Differentiation of brown adipose tissue and biogenesis of thermogenic mitochondria in situ and in cell culture

243

Biochimica et Biophysica Acta, 1018 (1990) 243-247 Elsevier BBAEBC 00026

Differentiation of brown adipose tissue and biogenesis of thermogenic mitochondria in situ and in cell culture J o s e f Hou~tEk, J a n K o p e c k S , M a r i e Baudy~ovfi, D a g m a r Janikovfi, S t a n i s l a v P a v e l k a and Petr Klement Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Received 1 May 1990)

Key words: Brown adipose tissue; Thermogenesis; Differentiation; Mitochondrion; Biogenesis;Uncoupling protein

Differentiation and biogenesis of mitochondria in brown adipose tissue (BAT) was studied in situ and in cell culture by Western blotting, enzyme activity measurements, [3SSlmethionine incorporation and immunofluorescence microscopy. In different rodent species the perinatal development of BAT thermogenic function resulted from the formation of thermogenic mitochondria which replaced the preexisting nonthermogenic mitochondria. Their biogenesis was characterized by the sudden appearance and rapid increase of the uncoupling protein (UCP), increase of cytochrome oxidase (COX) and decrease of H +-ATPase. In primary cell culture, differentiation of precursor cells from mouse BAT to typical multilocular adipocytes was accompanied by increasing content of COX and H +-ATPase. A selective synthesis of UCP was induced by activation of fl-adrenergic receptors or by elevated levels of cellular cAMP. UCP was quantitatively incorporated into mitochondria and within 24 h after stimulation reached near physiological concentration. Both in situ and in cell culture, the conditions enabling the expression of UCP gene were accompanied by activation of intracellular thyroxine 5'-deiodinase.

Introduction Thermal homeostasis of newborn mammals largely depends on thermogenesis in brown adipose tissue [1,2] which results from physiological H÷-short-circuiting of the mitochondrial membrane via the tissue-specific uncoupling protein [1,3]. The typical thermogenic mitochondria of functionally active BAT are characterized by high oxidative capacity, low ATPase activity and high content of the UCP [3,4]. BAT belongs to the most plastic mammalian tissues, and the long-term adaptive changes as well as the recruitment of BAT thermogenic potential are under hormonal control in which catecholamines, triiodothyronine and other hormones are probably involved (for review see Refs. 2 and 5). One of the main regulatory mechanisms appears to be fast and pronounced changes of the mitochondrial membrane

Abbreviations: UCP, uncoupling protein; BAT, brown adipose tissue; COX, cytochrome oxidase; F1, catalytic part of mitochondrial H +ATPase; SDS, sodium dodecyl sulfate. Correspondence: J. Hou~t~q¢, Institute of Physiology, Czechoslovak Academy of Science, Videtisk~ 1083, 14220 Prague 4, Czechoslovakia.

[1,2,5-8], where the content of UCP may serve as a sensitive marker of the thermogenic function and of terminal differentiation of brown adipocytes [6-8]. In this report we summarize our recent investigations (see also Refs. 7-9) which show that both in situ and in cell culture biogenesis of BAT thermogenic mitochondria results from highly differentiated synthesis of individual proteins. The major regulatory role exert catecholamines acting via fl-adrenergic receptors. Materials and Methods Interscapular BAT from mice (Balb/c), rats (Wistar I pav), and golden Syrian hamsters (Mesocricetus auratus) of the required age were used for the preparation of homogenates [7,8], isolation of mitochondria [10], and for morphological studies [7,8]. Stromal-vascular cells from BAT of 3-4-week-old mice were cultivated [9] in a modified ~11] Eagle's minimal essential medium containing Earl/~'s salts and supplemented with fetal calf serum, gentamicin, insulin, ascorbate, glutamine and glucose [9]. Cell counting, harvesting and measurements of [35S]methionine incorporation were performed as previously described [9]. When indicated, noradrenaline, isoproterenol or dibutyryl-cAMP were added to the cultivation medium.

