Changes in Scatchard plots for insulin binding to mammary epithelial cells from cycling, pregnant, and lactating mice.
Insulin binding to its receptors was studied in collagenase-dissociated mammary cells prepared from mice that were cycling, pregnant, or lactating. Mammary cells (3 X 10(6)) were incubated in medium 199 (0.4 ml) supplemented with iodinated insulin (0.8-1 ng/ml) for 45 min at room temperature. The dissociation of iodinated insulin from its receptors was accelerated by the presence of native insulin. The specific binding of insulin at a physiological concentration (1 ng/ml) increased on day 0 of pregnancy and on day 3 of lactation. The Scatchard plots showed predominantly high affinity binding during estrous cycles and in early pregnancy, and low affinity binding in late pregnancy and lactation. Scatchard plots were further analyzed for evidence of negative cooperativity and of two independent receptors. The negative cooperativity model gave well fitted curves for unconstrained parameters. However, dissociation constants for insulin binding to empty sites were too high during lactation. The independent two-receptor model gave also well fitted curves for unconstrained parameters at each stage. The dissociation constants for high affinity and low affinity receptors, which were optimal throughout the entire stage, were 1.0 and 20.0 nM, respectively. There was no significant difference between these dissociation constants and any of unconstrained constants at each stage. When dissociation constants were constrained throughout entire stages, the number of receptors for high affinity binding increased on day 0 of pregnancy and in early lactation, and the number for low affinity binding showed one peak on day 3 of lactation. Changes in these receptors were independent. From these results, we propose that at least two species of insulin receptors are present in mouse mammary epithelial cells; the high affinity receptor may be associated with the stimulation of DNA synthesis.
D000818:Animals; D004261:DNA Replication; D004848:Epithelium / Q000378:metabolism; D004971:Estrus; D005260:Female; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D011247:Pregnancy; D011270:Pregnancy, Animal; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; D011972:Receptor, Insulin