Thyroid hormone--sympathetic interaction and adaptive thermogenesis are thyroid hormone receptor isoform--specific

Abstract

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.

Figures

Figure 1

IBAT thermal response (ΔTIBAT) to NE infusion in euthyroid control mice. (a) Dose-response curve of maximal ΔTIBAT°C versus NE during 30-minute infusion. (b) Time course of ΔTIBAT °C during infusion of 5 × 103 pmol/min NE. Values are the mean ± SD of three to four mice.

Figure 2

IBAT thermogenic response (ΔTIBAT) during NE infusion in euthyroid and hypothyroid mice treated with T3 or GC-1. The data represent the maximal response every 5 minutes over a period of 30 minutes of NE infusion. Euthyroid (control) mice were compared with hypothyroid mice treated with vehicle, T3, or GC-1. The hypothyroid animals were kept on a low-iodine diet with propylthiouracil for 8 days and then received daily intraperitoneal injections of T3 for 10 days at 0 (vehicle only), or 7, 14, or 28 ng/g/day, corresponding to approximately two, four, and eight times the physiological replacement dose. Another five groups of mice were treated with 0, 3.6, 7.2, 14.4, and 28.8 ng/g/day GC-1. The GC-1 doses are equimolar to T3 doses. At the end of the treatment period, the IBAT thermal response to NE was measured. Values are the mean ± SD of three to four mice.

Figure 3

Mitochondrial UCP1 protein from IBAT of euthyroid control (C) or hypothyroid mice treated with T3, GC-1, or vehicle (–). (a) Western blot of mitochondrial protein using anti-UCP1 antibody. (b) Blots were analyzed by densitometry, and the results are shown. Values are the mean ± SD of three to four mice. The animals were treated as described in the legend to Figure 2. Equivalent protein loading was confirmed by Coomassie blue stain of gel. *P < 0.05 vs. control animals; **P < 0.05 vs. hypothyroid animals, by one-way ANOVA.

Figure 4

Markers of T3 or GC-1 actions in the liver. (a) Malic enzyme mRNA was quantitated in euthyroid control animals (C), and hypothyroid animals treated with vehicle (–), T3, or GC-1. (b) Representative Northern blot of malic enzyme mRNA from liver. GAPDH mRNA expression was used for normalization. Densitometric analysis of mRNA expression is shown in arbitrary units (malic enzyme mRNA/GAPDH mRNA). (c) Liver α-GPD was measured in the mitochondrial fraction. The animals were treated as described in the legend to Figure 2. Values are the mean ± SD of three to four mice. *P < 0.05 vs. control animals; **P < 0.05 vs. hypothyroid animals, by one-way ANOVA.

Figure 5

Heart rate in T3- versus GC-1–replaced hypothyroid animals. Resting heart rate was measured in anesthetized animals after a 10-minute stabilization period. The animals were treated as described in the legend to Figure 2. Values are the mean ± SD of three to four mice. *P < 0.05 vs. control animals; **P < 0.05 vs. hypothyroid animals, by one-way ANOVA.

Figure 6

Core temperature profile and IBAT UCP1 mRNA in mice kept in a cold environment (4°C) for 8 hours. The hypothyroid mice were treated with vehicle, T3 (7.2 ng/g/day) or GC-1 (3.6 ng/g/day) for 10 days and then transferred to a cold room. Animals were treated as described in the legend to Figure 2. (a) Core temperature response to cold exposure (4°C). Core temperature was measured with a rectal probe at indicated times. (b) Northern blot analysis of UCP1 mRNA from IBAT in euthyroid control and hypothyroid animals treated with vehicle, T3, or GC-1. Values are the mean ± SD of three to four mice. *P < 0.05 vs. control animals; **P < 0.05 vs. hypothyroid animals, by one-way ANOVA.

Figure 7

NE-stimulated cAMP accumulation in isolated brown adipocytes of euthyroid control, compared with vehicle (–) and T3- and GC-1–treated hypothyroid mice. Animals received T3 (14.4 ng/g/day) or GC-1 (7.2 ng/g/day) by intraperitoneal injection for 10 days. Cells were isolated as indicated in Methods. NE was added at 10–6 M, and the incubation lasted 1 hour. The media also contained 0.3 U/ml adenosine deaminase and 500 μM IBMX. Values are the mean ± SD of four tubes. *P < 0.05 vs. control cells, by one-way ANOVA.

Figure 8

Dose-response curve of cAMP accumulation in isolated brown adipocytes of vehicle (hypothyroid) or T3- or GC-1–treated hypothyroid mice. Cells were prepared as described in the legend to Figure 7, and were incubated with the indicated doses of NE (a), the β3 adrenergic receptor selective agonist CL (b), or forskolin (c). Values are the mean ± SD of three tubes. *P < 0.05 vs. hypothyroid and GC-1–treated cells, by one-way ANOVA.