binding capacity seen after a single injection of noradrenaline. surgical denervation.

Transcription

1 J. Physiol. (1984), 355, pp Printed in Great Britain EFFECTS OF DENERVATING BROWN ADIPOSE TISSUE ON THE RESPONSES TO COLD, HYPERPHAGIA AND NORADRENALINE TREATMENT IN THE RAT BY NANCY J. ROTHWELL AND MICHAEL J. STOCK From the Department of Physiology, St George's Hospital Medical School, Tooting, London SW17 ORE (Received 3 April 1984) SUMMARY 1. Surgical denervation of the five sympathetic nerves supplying one lobe of the interscapular brown fat of control rats caused small reductions in mass, protein content and the activity of the thermogenic mitochondrial proton conductance pathway (assessed from guanosine-5'-diphosphate (GDP) binding) when compared to the intact lobe. 2. Denervation did not affect the acute 100 % increase in mitochondrial GDP binding capacity seen after a single injection of noradrenaline. 3. Cold-adaptation (4 TC for 7 days) or over-feeding (cafeteria diet for 10 days) caused marked increases in the mass, protein content and specific mitochondrial GDP binding in intact brown adipose tissue, but these changes were totally prevented by surgical denervation. 4. These data indicate that the hypertrophy and the increased thermogenic capacity of brown fat induced by cold-adaptation or hyperphagia depend entirely upon the sympathetic innervation of the tissue. INTRODUCTION Heat production in brown adipose tissue (b.a.t.) is activated in rats exhibiting either non-shivering thermogenesis in response to cold exposure, or diet-induced thermogenesis in response to hyperphagia (see Girardier, 1983; Rothwell & Stock, 1983, 1984 for reviews). Both thermogenic stimuli produce rapid and sustained increases in the activity of the mitochondrial proton conductance pathway in brown fat (Nicholls & Locke, 1983; Brooks, Rothwell & Stock, 1982), and this is now considered to be the major thermogenic mechanism in the tissue (Nicholls & Locke, 1983). Cold exposure and hyperphagia also cause hypertrophy and hyperplasia of b.a.t. (Tulp, 1981; Brooks et al. 1982; Bukowiecki, Collet, Follea, Guay & Jahjah, 1982). All of these responses appear to be mediated largely by the sympathetic nervous system, and both non-shivering and diet-induced thermogenesis result directly from noradrenergic activation of b.a.t. The evidence supporting this sympathetic involvement has been reviewed recently (e.g. Girardier, 1983; Landsberg & Young, 1983; Rothwell & Stock, 1983, 1984) and includes such findings as: changes in heat

2 458 N. J. ROTHWELL AND M. J. STOCK production and b.a.t. activity associated with non-shivering and diet-induced thermogenesis can be mimicked byfl-adrenergic agonists and inhibited byf,-adrenergic antagonists or ganglionic blockers; noradrenaline turnover is elevated in b.a.t. from cold-adapted and hyperphagic rats; stimulation of the sympathetic nerves supplying brown fat results in activation of thermogenesis in the tissue. However, several other factors (e.g. insulin, glucagon, thyroid hormones and glucocorticoids) could be involved in the chronic adaptive changes in brown fat (see Rothwell & Stock, 1984), and the relative importance of sympathetic activity and these humoral influences is not known. One approach to this problem would be to test the effects of chronic administration of fl-adrenergic antagonists (e.g. propranolol) or chemical sympathectomy (e.g. guanethidine). However, these treatments affect sympathetic function throughout the body, and changes in food intake, gastrointestinal, cardiovascular and endocrine functions make the interpretation of results exceptionally difficult. An alternative approach is to study the effects of surgical denervation of b.a.t., and by using unilateral denervation of the bilobular interscapular depot, it is possible to quantify the neural influence on specific tissue responses to cold and hyperphagia. METHODS Male, Sprague-Dawley rats (200 g body wt., age 7 weeks; Charles River, Kent) were allowed free access to water and pelleted stock diet (PRD, Christopher Hill Group Ltd, Dorset) throughout. Animals were anaesthetized with halothane (2 % v/v) in an oxygen/nitrous oxide mixture, and an incision (approx. 1-5 cm) was made between the scapulae to expose the interscapular b.a.t. depot. The five nerves supplying the right lobe were carefully isolated and severed (denervated lobe) with minimal disturbance to the fat pad itself. Nerves supplying the left lobe were isolated but were not severed (intact lobe). The incision was closed with stitches and all animals regained consciousness within 20 min. In the first experiment, two groups of denervated rats (n = 4-5, each group caged together) were housed at 24 'C and another group at 4 TC (cold-adapted) for a period of 7 days after surgery. On the last day of the experiment, one group of control rats (24 TC) was injected with noradrenaline (NA) (40,g/100 g body wt., s.c.) 1 h before sacrifice (NA treated), and all others received an equal volume of saline. The rats were killed by cervical dislocation, and the two lobes of the interscapular b.a.t. depot were dissected separately, weighed and homogenized in 0-2 M-sucrose. Mitochondria were prepared by the method of Slinde, Pedersen & Flatmark (1975), and the activity of the proton conductance pathway was assessed from the binding of [3H]guanosine-5'-diphosphate (GDP) (0-25 /sm) in the presence of 2 /im-unlabelled nucleotide, or 200 1zM-GDP to assess non-specific binding (Brooks et al. 1982). Tissue and mitochondrial protein were determined by a dye-reagent method (Bio-Rad) using bovine serum albumin standards. In the second experiment, two groups (n = 6) of denervated rats were maintained at 24 TC (housed in pairs) and fed either stock diet alone (control) or stock diet plus a choice of four highly palatable human food items (e.g. cakes, biscuits, chocolate, meat, etc.) each day (cafeteria group). This 'cafeteria' diet is known to induce hyperphagia and diet-induced thermogenesis in young animals (see Rothwell & Stock, 1983). Ten days later, the animals were killed and the mass, protein content and mitochondrial GDP-binding capacity of each lobe of the interscapular b.a.t. depot were assessed as described above. RESULTS In the first experiment, interscapular b.a.t. mass and protein content were similar for intact lobes from NA-treated (40 #sg/ 100 g body wt.) and control rats, and

