DIFFERENTIATION-DEPENDENT EXPRESSION OF HEART TYPE FATTY-ACID-BINDINGPROTEIN IN C2C12 MUSCLE-CELLS

The aim of the present work was to establish a cell culture model for the investigation of the influence of heart type fatty acid-binding pr otein (H-FABP) on differentiation and lipid metabolism. Up to now no d ata have been reported on H-FABP in cell lines of skeletal muscle, one of the major sources of this protein in vivo. For this purpose mouse C2C12 cells were chosen, because these cells can be stimulated to diff erentiate in vitro from myoblasts to spontaneously contracting, multip ly nucleated myotubes expressing muscle-specific proteins Like creatin e kinase. Analysis of the cellular proteins by two-dimensional gel ele ctrophoresis and ELISA demonstrated that the expression of H-FABP is d ifferentiation dependent as well in these cells. Furthermore, immunofl uorescent labeling with H-FABP-specific antibodies revealed that induc tion of this protein occurred mainly in myotubes. Myoblasts contained only 7.1 +/- 3.1 ng H-FABP/mg soluble protein, however, upon different iation, this value increased about 60-fold to 420 +/- 90 ng/mg (n=4) i n a mixture of myoblasts and myotubes. H-FABP from C2C12 cells was sub sequently cloned and shown to be identical to the known mouse H-FABP. The induction of H-FABP during differentiation was also detected at mR NA level by probing with H-FABP-cDNA. Insulin, a known stimulator of i n vitro muscle cell differentiation, led to an increased differentiati on as referenced by creatine kinase activity, which is paralleled by a n increased H-FABP expression. The enhancement of H-FABP expression by insulin was found to be time- and dose-dependent. The increasing H-FA BP content may relate to an increasing fatty acid oxidation that has b een reported for differentiated L6 cells, a related muscle cell Line f rom rat. Such a correlation would favor a role of H-FABP in lipid meta bolism.