We have studied the effect of 3,5,3'-triiodothyronine (T3) on the respiration of adult rat hepatocytes in primary monolayer culture prepared from hypothyroid rat liver. After addition of T3 to the culture medium at a concentration of 2 x 10(-7) M, oxygen consumption of the cultured cells increased detectably at 24 h and was maximal at 72--96 h, relative to control cultures (38.0 +/- 1.8 vs. 25.0 +/- 1.5 microliter/h.mg protein). The thyroid-responsive enzymes, Na+ + K+-activated adenosine triphosphatase (NaK-ATPase) and alpha-glycerophosphate dehydrogenase (GPD), each exhibited increased activity in response to T3, in parallel with the change in oxygen consumption, whereas the activity of Mg-dependent ATPase was unaffected. These responses to T3 were dose dependent over similar concentration ranges, the half-maximal response for each occurring at ca 8 x 10(-10) M. In thyroid-treated cells, the observed increase in respiration was almost completely (90%) inhibited after addition of ouabain (10(-3) M) to the culture medium. It was found also that a 4-h exposure of the cultured hepatocytes to T3 was sufficient to elicit a significant thermogenic response, measured at a time (48 h later) when T3 was no longer present in the medium. The response to T3 occurred in fully defined culture medium and was independent of the presence or absence of hypothyroid rat serum, corticosterone, or insulin, and cellular ATP was unaffected by T3 in concentrations up to 2 x 10(-7) M. The findings document that adult rat hepatocytes in primary monolayer culture respond directly to thyroid hormone; the increases in respiration and NaK-ATPase activity elicited by T3 were cotemporal and apparently coordinate.

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