3 DENER VATED BROWN FAT although the mass of the denervated lobe was slightly reduced in both cases, this difference was not statistically significant (Table 1). Cold-adaptation (4 0C) caused hypertrophy of the intact lobe, as seen by the 39 % increase in mass and the 163 % increase in protein content. However, no hypertrophy was seen in the denervated lobes of the cold-adapted rats, and the mass and protein content were both reduced compared to the innervated lobe (Table 1). Mitochondrial protein isolated from brown fat, and specific mitochondrial GDP binding were not significantly altered by TABLE 1. Brown adipose tissue mass and protein content of control, NA-treated and cold-adapted rats (Expt. 1) Interscapular b.a.t. Protein content Mass (mg) (mg) (%) Control (1) (2) ± P16 NA-treated (3) (4) Cold-adapted (5) (6) 72+9*** ** 7X6+1i1 Significant differences 5 V8. all 5 V8. all 5 V Vs. 1, 3, 4 Mean values+s.e. of mean (n= 4-5). **P < 001, ***P < 0001 paired t test compared to intact lobe. Significant differences between groups assessed by Sheff6's critical range analysis using a level for significance of P < denervation in either control or NA-treated rats (Table 2), although total binding to the recoverable mitochondria was depressed in the denervated compared to the intact pad in control animals. Acute NA treatment did not affect mitochondrial protein but caused an increase in specific GDP binding in the innervated lobe (127 % above saline-injected controls) and a very similar increase in the denervated lobe (100% above saline control). Cold-adaptation resulted in a 55% increase in mitochondrial protein and significantly greater levels of specific and total mitochondrial GDP binding (438 and 710% above warm-adapted control values, respectively) in intact tissue. However, these changes were totally absent in the denervated lobe, and mitochondrial protein and GDP binding did not differ from controls. In Expt. 2 (cafeteria feeding), surgical denervation did not significantly alter the mass, protein content or mitochondrial protein of b.a.t. in control rats, but did produce a significant decrease in specific mitochondrial GDP binding (Table 3). Cafeteria feeding increased the mass (84% above control), protein content (34 %), mitochondrial protein (35 %) and specific GDP binding (100%) in the intact b.a.t. depot, although the percentage protein in the tissue was lower than that of controls. In contrast, these changes were completely absent in the denervated b.a.t. lobe from cafeteria-fed rats, and tissue mass, protein content, mitochondrial protein and GDP 459

