Thyroid Thermogenesis in Adult Rat Hepatocytes in Primary Monolayer Culture Direct Action of Thyroid Hormone In Vitro

We have studied the effect of 3,5,3 't r i iodothyronine (Ts) on the respiration of adul t rat hepatocytes in pr imary monolayer culture p repared f rom hypothyroid rat liver. After addit ion 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 g.1/h, mg protein). The thyroid-responsive enzymes, Na + + K+-activated adenosine t r iphosphatase (NaK-ATPase) and ot-glycerophosphate dehydrogenase (GPD), each exhibited increased activity in response to Ta, in parallel with the change in oxygen consumption, whereas the activity of Mg-dependent ATPase was unaffected. These responses to T3 were dose dependen t over similar concentration ranges, the half-maximal response for each occurr ing at ca 8 x 10 -l~ M. In thyroidt reated cells, the observed increase in respirat ion was almost completely (90%) inhibited after addi t ion o f ouabain (10 -3 M) to the culture medium. It was found also that a 4-h exposure o f the cul tured hepatocytes to Tn was sufficient to elicit a significant thermogenic response, measured at a t ime (48 h later) when T3 was no longer present in the medium. The response to T3 occurred in fully def ined culture medium and was independen t of the presence or absence o f hypothyroid rat serum, corticosterone, or insulin, and cellular ATP was unaffected by Ts in concentrations up to 2 x 10 -~ M. The findings document that adul t rat hepatocytes in pr imary monolayer cul ture respond directly to thyroid hormone; the increases in respirat ion and NaK-ATPase activity elicited by T3 were cotemporal and apparent ly coordinate. I N T R O D U C T I O N A d m i n i s t r a t i o n o f t h y r o i d h o r m o n e in vivo elici ts a t h e r m o g e n i c r e s p o n s e ( m e a s u r e d as i n c r e a s e d o x y g e n c o n s u m p t i o n ) wh ich can be a s c r i b e d to its a c t i on o n spec i f ic t a r g e t t i ssues ( B a r k e r , 1951). A m o n g these t issues is the l iver , wh ich has b e e n u t i l i zed e x t e n s i v e l y in s tud ies o f t he b i o c h e m i c a l basis o f t h y r o i d J. GEN. PHYSIOL. ~) The Rockefeller University Press 9 0022-1295/79/03/0369/15 $1.00 369 Volume 73 March 1979 369-383 on July 4, 2017 jgp.rress.org D ow nladed fom 370 THE JOURNAL OF GENERAL PHYSIOLOGY' VOLUME 7 3 ' 1979 thermogenesis. It was reported previously that stimulation of oxygen consumption (Qo2) by 3,5,3'-triiodothyronine (T3) was closely associated with increased activity of Na + + K+-activated adenosine triphosphatase (NaK-ATPase), suggesting that the thyroid-related change in energy expenditure is attributable to increased transmembrane active sodium transport (Ismail-Beigi and Edelman, 1970, 1971, 1974; Israel et al., 1973; Edelman and Ismail-Beigi, 1974; Asano et al., 1976; Rahimifar and Ismail-Beigi, 1977). Consistent with this hypothesis was the finding that the increases in Qo2 (elicited by injection of T3 in vivo and assayed in vitro, in hepatic slices and diaphragm segments) were inhibited 5090% by addition of ouabain to the media. The relevance of these findings to thyroid thermogenesis in vivo has been questioned recently (Folke and Sestoft, 1977). Some of these issues will be discussed below. Regardless of the merits of the arguments, however, ouabain-sensitive respiration provides an additional criterion of whether isolated cells challenged with T3 exhibit the same characteristics as freshly prepared liver slices from rats given T3 in vivo (Ismail-Beigi and Edelman, 1971, 1974; Asano et al., 1976). The existence of high affinity nuclear binding sites for T3 in responsive cells implies direct effects of thyroid hormone on target tissues (Oppenheimer et al., 1972; Samuels and Tsai, 1973). Moreover, T3 induces the synthesis of specific proteins (e.g., growth hormone) in cultured pituitary tumor cells (Martial et al., 1977). A direct effect of thyroid hormone on Qoz or NaK-ATPase in target tissues, however, has not been established previously. Moreover, after injection of thyroid hormone in vivo, it was unclear as to which liver cell type (parenchymal or nonparenchymal) was responsible for the increased Qo2 of liver slices. A direct approach to these problems requires isolated cells. To date, however, neither liver-derived cells nor other cell types have demonstrated a thermogenic response to thyroid hormone added in vitro. Tsai and Chen (1976) recently reported that fetal rat cardiac cells in culture exhibited increased glucose consumption after addition of T3 to the culture medium. This response, however, need not signify an effect on cellular respiration, because a correlation between these two processes has not been demonstrated. With regard to thyroid thermogenesis in isolated hepatocytes, the study requires a cell system that is viable for relatively long periods of time. Responses to T3 in vivo appear 12-18 h after administration of the hormone and rise to a peak at 48-72 h (Tata and Widnell, 1966; Ismail-Beigi and Edelman, 1974). In previous attempts, liverderived tumor cells in permanent culture did not exhibit a thermogenic response to thyroid hormone,1 and the use of liver slices, isolated perfused liver, or hepatocytes in suspension is excluded by the relatively brief viability of these preparations. Recently, however, an isolated hepatocyte system has been described that combines the stability of cell culture with the differentiated features of the intact liver: adult rat hepatocytes in primary monolayer culture (Bissell et al., 1973). By this technique, hepatic parenchymal cel ls-separated cleanly from nonparenchymal e l ements -a re established in nonproliferating primary culture and maintained in a viable state for several days, as judged both by ultrastructural Ismail-Beigi , F., and I. S. Ede lman . Unpub l i shed observat ions. on July 4, 2017 jgp.rress.org D ow nladed fom ISMAIL-BEIGI ET AL. Thyroid Thermogenesis in Primary Hepatocyte Culture 371 m o r p h o l o g y ( C h a p m a n et al., 1973) and by the p rese rva t ion o f n u m e r o u s specific hepa toce l lu l a r func t ions (Bissell et al., 1973; B o n n e y , 1974). I n the p re sen t s tudy , we e x a m i n e the ef fec t o f T3, a d d e d to the incuba t ion m e d i u m , on total a n d ouaba ininh ib i tab le resp i ra t ion , on N a K A T P a s e and on m i t o c h o n drial a g l y c e r o p h o s p h a t e d e h y d r o g e n a s e (L-g lycero l -3-phosphate oxidase) (GPD). T h e f indings in this cu l tu re system establish that the ef fec t o f Tn on hepa t ic p a r e n c h y m a l cells is d i rec t a n d involves c o o r d i n a t e increases in these p a r a m e t e r s o f t hy ro id t h e r m o g e n e s i s .