Departament Bioquímica i Biologia Molecular, Universitat de Barcelona, Barcelona, Spain
Submitted 7 June 2004 ; accepted in final form 6 January 2005
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ABSTRACT |
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palmitate; oleate; fatty acid binding proteins; skeletal muscle
In mice, FATP1 expression is highest in fat, skeletal muscle, and the heart (25). Intracellular FATP1 protein distribution has not been studied in muscle cells. Information about its physiological role in muscle comes exclusively from FATP1-knockout (FATP1-KO) mice (15). Deletion of the gene does not alter the metabolic phenotype of mice fed a starch-based diet but protects them from intramuscular accumulation of fatty acyl-CoA and triacylglyceride (TAG) induced by a fat diet without altering whole-body adiposity (15). Notably, in humans, homozygosity in a single nucleotide polymorphism within the FATP1 gene, consisting of an A/G change at position 48 in intron 8, is associated with increased plasma TAG levels (9, 21), probably due to impaired peripheral postprandial uptake of fatty acids.
In contrast, genetic models have revealed much regarding the role of the very ubiquitous membrane transporter FAT (31). Overexpression of the protein in muscle from transgenic mice (13) does not stimulate oxidation of palmitate in isolated resting soleus muscle but greatly enhances this process in response to contraction. Strikingly, palmitate incorporation into TAG is unaffected or lower in both resting and contracting muscles from these transgenic mice. On the other hand, deletion of the FAT gene in mice impairs fatty acid transport and incorporation into TAG in skeletal muscle, while it increases free fatty acid and TAG levels in plasma (6). Similarly, spontaneous inactivating mutation of the gene in the insulin-resistant hypertensive rat is associated with hyperlipidemia (10). FAT/CD36 has been localized unequivocally in the plasma membrane of muscle cells. Furthermore, this localization is stimulated in myocytes by insulin or contraction at the expense of intracellularly stored protein (5). However, insulin concomitantly enhances fatty acid esterification, whereas contraction prompts oxidation, suggesting that proteins other than membrane-recruited FAT restrict fatty acid metabolism.
In this study, we overexpressed FATP1 and FAT genes in human muscle cells, allowing us to compare their intracellular localization and role in the control of uptake and direction of palmitate and oleate toward lipid pools or oxidation.
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MATERIALS AND METHODS |
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We constructed the plasmids pFATPGFP and pFATGFP, which include NH2-terminal fusion protein constructs with EGFP. A 0.6-kb fragment of the FATP1 cDNA was isolated from pAdCMV-FATP by performing PCR. A mutation was introduced to knock out the stop codon while inserting a BamHI restriction site. 5'-GTGGGCCAGATCAACCAGC-3' and 5'-TGGATCCCCGGTACCTCCGAGTGAGA-3' were used as upstream and downstream oligonucleotides, respectively, in the PCR. The PCR-amplified fragment was digested with EspI (restriction site at 1,582 bp) and BamHI and thereby ligated with a 1,582-bp EcoRI-EspI fragment of the FATP1 cDNA, which was isolated from the pAdCMV-FATP, into EcoRI-BamHI-digested pEGFP-N1 vector to generate pFATPGFP. To prepare pFATGFP, a 0.35-kb fragment of the FAT cDNA was isolated from pSG5-FAT by performing PCR, in which the stop codon was replaced by an AgeI restriction site, using 5'-CATTTCCTACATGCAAGTC-3' and 5'-ATTTACCGGTTTTCCATTCTTAG-3' as upstream and downstream oligonucleotides. This fragment was cloned into pGEM-T to generate pGEM-T-FAT0.35. A second fragment of FAT cDNA was prepared by digestion of pSG5-FAT vector with AatII and NspI and ligated into pGEM-T-FAT0.35 to generate pGEM-T-FAT. Finally, pFATGFP was constructed by cloning the AatII-AgeI FAT cDNA from pGEM-T-FAT into pEGFP-N1 vector digested with the same restriction enzymes. pFATPGFP and pFATGFP were excised with EcoRI-AflII and BamHI-NotI, respectively. The isolated bands were inserted into pACCMVpLpA to generate the corresponding adenovirus: AdCMV-FATPGFP or AdCMV-FATGFP.
