl-CoA synthetase activity. The overexpression of either CPT1 or FATP1 alone produced an increase in fatty acid oxidation to CO2 compared with LacZ control cells (2.20 ± 0.11 nmol palmitoyl-CoA/mg protein × h), which was additive when both proteins were overexpressed together (Fig. 4A). However, no significant difference was observed in palmitoyl-CoA oxidation to ASPs in FATP1-overexpressing cells compared with LacZ control cells (27.6 ± 0.37 nmol palmitoyl-CoA/mg protein × h; Fig. 4B).

Bottom Line: The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1.However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects.These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.

ABSTRACT
Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for beta-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

Show MeSH
Related in: MedlinePlus