Role of insulin during cell cycle in a myeloid cell line

The characteristics of insulin receptor binding and structure and its proliferative and metabolic action in a human leukemic cell line was investigated during the cell cycle. Exponentially growing cells were separated by counterflow centrifugation which fractionates cells primarily on the basis of size into subpopulations representing G0/G1, S, and G2 + M cells. This method avoids disturbance of the cellular metabolism. After separation the cells showed a viability of at least 92%, underwent further proliferation, and remained morphologically unchanged, which was shown by electron microscopy. The cells could be enriched to 70-90% purity for G0/G1 phase and 50-60% purity for S and G2 + M phase, respectively, which was shown by DNA flow-cytometry. Specific binding of insulin could be demonstrated in G0/G1, S, and G2 + M enriched cells. Insulin binding sites decreased from 20-25,000 per cell in G0/G1 to 1-2,000 in S and increased to 30-50,000 in G2 + M. The affinity of insulin binding remained nearly constant during the cell cycle. The specificity of the insulin receptor could also be demonstrated by covalent crosslinking of the receptor to radiolabeled ligand in all enriched cell fractions. Glucose transport was stimulated by insulin independently of cell cycle. An increase to 140% of control was observed at an insulin concentration of 10 ng/ml. In contrast, glycogen synthesis could only be stimulated by insulin in the G0/G1 phase. An increase to 140% of control was already reached at 0.25 ng/ml insulin. Insulin in concentrations of 1 and 10 ng/ml stimulated the transit to S-phase in cycling, but not in resting, cells. The growth promoting action of insulin could be investigated by consecutive DNA analysis of the separated cells which had been stimulated by insulin.