Muscle cell cultures and adenoviral transduction.
Human muscle biopsies were obtained with the approval of the Research Committee of the Hospital Vall d'Hebrón, Barcelona, Spain. Human muscle primary cultures were begun from satellite cells of muscle biopsies from healthy individuals as described previously (2). Myoblasts were grown in DMEM/M-199 medium (3:1) with 10% FBS, 10 µg/ml insulin, 2 mM glutamine, 25 ng/ml fibroblast growth factor, and 10 ng/ml epidermal growth factor. Immediately after myoblast fusion, the medium was replaced by DMEM/M-199 devoid of growth factors and glutamine and with 10% FBS. Twelve-day-old myotubes were transduced with adenoviruses at a multiplicity of infection of 50 for 2 h. In these conditions, transduction efficiency was 90% (data not shown). In all studies, cells were used 3 days after transduction. Twenty-four hours before the metabolic experiments, cells were depleted of insulin and FBS and incubated with DMEM containing 5 mM glucose with or without fatty acids.
Fatty acid preparation and metabolic analysis. Sodium salts of palmitic and oleic acids were prepared in deionized water containing 1.2 equivalents of NaOH at 70°C until an optically clear dispersion was obtained. The fatty acid salt solution was added to DMEM containing fatty acid-free bovine serum albumin (BSA) with continuous agitation. The fatty acid-to-BSA molar ratio was 5:1 or 2.5:1, within the physiological range (6, 24, 27).
To measure fatty acid uptake, cells were rinsed with phosphate-buffered saline (PBS) containing 0.1 mM CaCl2 and 0.1% BSA and incubated in glucose-deprived DMEM with [1-14C]oleate (2.8 µCi/µmol) or [1-14C]palmitate (2.8 µCi/µmol) for 2 min. To stop the reaction, cells were rinsed with ice-cold PBS solution. Finally, monolayers were extracted in PBS that contained 1% SDS, and aliquots were measured for radioactivity in 5 ml of scintillation cocktail.
To detect the incorporation of fatty acids into lipids, cells were incubated with [1-14C]fatty acid (1 µCi/µmol). Monolayers were then rinsed with PBS, and lipids were extracted twice with hexane:isopropanol (3:2). After being dried under a nitrogen stream, the residual lipid extract was dissolved in chloroform:methanol (2:1) and TAG and phospholipids were separated using thin-layer chromatography (TLC) with hexane-diethyl ether-acetic acid (70:30:1). The lipid spots were identified by iodine vapor, scraped, and counted in a liquid scintillation cocktail.
To measure fatty acid oxidation rate, cells in 24-well plates were incubated with [1-14C]oleate (2.8 µCi/µmol) or [1-14C]palmitate (2.8 µCi/µmol). The incubation was terminated by addition of 125 µl of 3 M HClO4 to each well. Immediately, a Whatman no. 3 paper soaked with 25 µl of -phenylethylamine was placed over each well, which was then tightly covered and sealed with elastic film. After 1 h of incubation at room temperature, the filter paper was cut and the trapped 14CO2 was quantified in a scintillation cocktail. Cell extracts containing acid-soluble intermediates were centrifuged at 12,000 g, and the supernatant was counted for radioactivity.
To determine the TAG content, extracts were prepared by scraping cell monolayers into a buffer consisting of 50 mM Tris, 100 mM KCl, 20 mM KF, 0.5 mM EDTA, and 0.05% Lubrol PX, pH 7.9, and applying three 5-s pulses of sonication. Homogenates were centrifuged at 11,000 g for 15 min, and the resulting supernatants were collected. Protein concentration was measured with Bio-Rad protein assay reagent. TAGs were measured enzymatically with a Cobas Fara II autoanalyzer and a GPO-Trinder kit using triolein resuspended in the extraction buffer as a standard.
mRNA analysis.
