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Adaptive thermogenesis in small mammals and infants takes place in brown adipose tissue (BAT). Heat is produced via uncoupling protein 1 (UCP1)-mediated uncoupling between oxidation of energy substrates and adenosine 5'-triphosphate synthesis. Thyroid hormone (TH) signaling plays a role in this process. The deiodinases activate thyroxine (T4) to 3,5,3'-triiodothyronine (T3) (D2) or inactivate T4 and T3 to 3,3,5'-triiodothyronine and T2 (D3), respectively. Using a mouse model with selective inactivation of Dio3 in BAT (flox-Dio3 × UCP1-cre = BAT-D3KO), we now show that knocking out D3 resulted in premature exposure of developing brown adipocytes (embryonic days 16.5-18.5) to T3 signaling, leading to an earlier expression of key BAT genes, including Cidea, Cox8b, Dio2, Ucp1, and Pgc1α. Adult BAT-D3KO mice exhibited increased expression of 1591 genes as assessed by RNA sequencing, including 19 gene sets related to mitochondria, 8 related to fat, and 8 related to glucose homeostasis. The expression of 243 genes was changed by more than 1.5-fold, 36 of which play a role in metabolic/thermogenic processes. BAT-D3KO mice weigh less and exhibit smaller white adipocyte area, but maintain normal energy expenditure at room temperature (22 °C) and in the cold (4 °C). They also defend their core temperature more effectively and do not lose as much body weight when exposed to cold. We conclude that the coordinated actions of Dio3 in the embryonic BAT define the timing and intensity of T3 signaling during brown adipogenesis. Enhanced T3 signaling during BAT embryogenesis (Dio3 inactivation) results in selective life-long modifications in the BAT transcriptome., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Three types of beta adrenergic receptors (ARβ1-3) mediate the sympathetic activation of brown adipose tissue (BAT), the key thermogenic site for mice which is also present in adult humans. In this study, we evaluated adaptive thermogenesis and metabolic profile of a mouse with Arβ2 knockout (ARβ2KO). At room temperature, ARβ2KO mice have normal core temperature and, upon acute cold exposure (4 °C for 4 h), ARβ2KO mice accelerate energy expenditure normally and attempt to maintain body temperature. ARβ2KO mice also exhibited normal interscapular BAT thermal profiles during a 30-min infusion of norepinephrine or dobutamine, possibly due to marked elevation of interscapular BAT (iBAT) and of Arβ1, and Arβ3 mRNA levels. In addition, ARβ2KO mice exhibit similar body weight, adiposity, fasting plasma glucose, cholesterol, and triglycerides when compared with WT controls, but exhibit marked fasting hyperinsulinemia and elevation in hepatic Pepck (Pck1) mRNA levels. The animals were fed a high-fat diet (40% fat) for 6 weeks, ARβ2KO mice doubled their caloric intake, accelerated energy expenditure, and induced Ucp1 expression in a manner similar to WT controls, exhibiting a similar body weight gain and increase in the size of white adipocytes to the WT controls. However, ARβ2KO mice maintain fasting hyperglycemia as compared with WT controls despite very elevated insulin levels, but similar degrees of liver steatosis and hyperlipidemia. In conclusion, inactivation of the ARβ2KO pathway preserves cold- and diet-induced adaptive thermogenesis but disrupts glucose homeostasis possibly by accelerating hepatic glucose production and insulin secretion. Feeding on a high-fat diet worsens the metabolic imbalance, with significant fasting hyperglycemia but similar liver structure and lipid profile to the WT controls., (© 2014 Society for Endocrinology.)

The presence of brown adipose tissue (BAT) in adults has become increasingly well defined as a result of functional imaging studies of thermogenically active BAT. Findings from these studies have created a surge of scientific interest in BAT, because it represents a potential therapeutic target for obesity--a condition with profound health consequences and few successful therapies. BAT contributes to overall energy expenditure in small mammals and neonates through adaptive thermogenesis. Thyroid-hormone signalling, particularly through induction of type II deiodinase, has a central role in brown adipogenesis in vitro and BAT development in mouse embryos. Additionally, because of high intracellular expression of type II deiodinase, adult BAT has enhanced thyroid-hormone signalling with several thyroid-hormone-dependent thermogenic pathways, including expression of the genes Ppargc1a and Ucp1. BAT thermogenesis explains the essential part played by thyroid hormone in energy homoeostasis and adaptation to cold. Stimulation of BAT in adults, specifically through thyroid-hormone-mediated pathways, is a promising therapeutic target for obesity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple β-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the β(1) isoform in energy homeostasis. First, the 30  min i.v. infusion of norepinephrine (NE) or the β(1) selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the β(1) gene (KO of β(1) adrenergic receptor (β(1)KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, β(1)KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BAT Ucp1 mRNA levels and oxygen consumption. Furthermore, β(1)KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the β(1) signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis.

