The development of insulin receptors and responses in the differentiating nonfusing muscle cell line BC3H-1.

We have studied the development of high affinity insulin receptors and insulin-stimulated responses in the differentiating nonfusing muscle cell line BC3H-1. In the logarithmic growth phase, these myoblasts exhibit very low levels of insulin binding and no detectable insulin-stimulated glucose or amino acid uptake. Following the cessation of cell division and subsequent spontaneous differentiation, the resulting myocytes develop a 5-fold increase in specific 125I-insulin binding and demonstrate physiologic insulin-stimulated glucose and amino acid uptake (100% increase above baseline) with half-maximum stimulation at 1-3 nM in agreement with the known in vivo and in vitro insulin sensitivity of muscle tissue. Insulin stimulation of 2-deoxyglucose uptake is detectable within 3 min, becomes maximal within 15 min, and is mediated by a rapid increase of plasma membrane transport units, as determined by D-glucose-inhibitable cytochalasin B binding, resulting in a 2-fold increase in the Vmax for 2-deoxyglucose transport with no change in Km. Myocyte insulin binding is specific, reversible, and saturable, yielding equilibrium within 18 h at 4 degrees C. Scatchard analysis identified the high affinity insulin receptor with a Kd of 0.5 nM at 4 degrees C. The myocytes also demonstrate sensitive down-regulation of cell surface insulin receptors, with a maximum decrease of 50% in cell surface insulin binding following exposure to 20 nM insulin for 18 h at 37 degrees C. Since the differentiation of this muscle cell line from myoblasts to nonfusing myocytes is accompanied by the development of high affinity insulin receptors and physiologic insulin-stimulated glucose and alpha-methylaminoisobutyric acid uptake, this continuously cultured system provides an excellent model for the study of differentiation and mechanism of insulin action in muscle, its quantitatively most significant target tissue.

Effects of 12-O-tetradecanoylphorbol 13-acetate on glycerolipid metabolism in cultured myoblasts.

We recently reported that treatment of differentiated chick embryo myoblasts in culture with the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a 2-fold increase in the level of 1,2-diacylglycerol in the plasma membrane fraction within 15-30 min (Grove, R.I. and Schimmel, S.D. (1981) Biochem. Biophys. Res. Commun. 102, 158-164). This system has been characterized further and the metabolic origin and fate of the stimulated diacylglycerol have been investigated. The stimulation of 1,2-diacylglycerol was insensitive to alterations of Ca2+ concentration in the medium and to the presence of inhibitors of Ca2+ flux, protein synthesis and prostaglandin synthesis. The fatty acid composition of the newly formed diacylglycerol was similar to that of phosphatidylcholine. In addition, the glycerol moiety of the diacylglycerol was shown to be derived from a lipid with metabolic turnover similar to that of phosphatidylcholine. The tumor promoter was also found to stimulate rapidly synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine. A possible model is proposed, therefore, in which the tumor promoter stimulates a membrane-associated phospholipase C which generates 1,2-diacylglycerol via the hydrolysis of phosphatidylcholine. The newly formed diacylglycerol is then metabolized back to phosphatidylcholine or to phosphatidic acid and phosphatidylinositol.

Effect of Ca++ on triphosphoinositide extraction in fusing myoblasts.

Ca++-dependent degradation of triphosphoinositide has been postulated to regulate levels of membrane-bound Ca++ and to generate a 1,2-diacylglycerol fusogen in cell fusion. Triphosphoinositide metabolism was therefore studied during Ca++-induced fusion of cultured chick embryo myoblasts. Using a frequently cited extraction procedure, it was found that apparent Ca++-dependent triphosphoinositide degradation was actually due to inhibition of extraction. A new procedure using the ion-pairing reagent tetrabutylammonium sulfate was developed which was unaffected by Ca++ and gave 2- to 20-fold greater extraction of triphosphoinositide than existing procedures. With this procedure, no changes in triphosphoinositide metabolism were found during myoblast fusion.

Surface labeling studies of normal and dystrophic myogenic cells in culture.

Surface protein topographies of cultured normal and genetically dystrophic embryonic chick breast muscle cells and fibroblasts were compared by external labeling and size distribution analysis by sodium dodecyl sulfate-polyacrylamide gradient slab gel electrophoresis. Four labeling techniques were evaluated and all were found to be specific for the cell surface. Using radioiodination of tyrosine residues and periodate-barotritide labeling of sialic acid residues, no reproducible abnormalities could be detected in any of the dystrophic cell types--fibroblasts, fusion-inhibited differentiated myoblasts, and multinucleated differentiated myotubes. However, comparison of 3-day-old cultures of normal fusion-inhibited differentiated myoblasts with fused myotubes revealed that increased iodination of protein classes with nominal molecular weights of 230,000 and 88,000 was evident in the myoblasts. In addition, large structural differences were detected between differentiated myoblasts and myogenic cells grown in the presence of phorbol 12-myristate 13-acetate, a potent tumor promoter and reversible inhibitor of myogenic differentiation.

Binding of phorbol-12-myristate-13-acetate to cultured myoblasts.

The tumor promoter phorbol 12-myristate 13-acetate (PMA) binds in a rapid (maximal at 30 min), reversible, and non-saturable manner to cultured embryonic chick myoblasts. Serum was not required for binding although it promoted the release of PMA from the cells. This appears to be due to PMA binding to serum proteins. At any concentration tested, approx. 10% of the total PMA was bound at 5 min and 30% was bound at 30 min. It is estimated that a PMA concentration in the cell membrane of only 1 molecule of PMA/5000 molecules of membrane lipid elicits maximal biological responses in these cells.

Plasma membranes from cultured muscle cells: isolation procedure and separation of putative plasma-membrane marker enzymes.

Partially purified plasma membranes were obtained from chick-embryo muscle cells grown in tissue culture. The purification procedure involved homogenization in buffered isotonic sucrose followed by differential and sucrose density gradient centrifugations. The activities of five plasma-membrane markers, as well as microsomal and mitochondrial markers, were followed throughout the purification. When cultures were labeled with [(125)I]alpha-bungarotoxin, which binds to the surface of cultured muscle cells, the distributions of bound alpha-bungarotoxin and Na(+),K(+)-ATPase (EC 3.6.1.3) activity were nearly identical. The activities of these two plasma-membrane markers were maximal in the upper two fractions of the sucrose density gradient and were purified 5- to 7-fold with respect to total particulate protein. These fractions contained 20-30% of the Na(+),K(+)-ATPase activity and bound alpha-bungarotoxin, 4% of the microsomal marker TPNH-dependent cytochrome c reductase, 0.2% of the mitochondrial marker succinate-dependent cytochrome c reductase, 2.7% of the cellular RNA, and 0.02% of the DNA. The activity of the commonly used plasma-membrane marker, 5'-nucleotidase (EC 3.1.3.5), was low in the upper two sucrose gradient fractions and was maximal in a more dense fraction. The distributions of the other two plasma-membrane markers, leucyl beta-naphthylamidase and phosphodiesterase I, were intermediate between Na(+),K(+)-ATPase and 5'-nucleotidase. The distributions of all markers were similar in preparations from cultures containing mononucleated myogenic cells, multinucleated myotubes, fibroblasts, or all three cell types. Modification of the procedure to include homogenization in the absence of sucrose resulted in a 3.4-fold purification of the membranes containing 5'-nucleotidase, which were shifted to a lower density.