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244 Immunodetection of mitochondrial antigens by Western blotting [7], fluorography [12] of samples separated by SDS-polyacrylamide gel electrophoresis [13], measurements of thyroxine 5'-deiodinase [14] and protein determination [15] were performed as described. Immunofluorescence microscopy of cytoskeletal and mitochondrial proteins was performed after permeabilization of cells with Triton X-100 [16]. Specific rabbit antisera [7] to hamster UCP, rat heart COX and bovine heart F 1 and monoclonal antibodies to tubulin (Tu 01) [16] were used. Results and Discussion

Perinatal recruitment of B A T thermogenic function In both mouse and rat the thermogenic ability of BAT develops prenatally [2]. During the last week of fetal development rapid growth of BAT in mouse was accompanied by morphological differentiation of adipocytes, increase in number and complexity of mitochondria and pronounced changes in mitochondrial enzymes (Fig. 1 and Ref. 7). While the content of COX continuously increased between the 16th day of pregnancy and birth, F1-ATPase had already reached the maximum level on the 19th day. On the same day the UCP appeared for the first time and rapidly increased in next few days. The differing appearance and quantitative development of the three proteins reflecting the oxidative (COX), phosphorylating (ATPase) and thermogenic (UCP) capacities of BAT indicated remarkable asynchrony in the synthesis of individual mitochondrial proteins. Consequently, the typical thermogenic mitochondria appeared in mouse BAT (and similarly in rat BAT) 2 days before birth. Hamsters are born relatively immature [2,6] and the thermogenic function of BAT develops rather late [2,6,8]. During the first 2 postnatal weeks BAT showed intensive growth and differentiation of unilocular adipocytes

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with few small mitochondria, into multilocular adipocytes filled with numerous mitochondria containing multiple cristae [8]. Both in homogenates (Fig. 1) and in isolated mitochondria COX and F1-ATPase antigens were present during the first week while UCP appeared for the first time on the 8th day and then rapidly increased, reaching maximum on the 17th day. Also during this interval the content of COX increased rapidly, while ATPase content decreased. Quantification by Western blotting was fully confirmed by immunoelectron microscopy and by enzyme activity measurements [8]. The biogenesis of thermogenic mitochondria, thus, had very similar character in all species tested. BAT contained first less differentiated mitochondria with comparable amounts of COX and H ÷ATPase, qualitatively not different from mitochondria of phosphorylating tissues. The transformation of BAT energetics into the thermogenic mode apparently resulted from pronounced qualitative changes of the inner mitochondrial membrane, where UCP replaced H +ATPase, both structurally and functionally. In this process, different types of mitochondrion may exist in parallel and replace one another, and/or transformation of already existing nonthermogenic mitochondria may occur. As documented at transcriptional and translational levels [17-20], the timing of critical event of biogenesis, the initiation of the UCP synthesis is species-specific. The increase of specific mRNA for UCP and its activated synthesis, as well as the synthesis of several other proteins also require active 5'-deiodination of thyroxine directly in BAT (type II enzyme [21,22]). As shown in Fig. 2, in developing hamster the activation of thyroxine 5'-deiodinase corresponded closely with the onset and increase of UCP synthesis. Similarly, the appearance of UCP in BAT of rat and bovine correlated with the activation of 5'-deiodinase [23,24] and indicated the importance of active 5'-deiodination of thyroxine for biogenesis of thermogenic mitochondria.

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Differentiation of B A T in cell culture I n order to characterize biogenesis of thermogenic m i t o c h o n d r i a u n d e r controlled conditions, the differe n t i a t i o n of precursor cells from m o u s e B A T was studied in p r i m a r y cell culture [9]. After 1 day of cultivation the

cell p o p u l a t i o n consisted m a i n l y of u n d i f f e r e n t i a t e d interstitial ceils a n d preadipocytes. After 3 days cells showed rapid p r o l i f e r a t i o n a n d started to differentiate f o r m i n g p o l y h e d r a l cells (Fig. 3A). At confluence, a r o u n d day 6, almost all the cells had the typical

