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Hypothalamic control of brown adipose tissue in Zucker lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres
Brain Research, 405 (1987) 227-233
227
Elsevier BRE 12396
Hypothalamic control of brown adipose tissue in Zucker lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres S.J. H o l t 1, H.V. W h e a l 2 and D . A . Y o r k 1 1Department of Nutrition, School of Biochemical and PhysiologicalSciences, Southampton University, Southampton ( U. K.) and 2Departmentof Neurophysiology, School of Biochemical and Physiological Sciences, Southampton University, Southampton (U.K.) (Accepted 22 July 1986)
Key words: Ventromedial hypothalamus; Zucker rat; Brown adipose tissue; Sympathetic efferent; Supraoptic nucleus; Lateral hypothalamus; Dorsomedial nucleus
Electrophysiological stimulation of the hypothalamic ventromedial nucleus (VMN) resulted in an increase in interscapular brown adipose tissue (BAT) temperature in both lean and obese (fa/fa) rats. Graded stimulations resulted in progressively larger temperature increases in both lean and obese (fa/fa) groups. Both intraperitoneai injection of propranolol and surgical denervation (but not sham denervation) abolished the increase in BAT temperature following VMN stimulation, in both lean and obese (fa/fa) groups. Electrical stimulation of the supraoptic region, and certain anterior hypothalamic regions also resulted in increases in BAT temperature of lean and obese (fa/fa) rats, but stimulation of the dorsomedial nucleus and regions of the lateral hypothalamus did not affect BAT temperature. All hypothalamic regions capable of activating BAT gave a similar maximum rise in temperature for a given stimulus in lean and obese (fa/fa) rats. These results suggest that the efferent sympathetic pathway from the VMN and other hypothalamic regions of BAT is normal in the obese (fa/fa) rat. INTRODUCTION The ventromedial hypothalamus (VMH) has a well established role in the control of food intake in rats 31. Electrical stimulation of this region results in satiety and other sympathetic responses 3, whereas stimulation of the lateral hypothalamus initiates feeding 2'5 and parasympathetic responses 3. Bilateral lesions in the V M H area in young as well as adult rats causes pronounced accumulation of body fat. Obesity develops not only when animals are allowed to overeat, but also when their food intake is restricted to the level of control animals in pair-feeding experiments 6'7'19. This increase in energetic efficiency in the V M H lesioned rat is also characteristic of the genetically obese (fa/fa) Zucker rat 5,13 in which a reduction in metabolic rate is observed during the first
week of life, before either hyperphagia or hyperinsulinaemia is established 6,3°. Thus in both of these models of obesity a reduction in energy expenditure is an important causative factor in the accumulation of excess body fat. Thermogenesis in brown adipose tissue (BAT) has only recently become recognized as a significant component of diet-related expenditure (see ref. 34 for review). Its main function was thought to be the provision of heat for thermoregulatory thermogenesis 37. Both the VMN-lesioned rat and the obese (fa/fa) rat show reduced B A T thermogenesis when compared with their respective control groups 13'16'36, a result of the attenuated central sympathetic drive to the tissue. When the obese (fa/fa) rat is overfed by offering a 35% sucrose drinking solution in addition to a normal laboratory diet, it does not show the normal
Correspondence: S.J. Holt, Department of Nutrition, School of Biochemical and Physiological Sciences, Southampton University, Southampton, SO9 3TU, U.K.
228 increases in sympathetic activity or BAT function seen in the lean r a t 17'18'39 (dietary induced thermogenesis (DIT)). On the other hand the young obese (fa/fa) rat shows a normal increase in sympathetic activity and B A T thermogenesis when cold acclimated 17'39 (cold induced non-shivering thermogenesis (NST)) although this response may be impaired in the adult. The VMH-obese rat also displays a reduction in sympathetic stimulation of BAT, and this causes a failure to respond normally to both dietary and thermal stimuli 13'14'4°. The centres controlling energy and temperature homeostasis are thought to be located in differing regions of the hypothalamus 4'5. A functional link between the VMH and BAT has been demonstrated in normal rats T M . In addition, an acute reduction in sympathetic nerve activity has been reported after electrolytic lesions of the VMH 26. Furthermore, a functional disconnection is suggested in the VMH lesioned rat by their failure to show the normal changes in sympathetic activity and BAT thermogenesis in response to manipulations of diet and environmental temperature 14'26. The accumulated evidence suggests that the defect in the obese (fa/fa) rat may reside in the hypothalamus and be represented by either a failure to produce, recognize or respond to satiety signals. The aim of the present study was to examine electrophysiologically whether a dysfunction exists in the efferent pathway between the VMH and other hypothalamic regions and B A T in the obese (fa/fa) rat.
TABLE I
MATERIALS AND METHODS
%
Preparation o f animals Female lean (fa/?) and obese (fa/fa) rats at 7 weeks of age (150 + 5 g and 180 + 5 g b. wt., respectively) were used for all experiments. The animals were anaesthetized with urethane (25%) w/v solution injected i.p. at 1.2 g/kg) and placed in a stereotaxic instrument with the head orientated as described in the stereotaxic atlas of K6nig and Klippe123. Body temperature was maintained between 37 and 38 °C with a thermostatically controlled heating pad, and the experiments took place in a room kept between 24 and 26 °C.
