Triiodothyronine Causes Rapid Reversal of q/Cyclic Adenosine Monophosphate Synergism on Brown Adipocyte Respiration and Type II Deiodinase Activity zyxwvutsrqponml Michael Noronha, Atso Raasmaja, Norma Moolten, and P. Reed Larsen Previous studies have shown that thyroid status affects the response of brown adipose tissue (BAT) to the sympathetic nervous system. For example, hypothyroidism is associated with the development of a marked synergism between LX,- and p-adrenergic pathways to stimulate type II iodothyronine 5’-deiodinase activity. Hypothyroidism also attenuates the respiratory response (thermogenesis) of isolated brown adipocytes to norepinephrine. To explore the interactions of the sympathetic nervous system and thyroid status in these cells, we compared the thermogenic and 5’-deiodinase responses to adrenergic agonists in isolated brown adipocytes from hypothyroid rats during treatment with 3,5,3’-triiodothyronine (T,). The fivefold synergism of q- and 5-adrenergic catecholamines to increase the deiodinase activity was progressively reduced, reaching a control euthyroid value of unity after 5 days of T, treatment. Hypothyroidism reduced both the 0,max (twofold to threefold) and increased the concentration of agonist required for 50% stimulation (lo-fold) for both norepinephrine and forskolin. In hypothyroid cells, there was a twofold synergism between the o,-agonist cirazoline and forskolin to increase respiration, which was blocked by prazosin and reproduced by the calcium ionophore, A23187. This synergistic effect of the q-agonist was lost within 2 days of T, administration. These studies identify a second Ca’+-dependent intraadrenergic synergism, which functions to ameliorate the reduced cyclic adenosine monophosphate (CAMP) responsiveness of the hypothyroid brown adipocyte. zyxwvutsrqponmlk Copyright o 199 1 by zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA W. B. Saunders Company zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA B ROWN adipose tissue (BAT) is a site of facultative thermogenesis. It is stimulated by the sympathetic nervous system under conditions such as arousal from hibernation, the newborn, acclimation to cold, and adap- tion to different diets.‘-4 This results in marked increases in oxygen consumption, which is coupled to thermogenesis in thus tissue.s~7 Thyroid status influences the regulation of BAT thermogenesis by catecholamines.‘~” A key element in BAT thermogenesis is a unique protein, uncoupling protein (ITCP) or thermogenin, present in the inner membrane of BAT mitochondria. This protein dissipates the proton gradient created by the respiratory chain, thereby uncou- pling oxidation from phosphorylation6 Both CX~- and B-ad- renergic stimulation have an additive stimulatoty effect on thermogenesis in hamster brown adipocytes.“,‘3 Both adren- ergic agonists also enhance UCP mRNA transcription in mouse brown adipocytes in culture and optimal UCP synthesis in vivo requires thyroid hormone.i4.” In addition, long-chain fatty acids released as a consequence of cyclic adenosine monophosphate (CAMP)-mediated lipolysis in- teract with mitochondrial UCP to promote uncoupling.‘~’ In the hypothyroid rat, there is a marked reduction in B-adrenergic stimulation of brown adipocyte respiration.‘.” Several steps in the pathway of adrenergic stimulation of the synthesis and/or activation of UCP may contribute to this. Earlier studies have shown that the acute increase of intracellular CAMP in response to forskolin is not altered by hypothyroidism, suggesting that the impairment of the respiratory response occurs at a later step in this re- sponse.Y.‘hWhile Woodward and Saggerson confirmed the reduced respiratory response, they observed a greater suppressive effect of hypothyroidism on the lipolytic than on the respiratory response of rat brown adipocytes to norepinephrine.‘” This suggested that ol,-adrenergic path- ways might temper the reduction of the thermogenic response to B-adrenergic stimuli, which occurs in the hypothyroid state. We have previously noted the appearance of a threefold to fourfold synergism between (Ye- and B-adrenergic induc- tion of type II thyroxine 5’-deiodinase (S’D-II) as rats zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Met aboik m, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Vol40, No 12 (December), 1991: pp 1327-1332 become hypothyroid.“,” The appearance of maximal syner- gism requires 2 or more weeks of methimazole treatment and requires mRNA synthesis in vitro.‘-‘ In the present investigation, we compared the response of both S’D-II activity and respiration in brown adipocytes to determine whether there are similar effects in both systems in hypothy- roid rats before and during thyroid hormone replacement. MATERIALS AND METHODS M aterials All chemicals were obtained from Sigma (St Louis, MO) unless othenvise indicated. Bovine serum albumin (BSA), dithiothreitol (DTT), and A23187 were purchased from Calbiochem (La Jolla, CA), Minimum Essential Medium (MEM) from Flow Laboratories (Rockville, MD), 1-methyl-2-mercaptoimidazole from Fluka (Ronkonkona, NY), and collagenase (type I) from Cooper Diagnos- tics (Malvern, PA). Prazosin (an qantagonist) and cirazoline (a pure qadrenergic agonist”~“.lH)were generous gifts from Pfizer and LERS (Paris, France). respectively. Animals Male Sprague-Dawley rats (100 to 125 g) were obtained from Zivic-Miller Laboratories (Allison Park, PA). Animals were kept at 23°C in a 12-hour dark cycle, with free access to food (Purina rat chow, Ralston-Purina, St Louis, MO) and tap water. Hypothyroid- ism was induced by an addition of 0.02% 1-methyl-2-mercaptoimi- dazole in the drinking water for 2.5 to 3.5 weeks.‘” Isolation of Brown Adipocytes Brown adipocytes were isolated from interscapular BAT by collagenase as described by Fain et alX’ and modified by Pettersson and Vallin.” The tissue was digested with 2 mg/mL collagenase in From the Howard Hughes Medical Institute and Thyroid Division, Department of Medicine, Brigham and W omen’s Hospital and Har- vard Medical School Boston, MA. Supported by National Institutes of Health Grant No. Dh136256. Address reprint requests to P. Reed Larsen, MD, Thyroid Division, Brigham and W omen’s Hospital, 75 Francis St. Boston, MA 02115. Copyright 0 1991 by W .B. Saunders Company 0026049519114012- 0018$03.0010 1327