In order to define the mode of action of thyroid hormones with respect to their powerful influence on the short-term regulation of hepatic carbohydrate metabolism by catecholamines, glucagon, and insulin, we propose the following detailed investigations which focus upon three specific areas. Definition of the mechanism responsible for the 3-fold increase in the steady-state level of rat hepatocyte beta-adrenergic receptors observed in the hypothyroid state will be sought. Beta-adrenergic receptors will be purified in sufficient quantity to provide enough antigen to prepare anti-receptor antibodies for immunochemical studies. Utilizing a pulse-chase strategy (employing radio-labeled amino acids in an in vivo and in vitro design) and the immunochemical isolation of the beta-adrenergic receptors the rate of receptor turnover and the influence of altered thyroid states on this parameter will be determined. The irreversible, beta-adrenergic receptor antagonist,N-[2-hydroxy-3-(1-naphthoxy)propyl]-N'-bromoacetylethyl- enediamine, will be employed also to blockade hepatocyte beta-adrenergic receptors and subsequently allow determination of the rate of beta-receptor expression and synthesis in the intact hepatocytes isolated from hypothyroid and euthyroid rats. The modulation of hepatic phosphoprotein phosphatase activity by thyroid hormones will be characterized and the mechanism responsible for the increase in glycogen phosphorylase a dephosphorylation noted in hyperthyroid rats investigated. The influence of thyroid hormones on the activities of acetyl-CoA carboxylase, HMG-CoA reductase, and reductase kinase in the liver will be assessed to determine if changes in the phosphorylase phosphatase activity are reflected in these other putative substrates. The ability of insulin to regulate glycogen phosphorylase and synthase activities in hepatocytes from hyper-, hypo-, and euthyroid rats will be detailed. Thyroid hormone induced changes in the ability of insulin to regulate hepatic carbohydrate metabolism will be investigated at the level of the insulin receptor (binding and structure), intracellular enzymes, and perhaps the insulin effector system (probed using broken-cell insulin-sensitive assays recently developed by others), if indicated. These investigations will contribute to our knowledge of the actions of thyroid hormones in the liver and their influence on insulin action.
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