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Fig. 3. Immunofluorescencemicroscopy of mouse BAT cells differentiating in cell culture. Morphology of cells cultivated for 3 days (A-C) and for 6 days (D-F) was revealed by Nomarski interference contrast microscopy (A and D); and by immunofluorescence microscopy using rabbit antibody to COX (1:50, C and E) or to UCP (1:100, F) and FITC-labeled secondary antibody (swine anti-rabbit, 1:50). In B, simultaneous labeling of cells from C with monoclonal antibody to tubulin (1 : 50) and TRITC-labeled secondary antibody (swine anti-mouse, 1 : 50) is shown. In F, 1 #M noradrenaline was present for last 24 h of cultivation. Magnification was 780 × (B, C, E and F) or 200 × (A and D). Bar indicates 50 #m.

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Fig. 4. Catecholamine-induced synthesis of the uncoupling protein in mouse BAT cell culture. Cytochrome oxidase (COX), F1-ATPase (F1) and the uncoupling protein (UCP) were detected by Western blotting of whole cell extracts of mouse BAT stromal vascular cells (SV cells; 6 and 14 #g protein) used for inoculation and in cells cultivated for 8 days (15 btg protein). Isoproterenol (ISO) at 0.1 and 10 #M concentration was present 24 h before harvesting.

observed in C O X and F 1 antigens (Fig. 4). The optimal concentration of noradrenaline was a r o u n d 0.1/xM and isoproterenol was equally effective (Fig. 4), indicating that stimulation of t - r e c e p t o r s was involved in the induction of U C P synthesis. After 24 h the a m o u n t of U C P synthesized reached about the level of U C P f o u n d in B A T of moderately cold-stressed (22 ° C) mouse and almost all U C P was recovered in the crude mitochondrial fraction (12 000 × g sediment) indicating incorporation of U C P into mitochondria. The data were further confirmed by immunofluorescence microscopy (Fig. 3) which detected large quantities of U C P antigens in noradrenaline-stimulated confluent cells. Synthesis of U C P was also induced b y the presence of 1 m M dib u t y r y l - c A M P in cultivation m e d i u m for 24 h. In parallel, d i b u t y r y l - c A M P caused m o r e than 30-fold activation of 5'-deiodinase of thyroxine (from 0.005 + 0.001 to 0.158 + 0.005 p m o l / h per mg protein). In conclusion, in b o t h model systems, the biogenesis of thermogenic m i t o c h o n d r i a exhibited similar features. The most i m p o r t a n t ones are: (i) highly differentiated synthesis of individual mitochondrial proteins, including those that are c o m p o s e d of subunits of mitochondrial and nuclear origin; (ii) selective synthesis of U C P which is controlled b y fl-adrenergic receptors and requires active 5'-deiodination of thyroxine in the cell; and (iii) high turnover of U C P in which a selective degradation of U C P m a y be involved. References

appearance of multilocular adipocytes (Fig. 3D). The rate of protein synthesis, determined as [35S]methionine incorporation into cell proteins, was the highest at the exponential phase of growth (day 3) and about half of that in confluent cells. The pattern of the labeled proteins observed after the addition of cycloheximide suggested intensive synthesis of mitochondrially-encoded proteins. After 4 h of incubation with noradrenaline or isoproterenol, the total rate of protein synthesis and the labeling of most proteins, the fl-subunit of F a included, significantly decreased. However, the labeling of a few proteins was enhanced after the h o r m o n e addition. A m o n g them was the U C P of M r 32 kDa, which indicated that its synthesis was activated by catecholamines. The specific content of mitochondrial proteins changed substantially during cell differentiation. The a m o u n t of C O X increased continuously until day 6 (see i m m u n o fluorescence microscopy in Fig. 3). W h e n determined b y Western blotting (Fig. 4), a high content of C O X and also of Fa-ATPase were f o u n d a r o u n d confluence, indicating that synthesis of the latter enzyme was suppressed less than in vivo [4]. In contrast, the content of U C P was very low, unless high inoculation densities were used. After the addition of catecholamines the U C P antigen rapidly increased, while no change was

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