Deiodinase Activity of Brown Adipose Tissue in Daurian Ground Squirrel During Hibernation and Arousal

http://www.paper.edu.cn Comparative Biochemistry and Physiology Part A 120 (1998) 745–752 Uncoupling protein mRNA, mitochondrial GTP-binding, and T4 5%-deiodinase activity of brown adipose tissue in Daurian ground squirrel during hibernation and arousal Xiao-tuan Liu a, Qi-shui Lin b, Qing-fen Li a,*, Chen-xi Huang a, Ru-yong Sun a a Laboratory of Animal Ecology, Department of Biology, Beijing Normal Uni6ersity, 19 Xinwai Street, Beijing 100875, People’s Republic of China b State key laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, 320 Yueyang Road, Shanghai 200031, People’s Republic of China Received 21 November 1997; received in revised form 5 June 1998; accepted 11 June 1998 Abstract The mRNA level of uncoupling protein (UCP) specific for brown adipose tissue (BAT) in Daurian ground squirrel, was detected by using a [32P]-labeled oligonucleotide probe. The UCP concentration in mitochondria was indirectly determined by 3 % titration with its specific ligand [ H]-labeled GTP. Type II T4 5 -deiodinase of BAT was assayed concomitantly. We found two species of mRNA for UCP with lengths of about 1.9 and 1.5 kb, respectively, both occurring in almost the same concentration. UCP mRNA content was elevated significantly during hibernation, but the UCP concentration did not change compared with that of nonhibernating controls kept at room temperature. When hibernating squirrels were aroused, the UCP mRNA remained at the elevated level as during hibernation, but the UCP concentration increased in comparison with that of nonhibernating controls or % during hibernating. Changes in T4 5 -deiodinase activity in BAT were similar to the variations of the UCP mRNA level. These % results suggest that the activation of T4 5 -deiodinase in BAT may be an important factor for the up-regulation and maintenance of UCP mRNA content needed for the synthesis of sufficient UCP to acquire the thermogenic capacity for arousal from hibernation. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Arousal; Brown adipose tissue; Daurian ground squirrel; GTP-binding to mitochondria; Hibernation; mRNA for % uncoupling protein; Nonshivering thermogenesis; T4 5 -deiodinase 1. Introduction [5]. The key element for the energy dissipation capacity of BAT is uncoupling protein (UCP or thermogenin), a Brown adipose tissue (BAT) is the main site of 32 kDa protein uniquely expressed in the inner mem- facultative thermogenesis in small rodents and probably brane of BAT mitochondria. UCP dissipates the proton most eutherian mammals during the early postnatal gradient created by the respiratory chain, thereby accel- period. Furthermore, this tissue is also traditionally erating respiration. The uncoupling protein has a high regarded as being of particular significance in hibernating affinity for purine nucleotides such as GDP and GTP species, principally in relation to arousal from hiberna- [19,27]. Recently, Huang and Klingenberg reported that tion and maintenance of the euthermic state in the cold GTP binds to mitochondria or UCP more tightly than GDP [10]. In addition, the binding capacity determined * Corresponding author. Dept. of Biology, Beijing Normal Univer- represents the overall nucleotide binding sites on the sity. Tel.: +86 10 62209029; fax: +86 10 62200567; e-mail: BAT mitochondria, which limits the thermogenic poten- [email protected] tial of BAT to respond to environmental stimuli. 1095-6433/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)10095-8 转载中国科技论文在线 http://www.paper.edu.cn 746 X.-t. Liu et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 745–752 tion induced by cold exposure of laboratory rodents mine if they were hibernating. Each animal was allowed [32]. The optimal expression of UCP requires high to go through at least one spontaneous arousal before intracellular concentration of triiodothyronine (T3) being used. The hibernating squirrels were further di- [1,2]. The high T3 concentration required is provided by vided into two small groups. One group, the deeply hibernating ones (n=4), were decapitated immediately. the sympathetic activation of type II thyroxine (T4) % The second group, the arousing squirrels (n=4), were 5 -deiodinase which converts T4 to T3 in BAT [1,3,29]. % allowed to partially arouse before being sacrificed. Therefore, type II T4 5 -deiodinase is a site of synergism between the sympathetic nervous system and thyroid Arousal was induced by gentle subsequent disturbing hormones in the thermogenic activation of BAT [6], the hibernating animal by insertion of a rectal thermis- and is regarded as another marker of brown adipose tor. Squirrels were sacrificed after about 45 min when the rectal temperature had risen by about 6°C. tissue [20]. Rodent hibernators, which frequently exhibit body temperatures as low as 4–6°C, undergo remarkable 