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Volume 150, number 1 FEBS LETTERS December 1982

Exclusive occurrence of thermogenin antigen in brown

Barbara Cannon, Anders Hedin* and Jan Nedergaard

Departments of Metabolic Research and *Immunology, The Wenner-Gren Institute, University of Stockholm, Norrtullsgatan 16, 113 45 Stockholm, Sweden

Received 25 October 1982

Thermogenin is the purine-nucleotide binding polypeptide in mitochondria (Mr 32000) which confers upon these mitochondria the ability to produce heat. An enzyme-linked immunosorbent assay (ELISA) has been developed to demonstrate and quantitate the occurrence of thermogenin antigen in small amounts of tissue, and thus to characterize different depots of fat tissue as white or brown. The extreme sensitivity of the method allows determination of thermogenin in samples equivalent to < 1 mg tissue. The results indicate that thermogenin seems to be exclusively localised in brown fat mitochondria (as compared to white fat, liver or heart muscle mitochondria), and thermogenin antigen could only be found in brown adipocytes (as compared to white adipocytes). Thus, brown and white adipose tissue are probably ontogenetically different

Brown adipose tissue ELISA Mitochondria Nonshivering Thermogenin White adipose tissue

1. INTRODUCTION form of white adipose tissue, or whether it should be considered as a bona fide organ in itself, with its The demonstration that the thermogenic func- own ontogeny [lo]. tion of brown adipose tissue is directly related to The present interest in the connection between the presence in the mitochondria of a specific poly- the activity of brown adipose tissue and the ten- peptide - thermogenin - with a subunit A4, 32000 dency to evolve obesity in animals and man (review has made it possible to study thermogenesis at the [l 11) has made it necessary to be able to charac- molecular level [1,2], (reviews [3-51). terize different depots of fat tissue in terms of Thermogenin binds purine nucleotides (e.g., being white or brown. GDP) with a high affinity [6] and the estimation of We therefore found it necessary to develop a this binding has become the parameter most widely technique which could identify thermogenin even used to quantitate the state of the tissue [7]. in an inactive form, and which would allow quanti- However, there are indications that thermogenin fication of thermogenin in small amounts of tissue. may appear in an inactive form which may, e.g., be We present here results obtained with an en- activated by [S]. Further, the possi- zyme-linked immunosorbent assay system (ELISA) bility exists that thermogenin may occur in small which show that it is possible to quantitate the amounts or in an inactive form in tissues other amount of thermogenin in small samples. The re- than brown fat where, under certain conditions, sults indicate that thermogenin antigen is not some characteristics resembling those of brown fat found in tissues other than brown adipose tissue, mitochondria can be evoked [9]. not even in white, and thus brown adipose tissue Particularly, in the case of the distinction be- must be considered to be a distinct tissue, and tween brown and white adipose tissue, it has been probably the sole site where facultative non- discussed whether brown fat is but an ‘immature’ shivering thermogenesis occurs in the mammal.

Published by Elsevier Biomedical Press 00145793/82/0000-0000/$2.75 0 Federation of European Biochemical Societies 129 Volume 150, number 1 FEBS LETTERS December 1982

2. MATERIALS AND METHODS blood had coagulated overnight at 5°C. Control serum was obtained from rabbits which 2.1. Isolation of mitochondria had only been injected with Freund’s complete ad- Brown fat mitochondria were prepared from the juvant in buffer. pooled brown adipose tissue of cold-acclimated Serum was stored at -20°C until use. golden hamsters as in [ 121. They were stored in 0.25 M sucrose. 2.5. Indirect enzyme-linked immunosorbent assay White fat mitochondria were prepared from the (ELISA) for demonstration of thermogenin epididymal fat pads of cold-acclimated golden An indirect ELISA was performed as in [19] hamsters as in [13]. They were stored in 0.25 M using as conjugate sheep anti-rabbit immunoglobin sucrose, 10 mM Tris-HCI (pH 7). G conjugated to alkaline phosphatase. Thermo- Liver mitochondria were prepared from the genin, solubilised mitochondria or solubilised cells livers of cold-acclimated golden hamsters and from (in 0.05% Tween 20 and 5 mM mercaptoethanol) rats living at 21 “C as in [14,15] and stored in were allowed to coat plastic micro-ELISA plates 0.25 M sucrose. and these were evaporated to dryness. After Heart mitochondria were prepared from ox washing, the rabbit antiserum was added, the hearts obtained from a local slaughterhouse as in plates incubated and washed, and thereafter in- [16] and stored in 0.25 M sucrose. cubated with the conjugate. After further washing, the substrate for alkaline phosphatase, p- 2.2. Isolation of cells nitrophenylphosphate, was added and the plates Isolated brown fat cells from the pooled brown incubated until a satisfactory extinction was meas- adipose tissue of golden hamsters living at 21°C ured at 405 nm (routinely 30-45 min). were prepared as in [5] by the use of a collagenase In some cases a competitive indirect ELISA was digestion method. also performed. In these cases, thermogenin- Isolated white fat cells from the omental and coated plates were incubated with rabbit antiserum mesenteric white adipose tissue of golden hamsters containing known amounts of thermogenin, solu- living at 21°C were prepared as in [17] by the use bilised mitochondria or solubilised cells, and there- of a collagenase digestion method. after the assay was performed as above. Ascites cells were kindly provided by Dr Bertil Pettersson and washed thoroughly by centrifuga- 2.6. Protein tion before use. Protein was determined by the biuret method (mitochondria) or by the Lowry method [20]. 2.3. Isolation of thermogenin Thermogenin was isolated from the brown fat 2.7. Sodium dodecylsulphate-polyacrylamide gel mitochondria of 6 cold-acclimated golden ham- electrophoresis (SDS-PAGE) sters as in [18], except that in the final centrifuga- SDS-PAGE was run according to Weber and tion the detergent was exchanged for 0.1% Tween Osborne [21] or Laemmli [22] in 10% polyacryl- 20 and 5 mM mercaptoethanol was added. The iso- amide. The gels were stained with Coomassie blue. lated protein was stored at lo-20pg/ml at -20°C and used to prepare antiserum and in the immuno- logical assays. 3. RESULTS AND DISCUSSION