Thyroid hormone accelerates energy expenditure (EE) and is critical for cold-induced thermogenesis. To define the metabolic role played by thyroid hormone in the dissipation of calories from diet, hypothyroid mice were studied for 60 d in a comprehensive lab animal monitoring system. Hypothyroidism decreased caloric intake and body fat while down-regulating genes in the skeletal muscle but not brown adipose tissue thermogenic programs, without affecting daily EE. Only at thermoneutrality (30 C) did hypothyroid mice exhibit slower rate of EE, indicating a metabolic response to hypothyroidism that depends on ambient temperature. A byproduct of this mechanism is that at room temperature (22 C), hypothyroid mice are protected against diet-induced obesity, i.e. only at thermoneutrality did hypothyroid mice become obese when placed on a high-fat diet (HFD). This is in contrast to euthyroid controls, which on a HFD gained more body weight and fat at any temperature while activating the brown adipose tissue and accelerating daily EE but not the skeletal muscle thermogenic program. In the liver of euthyroid controls, HFD caused an approximately 5-fold increase in triglyceride content and expression of key metabolic genes, whereas acclimatization to 30 C cut triglyceride content by half and normalized gene expression. However, in hypothyroid mice, HFD-induced changes in liver persisted at 30 C, resulting in marked liver steatosis. Acclimatization to thermoneutrality dramatically improves glucose homeostasis, but this was not affected by hypothyroidism. In conclusion, hypothyroid mice are metabolically sensitive to environmental temperature, constituting a mechanism that defines resistance to diet-induced obesity and hepatic lipid metabolism.

Type 2 deiodinase (D2), which is highly expressed in brown adipose tissue (BAT), is an enzyme that amplifies thyroid hormone signaling in individual cells. Mice with inactivation of the D2 pathway (D2KO) exhibit dramatically impaired thermogenesis in BAT, leading to hypothermia during cold exposure and a greater susceptibility to diet-induced obesity. This was interpreted as a result of defective acute activation of BAT D2. Here we report that the adult D2KO BAT has a permanent thermogenic defect that stems from impaired embryonic BAT development. D2KO embryos have normal serum T3 but due to lack of D2-generated T3 in BAT, this tissue exhibits decreased expression of genes defining BAT identity [i.e. UCP1, PGC-1alpha and Dio2 (nonfunctional)], which results in impaired differentiation and oxidative capacity. Coinciding with a reduction of these T3-responsive genes, there is oxidative stress that in a cell model of brown adipogenesis can be linked to decreased insulin signaling and decreased adipogenesis. This discovery highlights the importance of deiodinase-controlled thyroid hormone signaling in BAT development, where it has important metabolic repercussions for energy homeostasis in adulthood.

Cold-induced adaptive (or nonshivering) thermogenesis in small mammals is produced primarily in brown adipose tissue (BAT). BAT has been identified in humans and becomes more active after cold exposure. Heat production from BAT requires sympathetic nervous system stimulation, T(3), and uncoupling protein 1 (UCP1) expression. Our previous studies with a thyroid hormone receptor-beta (TR beta) isoform-selective agonist demonstrated that after TR beta stimulation alone, adaptive thermogenesis was markedly impaired, although UCP-1 expression in BAT was normal. We used mice with a dominant-negative TR beta PV mutation (frameshift mutation in resistance to thyroid hormone patient PV) to determine the role of TR beta in adaptive thermogenesis and UCP1 expression. Wild-type and PV mutant mice were made hypothyroid and replaced with T(3) (7 ng/g x d) for 10 d to produce similar serum thyroid hormone concentration in the wild-type and mutant mice. The thermogenic response of interscapular BAT, as determined by heat production during iv infusions of norepinephrine, was reduced in PV beta heterozygous and homozygous mutant mice. The level of UCP1, the key thermogenic protein in BAT, was progressively reduced in PV beta(+/-) and PV beta(-/-) mutant mice. Brown adipocytes isolated from PV mutant mice had some reduction in cAMP and glycerol production in response to adrenergic stimulation. Defective adaptive thermogenesis in TR beta PV mutant mice is due to reduced UCP1 expression and reduced adrenergic responsiveness. TR beta mediates T(3) regulation of UCP1 in BAT and is required for adaptive thermogenesis.

Pctp(-/-) mice that lack phosphatidylcholine transfer protein (Pctp) exhibit a marked shift toward utilization of fatty acids for oxidative phosphorylation, suggesting that Pctp may regulate the entry of fatty acyl-CoAs into mitochondria. Here, we examined the influence of Pctp expression on the function and structure of brown adipose tissue (BAT), a mitochondrial-rich, oxidative tissue that mediates nonshivering thermogenesis. Consistent with increased thermogenesis, Pctp(-/-) mice exhibited higher core body temperatures than wild-type controls at room temperature. During a 24 h cold challenge, Pctp(-/-) mice defended core body temperature efficiently enough that acute, full activation of BAT thermogenic genes did not occur. Brown adipocytes lacking Pctp harbored enlarged and elongated mitochondria. Consistent with increased fatty acid utilization, brown adipocytes cultured from Pctp(-/-) mice exhibited higher oxygen consumption rates in response to norepinephrine. The absence of Pctp expression during brown adipogenesis in vitro altered the expression of key transcription factors, which could be corrected by adenovirus-mediated overexpression of Pctp early but not late during the differentiation. Collectively, these findings support a key role for Pctp in limiting mitochondrial oxidation of fatty acids and thus regulating adaptive thermogenesis in BAT.

