Thyroid Thermogenesis in Adult Rat Hepatocytes in Primary Monolayer Culture Direct Action of Thyroid Hormone In Vitro FARAMARZ ISMAIL-BEIGI, I S I D O R E S. E D E L M A N
D. M O N T G O M E R Y
BISSELL,
and
From the Cardiovascular Research Institute, the Liver Center of the Department of Medicine, and the Department of Biochemistry and Biophysics of the University of California, San Francisco, California 94143. Dr. Ismail-Beigi'spresent address is the Department of Medicine, Pahlavi University School of Medicine, Shiraz, Iran; he worked on the present material during sabbatical leave granted by Pahlavi University, spent at the University of California, San Francisco.
ABSTRACT We have studied the effect o f 3,5,3'-triiodothyronine (Ts) on the respiration of adult rat hepatocytes in primary monolayer culture p r e p a r e d from hypothyroid rat liver. After addition o f T3 to the culture m e d i u m at a concentration o f 2 x 10-7 M, oxygen consumption o f 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). T h e thyroid-responsive enzymes, Na + + K+-activated adenosine triphosphatase (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 o f M g - d e p e n d e n t ATPase was unaffected. These responses to T3 were dose d e p e n d e n t over similar concentration ranges, the half-maximal response for each occurring at ca 8 x 10-l~ M. In thyroidtreated cells, the observed increase in respiration was almost completely (90%) inhibited after addition o f ouabain (10 -3 M) to the culture medium. It was f o u n d also that a 4-h exposure o f the cultured hepatocytes to Tn was sufficient to elicit a significant thermogenic response, measured at a time (48 h later) when T3 was no longer present in the m e d i u m . T h e response to T3 occurred in fully defined culture m e d i u m and was i n d e p e n d e n t o f the presence or absence o f h y p o t h y r o i d rat serum, corticosterone, or insulin, and cellular A T P was unaffected by Ts in concentrations up to 2 x 10-~ M. T h e findings d o c u m e n t that adult rat hepatocytes in p r i m a r y monolayer culture r e s p o n d directly to thyroid hormone; the increases in respiration and NaK-ATPase activity elicited by T3 were cotemporal and a p p a r e n t l y coordinate. INTRODUCTION 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 elicits 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 ) w h i c h c a n b e a s c r i b e d to its a c t i o n o n s p e c i f i c t a r g e t tissues ( B a r k e r , 1951). A m o n g t h e s e tissues is t h e l i v e r , w h i c h h a s b e e n u t i l i z e d e x t e n s i v e l y in s t u d i e s o f t h e 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 Volume 73 March 1979 369-383
369
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 cells-separated cleanly from nonparenchymal e l e m e n t s - a r e established in nonproliferating primary culture and maintained in a viable state for several days, as judged both by ultrastructural Ismail-Beigi, F., a n d I. S. E d e l m a n . U n p u b l i s h e d observations.
ISMAIL-BEIGIET AL. ThyroidThermogenesisin PrimaryHepatocyteCulture
371
m o r p h o l o g y ( C h a p m a n et al., 1973) a n d by the p r e s e r v a t i o n o f n u m e r o u s specific h e p a t o c e l l u l a r f u n c t i o n s (Bissell et al., 1973; B o n n e y , 1974). I n the p r e s e n t s t u d y , we e x a m i n e the effect o f T3, a d d e d to the i n c u b a t i o n m e d i u m , o n total a n d o u a b a i n - i n h i b i t a b l e r e s p i r a t i o n , o n N a K - A T P a s e a n d o n 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 l y c e r o l - 3 - p h o s p h a t e oxidase) (GPD). T h e f i n d i n g s in this c u l t u r e system establish that the effect o f Tn o n h e p a t i c p a r e n c h y m a l cells is d i r e c t a n d involves c o o r d i n a t e increases in these parameters of thyroid thermogenesis. METHODS
Preparation and Maintenance of Hepatocyte Cultures Male Sprague-Dawley rats (150-180 g) were placed on iodide-deficient diet (Remington, iodide content
D. M O N T G O M E R Y
BISSELL,
and
From the Cardiovascular Research Institute, the Liver Center of the Department of Medicine, and the Department of Biochemistry and Biophysics of the University of California, San Francisco, California 94143. Dr. Ismail-Beigi'spresent address is the Department of Medicine, Pahlavi University School of Medicine, Shiraz, Iran; he worked on the present material during sabbatical leave granted by Pahlavi University, spent at the University of California, San Francisco.
