ABSTRACT
From the soluble protein fraction of lactating rat mammary epithelial cells, fatty acid-binding protein (FABP) was isolated by immunoaffinity chromatography. After digestion with trypsin, peptides were characterized with time-of-flight mass spectrometry and revealed identity with corresponding peptides derived from the heart-type FABP isolated from rat heart. In addition, by electrospray mass spectrometry the molecular mass has been determined to 14683.9 +/- 3 Da, further corroborating the identity. The content of FABP in mammary glands from virgin, pregnant and lactating rats was evaluated using two-dimensional gel electrophoresis and a FABP-specific immunosorbent assay. In the two-dimensional gels FABP was the apparently most abundant cytosolic protein in mammary epithelial cells from rats in late pregnancy as well as from lactating rats. The content of FABP was 59 +/- 19 microgram/mg (n = 11) of soluble proteins from the fully differentiated lactating mammary gland as determined by ELISA. This value represented an 80-fold increase compared with the FABP content of mammary gland from virgin rats, and is comparable with the level found in rat heart. Upon stimulation with insulin a small fraction of FABP was phosphorylated in lactating mammary epithelial cells. In conclusion, these findings indicate that the FABPs from rat mammary gland and heart are identical and further suggest that in mammary gland this FABP may play a role in signal transduction downstream from the insulin receptor.
Subject(s)
Carrier Proteins/metabolism , Mammary Glands, Animal/chemistry , Neoplasm Proteins , Nerve Tissue Proteins , Amino Acid Sequence , Animals , Carrier Proteins/chemistry , Carrier Proteins/isolation & purification , Cell Differentiation , Chromatography, Affinity , Electrophoresis, Gel, Two-Dimensional , Epithelium/chemistry , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Female , Homeostasis , Insulin/pharmacology , Lactation , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Phosphorylation , Pregnancy , Rats , Rats, Wistar , Trypsin/metabolismABSTRACT
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 protein (H-FABP) on differentiation and lipid metabolism. Up to now no data 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 differentiate in vitro from myoblasts to spontaneously contracting, multiply nucleated myotubes expressing muscle-specific proteins like creatine kinase. Analysis of the cellular proteins by two-dimensional gel electrophoresis and ELISA demonstrated that the expression of H-FABP is differentiation dependent as well in these cells. Furthermore, immunofluorescent labeling with H-FABP-specific antibodies revealed that induction of this protein occurred mainly in myotubes. Myoblasts contained only 7.1 +/- 3.1 ng H-FABP/mg soluble protein, however, upon differentiation, this value increased about 60-fold to 420 +/- 90 ng/mg (n = 4) in a mixture of myoblasts and myotubes. H-FABP from C2C12 cells was subsequently cloned and shown to be identical to the known mouse H-FABP. The induction of H-FABP during differentiation was also detected at mRNA level by probing with H-FABP-cDNA. Insulin, a known stimulator of in vitro muscle cell differentiation, led to an increased differentiation as referenced by creatine kinase activity, which is paralleled by an increased H-FABP expression. The enhancement of H-FABP expression by insulin was found to be time- and dose-dependent. The increasing H-FABP content may relate to an increasing fatty acid oxidation that has been reported for differentiated L6 cells, a related muscle cell line from rat. Such a correlation would favor a role of H-FABP in lipid metabolism.