4 460 N. J. ROTHWELL AND M. J. STOCK TABLE 2. Interscapular b.a.t. mitochondrial mass and GDP binding of control, NA-treated and cold-adapted rats B.a.t. mitochondrial Control NA-treated Cold-adapted Significant differences (1) (2) (3) (4) Protein (mg) ' Specific GDP binding (pmol/mg P) (5) (6) *** *** 5 v8. all 5 V8. all 3, 4 v8. 1, 2, 6 GDP binding to total recovered mitochondria (pmol) * *** 5 V8. all 3, 4, V8. 1, 2, 6 Mean values+s.e. of mean (n = 4-5). *P < 005, ***P < 0001 paired t test compared to intact lobe. Significant differences between groups assessed by Sheff6's critical range analysis using a level for significance of P < TABLE 3. Interscapular b.a.t. protein content and GDP binding of control and cafeteria-fed rats Protein content Mitochondrial Specific GDP Mass protein binding (mg) (mg) (%) (mg) (pmol/mg P) Control Cafeteria (1) (2) P ** (3) (4) * *** ** *** 44+9** Significant 3 v8. all 3 v8. all 3, 4 V8. 1, 2 3 v8. all 3 v8. all differences 4 v8. 1, 2 4 v8. 1, 2 Mean values+s.e. of mean (n = 5-6). *P < 0-05, **P < 001, ***P < paired t test compared to intact lobe. Significant differences between groups assessed by Sheff6's critical range analysis using a level for significance of P < binding were all lower than in their respective intact lobes. In addition, the total protein content and the mitochondrial protein of denervated b.a.t. from cafeteria-fed rats were significantly lower than in sympathectomized tissue from controls. DISCUSSION Five large nerves can be clearly identified entering each interscapular lobe rostrally, and these can be severed without direct disturbance to the tissue itself or of its blood supply, which enters via the caudal end of each lobe. Retention of normal

5 DENER VATED BROWN FAT blood supply and responses to humoral stimuli are supported by the findings of the first experiment, where acute treatment with NA caused almost identical stimulation of mitochondrial GDP-binding capacity (i.e. the proton conductance pathway) in the denervated and intact lobes. This was of a similar magnitude to that seen previously in unoperated animals (Brooks et at. 1982). Foster, Depocas & Zahror-Behrens (1982) have demonstrated that this technique for surgically denervating rat interscapular b.a.t. results in depletion of over 97 % of its NA content, and complete loss of the usual, 10-fold increase in blood flow seen in this tissue during acute exposure to cold (-6 TC). In addition, these workers demonstrated the strict unilateral sympathetic innervation of each pad, since denervation of one lobe did not affect NA content or responses to cold in the contralateral, intact lobe. In both experiments reported here, no significant effect of denervation was observed on b.a.t. mass or protein content in the tissue from control animals. However, the consistently small decreases in mitochondrial protein indicate that a more prolonged study might result in significant loss of mitochondrial protein. This suggestion is further supported by the fact that in the slightly longer study (Expt. 2, 10 days) denervation did result in a significant depression in GDP binding, whereas in the first study, denervation influenced only total mitochondrial GDP binding. Unfortunately, the duration of these experiments is limited by regrowth of sympathetic nerves. Seydoux, Tribollet & Girardier (1984) have reported a considerable restoration of NA levels in brown fat 2 months after denervation, and suggested that some regrowth may occur as soon as 2-3 weeks after surgery. The results of the first experiment confirm those of many previous studies (see Girardier, 1983; Nicholls & Locke, 1983; Rothwell & Stock, 1984, for reviews) on the effect of cold-adaptation on growth and activity of brown adipose tissue. In addition, they demonstrate that these changes can be totally prevented by surgical denervation of the tissue. The mass, protein content and mitochondrial GDP binding of the denervated brown fat from cold-adapted rats were all virtually identical to values obtained for control rats. These data complement those of Foster et al. (1982), who showed that the acute thermogenic response to cold in brown fat was abolished by denervation, and further indicate that any changes in circulating catecholamines arising from adrenal medullary release during cold exposure are ineffective in stimulating b.a.t. Himms-Hagen & Park (1984), however, have reported that unilateral denervation of b.a.t. did not prevent all of the increases in protein content and mitochondrial GDP binding associated with cold exposure (4 C). This may be because their denervation procedure involved severance of only four of the five nerves suppplying each lobe, and measurements were performed 21 days after surgery. Thus, it is possible that some of the sympathetic supply to the tissue remained intact, and the chances of regrowth under these conditions would be much greater. The absence of any changes in responsiveness to acute NA treatment after denervation (Table 2) is somewhat surprising, since interruption of the neural input to the tissue might be expected to cause sensitization to noradrenaline, possibly via an 'up-regulation' of /3-adrenoreceptor number. However, we have failed to detect any changes in the density of fl-adrenoreceptors (assessed from the binding of [3H]dihydroalprenolol) in b.a.t. membranes days after denervation of the tissue 461