2.0
Increase in BAT temperature (A °C) after electrical stimulation (8 V) of several hypothalamic areas' in lean and obese (fa/fa) rats Hypothalamic region
n
Lean
Obese (fa/fa)
Ventromedial Supraoptic Anterior Lateral Dorsomedial
10 6 5 10 8
0.89 + 0.05 0.90 + 0.04 0.86 + 0.05 No change No change
0.93 + 0.04 0.87 _+0.04 0.79 ___0.07 No change No change
B A T pad and the pad exposed by cutting through the overlying white adipose tissue and muscle. A thermocouple probe was inserted and secured between the two lobes of the tissue. The skin was then clipped together over the top of the depot. A thermocouple probe was also inserted 6 mm into the rectum to record core temperature. Temperatures were continuously monitored and recorded every 60 s with a mi-
2"0 Lean
18 1.6 1.4
I
0.8
t
0.6 o0-24t
(.9
Recordings o f B A T and rectal temperature A small incision was made above the interscapular
Obese
18 16 1-4 1.2 1.0 (1.8 06 04
Volts = 10 m i n
Time
Fig. 1. Change in BAT temperature following electrical stimulation of the hypothalamic VMN in lean and obese (fa/fa) rats: response to varying voltage. The figures on the time axis indicate the voltage of the stimulation. The duration of the stimulating voltage was 30 s at the arrow point.
229 croprocessing scanning thermometer and a digital printout (Comark Electronics, Rustingdon, Sussex).
Electrical stimulation Following exposure of the brain a fine (0.5 mm diameter) bipolar steel electrode (Clark Electromedical, Reading, Sussex) insulated except at the tip, was stereotaxically implanted from the dorsal approach, into the appropriate hypothalamic region using the coordinates of K6nig and Klippe123 with modifications for lean and obese (fa/fa) rats. An isolated stimulator was used to deliver a 30 s train of monophasic square wave pulses I ms, half cycle duration, 50 Hz at an intensity of 8 V. Graded intensities between 2 and 35 V were used. A stabilization period of 30 min was allowed before any stimulations or recordings began, and following stimulation, the BAT temperature was allowed to return towards baseline before the stimulus was repeated. At the termination of each experiment a small electrolytic lesion was made to mark the stimulus sites for subsequent histological examination. The rat was killed by cardiac perfusion with a 10% (v/v) formal saline solution containing 5 % (w/v) potassium ferrocyanide. The brain was removed and sections were cut at 50 pm on a freezing microtome and microscopically examined under low and medium power to determine the correct location of the electrode. The inclusion of potassium ferrocyanide in the perfusion solution resulted in an additional blue iron complex developing at the site of lesioning. The sections were stained with Cresyl violet to verify the nuclei containing regions and areas were identified using the atlas of K6nig and Klippe123.
initial component was a rapid increase in temperature which reached a maximum within 3-5 min at the lower voltages (<20) and 6-7 min at higher voltages. These effects were the same for each group. Graded stimulations of the VMN were followed by graded increases in BAT temperature in both lean an obese (fa/fa) groups. A minimum threshold of 3 V was usually observed, below which no increase in BAT temperature was recorded. No significance should be attached to the apparent difference, in Fig. 1, between lean and obese (fa/fa) rats in the voltage required for maximum response, since this was highly variable in both groups. However, the duration of the response to stimulation at a given voltage was characteristically longer in the obese (fa/fa) rat compared with the lean rat. In order to confirm that the BAT responses were mediated via the sympathetic innervation, the effects of the fl-adrenergic blocker propranolol and of denervation were investigated. Fig. 2 shows the typical changes in BAT and rectal temperatures following hypothalamic VMN stimulation. The rapid increase in BAT temperature preceded the less marked increase in rectal temperature. Treatment with propranolol completely abolished the response to subsequent VMN stimulation in both lean and obese (fa/fa) rats (Fig. 2). Fig. 3 shows the effect of severing the bilateral sympathetic nerve bundles on the response of BAT to VMN stimulation. After establishing a normal response, a sham denervation had no effect on the increase in BAT temperature following further VMN stimulation, whereas denervation abolished the capacity of the
Lean
RESULTS
Obese I
The effects of electrical stimulation of the VMN on BAT temperature in lean and obese (fa/fa) rats is shown in Table I. An 8 V stimulus train resulted in a similar temperature increase in both groups. Stimulation of the area 0.5 mm dorsal or ventral to the VMN did not evoke a change in BAT temperature (Fig. 4) indicating specificity to the nucleus region. Fig. 1 illustrates typical changes in BAT temperature induced by consecutive VMN stimulations of increasing voltage in lean and obese (fa/fa) rats. After a 30 s stimulus train there was a further 60 s delay before any increase in BAT temperature was observed. The
i I
380
I Rectal
~
370
I I I
E Control 3~0
t 10 rain
t
B.A]-
I I I
I I
Prx~oranalol
Control
t
t
t
I F~.ooranalo I I
t
Time
Fig. 2. The effect of stimulation of the hypothalamic VMN on BAT and rectal temperature in lean and obese (fa/fa) rats. The arrows indicate a 30 s electrical stimulation of 8 V. The dashed line shows the time point at which rats were injected (i.p.) with the 15-adrenergic antagonist propranolol (2 mg/kg).