Total RNA from myotubes was extracted using a guanidinium hydrochloride-based method. RNA (20 µg) was denatured, electrophoresed on 1.2% formaldehyde-agarose gels, and transferred to positively charged nylon membranes. Ethidium bromide (0.2 µg) was added to RNA samples to check equal loading of the gels and transfer efficiency. Prehybridization and hybridization were performed at 65°C using 0.25 M Na2HPO4 (pH 7.2), 1 mM EDTA, 7% SDS, and 1% blocking reagent solution. Blots were hybridized using as probes a 1.2-kb BglII-BamHI fragment of the mouse cDNA for FATP1, which is highly homologous to the human sequence between nucleotides 915 and 2,082 included in the probe, and the full-length cDNA (1.3 kb BamHI) of the rat FAT, which is highly homologous to the human mRNA. DNA probes were labeled with [-32P]dCTP using the random oligonucleotide primer method.
Confocal microscopy. Cells seeded onto coverslips were fixed with 3% paraformaldehyde, rinsed, and mounted in Immuno-Fluor. Fluorescence images were obtained with a Leica TCS four-dimensional confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope and x63 (NA x1.4 oil immersion) Leitz Plan-Apo lens objective. The light source was an argon/krypton laser (75 mW). Green fluorescence from GFP recombinants was excited at 488 nm, and optical sections (0.1 µm) were obtained. Lipid droplets were revealed by staining with Nile Red, which was included in the mounting medium, at 1:1,000 dilution from a saturated stock solution in acetone. The Nile Red image was captured as described previously (23).
Muscle cells seeded onto glass coverslips were fixed in PBS containing 3% (wt/vol) paraformaldehyde-60 mM sucrose. The cells were then permeabilized with 0.05% Triton X-100-20 mM glycine in PBS and blocked with 1% (wt/vol) BSA-10 mM glycine in PBS. Next, cells were incubated with antibody for the Golgi marker GM-130 and subsequently with the secondary antibody Alexa Fluor 546-conjugated goat anti-mouse IgG. Coverslips were mounted with Immuno-Fluor mounting medium.
Statistical analysis. Data are expressed as means ± SE. Statistical significance was established using an unpaired Student's t-test.
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RESULTS |
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DISCUSSION |
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We also report that FATP1 and FAT moderately stimulate long-term consumption of palmitate and oleate in the range of concentrations tested, with higher enhancement at 0.05 mM than at 0.5 or 1 mM. Notably, FATP1 strongly stimulated the utilization of palmitate and oleate for TAG synthesis. Stronger effects were exerted at the lower doses of fatty acids, and the stimulation was higher for palmitate than oleate, which is more efficiently used than palmitate for TAG synthesis (22). In contrast, incorporation of fatty acids in phospholipids was stimulated by FATP1 only at the lowest dose (0.05 mM). FATP1 data are consistent with the observation that in FATP1-KO mice, intramuscular accumulation of TAGs and acyl-CoA is severely impaired in animals fed fat diets, while no differences are observed in animals fed a standard chow diet (15). There are no other studies of FATP1 overexpression in muscle cells, but in growing epithelial HEK-293 cells it has been shown to also enhance oleic acid uptake and channel its incorporation into TAGs (11). The effects of FAT on the channeling of palmitate and oleate to TAGs and phospholipids synthesis were similar to those of FATP1. In transgenic mice (13), which show FAT overexpression in muscle, palmitate incorporation into TAGs was not enhanced, suggesting that this pathway is saturated in vivo. Nevertheless, in FAT/CD36-KO mice, incorporation of a fatty acid analog to muscle TAG is restricted (6), which indicates the involvement of FAT.
The effects of FATP1 and FAT on fatty acid oxidation in skeletal muscle cells were studied. In all cell types and at all concentrations of fatty acid tested, palmitate was more readily oxidized than oleate to CO2 and to acid-soluble metabolites. ASI accounted for a higher proportion of the oxidation products than CO2 (1, 28). FATP1 and FAT overexpression reduced the proportion of palmitate that was completely oxidized, while the production of intermediate metabolites was not markedly altered. In transgenic mice, FAT did not stimulate palmitate oxidation in resting muscle (13), although after contraction, a marked enhancement was detected. CO2 production from oleate was also inhibited by FATP1 and FAT but only at concentrations <1 mM, while the levels of intermediate metabolites were unchanged as well. The finding that FATP1 and FAT reduce CO2 production without markedly changing acid-soluble metabolites suggests that -oxidation is not specifically inhibited, while disposal of acetyl-CoA to oxidation is affected. Further work is necessary to elucidate the mechanism of action of FATP1 and FAT.