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting ( approximately 70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase ( approximately 3.0 degrees C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.

The Dio2 gene encodes the type 2 deiodinase (D2) that activates thyroxine (T4) to 3,3',5-triiodothyronine (T3), the disruption of which (Dio2(-/-)) results in brown adipose tissue (BAT)-specific hypothyroidism in an otherwise euthyroid animal. In the present studies, cold exposure increased Dio2(-/-) BAT sympathetic stimulation approximately 10-fold (normal approximately 4-fold); as a result, lipolysis, as well as the mRNA levels of uncoupling protein 1, guanosine monophosphate reductase, and peroxisome proliferator-activated receptor gamma coactivator 1, increased well above the levels detected in the cold-exposed wild-type animals. The sustained Dio2(-/-) BAT adrenergic hyperresponse suppressed the three- to fourfold stimulation of BAT lipogenesis normally seen after 24-48 h in the cold. Pharmacological suppression of lipogenesis with betabeta'-methyl-substituted alpha-omega-dicarboxylic acids of C14-C18 in wild-type animals also impaired adaptive thermogenesis in the BAT. These data constitute the first evidence that reduced adrenergic responsiveness does not limit cold-induced adaptive thermogenesis. Instead, the resulting compensatory hyperadrenergic stimulation prevents the otherwise normal stimulation in BAT lipogenesis during cold exposure, rapidly exhausting the availability of fatty acids. The latter is the preponderant determinant of the impaired adaptive thermogenesis and hypothermia in cold-exposed Dio2(-/-) mice.

Brown adipose tissue (BAT; brown fat) is the principal site of adaptive thermogenesis in the human newborn and other small mammals. Of paramount importance for thermogenesis is vascular perfusion, which controls the flow of cool blood in, and warmed blood out, of BAT. We have developed an optical method for the quantitative imaging of BAT perfusion in the living, intact animal using the heptamethine indocyanine IR-786 and near-infrared (NIR) fluorescent light. We present a detailed analysis of the physical, chemical, and cellular properties of IR-786, its biodistribution and pharmacokinetics, and its uptake into BAT. Using transgenic animals with homozygous deletion of Type II iodiothyronine deiodinase, or homozygous deletion of uncoupling proteins (UCPs) 1 and 2, we demonstrate that BAT perfusion can be measured noninvasively, accurately, and reproducibly. Using these techniques, we show that UCP -1/-2 knockout animals, when compared to wild-type animals, have a higher baseline perfusion of BAT but a similar maximal response to beta 3-receptor agonist. These results suggest that compensation for UCP deletion is mediated, in part, by the control of BAT perfusion. Taken together, BAT perfusion can now be measured noninvasively using NIR fluorescent light, and pharmacological modulators of thermogenesis can be screened at relatively high throughput in living animals.

Type 2 iodothyronine deiodinase (D2) is a selenoenzyme, the product of the recently cloned cAMP-dependent Dio2 gene, which increases 10- to 50-fold during cold stress only in brown adipose tissue (BAT). Here we report that despite a normal plasma 3,5,3'-triiodothyronine (T3) concentration, cold-exposed mice with targeted disruption of the Dio2 gene (Dio2(-/-)) become hypothermic due to impaired BAT thermogenesis and survive by compensatory shivering with consequent acute weight loss. This occurs despite normal basal mitochondrial uncoupling protein 1 (UCP1) concentration. In Dio2(-/-) brown adipocytes, the acute norepinephrine-, CL316,243-, or forskolin-induced increases in lipolysis, UCP1 mRNA, and O(2) consumption are all reduced due to impaired cAMP generation. These hypothyroid-like abnormalities are completely reversed by a single injection of T3 14 hours earlier. Recent studies suggest that UCP1 is primarily dependent on thyroid hormone receptor beta (TR beta) while the normal sympathetic response of brown adipocytes requires TR alpha. Intracellularly generated T3 may be required to saturate the TR alpha, which has an approximately fourfold lower T3-binding affinity than does TR beta. Thus, D2 is an essential component in the thyroid-sympathetic synergism required for thermal homeostasis in small mammals.