230 LeQo
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t r< ~
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15 t_ E
'h
',,'J~1%
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! I'~
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I I I Sham
jDenervation
,
i
Rectal
I
\ !
i ! [Control Sham
J
Obese
Denenvation
i
t lOmin
t
t
t
Time
Fig. 3. The effect of electrical stimulation of the hypothalamic VMN on BAT and rectal temperature in lean and obese (fa/fa) rats. The arrows indicate the times at which a 30 s 8 V stimulus was applied. The solid ( .....) line indicates the time of bilateral denervation of the BAT and the dashed line (---) indicates the time of sham denervation. 6060~
5910 u
5150~
so
4620.u
tissue to respond to further stimulation. This effect was identical in both lean and obese (fa/fa) rats, B A T is known to be responsive to environmental temperature changes and neurons controlling this response are present in several hypothalamic regions. Thus we studied the effect of electrically stimulating a number of these nuclei on B A T temperature in lean and obese (fa/fa) rats. Table I shows that B A T temperature increases obtained after stimulation of both the supraoptic and certain regions of the anterior hypothalamus were similar in both lean and obese (fa/fa) rats. Fig. 4 illustrates the regions in which a positive response was obtained. The increases in B A T temperature are similar to those observed after VMN stimulation for a given voltage. Conversely no increases in B A T temperature were obtained after stimulation in either the lateral hypothalamic areas examined or in the dorsomedial nuclei in either genotype. The negative responses are also illustrated in Fig. 4. These results indicate that in the hypothalamic regions examined the capacity to evoke an activation of B A T thermogenesis is essentially similar in lean and obese O%/fa) rats. DISCUSSION
5660~J
5340 m
4380u
4 2 3 0 ,u
Fig. 4. Schematic representation of partial coronal sections (anterior to posterior according to the coordinates of K6nig and Klippe123). Circles show position of electrode tip which evoked an increase (0) or no change (O) in BAT temperature following an 8 V stimulation. Abbreviations: FMP, fasciculus medialis prosencephali; OT, optic tract; IIIV, third ventricle; so, nucleus supraopticus; ha, nucleus anterior; hi, nucleus lateralis; hvm, nucleus ventromedialis; hd, nucleus dorsomedialis.
Brown adipose tissue is the major site for both NST and D I T in small rodents 11"32"33.Its thermogenic activity is controlled principally by its abundant sympathetic innervation 24. Previous work in our laboratory and by others has shown that the genetically obese (fa/fa) rat is able to activate B A T thermogenesis in response to cold, but the response to dietary stimuli is absent or severely impaired 17'39. This inability to activate D I T has been related to the absence of sympathetic stimulation of B A T 43'44. The hypothalamic centres regulating thermoregulatory and dietary induced sympathetic activity are thought to differ. In this work we have shown that stimulation of a number of hypothalamic regions will activate BAT. This confirms previous observations that the VMN appears to have a functional connection with B A T 15' 29,41. The rapid increase in temperature after VMN stimulation is similar to that previously described 29,41. However, our results differed from those published in two respects: firstly, we did not observe the initial, transitory decrease in temperature re-
231 ported, despite continuous monitoring of BAT temperature; secondly the duration of the response was shorter in the present study. There may be many possible differences, including the anaesthetic used, different mode of stimulation, rat species or age and the room temperature at which the experiment took place, which may account for the discrepancies observed. The functional connection between the VMN and BAT is clearly mediated through the sympathetic innervation to the tissue, since both pretreatment with the fl-adrenergic antagonist propranolol and sympathetic nerve transection blocked the response. We have made similar observations after stimulation of the supraoptic area (unpublished observations). The responses of the obese (fa/fa) rats to regional hypothalamic stimulation were similar to those observed in the lean rat with regard to the rapid onset and maximal size of the response. However the duration of the response was longer in the obese (fa/fa) rat. Blood flow to BAT in the adult obese (fa/fa) rat is reduced compared with the lean animal 42 and this reduced capacity to dissipate the heat produced would potentiate the duration of the response. Additionally the obese (fa/fa) rat has an insulating layer of subcutaneous fat overlying the interscapular BAT depot which may be expected to reduce radiant heat losses. In these studies we have shown that electrical stimulation of the VMN, supraoptic area and regions of the anterior hypothalamus activated BAT thermogenesis. Lesions of the VMN have previously been shown to reduce sympathetic activation to BAT and to be associated with loss of DIT 13A4'35'4°. In contrast, the anterior hypothalamus is thought to be involved with the control of NST 8. A functional connection of the supraoptic area and BAT has not previously been demonstrated. The main function of this region is thought to be neurosecretory, with efferent pathways projecting mainly to the hypophysis 38. It is possible that the observed response may be due to stimulation of the 'supraoptic decussations', which are known to project to the VMN 28. The fibres of this pathway also contain aminergic neurons 12,21,28 and stimulation may activate one of the several ascending or descending aminergic fibre tracts coursing through the hypothalamus 4,28. Further experiments are therefore required to elucidate the hypothalamic pathways involved with sym-
pathetic activation of BAT. Stimulation of regions between the anterior hypothalamus at the supraoptic level and the anterior VMN did not result in a change in BAT temperature. Similarly in the lateral hypothalamic areas examined and in the dorsal medial nucleus no functional connection was demonstrated. These regions are not known to have any direct role in either DIT or NST. Our results have clearly demonstrated that all hypothalamic centres capable of activating BAT in lean rats were similarly responsive in obese (fa/fa) rats. We may thus hypothesize that the inability of the obese (fa/fa) rat to activate sympathetic function in response to diet, may be due to an absence of afferent signals, or the inability to recognize signal inputs or couple them to the appropriate efferent output. Glucose sensitive, insulin responsive neurons are located in the VMN 27. The recent demonstration of specific insulin receptors in the hypothalamus and other brain regions lends support to the concept of a physiological role for insulin in controlling brain function. It is of particular interest to note that the level of insulin binding is severely depressed in the hypothalamus of the obese (fa/fa) rat and this reduction shows a gene dosage effect1°. In addition it is known that the obese (fa/fa) rat does not show the normal feeding response, nor the inhibition of BAT thermogenesis in response to either centrally or peripherally administered 2-deoxy-D-glucose (2DG) 1'2°. Since 2-DG impairs sympathetic drive to BAT, it is possible that the absence of diet induced BAT thermogenesis in the obese (fa/fa) rat may reflect a central impairment of glucose metabolism. Such a hypothesis is also supported by a recent demonstration that a restoration of diet induced BAT thermogenesis in the obese (fa/fa) rat following adrenalectomy 17'25is associated with a restoration of normal responses to 2-DG administration 1. Further work to investigate the response of hypothalamic centres of the obese (fa/fa) rat to nutrient and endocrine stimulation is required to substantiate this hypothesis.