Moreover, we have demonstrated that reducing the ratio of 0.5 mM palmitate or oleate to BSA did not modify the metabolic effects of FATP1 or FAT. Neither transport nor fatty acid oxidation or ASI or CO2 production was affected by the BSA ratio. However, palmitate and oleate depletion were markedly reduced, as was the incorporation of either fatty acid to TAGs, but not in phospholipids. These data suggest that whereas transport, incorporation into phospholipids, and oxidation are saturated by unbound fatty acid at 0.5 mM fatty acid-0.2 mM BSA, consumption and channeling toward TAG synthesis are not, as shown for other fatty acid actions (29).
The cellular localization of FATP1 and FAT was analyzed by means of fusion constructs with GFP. FATPGFP was dispersed mainly through the cytosol in a reticular pattern and concentrated in the perinuclear region and was partially colocalized with a specific marker of the Golgi system, GM-130. Although FATP1 was originally considered an integral plasma membrane protein (25), its single transmembrane domain and very short amino-terminal stretch exposed to the exterior of the cell provide an unusual structure for facilitating transport across the membrane (17). FATP1 has been localized on the plasma membrane of HEK-293 cells (11, 25), but immunofluorescence studies of endogenous FATP1 in 3T3-L1-adipocytes (25, 26) and transfected GFP fusion constructs in COS-7 cells (17) show a distribution consistent with an internal membrane-associated protein, with endoplasmic reticulum and reticular pattern, and with great overlap with the Golgi marker GM-130 (26), in striking similarity to the muscle cell. In separate experiments, we incubated cells with oleate to induce maximal TAG accumulation, and lipid droplets were revealed by staining with Nile Red. Lipid droplets were found throughout the cytoplasm in linear arrangements or concentrated in masses around the nuclei. FATP1 localization was not affected by oleate treatment, nor was the lipid droplet pattern modified by FATP1 overexpression. Indeed, distribution of lipid droplets did not overlap with that of FATP1. Overall, our data indicate that the translocation of FATP1 to the extracellular membrane is not required for the promotion of fatty acid uptake. Nevertheless, we cannot rule out the possibility that if FATP1 had been translocated in an endosome-like structure to the cell periphery as described in adipocytes (26), a greater effect might have been observed. We suggest that FATP1 may instead function within an intracellular protein complex that may include acyl-CoA synthase, which colocalizes with FATP1 in adipocytes (8) and other currently undefined proteins. FATP1 may enhance fatty acid import to this protein complex, trapping fatty acids and in that way facilitating their access to the specific acyl-CoA synthase or other enzymes in the synthetic pathway of TAG. It is also possible that FATP1 accounts for part of the synthesis of acyl-CoA by means of its reported acyl-CoA synthase activity in vitro (7, 8). The suggestion that FATP1 may not function as a fatty acid transporter has been made previously after expression of the functional FATP1-Myc/His fusion protein in COS1 cells (7).
FATGFP, independently of fatty acid availability, was predominantly localized in the extracellular membrane of muscle cells and intracellularly in the periphery of vesicles, which did not overlap with lipid droplets or the Golgi marker. Other studies, in which cell fractionation and subsequent immunodetection techniques were applied, reported a similar distribution profile in skeletal (4, 19) and cardiac (18) myotubes. FAT contains multiple lipid modification sites (16) and has been found in Chinese hamster ovary and C32 cells in association with lipid rafts and membrane domains rich in cholesterol and sphingolipids and absent from caveolae and clathrin-coated pits (30). Indeed, in myocytes, FAT, like other lipid raft-associated proteins, translocates to the plasma membrane upon stimulation by insulin (19) or contraction (18).
In summary, we have demonstrated that in cultured human myotubes FATP1 is found in the cytosol, while FAT is overtly present in the extracellular membrane. Nevertheless, both FATP1 and FAT enhance palmitate and oleate transport and consumption and direct these fatty acids toward TAG synthesis while inhibiting their aerobic oxidation. Our findings indicate that FATP1 may trap and draw fatty acid toward their accumulation rather than restrict their transport.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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