In newborns and small mammals, cold-induced adaptive (or nonshivering) thermogenesis is produced primarily in brown adipose tissue (BAT). Heat production is stimulated by the sympathetic nervous system, but it has an absolute requirement for thyroid hormone. We used the thyroid hormone receptor-beta--selective (TR-beta--selective) ligand, GC-1, to determine by a pharmacological approach whether adaptive thermogenesis was TR isoform--specific. Hypothyroid mice were treated for 10 days with varying doses of T3 or GC-1. The level of uncoupling protein 1 (UCP1), the key thermogenic protein in BAT, was restored by either T3 or GC-1 treatment. However, whereas interscapular BAT in T3-treated mice showed a 3.0 degrees C elevation upon infusion of norepinephrine, indicating normal thermogenesis, the temperature did not increase (<0.5 degrees C) in GC-1--treated mice. When exposed to cold (4 degrees C), GC-1--treated mice also failed to maintain core body temperature and had reduced stimulation of BAT UCP1 mRNA, indicating impaired adrenergic responsiveness. Brown adipocytes isolated from hypothyroid mice replaced with T3, but not from those replaced with GC-1, had normal cAMP production in response to adrenergic stimulation in vitro. We conclude that two distinct thyroid-dependent pathways, stimulation of UCP1 and augmentation of adrenergic responsiveness, are mediated by different TR isoforms in the same tissue.

Unlabelled: To study the thermal response of interscapular brown fat (IBF) to norepinephrine (NE), urethan-anesthetized rats (1.2 g/kg ip) maintained at 28-30 degrees C received a constant venous infusion of NE (0-2 x 10(4) pmol/min) over a period of 60 min. IBF temperatures (T(IBF)) were recorded with a small thermistor fixed under the IBF pad. Data were plotted against time and expressed as maximal variation (Deltat degrees C). Saline-injected rats showed a decrease in T(IBF) of approximately 0.6 degrees C. NE infusion increased T(IBF) by a maximum of approximately 3.0 degrees C at a dose of 10(4) pmol x min(-1) x 100 g body wt(-1). Surgically thyroidectomized (Tx) rats kept on 0.05% methimazole showed a flat response to NE. Treatment with thyroxine (T(4), 0.8 microg x 100 g(-1) x day(-1)) for 2-15 days normalized mitochondrial UCP1 (Western blotting) and IBF thermal response to NE, whereas iopanoic acid (5 mg x 100 g body wt(-1) x day(-1)) blocked the effects of T(4). Treatment with 3,5, 3'-triiodothyronine (T(3), 0.6 microg x 100 g body wt(-1) x day(-1)) for up to 15 days did not normalize UCP1 levels. However, these animals showed a normal IBF thermal response to NE. Cold exposure for 5 days or feeding a cafeteria diet for 20 days increased UCP1 levels by approximately 3.5-fold. Nevertheless, the IBF thermal response was only greater than that of controls when maximal doses of NE (2 x 10(4) pmol/min and higher) were used., Conclusions: 1) hypothyroidism is associated with a blunted IBF thermal response to NE; 2) two- to fourfold changes in mitochondrial UCP1 concentration are not necessarily translated into heat production during NE infusion.

To investigate the role of type II 5'-deiodinase (5'D-II) in the expression of mitochondrial uncoupling protein (UCP) in brown adipose tissue (BAT), we injected intact male rats with reverse (r) 3,5,3'-triiodothyronine (T3; 100 micrograms. 100 g body wt-1. day-1), an inhibitor of 5'D-II, for 2-5 days. UCP decreased by approximately 20% in rats kept at 28 degreesC and failed to increase during cold exposure (4 degreesC). Next, thyroxine treatment (1-10 micrograms. 100 g body wt-1. day-1) increased nuclear T3 in rats kept at 28 or 4 degreesC. In these rats, nuclear T3 correlated positively with UCP. In addition, T3 (1-50 micrograms. 100 g body wt-1. day-1) given to intact rats (5-15 days; 28 degreesC) induced an approximately twofold increase in UCP. In these T3-treated animals, the interscapular BAT thermal response to norepinephrine infusion also correlated positively with T3 dose and UCP content. Treatment with propranolol or reserpine failed to block the T3 induction of UCP (approximately 1.8- and approximately 2.3-fold). The results emphasize the importance of local 5'D-II and reveal an independent role of T3 in the expression of UCP.