ACKNOWLEDGEMENTS This research was supported by the Wellcome Trust.
232 REFERENCES 1 Allars, J. and York, D.A., The effects of 2-deoxy-D-glucose on brown adipose tissue of lean and obese Zucker rats, Int. J. Obesity, in press. 2 Anand, B.K. and Brobeck, J.K., Hypothalamic control of food intake in rats and cats, Yale J. Biol. Med., 24 (1951) 128-140. 3 Ban, T., Fibre connections in the hypothalamus and some autonomic functions, Pharmacol. Biochem. Behav., 3 (1975) 3-13. 4 Boulant, Y.A., Hypothalamic control of thermoregulation. In P.J. Morgan and J. Panksepp (Eds.), Handbook of the Hypothalamus. Vol 3 Part A, Marcel Dekker, New York, 1981, pp. 1-82. 5 Bray, G.A. and York, D.A., Hypothalamic and genetic obesity in experimental animals. An autonomic and endocrine hypothesis, Physiol. Rev., 59 (1979) 719-809. 6 Bray, G.A., York, D.A. and Swerloff, R.S., Genetic obesity in rats. 1. The effects of food restriction on body composition and hypothalamic function, Metabolism, 22 (1973) 435-442. 7 Brooks, C.M. and Lambert, E.F., A study of the effects of food intake and the method of feeding on the rate of weight gain during hypothalamic obesity in the albino rat, Am. J. Physiol., 147 (1946) 695-707. 8 Bruck, K. and Wunnenburg, W., 'Meshed' control of two effector systems: non shivering and shivering thermogenesis. In J.D. Hardy, A.P. Gagge and J.A.J. Stolwijk (Eds.), Physiological and Behavioural Temperature Regulation, Thomas Springfield, III, 1970, pp. 562-580. 9 Chi, C.C., Afferent connections to the ventromedial nucleus of the hypothalamus in rats, Brain Research, 17 (1970) 439-445. 10 Figlewicz, D.P., Dorsa, D.M., Stein, L.J., Baskin, D.G., Paquette, T., Greenwood, M.R., Woods, S.C. and Porte, D., Brain and liver insulin binding is decreased in Zucker rats carrying the 'fa' gene, Endocrinology, 117 (1985) 1537-1543. 11 Foster, D.O. and Frydman, M.L., Tissue distribution of cold-induced thermogenesis in conscious warm- or cold-acclimated rats reevaluated from changes in tissue blood flow. The dominant role of BAT in the replacement of shivering by non-shivering thermogenesis, Can. J. Physiol. Pharmacol.. 57 (1979) 257-270. 12 Fuxe, K., Evidence for the existence of monoaminergic neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the CNS, Acta Physiol. Scand., 64 Suppl. 247 (1965) 37-85. 13 Hogan, S., Coscina, D.V. and Himms-Hagen, J., Brown adipose tissue of rats with obesity inducing ventromedial hypothalamic lesions, Am. J. Physiol., 243 (1982) E338-E344. 14 Hogan, S., Himms-Hagen, J. and Coscina, D.V., Lack of diet-induced thermogenesis in brown adipose tissue of obese medial hypothalamic lesioned rats, Physiol. Behav., 35 (1983) 287-294. 15 Holt, S.J., Wheal, H. and York, D.A., Hypothalamic control of brown adipose tissue in lean and obese Zucker rats, Proc. Nutr. Soc., 44 (1985) 124A. 16 Holt, S.J. and York, D.A., The effect of adrenalectomy on GDP binding to brown adipose tissue mitochondria of obese rats, Biochem. J., 208 (1982) 819-822. 17 Holt, S.J.. York, D.A. and Fitzsimmons, J.T.R., The ef-
fects of corticosterone, cold exposure and overfeeding with sucrose on brown adipose tissue of obese Zucker rats (fa/fa), Biochem. J., 214 (1983) 215-223. 18 Holt, S.J., York, D.A., Rothwell, N.J. and Stock, M.J., The effect of age and gene dosage in brown adipose tissue function of obese Zucker rats, Am. J. Physiol., 246 (1984) E391-E396. 19 Hustveda, B., Jeszka, J., Christopherson, A. and Lovo, A., Energy metabolism in rats with ventromedial hypothalamic lesions, Am. J. Physiol., 246 (1984) E319-E326. 20 Ikeda, H., Nishikawa, K. and Matsuo, T., Feeding responses of Zucker fatty rat to 2-deoxy-D-glucose norepinephrine and insulin, Am. J. Physiol., 239 (1980) E379-E384. 21 Jacobowitz, D.M., Fluorescence microscopic mapping of CNS norepinephrine systems in the rat forebrain. In W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, pp. 368-380. 