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. 2009 Sep;50(9):1789-99.
doi: 10.1194/jlr.M800535-JLR200. Epub 2009 May 9.

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

David Sebastián  1 , Maria Guitart, Celia García-Martínez, Caroline Mauvezin, Josep M Orellana-Gavaldà, Dolors Serra, Anna M Gómez-Foix, Fausto G Hegardt, Guillermina Asins
Affiliations

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

David Sebastián et al. J Lipid Res. 2009 Sep.

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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Figures

Fig. 1.
Fig. 1.
Overexpression of FATP1, FAT/CD36, and CPT1A in L6E9 myotubes. L6E9 myotubes were transduced with Ad-FATP1, Ad-FAT/CD36, Ad-CPT1A, or Ad-LacZ as a control, and a Western blot using a specific antibody against FATP1 (A), FAT/CD36 (B), or CPT1A (C) was performed in 50 μg of total extract.
Fig. 2.
Fig. 2.
Effect of FATP1 overexpression on palmitate metabolism and acyl-CoA synthetase activity. A: Palmitate incorporation into complex lipids. L6E9 cells that were infected with Ad-LacZ, Ad-FATP1, and Ad-FAT/CD36 were incubated for 16 h in serum-free medium containing 1 μCi/ml [1-14C]palmitic acid and 0.25 mM unlabeled palmitate complexed to 1% (w/v) BSA. Fatty acid incorporation into TGs, DAG, PLs, and NEPalm was assessed using TLC after lipid extraction. B: Fatty acid oxidation. Cells were infected with Ad-LacZ, Ad-FATP1, or Ad-FAT/CD36, and palmitate oxidation to CO2 was measured for 3 h. C: Acyl-CoA synthetase activity was measured in both total and mitochondrial extracts coming from L6E9 myotubes transduced with Ad-LacZ or Ad-FATP1. Data are the mean SE of five experiments performed in triplicate. *P 0.01 versus Ad-LacZ; **P 0.001 versus Ad-LacZ.
Fig. 3.
Fig. 3.
Palmitate oxidation in mitochondrial fractions. Mitochondrial fractions that were obtained from cells infected with Ad-LacZ, Ad-FATP1, Ad-CPT1A, or Ad-FAT/CD36 were incubated for 1 h with agitation in 400 μl of pregassed complete MKRH buffer and 50 μl of a 2.5 mM 5:1 palmitate-BSA complex containing 10 μCi/ml [1-14C]palmitic acid, and palmitate oxidation to CO2 (A) and to ASPs (B) was measured. The ratio of ASPs/CO2 was also calculated (C). Data are the mean SE of five experiments performed in duplicate. *P < 0.01 versus Ad-LacZ, **P < 0.001 versus Ad-LacZ, &P < 0.001 versus the overexpression of only one protein (CPT1A, FATP1, or FAT/CD36), and #P < 0.01 versus control without etomoxir.
Fig. 4.
Fig. 4.
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).
Fig. 5.
Fig. 5.
Protein expression levels of CPT1A, FATP1, and FAT/CD36 in L6E9 myotubes. Western blot for CPT1A, FATP1, and FAT/CD36 proteins was performed in 50 μg of mitochondrial fractions obtained from cells infected with Ad-LacZ, Ad-CPT1A, Ad-FATP1, and Ad-FAT/CD36. Western blot against porin was used as a mitochondrial loading control.
Fig. 6.
Fig. 6.
FATP1 localizes in mitochondria in L6E9 myotubes. A: L6E9 myoblasts were transfected with pFATP1-GFP or pFAT/CD36-GFP and induced to differentiate. Western blot analyses were performed 48 h posttransfection on total cell extracts (TE) (35 μg protein), mitochondrial enriched fractions (mit) (35 g protein) after centrifugation at 1,500 g and 10,000 g, or the supernatant (Sup.) of the 10,000 g pellet (35 μg) and membranes hybridized with antibodies against FAT/CD36, FATP1, GFP, or porin as stated. B: Colocalization analysis of the mitochondrial marker COI and FATP1-GFP. L6E9 cells were transfected with pFATP1-GFP and studied 48 h posttransfection. a: Image of the FATP1-GFP protein observed under a confocal microscope. b: Image of mitochondrial network by staining with antibodies against COI. c: Colocalization of the signal of GFP and COI. C: Qualitative analyses of colocalization were assessed using the plugin RGB Profiler from the WCIF ImageJ software. A line was drawn arbitrarily over an area of interest, and the plot presents the overlap of the intensity of each laser (from distinct labeled proteins).
Fig. 7.
Fig. 7.
Coimmunoprecipitation of FATP1 and CPT1. L6E9 myotubes were transduced with Ad-LacZ, Ad-CPT1A, Ad-FATP1, and Ad-FAT/CD36, and FATP1 or FAT/CD36 proteins were immunoprecipitated using specific antibodies in either total extracts (1,000 μg) (A) or mitochondrial extracts (150 μg) (B), and CPT1A protein was detected in these immunoprecipitates by Western blot. C: FATP1 protein was immunoprecipitated in either total extracts (2,000 μg) or mitochondrial-enriched extracts (300 μg) from L6E9 myotubes, and CPT1A protein was detected by Western blot. D: FATP1 protein was immunoprecipitated in either total extracts (1,000 μg) or mitochondria-enriched extracts (150 μg) from both gastrocnemius and soleus muscles, and CPT1B protein was detected by Western blot using a specific antibody. Total expression of CPT1B in 50 μg of total and mitochondrial extracts is also shown (Input). A negative control with an unspecific antibody is also shown. IP, immunoprecipitation; IB, immunoblot.
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