The proposed research involves defining the detailed mechanisms by which glucocorticoids and other hormones interact with thyroid hormone to control biological processes in mammalian cells. The cell culture systems to be investigated are GH1 cells, two clonal cells of rat pituitary cell lines which synthesize growth hormone. With these cells physiological concentrations of thyroid hormones increase growth hormone synthesis and the effect of glucocorticoids on regulating growth hormone synthesis is highly dependent on the action of thyroid hormone. These cells contain nuclear receptors for thyroid hormone and also contain glucocorticoid receptors and the regulation of the synthesis of growth hormone by thyroid hormone and glucocorticoid appears to be modulated by their respective regular receptors. In addition, GH1 cells and GC cells contain insulin receptors with an estimated Kd of 2-5 x 10 to the minus 10th power M with approximately 8,000 insulin binding sites per cell. In GC cells thyroid hormone and glucocorticoid induce increases in insulin receptor levels and the influence of glucocorticoid is additive but not synergistic with that of thyroid hormone differing from the regulation of the growth hormone response. Furthermore, thyroid hormone and glucocorticoid independently inhibit insulin degradation by these cells by a process which appears to involve a decrease in cell uptake of insulin and therefore the inhibition of insulin degradation appears to be modulated by the influence of thyroid hormone and or glucocorticoid on inhibiting insulin internalization. The mechanism of inhibition of insulin metabolism in GH1 cells will be investigated by assessing the kinetics of internalization of insulin and the kinetics of internalization of surface membrane insulin receptors. This will be determined from a biochemical point of view as well as by direct visualization using fluorescent insulin derivitives by Video Intensification Microscopy to monitor the kinetics of internalization. The influence of insuin action and internalization of these cells will be assessed in terms of the influence of insulin on controlling the growth hormone response in concert with thyroid and glucocorticoid hormones.