22 Jansky, L., Nonshivering thermogenesis and its thermoregulatory significance, Biol. Rev., 48 (1973) 85-132. 23 KOnig, J.F.R. and Klippel, R.A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, MD, 1963. 24 Landsberg, L. and Young, J.B., Autonomic regulation of thermogeneis. In L. Girardier and M.J. Stock (Eds.), Mammalian Thermogenesis, Chapman and Hall, London, 1983, pp. 99-140. 25 Marchington, D., Rothwell, N.J., Stock, M.J. and York, D.A., Energy balance, diet induced thermogenesis and brown adipose tissue in lean and obese (fa/fa) Zucker rats after adrenalectomy, J. Nutr., 113 (1983) 1395-1400. 26 Niijima, A., Rohner-Jeanrenaud, F. and Jeanrenaud, B., Role of ventromedial hypothalamus on sympathetic efferents of brown adipose tissue, Am. J. Physiol., 247 (1984) R650-R654. 27 Oomura, Y., Ohta, M., Ishibashi, S., Kita, H., Okajima, T. and Ono, T., In G.A. Bray (Ed.), Recent Advances in Obesity Research, Vol. 2, Newman Publishing, London, 1978, pp. 17-26. 28 Palkovits, M. and Zaborszky, L., Neural connections of the hypothalamus. In P.J. Morgan and J. Panksepp (Eds.), Handbook of the Hypothalamus, Vol. 1, Marcel Dekker, New York, 1979, pp. 379-510. 29 Perkins, M.N., Rothwell, N.J., Stock, M.J. and Stone, T.W., Activation of brown adipose tissue thermogenesis by the ventromedial hypothalamus, Nature (London), 289 (1981) 401-402. 30 Planche, E., Joliff, M., De Gasquet, P. and LeLiepvre, X., Evidence of a defect in energy expenditure in 7 day old Zucker rats (fa/fa), Am. J. Physiol., 245 (1983) E107-El13. 31 Powley, T.L., Opsahl, C.A., Cox, J.E. and Weingarten, H.P., The role of the hypothalamus in energy homeostasis. In P.J. Morgan and J. Panksepp (Eds.), Handbook of the Hypothalamus, Vol. 3, Part A, Marcel Dekker, New York, 1981, pp. 211-298. 32 Rothwell, N.J. and Stock, M.J., Similarities between cold and diet induced thermogenesis in the rat, Can. J. Physiol. PharmacoL, 58 (1980) 842-848. 33 Rothwell, N.J. and Stock, M.J., Influence of noradrenaline on blood flow to brown adipose tissue in rats exhibiting diet induced thermogenesis, Pflagers Arch., 389 (1981) 237-242. 34 Rothwell, N.J. and Stock, M.J., Diet induced thermogene-
233 sis. In J. Girardier and M.J. Stock (Eds.), Mammalian Thermogenesis, University Press, Cambridge, 1983, pp. 208-233. 35 Saito, M., Minokoshi, Y. and Shimazu, T., Brown adipose tissue after ventromedial hypothalamic lesions in rats, Am. J. Physiol., 248 (1985) E20-E25. 36 Seydoux, J., Ricquier, D., Rohner-Jeanrenaud, F., Assimacopouios-Jeannet, F., Jeanrenaud, B. and Girardier, L., Functional disconnection of brown adipose tissue in hypothalamicallyobese rats, Pflagers Arch., 390 (1981) 1-4. 37 Smith, R.E. and Horwitz, B.A., Brown fat and thermogenesis, Physiol. Rev., 49 (1969) 330-425. 38 Swanson, L.W. and Sawchenko, P.E., Hypothalamic integration, organisation of the paraventricular and supraoptic nuclei, Ann. Rev. Neurosci., 6 (1983) 269-324. 39 Triandafillou, J. and Himms-Hagen, J., Brown adipose tissue in genetically obese (fa/fa) rats: response to cold and
diet, Am. J. Physiol., 244 (1983) E145-E150. 40 Vander Tuig, J.G., Knehans, A.W. and Rosmos, D.R., Reduced sympathetic nervous system activity in rats with ventromedial hypothalamic lesions, Life Sci., 30 (1982) 902-913. 41 Vander Tuig, J., Kerner, J. and Rosmos, D.R., Hypothalamic obesity, brown adipose tissue and sympathoadrenal activity in rats, Am. J. Physiol., 248 (1985) E607-E617. 42 Wickler, S.J., Horwitz, B.A. and Stern, J.S., Regional blood flow in genetically obese rats during non-shivering thermogenesis, Int. J. Obesity, 6 (1982) 481-490. 43 York, D.A., Holt, S.J. and Marchington, D., Regulation of brown adipose tissue thermogenesis by corticosterone in obese (fa/fa) rats, Int. J. Obesity, 9 Suppl. 2 (1985) 89-96. 44 York, D.A., Marchington, D., Holt, S.J. and Allars, J., Regulation of sympathetic activity in lean and obese Zucker (fa/fa) rats, Am. J. Physiol., 249 (1985) E299-E303.
