This renewal evaluates the insulin receptor on immunocompetent cells with two specific aims flowing from the studies of the last grant cycle: 1) characterization of the fine regulation of the insulin receptor on immunocompetent cells; and 2) the immunoregulatory role of insulin and its receptor on these cells. A model is envisioned of three distinct mechanisms by which the lymphocyte insulin receptor is regulated. Mechanism 1: antigen driven on-off signals for the de novo synthesis of the insulin receptor, three pathways are studied by protocols in this application: 1) cryptic-receptors in the unactivated lymphocyte are sought by relating the appearance of the receptor to mRNA synthesis timed by cytofluorgraphic analysis of acridine orange staining as affected by the RNA polymerase II inhibitor Alpha amanitin; 2) the role of glycosylation for the insertion of the receptor is discerned using the inhibitor tunicamycin; and 3) the structure of the synthesized receptor is determined by ligand binding to nitrocellulose transblots of solubilized receptor. Mechanism 2: classical down regulation by ligand binding and internalization extant only for activated, receptor (+) lymphocytes is characterized by photoaffinity labeling using B29-lysine N-(2-nitro-4-azidophenyl)glycyl insulin. Mechanism 3: the modulation of activated lymphocyte insulin receptor synthesis by ambient insulin by a mechanism which does not involve insulin binding. Four hour euglycemic clamps in man are proposed to test the hypothesis that the receptor (+) monocyte passes to the receptor (-) T-cell signals concerning insulin concentrations for T-cell insulin receptor regulation. The lymphocyte insulin receptor model helped create the new field of hormonal immunoregulation to which several protocols are directed. The biologic importance of ligand directed receptor regulation is sought by measuring insulin directed glucose transport and cytotoxic T-cell function on lymphocytes removed from glucose clamped subjects. To test the hypothesis that insulin through its receptor maintains the active state of the lymphocyte, a submaximal alloantigen activation model is created and used. To test a new model of lymphocyte activation, acquisition of activation receptors including that for insulin is timed using monoclonal antibodies and flow cytometric analysis of the lymphocyte cell cycle. With an understanding of hormone receptors on immune cells one can begin to understand immune dysfunction in patients with disorders of carbohydrate metabolism.
