A major long-term objective of this proposal is to evaluate the regulation of the Type II iodothyronine 5'-deiodinase in brown adipose tissue, pituitary, and cerebral cortex. The enzymatic mechanism of T4 to T3 conversion will be examined by isolating the enzyme and the regulation of its activation elucidated by studying intracellular processes leading to alterations in enzyme activity. These may be due to changes in the rate of enzyme synthesis or to activation of existing enzyme or co-factors. The effects of catecholamines, insulin, glucagon, and thyroid hormone on 5'-deiodinase will be evaluated and the mechanisms elucidated in brown adipocytes from rat or hamster. In addition, we will examine the effects of hypo- and hyperthyroidism (T3 induced) and hypopituitarism on the response of their cells to those hormones. The chronology of the changes in enzyme responsiveness with these manipulations will be examined and correlated with changes in the intermediary metabolism of the brown fat cell. Immunization of rabbits and mice will be performed to produce polyclonal or monoclonal antibodies to be used as enzyme probes. Isolation of the enzyme for immunizations will be done by ion exchange and hydrophobic interaction chromatography. These products will also be used to screen expression libraries of cDNA prepared from the brown adipose tissue of rats which have been shown to have high rates of enzyme synthesis. The amino acid sequence of the enzyme will be determined from the cDNA sequence. In other studies, the role of thyroid hormone in regulation of TSH release from isolated pituitary cells will be investigated by techniques employing somatic cell fusion of thyrotroph enriched rat pituitaries with pituitary tumor cells. Permanent lines developed from such fusions have been found to replicate and continuously secrete TSH giving them a significant advantage over either primary cultures or the mouse thyrotroph tumors which do not replicate. The effects of TRH, thyroid hormone, and other secretagogues on TSH secretion from these cells will be evaluated to determine the mechanism for the thyroid hormone inhibition of TSH release from these cells. This research is relevant to the treatment of hypothyroidism (especially in newborns), the problem of obesity, survival during cold stress and the design and propoer interpretation of tests of thyroid function.
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