227
Elsevier BRE 12396
Hypothalamic control of brown adipose tissue in Zucker lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres S.J. H o l t 1, H.V. W h e a l 2 and D . A . Y o r k 1 1Department of Nutrition, School of Biochemical and PhysiologicalSciences, Southampton University, Southampton ( U. K.) and 2Departmentof Neurophysiology, School of Biochemical and Physiological Sciences, Southampton University, Southampton (U.K.) (Accepted 22 July 1986)
Key words: Ventromedial hypothalamus; Zucker rat; Brown adipose tissue; Sympathetic efferent; Supraoptic nucleus; Lateral hypothalamus; Dorsomedial nucleus
Electrophysiological stimulation of the hypothalamic ventromedial nucleus (VMN) resulted in an increase in interscapular brown adipose tissue (BAT) temperature in both lean and obese (fa/fa) rats. Graded stimulations resulted in progressively larger temperature increases in both lean and obese (fa/fa) groups. Both intraperitoneai injection of propranolol and surgical denervation (but not sham denervation) abolished the increase in BAT temperature following VMN stimulation, in both lean and obese (fa/fa) groups. Electrical stimulation of the supraoptic region, and certain anterior hypothalamic regions also resulted in increases in BAT temperature of lean and obese (fa/fa) rats, but stimulation of the dorsomedial nucleus and regions of the lateral hypothalamus did not affect BAT temperature. All hypothalamic regions capable of activating BAT gave a similar maximum rise in temperature for a given stimulus in lean and obese (fa/fa) rats. These results suggest that the efferent sympathetic pathway from the VMN and other hypothalamic regions of BAT is normal in the obese (fa/fa) rat. INTRODUCTION The ventromedial hypothalamus (VMH) has a well established role in the control of food intake in rats 31. Electrical stimulation of this region results in satiety and other sympathetic responses 3, whereas stimulation of the lateral hypothalamus initiates feeding 2'5 and parasympathetic responses 3. Bilateral lesions in the V M H area in young as well as adult rats causes pronounced accumulation of body fat. Obesity develops not only when animals are allowed to overeat, but also when their food intake is restricted to the level of control animals in pair-feeding experiments 6'7'19. This increase in energetic efficiency in the V M H lesioned rat is also characteristic of the genetically obese (fa/fa) Zucker rat 5,13 in which a reduction in metabolic rate is observed during the first
week of life, before either hyperphagia or hyperinsulinaemia is established 6,3°. Thus in both of these models of obesity a reduction in energy expenditure is an important causative factor in the accumulation of excess body fat. Thermogenesis in brown adipose tissue (BAT) has only recently become recognized as a significant component of diet-related expenditure (see ref. 34 for review). Its main function was thought to be the provision of heat for thermoregulatory thermogenesis 37. Both the VMN-lesioned rat and the obese (fa/fa) rat show reduced B A T thermogenesis when compared with their respective control groups 13'16'36, a result of the attenuated central sympathetic drive to the tissue. When the obese (fa/fa) rat is overfed by offering a 35% sucrose drinking solution in addition to a normal laboratory diet, it does not show the normal
Correspondence: S.J. Holt, Department of Nutrition, School of Biochemical and Physiological Sciences, Southampton University, Southampton, SO9 3TU, U.K.
228 increases in sympathetic activity or BAT function seen in the lean r a t 17'18'39 (dietary induced thermogenesis (DIT)). On the other hand the young obese (fa/fa) rat shows a normal increase in sympathetic activity and B A T thermogenesis when cold acclimated 17'39 (cold induced non-shivering thermogenesis (NST)) although this response may be impaired in the adult. The VMH-obese rat also displays a reduction in sympathetic stimulation of BAT, and this causes a failure to respond normally to both dietary and thermal stimuli 13'14'4°. The centres controlling energy and temperature homeostasis are thought to be located in differing regions of the hypothalamus 4'5. A functional link between the VMH and BAT has been demonstrated in normal rats T M . In addition, an acute reduction in sympathetic nerve activity has been reported after electrolytic lesions of the VMH 26. Furthermore, a functional disconnection is suggested in the VMH lesioned rat by their failure to show the normal changes in sympathetic activity and BAT thermogenesis in response to manipulations of diet and environmental temperature 14'26. The accumulated evidence suggests that the defect in the obese (fa/fa) rat may reside in the hypothalamus and be represented by either a failure to produce, recognize or respond to satiety signals. The aim of the present study was to examine electrophysiologically whether a dysfunction exists in the efferent pathway between the VMH and other hypothalamic regions and B A T in the obese (fa/fa) rat.
TABLE I
MATERIALS AND METHODS
%
Preparation o f animals Female lean (fa/?) and obese (fa/fa) rats at 7 weeks of age (150 + 5 g and 180 + 5 g b. wt., respectively) were used for all experiments. The animals were anaesthetized with urethane (25%) w/v solution injected i.p. at 1.2 g/kg) and placed in a stereotaxic instrument with the head orientated as described in the stereotaxic atlas of K6nig and Klippe123. Body temperature was maintained between 37 and 38 °C with a thermostatically controlled heating pad, and the experiments took place in a room kept between 24 and 26 °C.
