Increased response to noradrenaline of isolated brown adipocytes

BIOCHEMICAL SOCIETY TRANSACTIONS

282 while the oxidation of saturating concentrations of D,-isocitrate is less sensitive. This data indicate that primary flux control of palmitoyl-L-carnitine oxidation in uncoupled BAT mitochondria resides in Complex I of the respiratory chain, with a flux control coefficient (CAi) (Westerhoff et al., 1984) near to 1.0. However, since matrix [NADH] does not continuously rise during steady-state fatty acylcarnitine oxidation, this flux control might operate through the vehicle of another factor, in equilibrium with the flux across Complex I (possibly the [NADH] itself). This factor would then control flux through discrete steps in the fatty acid oxidation pathway, before the respiratory chain.

Alexander, G. (1979) in Environmental Physiology 111 (Robertshaw, D., ed.), vol. 20, International Review of Physiology, pp. 4 6 1 55, University Park Press, Baltimore Flatmark, T. & Pedersen, J. I. (1975) Biochim. Biophys. Acta 416, 53-103 McCormack, J. G. & Denton, R. (1980) Biochem. J . 190, 95-105 Nicholls, D. G. (1979) Biochim. Biophys. Acta 549, 1-29 Nicholls, D. G. & Garland, P. B. (1969) Biochem. J . 114, 115-125 Normann, P. T., Ingebretsen, 0. C. & Flatmark, T. (1978) Biochim. Biophys. Acta 501, 286295 Ryberg, D., Berg, K. & Grav, H. J. (1981) Biochem. SOC.Trans. 9, 294P Westerhoff, H. V., Groen, A. K. & Wanders, R. J. A. (1984) Biosci. Rep. 4, 1-22

Increased response to noradrenaline of isolated brown adipocytes from guinea pigs during cold-adaptation JOHANNES RAFAEL, WALTRAUD FESSER and DAVID G. NICHOLLS Institut fur Biochemie I, Universitat Heidelberg, 0-6900 Heidelberg, Federal Republic of Germany, and Neurochemistry Laboratory, Department of Psychiatry, University of Dundee, Dundee DD1 95Y, U.K. The thermogenic capacity of brown adipose tissue depends on developmental, temperature or dietary stimuli (reviewed by Nicholls & Locke, 1984). Nonshivering thermogenesis in the guinea pig decreases rapidly after birth at thermoneutral temperature and can subsequently be greatly enhanced by cold-adaptation (Briick, 1967). This is reflected in the brown adipose tissue as a major source of non-shivering heat production: cold-adaptation is accompanied by increased respiratory capacity in the tissue and loss of mitochondrial respiratory control (Rafael et al., 1968). The amount of 32 kDa mitochondrial membrane protein mediating the specific proton-conductance pathway in brown adipose tissue (Nicholls & Locke, 1984) is increased (Rial & Nicholls, 1984). Adipocytes were isolated from the combined brown fat deposits of 4-week-old Dunkin-Hartley strain guinea pigs kept at thermoneutral temperature (28°C) and during 1-21 days of adaptation to 5°C. The method of brown adipocyte isolation and incubation described earlier (Locke et al., 1982) was modified: Ficoll was omitted from the medium and the collagenase digestion was limited to 45 min; pyruvate was added in respiratory experiments. Amounts of adipocytes in the tissue and mitochondrial protein in brown fat and cells were calculated from the specific cytochrome oxidase activity. In light microscopy, cells from thermoneutral controls (W-cells) appear almost exclusively unilocular, whereas 80% of the cells are multilocular after 3 weeks of coldadaptation (C-cells). The volume per cell decreases by 7&75% during cold-adaptation. The mitochondrial protein content per adipocyte is doubled in C-cells if compared with W-cells. This is reflected in the basal respiration of the cells as well as in respiration maximally released by FCCP (Fig. I). Respiration is controlled both in W-cells and in C-cells in the presence of sufficient external respiratory substrate (pyruvate), thus confirming recent results from measuring the membrane potential dependent uptake of a cationic fluorescent dye by brown adipocytes (Rafael & Nicholls, 1984). Respiration Abbreviation used: FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