In euthyroid rats, maximal sympathetic nervous system stimulation (e.g. during cold exposure) results in a 3- to 4-fold increase in brown adipose tissue lipogenesis, a response that is blunted in hypothyroid rats. To further investigate this phenomenon, the role of local type II 5'-deiodinase (5'-DII) was studied in freshly isolated brown adipocytes. In a typical experiment, 1.5 x 10(6) cells were incubated for up to 48 h in a water-saturated 5% CO2-95% O2 atmosphere. After incubation with medium alone or with different concentrations of T4, T3, and/or norepinephrine (NE), lipogenesis was studied by measuring 1) the rate of fatty acid synthesis as reflected by 3H2O incorporation into lipids and 2) the activity of key rate-limiting enzymes, i.e. acetyl coenzyme A carboxylase and malic enzyme, and the results are reported in terms of DNA content per tube. Lipogenesis decreased progressively over time (approximately 40%) when no additions were made to the incubation medium. T4 or T3 partially prevented that inhibition at physiological concentrations (65 x 10[-9] and 0.77 x 10[-9] M, respectively), whereas a receptor-saturating concentration of T3, (154 x 10[-9] M) doubled the lipogenesis rate. The addition of 10(-6) M NE inhibited lipogenesis acutely (approximately 50% by 12 h) and was followed by a progressive stimulation that reached approximately 2-fold by 48 h, but only in the presence of T4. Furthermore, NE did not attenuate T3 (154 x 10[-9] M)-induced lipogenesis. Both the inhibition and the stimulation of lipogenesis caused by NE showed a strong dose-response relationship within the range of 10(-11)-10(-5) M. The role of local 5'-DII was further tested by incubating brown adipocytes with 10(-6) M NE and T4 (65 x 10[-9] M) in the presence of 100 microM iopanoic acid, a potent inhibitor of 5'-DII. Although iopanoic acid did not affect the T3 stimulation of lipogenesis, it did block the approximately 2-fold stimulation of lipogenesis triggered by NE in the presence of T4, confirming the mediation of 5'-DII in this process. In conclusion, lipogenesis in brown adipose tissue is under complex hormonal control, with key roles played by NE, thyroid hormones, and local 5'-DII. As in other tissues, NE-generated signals acutely (12 h) inhibited lipogenesis. However, the presence of the 5'-DII generated enough T3 to stimulate lipogenesis and gradually reverse the short-lived NE-induced inhibition, leading to the 2- to 3-fold response observed at later time points.

Hypothyroidism profoundly reduces the capacity of brown adipose tissue (BAT) to generate cAMP in response to adrenergic stimulation. Evidence obtained with isolated brown adipocytes suggests a postreceptor defect that offsets the hypothyroidism-induced increase in beta3-adrenergic receptors. The goal of the present studies was to identify the defect in the cAMP generation pathway for which we studied cAMP generation in isolated cells and purified BAT membranes from normal and hypothyroid rats. Studies with adenosine deaminase and the adenosine receptor-1 agonist r-phenyl isopropyl adenosine (R-PIA) show that hypothyroid cells are not more sensitive to adenosine (same EC50) but more inhibited by high concentrations of R-PIA. Pretreatment with pertussis toxin reduced the gap in cAMP generation between eu- and hypothyroid cells and the inhibition mediated by R-PIA, but did not normalize the cAMP response to forskolin in hypothyroid cells. Although purified euthyroid BAT membranes increased cAMP production with GTP concentrations up to submillimolar range, to plateau or slightly decrease at higher levels, hypothyroid membranes were weakly stimulated by low concentrations of GTP and markedly inhibited (>50%) at concentrations > or = 10(-4) M. When assayed at 0.3 mM ATP and 1 microM GTP, hypothyroid membranes actually generated more cAMP in response to forskolin, but this was reversed when GTP concentration was 1 mM. Immunoblotting studies showed no significant effects of hypothyroidism on the abundance of G(alpha)i or Gbeta subunits, and ADP ribosylation of G(alpha)i was only 45% increased in hypothyroidism in contrast to a 2.5-fold increase in hypothyroid white adipose tissue membranes from the same rats. Hypothyroid membranes also exhibited different kinetics regarding ATP, with higher cAMP generation at submillimolar concentrations but less at >1 mM ATP. Actually, at ATP concentrations >0.6 mM, cAMP generation was markedly inhibited in hypothyroid membranes. Fixing the concentration of free Mg++ in these experiments indicates that most of the inhibition seen in hypothyroid membranes is caused by ATP, whereas euthyroid membranes are more sensitive to changes in free Mg++. Ca++ +/- calmodulin did not stimulate adenylyl cyclase (AC) activity. On the contrary, AC activity was inhibited by Ca++ in a concentration-dependent manner, by as low as 100 nM free Ca++, and to greater extent in hypo- than in euthyroid membranes (maximal inhibition 60 vs. 25-30%). Our results suggest that, functionally, hypothyroidism causes a change in the AC of BAT membranes consistent with a relative or absolute increase in the type VI AC (AC-VI). The effects on this AC of nucleotides, Ca++, and Mg++ at concentrations prevailing in the hypothyroid brown adipocyte are probably the major factor in the reduced capacity of these cells to generate cAMP. These results also open the possibility of a novel, differential effect of thyroid hormone on AC expression, and support the concept that thyroid hormone affects the adrenergic signal transduction pathways in a tissue-selective manner.