2.0
Increase in BAT temperature (A °C) after electrical stimulation (8 V) of several hypothalamic areas' in lean and obese (fa/fa) rats Hypothalamic region
n
Lean
Obese (fa/fa)
Ventromedial Supraoptic Anterior Lateral Dorsomedial
10 6 5 10 8
0.89 + 0.05 0.90 + 0.04 0.86 + 0.05 No change No change
0.93 + 0.04 0.87 _+0.04 0.79 ___0.07 No change No change
B A T pad and the pad exposed by cutting through the overlying white adipose tissue and muscle. A thermocouple probe was inserted and secured between the two lobes of the tissue. The skin was then clipped together over the top of the depot. A thermocouple probe was also inserted 6 mm into the rectum to record core temperature. Temperatures were continuously monitored and recorded every 60 s with a mi-
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Recordings o f B A T and rectal temperature A small incision was made above the interscapular
Obese
18 16 1-4 1.2 1.0 (1.8 06 04
Volts = 10 m i n
Time
Fig. 1. Change in BAT temperature following electrical stimulation of the hypothalamic VMN in lean and obese (fa/fa) rats: response to varying voltage. The figures on the time axis indicate the voltage of the stimulation. The duration of the stimulating voltage was 30 s at the arrow point.
229 croprocessing scanning thermometer and a digital printout (Comark Electronics, Rustingdon, Sussex).
Electrical stimulation Following exposure of the brain a fine (0.5 mm diameter) bipolar steel electrode (Clark Electromedical, Reading, Sussex) insulated except at the tip, was stereotaxically implanted from the dorsal approach, into the appropriate hypothalamic region using the coordinates of K6nig and Klippe123 with modifications for lean and obese (fa/fa) rats. An isolated stimulator was used to deliver a 30 s train of monophasic square wave pulses I ms, half cycle duration, 50 Hz at an intensity of 8 V. Graded intensities between 2 and 35 V were used. A stabilization period of 30 min was allowed before any stimulations or recordings began, and following stimulation, the BAT temperature was allowed to return towards baseline before the stimulus was repeated. At the termination of each experiment a small electrolytic lesion was made to mark the stimulus sites for subsequent histological examination. The rat was killed by cardiac perfusion with a 10% (v/v) formal saline solution containing 5 % (w/v) potassium ferrocyanide. The brain was removed and sections were cut at 50 pm on a freezing microtome and microscopically examined under low and medium power to determine the correct location of the electrode. The inclusion of potassium ferrocyanide in the perfusion solution resulted in an additional blue iron complex developing at the site of lesioning. The sections were stained with Cresyl violet to verify the nuclei containing regions and areas were identified using the atlas of K6nig and Klippe123.
initial component was a rapid increase in temperature which reached a maximum within 3-5 min at the lower voltages (<20) and 6-7 min at higher voltages. These effects were the same for each group. Graded stimulations of the VMN were followed by graded increases in BAT temperature in both lean an obese (fa/fa) groups. A minimum threshold of 3 V was usually observed, below which no increase in BAT temperature was recorded. No significance should be attached to the apparent difference, in Fig. 1, between lean and obese (fa/fa) rats in the voltage required for maximum response, since this was highly variable in both groups. However, the duration of the response to stimulation at a given voltage was characteristically longer in the obese (fa/fa) rat compared with the lean rat. In order to confirm that the BAT responses were mediated via the sympathetic innervation, the effects of the fl-adrenergic blocker propranolol and of denervation were investigated. Fig. 2 shows the typical changes in BAT and rectal temperatures following hypothalamic VMN stimulation. The rapid increase in BAT temperature preceded the less marked increase in rectal temperature. Treatment with propranolol completely abolished the response to subsequent VMN stimulation in both lean and obese (fa/fa) rats (Fig. 2). Fig. 3 shows the effect of severing the bilateral sympathetic nerve bundles on the response of BAT to VMN stimulation. After establishing a normal response, a sham denervation had no effect on the increase in BAT temperature following further VMN stimulation, whereas denervation abolished the capacity of the
Lean
RESULTS
Obese I
The effects of electrical stimulation of the VMN on BAT temperature in lean and obese (fa/fa) rats is shown in Table I. An 8 V stimulus train resulted in a similar temperature increase in both groups. Stimulation of the area 0.5 mm dorsal or ventral to the VMN did not evoke a change in BAT temperature (Fig. 4) indicating specificity to the nucleus region. Fig. 1 illustrates typical changes in BAT temperature induced by consecutive VMN stimulations of increasing voltage in lean and obese (fa/fa) rats. After a 30 s stimulus train there was a further 60 s delay before any increase in BAT temperature was observed. The
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Fig. 2. The effect of stimulation of the hypothalamic VMN on BAT and rectal temperature in lean and obese (fa/fa) rats. The arrows indicate a 30 s electrical stimulation of 8 V. The dashed line shows the time point at which rats were injected (i.p.) with the 15-adrenergic antagonist propranolol (2 mg/kg).
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Fig. 3. The effect of electrical stimulation of the hypothalamic VMN on BAT and rectal temperature in lean and obese (fa/fa) rats. The arrows indicate the times at which a 30 s 8 V stimulus was applied. The solid ( .....) line indicates the time of bilateral denervation of the BAT and the dashed line (---) indicates the time of sham denervation. 6060~
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tissue to respond to further stimulation. This effect was identical in both lean and obese (fa/fa) rats, B A T is known to be responsive to environmental temperature changes and neurons controlling this response are present in several hypothalamic regions. Thus we studied the effect of electrically stimulating a number of these nuclei on B A T temperature in lean and obese (fa/fa) rats. Table I shows that B A T temperature increases obtained after stimulation of both the supraoptic and certain regions of the anterior hypothalamus were similar in both lean and obese (fa/fa) rats. Fig. 4 illustrates the regions in which a positive response was obtained. The increases in B A T temperature are similar to those observed after VMN stimulation for a given voltage. Conversely no increases in B A T temperature were obtained after stimulation in either the lateral hypothalamic areas examined or in the dorsomedial nuclei in either genotype. The negative responses are also illustrated in Fig. 4. These results indicate that in the hypothalamic regions examined the capacity to evoke an activation of B A T thermogenesis is essentially similar in lean and obese O%/fa) rats. DISCUSSION
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Fig. 4. Schematic representation of partial coronal sections (anterior to posterior according to the coordinates of K6nig and Klippe123). Circles show position of electrode tip which evoked an increase (0) or no change (O) in BAT temperature following an 8 V stimulation. Abbreviations: FMP, fasciculus medialis prosencephali; OT, optic tract; IIIV, third ventricle; so, nucleus supraopticus; ha, nucleus anterior; hi, nucleus lateralis; hvm, nucleus ventromedialis; hd, nucleus dorsomedialis.