of W-cells is twofold increased when stimulated with noradrenaline, but ten-fold enhanced by the addition of FCCP. Respiration a.ttained in the presence of noradrenaline increases rapidly during the first days of coldadaptation, and after 7 days is no longer limited by mitochondrial respiratory control, i.e. noradrenalineinduced uncoupling is constrained only by the maximal respiratory capacity of the mitochondria (Fig. I). Guinea-pig C-cells are thus clearly different from adipocytes isolated from cold-adapted rats (Bukowiecki et al., 1981) and hamsters (Nedergaard & Lindberg, 1979), which have been found to be less responsive to noradrenaline than cells from warm-adapted animals. Guinea-pig brown adipocytes are a good model to test cold-adaptation of brown adipose tissue on a cellular level. The specific cellular respiration released by noradrenaline is increased by a factor of 12 after 3 weeks of cold-adaptation (Fig. I). Since the total number of brown adipocytes in the guinea pig is approximately

, 0 '

2

4

7

14

21

Time of cold-adaptation (days)

Fig. 1. Effect of cold-adaptation on the respiration of guinea-pig brown adipocytes

Cells (45 000/ml) were incubated at 37°C. 0 , Control; 0,(+)noradrenaline ( 2 p ~ ) B, ; (+)-noradrenaline ( 2 p ~ ) FCCP (10 p ~ )Results . are means fS.E.M. from five to 12 experiments. Error bars not shown are smaller than the symbols.

+

1986

283

614th MEETING, OXFORD doubled concomitantly, the total noradrenalinestimulated heat production in brown fat is increased by a factor of 24. The conductance of the uncoupling pathway in isolated brown fat mitochondria is proportional to the content of uncoupling protein which shows a seven-fold increase after 3 weeks of cold-adaptation (Rial & Nicholls, 1984). The increase of noradrenaline-stimulated respiration in brown adipocytes correlates with the induction of uncoupling protein in the mitochondria. The authors wish to thank the Royal Society, the Deutsche

Forschungsgemeinschaft, Federal Republic of Germany, and the British Medical Research Council for supporting this work. Briick, K. (1967) Naturwissenschaften 54, 156-161 Bukowiecki, L. J., Follea, N., Lupien, J. & Paradis, A. (1981) J. Biol. Chem. 256, 1284CI 2848 Locke, R. M., Rial, E. & Nicholls, D. G. (1982) Eur. J . Biochem. 129, 381-387 Nedergaard, J. & Lindberg, 0. (1979) Eur. J . Biochem. 95, 139-145 Nicholls, D. G. & Locke, R. M. (1984) Physiol. Rev. 64,1-64 Rafael, J., Klaas, D. & Hohorst, H. J. (1958) Hoppe Seyler’s Z. Physiol. Chem. 349, 1711-1724 Rial, E. & Nicholls, D. G. (1984~)Biochem. J . 222, 685493 Rafael, J. & Nicholls, D. G. (19846) FEBS Lett. 170, 181-185