Uncoupling protein (UCP) mRNA levels were studied in the interscapular brown adipose tissue (BAT) of rats undergoing different manipulations of the adrenal function and BAT adrenergic stimulation. Adrenalectomy did not affect UCP mRNA levels for up to 8 days post-surgery. However, adrenalectomized rats underwent a greater increase in UCP mRNA levels (26%) than intact rats after 4 h of cold exposure. Administration of corticosterone (500 micrograms.100 g body wt-1.day-1 sc) to intact or adrenalectomized rats, kept at 28 degrees C, produced a marked decrease of UCP mitochondrial content and cellular mRNA levels in a time-dependent manner (30% by 12 h and 50% by 24 h). Pretreatment of intact rats with corticosterone virtually abolished the UCP mRNA response to cold and norepinephrine (NE). In contrast, when rats had been preexposed to cold for 96 h, the injection of corticosterone did not affect UCP mRNA. These results show that corticosterone is a powerful inhibitor of UCP gene expression in vivo. Corticosterone inhibits both basal gene expression at thermoneutrality and the response to adrenergic stimulation either by cold or exogenous NE, suggesting a direct action on BAT. The data further suggest that corticosterone inhibits the initial accumulation of UCP mRNA mediated by UCP gene transcription, rather than accelerating the degradation of UCP mRNA.

The activities of malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G-6-PDH), two NADPH-generating lipogenic enzymes, were measured in brown adipose tissue (BAT) of rats undergoing various neurohormonal manipulations. Methimazole-induced hypothyroidism doubled the activity of these two enzymes but, surprisingly, triiodothyronine (T3) given to hypothyroid rats caused a time- and dose-dependent stimulation of up to three- to fourfold. Unilateral BAT denervation modestly reduced the activity of these enzymes (approximately 30%) and failed to prevent the stimulation induced by hypothyroidism, whereas growth hormone (GH) successfully blocked this effect of hypothyroidism. Insulin stimulated both enzymes regardless of the thyroid status but failed to abolish the inhibitory effect of GH. In intact rats, cold exposure caused a time-dependent increase in the activity of both ME and G-6-PDH, which reached 5.2- and 3-fold, respectively, after 96 h. This cold-induced stimulation was not observed in hypothyroid rats, but it was restored by physiological doses of thyroxine (800 ng.100 g body wt-1.24 h-1). Replacement with T3 (300 ng.100 g body wt-1.24 h-1), in contrast, did not have this effect. In hypothyroid rats with hemidenervation of BAT, norepinephrine (NE) modestly increased ME and G-6-PDH activities in the denervated side, with little or no effect in the intact side. Receptor-saturating doses of T3 (50 micrograms.100 g body wt-1.day-1 over 48 h) stimulated two- and threefold both enzymes in both sides, reducing or obliterating the effect of denervation. The data suggest a complex neurohormonal regulation of the activity of ME and G-6-PDH in BAT.(ABSTRACT TRUNCATED AT 250 WORDS)

We intend to develop in vitro model systems to study the hormonal regulation of uncoupling protein (UCP) and its role in brown adipose tissue (BAT) thermogenesis. We report here that UCP mRNA responses to adrenergic and thyroid hormone manipulations in freshly dispersed, mature brown adipocytes mimic in vivo observations. Studies were performed in brown adipocytes obtained from interscapular brown fat of euthyroid or hypothyroid rats. The tissue was dispersed with collagenase, and cells were isolated by floatation over 4% BSA. UCP mRNA in these cells is 2-3 times more abundant than that in the whole tissue, indicating a selection of cells expressing the gene. In cells from euthyroid rats, UCP mRNA is maximally elevated within 2 h of exposure to 1 microM forskolin and 50 ng T3/ml (77 nM total, 0.43 nM free). T3 significantly enhances the effect of forskolin. In the absence of stimulation, UCP mRNA rapidly disappears from euthyroid brown adipocytes, and this can be prevented with the addition of T3 by a mechanism not requiring on-going transcription. In cells from hypothyroid rats, forskolin or isoproterenol plus phenylephrine fail to stimulate UCP mRNA, but within 3 h of exposure to T3, cells recover full responsiveness. As in vivo, a high extracellular concentration of T3 is required for maximal responsiveness of UCP mRNA to cAMP, while T4 can restore responsiveness in physiological concentrations (40 pM). This effect of T4 is prevented by iopanoic acid, a compound that blocks the type II T4 5'-deiodinase. In conclusion, 1) freshly dispersed brown adipocytes retain all of the properties concerning UCP regulation by thyroid hormone and sympathetic nervous system described for brown fat in vivo; 2) the observations made in vivo, thus, represent direct action of norepinephrine and thyroid hormone on these cells; 3) as in vivo, T4 is a better source of intracellular T3 than extracellular T3 for brown adipocytes; hence, the in vivo findings are the result of the cell biology of 5'-deiodinase type II rather than dynamic factors inherent to the in vivo condition; 4) stabilization of mature UCP mRNA by T3 is an important mechanism to maintain the levels of this mRNA elevated under sustained stimulation; and 5) dispersed brown adipocytes and UCP gene products constitute a powerful model to study interactions between the sympathetic nervous system and thyroid hormone at a cellular or molecular level.

