Decrease in mitochondrial GDP binding without parallel changes in ...

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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

BIOCHEMICAL SOCIETY TRANSACTIONS

284 change in uncoupling protein linked to the activation of thermogenesis. Ashwell, M., Holt, S., Jennings, G., Stirling, D. M., Trayhurn, P. & York, D. A. (1985) FEBS Lett. 179, 233-237 Desautels, M. & Himms-Hagen, J. (1979) Can. J. Biochem. 56,378-383 Desautels, M., Zaror-Behrens, G. & Himms-Hagen, J. (1978) Can. J .

Biochem. 56, 378-383 Goodbody, A. E. & Trayhurn, P. (1981) Biochem. J. 194, 1019-1022 Lean, M. E. J., Branch, W. J., James, W. P. T., Jennings, G. & Ashwell, M. (1983) Biosci. Rep. 3, 61-71 Nedergaard, J. & Cannon, B. (1985) Am. J. Physiol. 248, C3654371 Nicholls, D. G. & Locke, R. M. (1984) Physiol. Rev. 64, 1 4 Rial, E. & Nicholls, D. G. (1984) Biochem. J . 222, 685-693

Rates of lipogenesis in isolated brown adipocytes from newborn rats CESAR RONCERO, MATILDE PERAS, M. J. DOMINGUEZ, MARGARITA FERNANDEZ and MANUEL BENITO* Departamento de Bioquimica, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040-Madrid, Spain During late foetal life, the rate of lipogenesis decreases in liver and increases in brown adipose tissue (Lorenzo et al., 1981; Pillay & Bailey, 1982). At birth, the rate of lipogenesis in brown fat becomes maximally stimulated, decreasing sharply during the first hour of life (Benito et al., 1984). The injection of glucose fails to increase the lipogenic rate 1 h after birth even though peak plasma insulin concentration is induced (Benito et al., 1984). In addition, we have developed a method for isolation of foetal adipocytes as a tool for metabolic studies (Elliott et al., 1984). Accordingly, the aim of this work was to investigate the rate of lipogenesis in isolated adipocytes from foetal rats and its short-term regulation by hormones like insulin, adrenaline and glucagon, whose plasma levels change profoundly immediately after birth (Martin et al., 1981; Cuezva et al., 1982). Albino Wistar pregnant rats (20&300 g), fed on a stock laboratory diet, were killed for the experiments between 09.00 and 10.00 h on day 22 of gestation. Conception was assumed by the presence of spermatozoa in the vagina, and gestational age was confirmed by the foetal weight. Isolated adipocytes were prepared essentially by the methods of Berry & Friend (1969) and Seglen (1972), as modified by Elliott et af. (1 984). Lipogenesis was measured with ' H 2 0 by the method described by Harris (1975). The rates of lipogenesis were calculated from the 60min values and are expressed as pmol of 'H,O incorporated into lipid/h per lo7 cells. The rate of lipogenesis in isolated adipocytes from endogenous substrates increased significantly in the presence of 10-9~-insulin (from 0.077 f 0.002 to 0.096 f 0.005pmol of 'H,O/h per lo7 cells). However, no effects were seen when M-adrenaline or lop6Mglucagon were added to the incubation medium under the same experimental conditions (from 0.077 & 0.002 to 0.079pmol of 'H,O/h per lo7 cells in the case of adrenaline or to 0.081 pmol of 'H,O/h per lo7cells in the case of glucagon respectively). The presence of 10mMlactate plus 1 mM-pyruvate as exogenous substrates increased 3-fold the rate of lipogenesis observed in isolated brown adipocytes from newborn rats in its absence (from 0.077 f 0.002 to 0.251 f 0.011 pmol of 'H,O/h per lo7 cells). Addition of lactate plus pyruvate was found to be the best substrate for lipogenesis in brown adipocytes at the late foetal stage in the absence of hormones, as observed in foetal hepatocytes (results not shown). However, the presence of lactate plus pyruvate increased 2-fold the rate of lipogenesis in foetal hepa*To whom correspondence and requests for reprints should be addressed.

tocytes. These results clearly indicate that the rate of lipogenesis in isolated brown adipocytes is significantly higher than that obtained in isolated hepatocytes from foetal rats, and is consistent with the value obtained in vivo at that time (Benito et al., 1984). In the presence of insulin, the rate of lipogenesis with lactate plus pyruvate as a substrate in isolated brown adipocytes slightly increases as compared with the value obtained in its absence (from 0.251 0.01 1 to 0.284 +_ 0.025 pmol of 'H,O/h per lo7cells). Conversely, the rate of lipogenesis from isolated brown adipocytes, in the presence of lactate plus pyruvate as a substrate, decreased significantly on the addition of lo-' M-adrenaline to the medium (from 0.251 & 0.01 1 to 0.189 f 0.009pmol of 'H,O/h per lo7 cells). Finally, in the presence of lop6M-glucagon the rate of lipogenesis in isolated brown adipocytes decreased slightly as compared with the value obtained in the absence of hormone (from 0.251 f 0.011 to 0.230 f 0.009 pmol of 'HZO/hper lo7cells). These results seem to indicate that insulin works on the rate of lipogensis in isolated brown adipocytes, as suggested in vivo by Pillay & Bailey (1982). In addition, glucagon does not seem to regulate the rate of lipogenesis in brown adipocytes, as already indicated by Elliott et al. (1984), where glucagon did not produce any effect on cyclic AMP accumulation in the cells nor on the rate of lipolysis studied. However, the slight inhibitory effect found by glucagon on the rate of lipogenesis in brown adipocytes in the presence of lactate plus pyruvate as a substrate clearly correlates with the results obtained in vivo in the foetal rats (Benito et al., 1984). Finally, the inhibitory effect by adrenaline on the rate of lipogenesis in brown adipocytes in the presence of lactate plus pyruvate as exogenous substrate is consistent with the adrenergic stimulation of thermogenesis in brown adipose tissue at birth (Nichols & Locke, 1984). In conclusion, the rate of lipogenesis in brown adipocytes seem to be regulated by insulin and adrenaline in the foetal-neonatal transition in the rat, as they are in vivo under physiological conditions. Glucagon, however, does not seem to regulate the lipogenic rate in brown adipocytes as it does not have any effect on cyclic AMP accumulation in the cells. This work was supported by a grant from Consejo Superior de Investigaciones Cientificas, Spain. Benito, M., Lorenzo, M. & Caldes, T. (1984) Biochem. J . 224,823-828 Berry, M. N. & Friend, D. S. (1969) J. Cell Biol. 43, 506520 Cuezva, J. M., Burkett, E. S., Kerr, D. S., Rodman, H. M. & Patel, M. S. (1982) Pediutr. Res. 16, 632437 Elliott, K. R. F., Roncero, C., Fernandez, M. & Benito, M. (1984) Biosci. Rep. 4, 397401 Harris, R. A. (1975) Arch. Biochem. Biophys. 169, 168-180 Lorenzo, M., Caldks, T., Benito, M. & Medina, J. M. (1981) Biochem. J. 198, 425428 Martin, A., Caldts, T., Benito, M. & Medina, J. M. (1981) Biochim. Biophys. Acta 672, 262-267 Nichols, D. & Locke, R. M. (1984) Physiol. Rev. 64, 1-64 Pillay, D. & Bailey, E. (1982) Int. J. Biochem. 14, 51 1-517 Seglen, P. 0. (1972) Exp. Cell Res. 74, 450-454

1986