2.4. Preparation of anti-thermogenin antiserum 3.1. Purity of the antigen Rabbits were injected with -15 pg thermogenin Thermogenin, prepared by the hydroxyapatite antigen mixed with Freund’s complete adjuvant in method [ 181, even in heavily overloaded SDS- the thermogenin isolation buffer. Booster injec- PAGE gels showed only one band (not shown), tions were given after 3 and 4 weeks and the rabbits indicating that this is the only major protein in the were bled from the ear vein 1 week later. There- preparation. Although the presence of very minor, after booster injections were given every 6 weeks. highly antigenic contaminants cannot be entirely Serum was obtained by centrifugation after the excluded, their presence seems unlikely.

130 Volume 150, number 1 FEBS LETTERS December 1982

Fig. 1. Dose-response curve of anti-thermogenin anti- serum in indirect ELISA. Micro-ELISA plates were I . 102 10-L 1U3 10-z 101 coated with 25-50 ng thermogenin/hole, incubated for sample dll”tlm 3 h at 37”C, dried and further coated with 1% bovine serum albumin. After washing, the plates were incubated Fig. 2. Inhibition of thermogenin/anti-thermogenin with serially diluted serum, followed by conjugate as in ELISA by varying concentrations of isolated mitochon- section 2. Incubation with substrate was for 30 min at dria from different tissues or by thermogenin. Micro- room temperature. ELISA plates were coated with thermogenin as above. Antiserum and control serum were used at a dilution of 1: 1000, and mixed with varying concentrations of mito- chondria and thermogenin. Thermogenin was here ini- 3.2. Titration of anti-thermogenin antiserum tially at 10 pg/ml, and the mitochondria at 1.5 mg/ml. against thermogenin in indirect ELISA The mitochondria were solubilised in 0.9% NaCl, 0.05% Tween 20, 5 mM mercaptoethanol. Incubation with Fig. 1 demonstrates the reactive strength of the substrate was for 45 min at room temperature. WAT and anti-thermogenin antiserum against thermogenin BAT indicate mitochondria from white and brown adi- in indirect ELBA. High activity was found at pose tissue, respectively; (- ) incubations with anti- serum dilutions of 1: 1000 and no activity was thermogenin antiserum; (- - -) incubations with control found with control serum. serum.

3.3. Demonstration of thermogenin antigen in brown fat mitochondria In fig. 2 the results of competitive indirect tained; thus the values obtained here are in good ELBA are shown, clearly demonstrating the pre- agreement with those from other studies, indi- sence of the antigen in brown fat mitochondria but cating that this method can be used as a sensitive its absence in white fat mitochondria and liver assay for the presence of thermogenin antigen mitochondria from hamster. At high concentra- (C 100 ng protein is required for quantitive results). tions, white fat mitochondria gave a slight reaction which is probably unspecific, but at least indicates 3.4. Demonstration of thermogenin antigen in that the amount of thermogenin is 100-1000 times brown fat cells lower in white fat mitochondria than in brown fat Isolated brown fat cells also reacted well with mitochondria. anti-thermogenin antiserum as shown in fig. 3. Ox heart mitochondria and rat liver mitochon- Here an equivalent number of white fat cells did dria gave entirely negative results (not shown). not give any specific response. Ascites cells were Determination of the concentration of ther- also completely without effect (not shown). mogenin antigen in brown fat mitochondria by The results above indicate a concentration of comparison with isolated thermogenin revealed thermogenin in the cells of -2-10pg/106 cells. 50- 100 pg thermogenin/mg mitochondrial pro- This can be compared with an estimated presence tein. This value should be compared with the one of - 1 mg mitochondria/ lo6 cells [25] and a nucleo- obtained from a calculation based upon a dimeric tide-binding value of -0.4 nmol in brown fat mito- Mr of 64000 [23] and a nucleotide-binding value of chondria from control hamsters [24], resulting in 0.8 nmol/mg protein in these mitochondria [24]. an expected concentration of 25 ,ug/106 cells. It Through this a concentration of -55 pg/mg is ob- should be noted that with this system the presence