As judged by the response of uncoupling protein and key enzymes, brown adipose tissue (BAT) is highly dependent upon the local generation of T3 catalyzed by the tissue type II T4 5'-deiodinase (5'-D-II). In hypothyroid rats treated with T3 or T4, the capacity to withstand cold seems better correlated with the normalization of BAT responses than with the liver thyroid status. 5'D-II is activated by cold via sympathetic nervous system (SNS) stimulation, and the activation generates enough T3 to nearly saturate BAT nuclear T3 receptor (NTR) in euthyroid rats. In hypothyroidism, 5'D-II is highly stimulated by the SNS and hypothyroxinemia. In the present studies we have taken advantage of this situation to test 1) the capacity of 5'D-II to maintain nuclear T3 in rats with various degrees of hypothyroxinemia, and 2) the hypothesis that thyroid hormone-dependent BAT-facultative thermogenesis, rather than the effect of thyroid hormone on obligatory thermogenesis (basal metabolic rate), is the basic mechanism by which thyroid hormone confers protection against acute cold exposure. We treated methimazole-blocked rats (undetectable plasma T4 and T3) for a week with either subreplacement doses of T4 (0.5, 1, 2, and 4 micrograms/kg.day) or replacement doses of T4 or T3 (8 or 3 micrograms/kg.day, respectively). Sources and content of BAT nuclear T3 were studied at 25 C and after 48 h at 4 C by labeling the plasmaborne T3 (T3[T3]) with [131I]T3 and the locally generated T3 (T3[T4]) with [125I]T4. Neither the kinetics of nuclear-plasma exchange of T3[T3], the time of appearance of T3[T4] in BAT nuclei, nor NTR maximal binding capacity (0.71 ng T3/mg DNA) was affected by hypothyroidism. Kinetic analyses indicated a maximal BAT NTR occupancy of 40% at euthyroid serum T3 concentrations if T4 is not present. Replacement with T4 normalized both serum T4 and T3, while replacement with T3 normalized serum T3; for all other doses of T4, serum T4 and T3 concentrations were predictably related to the dose. 5'D-II activity decreased with increasing doses of T4, but for each dose of T4, this activity was 2-4 times greater at 4 C than at 25 C. BAT NTR occupancy normalized with 2 micrograms T4/kg in rats maintained at 25 C and with 4 micrograms T4/kg in cold-exposed rats, although in neither condition were serum T4 and T3 normalized nor more than 30% of NTR occupied by plasma T3.(ABSTRACT TRUNCATED AT 400 WORDS)

The amount and sources of T3 associated with high affinity, low capacity cellular nuclear receptors in brown adipose tissue (BAT) have been estimated by in vivo pulse-labeling techniques. Maximal binding capacity was measured by in vivo saturation analysis. Nuclear receptor occupancy at endogenous levels of T3 and T4 in euthyroid rats was estimated from the equilibrium nuclear to serum ratio of tracer T3, and the locally generated nuclear T3 to serum T4 ratio after injecting tracer T3 and T4. These ratios were multiplied, respectively, by the endogenous concentrations of T3 and T4 as measured by RIA. The maximal binding capacity was 0.65 ng T3/mg DNA, and saturation was 71%. Fifty-five percent of the nuclear T3 was generated locally, and 45% was derived from circulating T3. BAT is, hence, comparable to the liver in number of receptors (approximately 5000/cell) and to the pituitary with regard to saturation and relative contributions of locally generated T3 and plasma T3 to nuclear T3. These results suggest that BAT may be an important target for thyroid hormones and, along with other data, that alterations in the activity of the type II 5'-deiodinase of this tissue may influence the saturation of nuclear T3 receptors.

