Abstract
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.
Highlights
We have studied the development of high affinity development of insulinreceptorsandinsulin-mediatedreinsulin receptors and insulin-stimulated responses in sponses duringpharmacologically induced celluladrifferentiathe differentiating nonfusingmuscle cell line BC3H-1. tion in the 3T3-Ll fibroblast/adipocyte (17-19)
Insulin stimulation of 2deoxyglucose uptake is detectable within 3 min, betion of insulin-receptor interactions, receptor development, regulation, intracellular processing, and insulin-induced biochemical effects has not been available, we report here the initial characterization of the continuously cultured murine muscle cell line BC3H-1 as sucha system
Development of Insulin Receptors during Myocyte Differentiation-We have studied the development of insulin-binding activity as a function of growth and differentiation of BCSH1cells
Summary
Was obtained from ICN Pharmaceuticals Inc. (Plainview, NY) and cytochalasinB was obtained from Aldrich. For insitu binding cells were pCi of 2-deoxy-~-[~H]glucowseas added/ml and the uptake termicultured for the indicated times on 35-mm plates, the culture media was removed and the platews ere rinsed with 4ml of RRA buffer (100 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid, 120 mM NaCl, 1.2 mM MgS04,2.5 mM KC1,15 mM Na acetate, mM glucose, 1 mM EDTA, and 1% bovine serum albumin adjusted to pH 7.6 at 4 "C).Binding was initiated by the addition of 0.7-1 ml of RRA buffer at 4 "C containing10"" M '251-insulinto theplates, and thecells were nated by the addition of 0.5 ml of 3 mM phloretin in PBS, aspiration of the uptake mixture, and 3-4 rapid washes with 0.1 mM phloretin in cold PBS. The counting efficiency of the "C-labeled compounds was approximately 90% and thatof the tritiated compounds approximately 40%
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