Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion

Abstract

We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette. Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs. The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells. In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity. Uptake rates of palmitate were equal for all clone types, in spite of different FABP INTRODUCTIONFatty acid-binding proteins (FABPs) belong to a conserved family of intracellular lipid-binding proteins with low molecular mass (14-15 kDa) [1][2][3]. Eight different FABP types have been described : heart, liver, adipose, myelin, intestinal, epidermal, brain and ileal. Many FABP cDNAs have been isolated, cloned and the proteins expressed in bacterial systems. In this way, large amounts of proteins have become available for three-dimensional analysis by crystallography and\or NMR (reviewed in [1,3]). All FABPs studied thus far contain a folding motif designated an up-and-down β-barrel [3].The main function of FABP is thought to be intracellular binding and targeting of fatty acids. FABPs may also modulate the effect of fatty acids on various metabolic enzymes and receptors and cellular processes such as signal transduction and gene expression [1,4]. The function significance of the different FABP types is not clear and may vary with the tissues and the physiological conditions. Adaptation may be related to metabolic requirements of the cell, ligand trafficking and\or modulatory functions.A relation has been observed between fatty acid-oxidation capacity and FABP content of various rat tissues and various human and rat muscles [5][6][7]. FABP content of muscle showed no change under various physiological conditions, except an increase in fast-twitch muscles on chronic electrostimulation and endurance training [7]. Heart FABP (H-FABP) content and fatty acid-oxidation capacity increase in skeletal and heart muscle during postnatal development [7,8]. Some indications of a role Abbreviations used : A-FABP, adipocyte fatty acid-binding protein ; CHO, Chinese hamster ovary ; CK, creatine kinase ; CS, citrate synthase ; D-PBS, Dulbecco's modified PBS ; H-FABP, heart fatty acid-binding protein ; I-FABP, intestinal fatty acid-binding protein ; L-FABP, liver fatty acid-binding protein ; PC, phosphatidylcholine ; PE, phosphatidylethanolamine ; RT, reverse transcriptase ; DMEM, Dulbecco's modified Eagle's medium ; SV40, simian virus 40 ; GAPDH, glyceraldehyde-3-phosphate dehydrogenase ; pac, puromycin acetyltransferase.1 To whom correspondence shou...

New insights into the structure and function of fatty acid-binding proteins

“…Interestingly, the expression of H-FABP in C 2 C 12 muscle cells appeared to be differentiation dependent [243]. Cultured human and rat muscle cells also showed an increase in H-FABP at differentiation [242,244]. The increasing H-FABP content upon differentiation may relate to increasing FA oxidation that has been reported for differentiated L6 cells [245].…”

“…Differentiation-promoting effects of MDGI/H-FABP were observed in pluripotent mouse embryonic stem cells [239], mammary epithelial cells [233] and cardiac myocytes [240]. Expression of bovine H-FABP in yeast caused inhibition of growth [241], but L6 muscle cells transfected with H-FABP cDNA did not show changes in growth or differentiation compared to mocktransfected cells [242]. Interestingly, the expression of H-FABP in C 2 C 12 muscle cells appeared to be differentiation dependent [243].…”

“…The increasing H-FABP content upon differentiation may relate to increasing FA oxidation that has been reported for differentiated L6 cells [245]. Transfection of L6 cells with A-FABP cDNA increased the proliferation rate and blocked the fusion of these cells, simultaneous with changes in their phospholipid composition [242]. Transfection of MDCK cells with either H-FABP, L-FABP or A-FABP cDNA did not influence proliferation [246].…”

The cytoplasmic fatty-acid-binding proteins: thirty years and counting

“…Data from such experiments have been summarized (table 2 in [7]). Whereas some experiments show no increase in LCFA uptake as in A-FABP C transfection L6 myoblasts [163], human breast cancer cells transfected with H-FABP C , increased palmitate uptake 67% with no changes in targeting of ligand to complex lipid synthesis [164]. Similar increases in palmitate uptake have been reported in rat hepatocytes [165], but overexpression of I-FABP C in Caco-2 cells inhibited LCFA absorption by 34%, again with no alteration in the triglyceride pool [166].…”

Inversely related expression of epidermal- and heart-type fatty acid binding proteins during myogenic differentiation of C2C12 myoblasts

“…We conclude that E-and H-FABP together ensure that oleic acid uptake in myoblasts and myotubes remains constant. In the light of this result one may take a new look at data reported for the differentiating rat muscle cell line L6, in which H-FABP content [53] and the fatty acid oxidation rate [54] increased, whilst the uptake of fatty acids remained unchanged [53]. A compensatory increase in E-FABP levels may account for this observation.…”