Decrease in mitochondrial GDP binding without parallel changes in uncoupling protein in brown adipose tissue of the guinea pig MARGARET ASHWELL, GRAHAM JENNINGS, DOROTHY M. STIRLING and PAUL TRAYHURN Dunn Nutrition Laboratory. Medical Research Council and University of Cambridge, Downham’s Lane, Milton Road, Cambridge CB4 I X J , U.K. Brown adipose tissue contains a tissue-specific protein, M, 32 000, which regulates a proton conductance pathway across the mitochondrial inner membrane (Nicholls & Locke, 1984). The activity of the proton conductance pathway has been widely assessed by determining the extent to which brown adipose tissue mitochondria bind radioactively labelled purine nucleotides, particularly GDP, in vitro. There is, however, concern as to whether binding studies are an index of the activity of the proton conductance pathway or whether such studies are a measure of the amount of uncoupling protein. Evidence for a dissociation between the level of GDP binding and the amount of uncoupling protein was first observed in rats during acute cold exposure or following the administration of noradrenaline (Desautels et al., 1978; Desautels & Himms-Hagen, 1979). In these studies rapid increases in GDP binding were obtained without apparent changes in the concentration of uncoupling protein, and it was consequently suggested that GDPbinding sites could be ‘masked’. However, uncoupling protein was quantified on the basis of the size of the bands in the M, 32000 region after separation of mitochondrial proteins by polyacrylamide electrophoresis with sodium dodecyl sulphate, and this is neither sensitive nor specific. Recent studies using a radioimmunoassay to quantify the amount of uncoupling protein in brown adipose tissue of young oblob mice and fa/’@ rats has provided clear support for a dissociation between GDP binding and the concentration of uncoupling protein (Ashwell et al., 1985). In contrast, other recent studies have reported parallel changes in binding and the amount of uncoupling protein after acute exposure of rats to cold (Nedergaard & Cannon, 1985). It has also been argued that guinea pigs, in particular, do not exhibit any masking/unmasking of GDP-binding sites (Rial & Nicholls, 1984), but an independent measure of uncoupling protein has not been employed with this species. In the present study GDP binding and uncoupling protein have been measured in cold-acclimated guinea pigs exposed acutely to the warm, uncoupling protein being determined by radioimmunoassay. Sixteen guinea pigs, aged 1-2 months, were caged singly in metal cages for 3 4 weeks at 4”C, with a 12 h light/l2 h dark cycle (lights from 07.00 h). Half of the

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guinea pigs were transferred to plastic cages and housed overnight (17h) at 26°C. The animals were killed by cervical dislocation and interscapular brown adipose tissue removed. The tissue was homogenized and samples taken for the measurement of tissue protein and total cytochrome oxidase activity (Goodbody & Trayhurn, 1981). The bulk of the homogenate was used to prepare mitochondria. Mitochondria1 GDP binding and the radioimmunoassay of uncoupling protein were performed as previously (Goodbody & Trayhurn, 1981; Lean et al., 1983). A rabbit anti-guinea pig uncoupling protein serum was employed in the radioimmunoassay, together with a guinea pig protein standard. The results in Table 1 show that warm exposure for 17 h had no significant effect (P > 0.05) on the weight of interscapular brown adipose tissue, nor was there any effect on the protein content and total cytochrome oxidase activity of the tissue. GDP binding was, however, significantly decreased after exposure to the warm, and this change took place without any alteration in the concentration of uncoupling protein. Scatchard analysis of GDP-binding data showed similar affinities for the warm exposed animals (Kd2.2 PM) and the animals maintained at 4°C (Kd 2 . 8 ~ ~ ) . It is concluded that guinea pigs can exhibit the same dissociation between the level of GDP binding and the concentration of uncoupling protein as has been reported for rats and mice. It is also concluded that unmasking of GDP-binding sites is likely to reflect a conformational

Table 1. Brown adipose tissue in cold-acclimated (4°C) guinea pigs exposed acutely ( 1 7 h ) to the warm (26°C) For experimental details see the text. The results are given as mean values ~ s . E . M .for eight determinations in each group except for uncoupling protein where four determinations were made. * P < 0.001 compared with cold-acclimated group. Body wt. (g) Interscapular brown adipose tissue wt. (8) Brown adipose tissue protein content (mg) Cytochrome oxidase activity (pmol of cytochrome c oxidized/min per tissue) GDP binding (pmol/mg of mitochondrial protein) Uncoupling protein (pg/mg of mitochondrial protein)

Cold-acclimated

Warm-exDosed

379.3 17.3 2.32 & 0.19

398.6 & 22.2 2.67 & 0.30

63.3 & 5.4

69.3 & 7.5

308.2 k 18.1

268.6 & 18.3

505.6

333.8

30.7

23.5 & 3.1

k 26.2*

22.8 & 1.7