Adrenergic control of induction of type II iodothyronine 5′-deiodinase activity in cultured mouse brown adipocytes

Abstract

Iodothyronine 5'-deiodinase (5'D) of mouse brown adipocytes differentiated in cell culture was characterized in detail with respect to the adrenergic control of its biosynthesis. The stimulation of 5'D required mRNA and protein synthesis and was dependent on the stage of differentiation of the cells. The maximum induction was observed around confluence (7-day-old cells), in pre- and post-confluent cells the 5'D activity was significantly less induced. The transient responsiveness of brown fat-cells to the stimulatory effect of adrenergic agents was reflected also in the time course of the induction of 5'D by different concentrations of agonists. The maximum response occurred regularly after an 8 h incubation and implicated a rather fast turnover of the induced enzyme. On the basis of the inhibitory effects of cycloheximide and actinomycin D, the half-life of the induced 5'D and its mRNA were estimated to be 1.5 and 3.3 h respectively. The noradrenaline-induced 5'D activity was shown to be that of the type II enzyme, insensitive to propylthiouracil (PTU). The estimated values of its apparent Km for thyroxine, Km for the co-substrate dithiothreitol, and Vmax. in the presence of 1 mM PTU were 2 nM, 2.6 mM, and 0.1 pmol of I-/h per mg of protein respectively. The 5'D activity was effectively induced by forskolin and dibutyryl cyclic AMP, as well as by isoprenaline, noradrenaline and CGP-12177, but not by phenylephrine, cirazoline or oxymetazoline. This indicates that, contrary to previous observations in vivo, stimulation of 5'D in cultured brown fat-cells involves elevated cyclic AMP levels and is mediated predominantly via beta-receptors, particularly via the so-called beta 3-adrenoceptors.

The in vivo effects of beta-3-receptor agonist CGP-12177 on thyroxine deiodination in cold-exposed, sympathectomized rat brown fat

“…The 4 h effects of CGP-12177 were abolished by propranolol but not by prazosin, thus con®rming that beta-receptors were mostly or solely involved in the in vivo activation of 5 H -DII. These results correlate with those obtained in isolated brown adipocytes in vitro by Pavelka et al (24) and Hernandez & Obrego Ân (25). In rats injected with NE, however, prazosin ± and to a lesser extent propranolol ± blocked the NE effect, thereby suggesting that the natural agonist activated 5 H -DII in vivo via alpha-1-receptors and to a lesser extent through beta-receptors.…”

“…The alpha-1-receptor appeared to potentiate the ability of cAMP to stimulate thermogenesis synergistically with the beta-3-receptor. A synergism between beta-and alpha-1-receptors in the stimulation of 5 H -DII activity was observed in isolated adipocytes (9) although other in vitro studies (24,25) conditions. The present data indicate that the in vivo administration of CGP-12177 can activate BAT 5 H -DII via beta-receptors without a signi®cant participation of alpha-1-receptors.…”

Triiodothyronine is required for the stimulation of type II 5′-deiodinase mRNA in rat brown adipocytes

“…D2 is stimulated adrenergically in floating adipocytes (32), and a synergy between the ␤-and ␣ 1 -adrenergic pathways was found (38), the adrenergic stimulation of D2 being mostly ␤-adrenergic in mouse brown adipocytes (35). We have shown that T 3 is an absolute requirement for the adrenergic stimulation of D2 and amplifies this response (19).…”

“…Besides, BAT D2 activity is upregulated by insulin (46), as shown after insulin injection, or in diabetic rats, as well as in floating rat brown adipocytes (29). In these cells, D2 activity is stimulated by adrenergic agents, and a synergy between ␣ 1 -and ␤-adrenergic pathways has been described (32,38), whereas in cultures of mouse brown adipocytes the main pathway is ␤-adrenergic (35).…”

“…D2 activity is localized in brain, adenohypophysis, BAT, pineal gland, and the maternal side of the placenta, among other tissues (24,26,39,44,53). D2 activity is regulated in rat tissues, glial cells, or brown adipocytes by a number of factors such as thyroid hormones (26,27,47), adrenergic agents (19,22,32,35,38,39,44), cAMP (10,17,22), growth factors (11), and insulin (29,46).The cDNAs coding for the three deiodinases have been isolated in rat, human, and other species (2,12,13). Sequence analysis has demonstrated that all of them contain an in-frame TGA codon that is translated as selenocysteine due to the presence of a specific structure, named selenocysteine insertion sequence (SECIS), in the 3Ј untranslated region of their mRNAs (2, 6).…”