2.2. Brown adipose tissue rewarming during periodic arousals, with body temperature increasing to 37°C within 2–3 h [18]. Recently, After sacrifice, the bilateral axillary brown fat pads Spermophilus duaricus, a typical hibernator inhabiting (the main BAT depot in ground squirrels) were rapidly North China, has been studied in our laboratory. It was dissected, cleaned of any adhering connective tissue, confirmed that BAT was the major source of nonshiver- weighed, quickly frozen in liquid N2, then stored at 70°C until use, except that T 5%-deiodinase was ing thermogenesis induced by cold acclimation in eu- − 4 immediately assayed with about 0.3 g fresh tissue as thermic squirrels [35]. But it remains unclear how BAT described below. functions during the bouts of hibernation and arousal in this species. In the present study, we further investi- gated the thermogenic capacity of brown adipose tissue 2.3. Isolation of BAT mitochondria and assay of total as measured by mitochondrial GTP binding, and the and mitochondrial protein relation between expression of UCP mRNA and type II T 5%-deiodinase during hibernation and partial arousal. BAT mitochondria were isolated as described previ- 4 ously [14]. BAT was homogenized and samples of homogenate were taken for the measurement of total 2. Material and methods tissue protein. The remainder of the homogenate was used to prepare mitochondria. Protein in homogenates and in isolated mitochondria was determined by the 2.1. Animals Lowry’s method [15] with bovine serum albumin as standard. Daurian ground squirrels were trapped on farmland on the outskirts of Yanggao county (40°51%N, 113°72%E), Shanxi province, China, between September 2.4. GTP-binding to BAT Mitochondria and October, 1996. They were transported to the animal facility of the Biological Sciences Building, Beijing The specific number of GTP binding sites was determined from a seven or eight-point Scatchard plot of Normal University, and four to five squirrels were 3 housed in one steel wire cage at a temperature of [ H]-GTP binding in single determination, as described by Lin [13]. Briefly, protein (0.3–0.5 mg) and 0.5 to 5 2292°C and kept on a 12 L:12 D light cycle (lights on mM[3H]-GTP (2.7 mCi ml−1) and [14C]-sucrose (0.27 at 06:00) with food (for laboratory mice and rat pro- mCi ml−1) were incubated for 15 min at 25°C in total duced by Beijing Institute of Laboratory Animal, Bei- volume of 150 ml buffer containing 20 mM MOPS, 0.16 jing) and water ad libitum. mM EDTA, 20 mM Na SO , pH 6.7. Incubation was Squirrels were divided into two groups in January 2 4 terminated by centrifugation for 10 min (16000×g), 1997. Both males and females, equally divided among the supernatant was carefully aspirated and the tube each group, were used. One was the nonhibernating wall was then washed quickly and gently by using 250 9 control (n=6) which were held at 22 2°C through- ml buffer without [3H]-GTP and [14C]-sucrose again by out, and the other was the experimental group (n=8). centrifugation for 5 min. This washing supernatant was The body mass of the latter group (n=8) was moni- discarded and the pellet was suspended in 50 mlof20% tored regularly. When squirrels had increased above Triton X-100. Radioactivity of both the dissolved pellet 250 g, they were placed into individual plastic cages and the supernatant after the first centrifugation was with sawdust and transferred to a room of 492°C in counted in a liquid scintillation counter. [14C]-Sucrose total darkness with neither food nor water. The animals was used to trace the amount of contaminating super- at 4°C were checked visually every morning to deter- natant in the pellet fraction. 中国科技论文在线 http://www.paper.edu.cn X.-t. Liu et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 745–752 747 2.5. Assessment of uncoupling protein mRNA le6el buffer. The homogenized tissue was centrifuged at 800×g for 15 min at 4°C and the infranatant was 2.5.1. Oligonucleotide probe directly used to assay the activity of enzyme as previ- A 32-mer antisense oligonucleotide with a ho- ously described [12]. Protein content of the BAT in- mologous sequence specific for UCP mRNA from a franatant was quantitated as above [15]. The T4 (6.4 wide variety of species was selected [32]. The oligonu- nM) was used as substrate. Individual incubation ves- % cleotide with 5 -hydroxyl termini was synthesized in sels contained 0.1 ml infranatant and T4 in HEPES Shanghai Institute of Biochemisty (with DNA Synthe- buffer were incubated at 37°C for 60 min. At the end of sizer, Oligo 1000, Beckman) and labeled with [32P]- incubation, 2 vols. of ice-cold absolute ethanol were dATP and T4 polynucleotide kinase. added to each incubation mixture which was then vor- RNA extraction and Northern blotting analysis: texed and kept at −20°C overnight before centrifuga- Frozen BAT in liquid N2 was pulverized immediately tion at 800×g for 20 min.

Load more