131 Volume 150, number 1 FEBS LETTERS December 1982

REFERENCES

111 Ricquier, D. and Kader, J.-C. (1976) Biochem. Bio- phys. Res. Commun. 73, 577-583. PI Heaton, G.M., Wagenvoord, R. J., Kemp, A. jr and Nicholls, D.G. (1978) Eur. J. Biochem. 82, 515-521. (31 Nicholls, D.G. (1979) Biochim. Biophys. Acta 549, l-29. 141 Cannon, B., Nedergaard, J. and Sundin, U. (1981) in: Survival in the Cold (Musacchia, X.J. and Jansky, L., eds) pp. 99-120, Elsevier Biomedical, Amsterdam, New York. PI Nedergaard, J. and Lindberg, 0. (1982) Int. Rev. Cytol. 74, 187-286. [61 Nicholls, D.G. (1976) Eur. J. Biochem. 62, 223- Fig. 3. Indirect ELISA of brown and white fat cells from 228. hamster. 20000 solubilised cells were used/hole and were 171 Cannon, B. and Nedergaard, J. (1983) J. Thermal incubated and dried as thermogenin in fig. 1, and the Biol. in press. ELISA carried out as in that figure. Incubation with sub- PI Desautels, M. and Himms-Hagen, J. (1979) Can. J. strate was for 30 min at room temperature. WAT and Biochem. 57, 968-976. BAT indicate cells from white and brown adipose tissue, 191 Selwyn, M. J., Dawson, A.P. and Fulton, D.V. respectively; (- ) incubations with anti-thermogenin (1979) Biochem. Sot. Trans. 7, 216-219. antiserum; (- - -) incubations with control serum. The [lOI Hahn, P. and Novak, M. (1975) J. Lipid Res. 16, points shown are means f SE from 3 expt on as many 79-91. different preparations, and each performed in duplicate. 1111 Himms-Hagen, J. (1979) Can. Med. Ass. J. 121, 1361-1364. WI Cannon, B. and Lindberg, 0. (1979) Methods En- zymol. 55, 65-78. 1131 Martin, B.R. and Denton, R.M. (1970) Biochem. J. of thermogenin can be demonstrated with < 100000 117, 861-877. cells (equivalent to Cl mg tissue). 1141 Hogeboom, G.H., Schneider, W.C. and Palade, G.H. (1948) J. Biol. Chem. 172, 619-635. WJI Nedergaard, J. and Cannon, B. (1979) Methods 4. CONCLUSIONS Enzymol. 55, l-28. Thermogenin seems to be exclusively localised in 1161 Low, H. and Vallin, I. (1963) Biochim. Biophys. brown fat mitochondria. There is no evidence of Acta 69, 361-374. any latent form of the antigen in any other tissue. [I71 Fain, J.N. (1975) Methods Enzymol. 35, 555-561. Lin, C.S. and Klingenberg, M. (1980) FEBS Lett. The sensitive ELISA technique can be used to 1181 113, 299-303. identify and quanify the thermogenin antigen in [I91 Engvall, E. and Perlmann, P. (1972) J. Immunol. small samples. 109, 1299135. The absence of any thermogenin antigen in epi- PO1 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and didymal, omental or mesenteric white adipose Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. tissue would support the contention that white adi- WI Weber, K. and Osborne, M. (1969) J. Biol. Chem. pose tissue and brown adipose tissue are separate 244, 440664412. organs with different ontogeny. PI Laemmli, U.K. (1970) Nature 227, 680-685. 1231 Lin, C.S., Hackenberg, H. and Klingenberg, M. (1980) FEBS Lett. 113, 304-306. ACKNOWLEDGEMENTS [241 Sundin, U. Moore, G. and Cannon, B. (1982) sub- mitted. The authors thank Agneta Bergstrom and Barbro PI Lindberg, O., Bieber, L.L. and HouSttk (1976) in: Svensson for technical assistance. This work was Regulation of Depressed Metabolism and Thermo- supported by a grant from the Swedish Natural genesis (Jansky, L. and Musacchia, X.J. eds) Science Research Council. pp. 117-136, Thomas, Springfield OH.

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