ABSTRACT We have studied the effect o f 3,5,3'-triiodothyronine (Ts) on the respiration of adult rat hepatocytes in primary monolayer culture p r e p a r e d from hypothyroid rat liver. After addition o f T3 to the culture m e d i u m at a concentration o f 2 x 10-7 M, oxygen consumption o f 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). T h e thyroid-responsive enzymes, Na + + K+-activated adenosine triphosphatase (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 o f M g - d e p e n d e n t ATPase was unaffected. These responses to T3 were dose d e p e n d e n t over similar concentration ranges, the half-maximal response for each occurring at ca 8 x 10-l~ M. In thyroidtreated cells, the observed increase in respiration was almost completely (90%) inhibited after addition o f ouabain (10 -3 M) to the culture medium. It was f o u n d also that a 4-h exposure o f the cultured hepatocytes to Tn was sufficient to elicit a significant thermogenic response, measured at a time (48 h later) when T3 was no longer present in the m e d i u m . T h e response to T3 occurred in fully defined culture m e d i u m and was i n d e p e n d e n t o f the presence or absence o f h y p o t h y r o i d rat serum, corticosterone, or insulin, and cellular A T P was unaffected by Ts in concentrations up to 2 x 10-~ M. T h e findings d o c u m e n t that adult rat hepatocytes in p r i m a r y monolayer culture r e s p o n d directly to thyroid hormone; the increases in respiration and NaK-ATPase activity elicited by T3 were cotemporal and a p p a r e n t l y coordinate. INTRODUCTION 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 elicits 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 ) w h i c h c a n b e a s c r i b e d to its a c t i o n o n s p e c i f i c t a r g e t tissues ( B a r k e r , 1951). A m o n g t h e s e tissues is t h e l i v e r , w h i c h h a s b e e n u t i l i z e d e x t e n s i v e l y in s t u d i e s o f t h e 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 Volume 73 March 1979 369-383
369
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 cells-separated cleanly from nonparenchymal e l e m e n t s - a r e established in nonproliferating primary culture and maintained in a viable state for several days, as judged both by ultrastructural Ismail-Beigi, F., a n d I. S. E d e l m a n . U n p u b l i s h e d observations.
ISMAIL-BEIGIET AL. ThyroidThermogenesisin PrimaryHepatocyteCulture
371
m o r p h o l o g y ( C h a p m a n et al., 1973) a n d by the p r e s e r v a t i o n o f n u m e r o u s specific h e p a t o c e l l u l a r f u n c t i o n s (Bissell et al., 1973; B o n n e y , 1974). I n the p r e s e n t s t u d y , we e x a m i n e the effect o f T3, a d d e d to the i n c u b a t i o n m e d i u m , o n total a n d o u a b a i n - i n h i b i t a b l e r e s p i r a t i o n , o n N a K - A T P a s e a n d o n 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 l y c e r o l - 3 - p h o s p h a t e oxidase) (GPD). T h e f i n d i n g s in this c u l t u r e system establish that the effect o f Tn o n h e p a t i c p a r e n c h y m a l cells is d i r e c t a n d involves c o o r d i n a t e increases in these parameters of thyroid thermogenesis. METHODS
Preparation and Maintenance of Hepatocyte Cultures Male Sprague-Dawley rats (150-180 g) were placed on iodide-deficient diet (Remington, iodide content