Brown adipose tissue is the major site for both NST and D I T in small rodents 11"32"33.Its thermogenic activity is controlled principally by its abundant sympathetic innervation 24. Previous work in our laboratory and by others has shown that the genetically obese (fa/fa) rat is able to activate B A T thermogenesis in response to cold, but the response to dietary stimuli is absent or severely impaired 17'39. This inability to activate D I T has been related to the absence of sympathetic stimulation of B A T 43'44. The hypothalamic centres regulating thermoregulatory and dietary induced sympathetic activity are thought to differ. In this work we have shown that stimulation of a number of hypothalamic regions will activate BAT. This confirms previous observations that the VMN appears to have a functional connection with B A T 15' 29,41. The rapid increase in temperature after VMN stimulation is similar to that previously described 29,41. However, our results differed from those published in two respects: firstly, we did not observe the initial, transitory decrease in temperature re-
231 ported, despite continuous monitoring of BAT temperature; secondly the duration of the response was shorter in the present study. There may be many possible differences, including the anaesthetic used, different mode of stimulation, rat species or age and the room temperature at which the experiment took place, which may account for the discrepancies observed. The functional connection between the VMN and BAT is clearly mediated through the sympathetic innervation to the tissue, since both pretreatment with the fl-adrenergic antagonist propranolol and sympathetic nerve transection blocked the response. We have made similar observations after stimulation of the supraoptic area (unpublished observations). The responses of the obese (fa/fa) rats to regional hypothalamic stimulation were similar to those observed in the lean rat with regard to the rapid onset and maximal size of the response. However the duration of the response was longer in the obese (fa/fa) rat. Blood flow to BAT in the adult obese (fa/fa) rat is reduced compared with the lean animal 42 and this reduced capacity to dissipate the heat produced would potentiate the duration of the response. Additionally the obese (fa/fa) rat has an insulating layer of subcutaneous fat overlying the interscapular BAT depot which may be expected to reduce radiant heat losses. In these studies we have shown that electrical stimulation of the VMN, supraoptic area and regions of the anterior hypothalamus activated BAT thermogenesis. Lesions of the VMN have previously been shown to reduce sympathetic activation to BAT and to be associated with loss of DIT 13A4'35'4°. In contrast, the anterior hypothalamus is thought to be involved with the control of NST 8. A functional connection of the supraoptic area and BAT has not previously been demonstrated. The main function of this region is thought to be neurosecretory, with efferent pathways projecting mainly to the hypophysis 38. It is possible that the observed response may be due to stimulation of the 'supraoptic decussations', which are known to project to the VMN 28. The fibres of this pathway also contain aminergic neurons 12,21,28 and stimulation may activate one of the several ascending or descending aminergic fibre tracts coursing through the hypothalamus 4,28. Further experiments are therefore required to elucidate the hypothalamic pathways involved with sym-
pathetic activation of BAT. Stimulation of regions between the anterior hypothalamus at the supraoptic level and the anterior VMN did not result in a change in BAT temperature. Similarly in the lateral hypothalamic areas examined and in the dorsal medial nucleus no functional connection was demonstrated. These regions are not known to have any direct role in either DIT or NST. Our results have clearly demonstrated that all hypothalamic centres capable of activating BAT in lean rats were similarly responsive in obese (fa/fa) rats. We may thus hypothesize that the inability of the obese (fa/fa) rat to activate sympathetic function in response to diet, may be due to an absence of afferent signals, or the inability to recognize signal inputs or couple them to the appropriate efferent output. Glucose sensitive, insulin responsive neurons are located in the VMN 27. The recent demonstration of specific insulin receptors in the hypothalamus and other brain regions lends support to the concept of a physiological role for insulin in controlling brain function. It is of particular interest to note that the level of insulin binding is severely depressed in the hypothalamus of the obese (fa/fa) rat and this reduction shows a gene dosage effect1°. In addition it is known that the obese (fa/fa) rat does not show the normal feeding response, nor the inhibition of BAT thermogenesis in response to either centrally or peripherally administered 2-deoxy-D-glucose (2DG) 1'2°. Since 2-DG impairs sympathetic drive to BAT, it is possible that the absence of diet induced BAT thermogenesis in the obese (fa/fa) rat may reflect a central impairment of glucose metabolism. Such a hypothesis is also supported by a recent demonstration that a restoration of diet induced BAT thermogenesis in the obese (fa/fa) rat following adrenalectomy 17'25is associated with a restoration of normal responses to 2-DG administration 1. Further work to investigate the response of hypothalamic centres of the obese (fa/fa) rat to nutrient and endocrine stimulation is required to substantiate this hypothesis.
ACKNOWLEDGEMENTS This research was supported by the Wellcome Trust.
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