6 N. J. ROTHWELL AND M. J. STOCK (N. J. Rothwell, M. J. Stock & D. K. Sudera, unpublished data). This may be due to the relatively short duration of the experiments, but the observation is consistent with the results obtained in the present study. As in many previous studies, cafeteria feeding produced marked increases in the mass, protein content and mitochondrial GDP binding in intact brown fat. However, these changes were totally prevented by denervation, and in fact the protein content of denervated tissue from cafeteria rats was actually lower than in sympathectomized lobes from control animals. Why this should occur is not known, but it may be indirectly related to large increases in fat deposition in the tissue affecting protein turnover. The GDP-binding results show that activation of diet-induced thermogenesis via the proton conductance pathway of brown fat mitochondria is entirely dependent on the sympathetic innervation. It has been suggested that the stimulation of b.a.t. by cold and diet involves different mechanisms, since, unlike cold-adapted rats, increases in GDP binding by b.a.t. mitochondria from cafeteria-fed rats are not associated with increases in the amount of the 'uncoupling' protein (Mr 32000) that binds GDP (Hogan, Himms- Hagen, Triandafillou & Gwilliam, 1981). However, the protein was measured using sodium dodecyl sulphate (SDS)-polyacrylamide gels, and a more sensitive radioimmunoassay has revealed increases in the concentration of the protein in mitochondria from cafeteria-fed rats which correlate with changes in specific GDP binding (Ashwell, Rothwell, Stirling, Stock & Winter, 1984). This finding, together with the results of the present study, indicate that the induction of mitochondrial thermogenesis by cold and hyperphagia involves the same mechanisms, and both are dependent almost entirely on an intact sympathetic innervation. Nevertheless, cold exposure provides a more potent stimulus to brown fat thermogenesis than overfeeding, and responses to both stimuli could obviously be modified by circulating hormones and/or substances other than NA released from the sympathetic nerves. We are grateful to Kevin Bryant and Mike Lacey for their excellent technical assistance. This work was supported by grants from ICI (Joint Research Scheme) and the Royal Society (Fellowship to N.J.R.). REFERENCES ASHWELL, M., ROTHWELL, N. J., STIRLING, D., STOCK, M. J. & WINTER, P. D. (1984). Changes in mitochondrial uncoupling protein and GDP-binding in brown adipose tissue of cafeteria-fed rats. Proceedings of the Nutrition Society (in the Press). BROOKS, S. L., ROTHWELL, N. J. & STOCK, M. J. (1982). Effects of diet and acute noradrenaline treatment on brown adipose tissue development and mitochondrial purine nucleotide binding. Quarterly Journal of Experimental Physiology 67, BUKOWIECKI, L., COLLET, A. J., FOLLEA, N., GUAY, G. & JAHJAH, L. (1982). Brown adipose tissue hyperplasia a fundamental mechanism of adaptation to cold and hyperphagia. American Journal of Physiology 242, E FOSTER, D. O., DEPOCAS, F. & ZAROR-BEHRENS, G. (1982). Unilaterality of the sympathetic innervation ofeach pad of rat interscapular brown adipose tissue. Canadian Journal of Physiology and Pharmacology 60, GIRARDIER, L. (1983). Brown fat: an energy dissipating tissue. In Mammalian Thermogenesis, ed. GIRARDIER, L. & STOCK, M. J., pp London: Chapman & Hall. HIMMS-HAGEN, J. & PARK, I. (1984). Effect of denervation on brown adipose tissue growth and thermogenesis in cold-acclimated and cafeteria-fed rats. Proceedings of the Thermal Physiology

7 DENER VATED BROWN FAT 463 Satellite, 29th IUPS Congre88, AU8tralia, ed. HALES, J. R. S. New York: Raven Press (in the Press). HOGAN, S., HiMMs-HAGEN, J., TRIANDAFILLOU, J. & GWILLIAM, C. (1981). Brown adipose tissue of cafeteria-fed rats. American Journal of Phy8iology 239, C LANDSBERG, L. & YOUNG, J. B. (1983). Autonomic regulation of thermogenesis. In Mammalian Thermogene8i8, ed. GIRARDIER, L. & STOCK, M. J., pp London: Chapman & Hall. NICHOLLS, D. G. & LOCKE, R. (1983). Cellular mechanisms of heat dissipation. In Mammalian Thermogenesi8, ed. GIRARDIER, L. & STOCK, M. J., pp London: Chapman & Hall. ROTHWELL, N. J. & STOCK, M. J. (1983). Diet-induced thermogenesis. In Mammalian Thermogene8i8, ed. GIRADIER, L. & STOCK, M. J., pp London: Chapman & Hall. ROTHWELL, N. J. & STOCK, M. J. (1984). Brown adipose tissue. In Recent Advance8 in Phy8iology, vol. 10, ed. BAKER, P. F., pp Edinburgh: Churchill Livingstone. SEYDOUX, J., TRIBOLLET, E. & GIRARDIER, L. (1984). The effectiveness of surgical denervation of brown adipose tissue in the rat: further observations. Proceedings of the Thermal Physiology Satellite, 29th IUPS Congress, Australia, ed. HALES, J. R. S. New York: Raven Press (in the Press). SLINDE, G., PEDERSEN, J. I. & FLATMARK, T. (1975). Sedimentation coefficient and buoyant density of brown adipose tissue mitochondria from guinea-pigs. Analytical Biochemistry 65, TULP, 0. L. (1981). The development of brown adipose tissue during experimental overnutrition in rats. International Journal of Obesity 5,