Changes in Scatchard Plots for Insulin Binding to Mammary Epithelial Cells from Cycling, Pregnant, and Lactating Mice*

Abstract

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.

Differential transformation of mammary epithelial cells by Wnt genes.

“…2. -, nontransforming; +, low-level transformation potential; + +, high-level transformation potential. and potentiates the lactogenic effects of prolactin (2), and high-affinity insulin receptors are expressed at elevated levels in early-pregnancy mammary gland (16). Epidermal growth factor performs a dual function, stimulating epithelial cell proliferation while inhibiting functional differentiation (48, 49; reviewed in reference 3).…”

Endocrine control of metabolic adaptation during lactation

“…The mammary gland appears to be much more sensitive to insulin (Jones er Burnol et al 1987) than adipose tissue and muscle, hence it is able to maintain high rates of insulin-sensitive processes during lactation despite the prevailing hypoinsulinaemia. The mechanism responsible for this enhanced sensitivity to insulin is unknown, but in contrast to the other tissues described previously, the number of insulin receptors of mammary epithelial cells is increased during lactation (Flint, 1982;Inagaki & Kohmoto, 1982). As in adipocytes, liver and muscle, insulin also promotes the release of a putative second messenger from the plasma membrane of rat mammary cells (E. Kilgour and R. G. Vernon, unpublished results).…”

The Regulation of the Prolactin Receptor Gene Expression in the Mammary Gland of Early Pregnant Mouse.

“…Lee and Sakai [10] observed that the mammary glands in pregnant mice at 7 to 9 days showed neither an increase in PRL binding nor the onset of casein synthesis caused by ovariectomy. The rate of DNA synthesis peaks on day 3 of pregnancy and thereafter decreases in the mammary gland [26]. During early pregnancy, ductal growth occurs extensively, and the ducts undergo further branching [1].…”