We have examined the activity of three lipogenic enzymes [malic enzyme (ME), glucose-6-phosphate dehydrogenase (G-6-PD), and acetyl coenzyme A (CoA) carboxylase], the activity of the mitochondrial FAD-dependent alpha-glycerolphosphate dehydrogenase (alpha-GPD), and the mitochondrial concentration of uncoupling protein (UCP) in brown adipose tissue (BAT) of euthyroid and hypothyroid rats, both at room temperature and in response to acute cold stress. These enzymes and UCP are important for the thermogenic response of BAT in adaptation to cold. The basal level of the lipogenic enzymes was normal or slightly elevated in hypothyroid rats maintained at 23 degrees C, but the levels of alpha-GPD and UCP were markedly reduced. Forty-eight hours at 4 degrees C resulted in an increase in the activity of G-6-PD, acetyl-CoA carboxylase, and alpha-GPD and in the concentration of UCP both in euthyroid and hypothyroid animals, but the levels reached were invariably less in hypothyroid animals, indicating that thyroid hormone is necessary for a full metabolic response of BAT under maximal demands. Of all variables measured, the most affected was UCP (only one-fifth of the response of euthyroid rats to cold) followed by alpha-GPD (approximately 50% the euthyroid response). The administration of replacement doses of triiodothyronine (T3) to hypothyroid rats for 5-7 days did not normalize any of the BAT responses, whereas the replacement of thyroxine (T4) for only 2 days sufficed to normalize them all. This effect of T4 was abolished by preventing its conversion to T3 with iopanoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcription of the uncoupling protein (UCP) gene in rat brown adipose tissue has been evaluated by a nuclear run-on transcription assay. Nuclei from euthyroid rats treated with norepinephrine (NE) exhibited a 3-4-fold increase in transcription 2 h after the injection, whereas the UCP mRNA abundance increased 2-3-fold during the same interval. In contrast, neither UCP mRNA nor the transcription rate increased in response to the same treatment in hypothyroid rats, but the transcriptional response was recovered 2 h after the injection of a receptor saturating dose of 3,5,3'-L-triiodothyronine (T3). When injected to rats given T3 4 h before, NE increased UCP gene transcription and mRNA abundance by 4-5 and 3-4-fold, respectively, by 2 h. Neither cycloheximide nor emetine prevented this effect of T3. In euthyroid rats, the UCP gene transcription rate increased within 15 min of exposure to 4 degrees C. In contrast, in hypothyroid rats exposed to 4 degrees C overnight the transcription rate remained unchanged, but it started to increase steeply 2 h after the intravenous injection of a receptor saturating dose of T3 with UCP mRNA lagging approximately 1 h behind. Unilateral denervation of interscapular brown adipose tissue in hypothyroid rats caused a 60-70% ipsilateral reduction in transcription rate and UCP mRNA. When T3 was given to these rats, it only stimulated UCP gene transcription and UCP mRNA accumulation in the intact side, but there was a symmetrical stimulation when NE was injected following the T3. We conclude that the UCP gene is under dual control by T3 and NE. The primary signal seems to be generated by NE. This signal increases UCP mRNA by stimulating the transcription rate of the UCP gene. From the denervation experiments and the lack of response of UCP to NE in hypothyroid rats, we estimate that T3 amplifies 4-5-fold the transcriptional response to NE. No stabilization of UCP mRNA needs to be invoked to explain the acute effect of T3. The T3-dependent signal amplifying the NE effect is consistent with being the T3-receptor complex or another T3-derived signal that is neither a newly synthesized protein nor a rapidly turned-over pre-existing protein, activated by T3.

The effect of thyroxine (T4) and triiodothyronine (T3) on the expression of uncoupling protein (UCP) in rat brown adipose tissue (BAT) has been examined. Thyroidectomized rats have a threefold reduction in basal UCP levels. When exposed to cold, they become hypothermic and show a fivefold lower response of UCP than euthyroid controls. T3 augments the basal levels and the response of UCP and its mRNA to cold in a dose-dependent manner. However, to normalize the response of UCP, T3 has to be given in a dosage that produces systemic hyperthyroidism. Mere T3 replacement corrects the systemic hypothyroidism but not the hypothermia or the low levels of UCP. In contrast, replacement doses of T4 prevent the hypothermia and correct the UCP level. Both effects of T4 are blocked by preventing T4 to T3 conversion in BAT. Thus, the optimal UCP response to cold and protection against hypothermia require a high BAT T3 concentration, which is attained from euthyroid levels of T4 via the activation of the BAT T4 5'deiodinase during cold exposure, but not from euthyroid plasma T3 levels.

Cold exposure induces a rapid increase in uncoupling protein (UCP) concentration in the brown adipose tissue (BAT) of euthyroid, but not hypothyroid, rats. To normalize this response with exogenous 3,5,3'-triiodothyronine (T3), it is necessary to cause systemic hyperthyroidism. In contrast, the same result can be obtained with just replacement doses of thyroxine (T4) and, in euthyroid rats, the normal response of UCP to cold occurs without hyperthyroid plasma T3 levels. Consequently, we explored the possibility that the cold-induced activation of the type II 5'-deiodinase resulted in high levels of nuclear T3 receptor occupancy in euthyroid rats. Studies were performed with pulse injections of tracer T3 or T4 in rats exposed to 4 degrees C for different lengths of time (1 h-3 wk). Within 4 h of cold exposure, we observed a significant increase in the nuclear [125I]T3 derived from the tracer [125I]T4 injections (T3[T4]) and a significant reduction in the nuclear [125I]T3 derived from [125I]T3 injections (T3[T3]). The number of BAT nuclear T3 receptors did not increase for up to 3 wk of observation at 4 degrees C. The mass of nuclear-bound T3 was calculated from the nuclear tracer [125I]T3[T3] and [125I]T3[T4] at equilibrium and the specific activity of serum T3 and T4, respectively. By 4 h after the initiation of the cold exposure, the receptors were greater than 95% occupied and remained so for the 3 weeks of observation. This effect of cold was hindered by prazosin, a drug that prevents the cold-induced activation of 5'-deiodinase. We conclude that the simultaneous activation of the deiodinase with adrenergic BAT stimulation serves the purpose of nearly saturating the nuclear T3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)