Essentially no specific binding sites for insulin are detected in small lymphocytes freshly isolated from human blood. Insulin-binding sites appear on the lymphocyte surface during transformation in vitro with concanavalin A, and the number of these receptors increases sharply to reach a maximum between 24 and 46 hr after exposure to the mitogen. The The binding of insulin to its receptor sites on fat cells and on liver and fat-cell membranes has been studied extensively (1-8). Insulin-receptor sites on cells other than the typical target cells for insulin, namely human circulating cells and cultured fibroblasts, have recently been described (9). The present studies demonstrate that while no significant specific binding of insulin is detectable on normal peripheral blood lymphocytes of humans, a dramatic appearance of insulin receptors occurs during lymphocyte transformation induced by the plant mitogen concanavalin A. The short-term lymphocyte culture provides a useful model for investigation of changes in the number and properties of surface receptor sites of cells as they undergo functional changes such as metabolic activation, differentiation, and cell division. The present studies are also of interest in view of the profound insulin-like biological properties of concanavalin A (10), the inhibition by insulin (11, *) and by concanavalin A (10, *) of * Tell, G. & Cuatrecases, P., submitted to J. Biol. Chem. 2604 adenylate cyclase activity in isolated membrane preparations, the growth-promoting properties of insulin in various mammalian cells in tissue culture (12,15), and the important relationships that apparently exist between cyclic AMP and cell growth (15-18).
METHODSLymphocytes were separated from other leukocytes by passage of freshly drawn, heparinized blood (200-500 ml) from a healthy donor through a sterile, disposable nylon-fiber column (Fenwal Laboratories) that was immediately prewashed (at 370) with 250 ml of sterile normal saline. The erythrocytes (in 10-ml aliquots) were allowed to settle by gravity at 370 and the lymphocyte-rich plasma was collected before the appearance of a visible buffy coat. For removal of excess platelets, the plasma was centrifuged at 150 X g for 15 min, and the pellet was washed 2-3 times with Medium 199 containing 5% fetal-bovine serum. The lymphocytes, recovered in 60% yield, constituted 95-98% of the cells; 1-5% were granulocytes and less than 1% were monocytes. Platelets did not exceed 50, and erythrocytes did not exceed 200 per 100 leukocytes. Leukocyte counts (modified Neubauer hemocytometer) were based on a count of at least 700 cells. For differential counts (500 cells, three slides), samples on pulled coverslips were fixed in 99% methanol and stained with Giemsa stain. Increased cell size, characteristic nuclear staining, multiple nucleoli, and cytoplasmic basophilia were used to judge blastic transformation.For culture the cells were suspended in Medium 199 supplemented with 8% fresh homologous serum containing 100 units of penicillin and 50 jig of s...