Ovarian hormone effects on activity, glucoregulation and thyroid hormones in the rat

Physiology & Behavior, Vol. 36, pp. 567-573. Copyright©PergamonPress Ltd., 1986. Printed in the U.S.A.

0031-9384/86$3.00 + .00

Ovarian Hormone Effects on Activity, Glucoregulation and Thyroid Hormones in the Rat D. K. T H O M A S , L. H. S T O R L I E N , *l W. P. B E L L I N G H A M

AND KATY GILLETTE

Department o f Psychology, Australian National University, Canberra, 2600, Australia and *Garvan Institute o f Medical Research, St. Vincent's Hospital, Sydney. 2010, Australia R e c e i v e d 14 M a y 1985 THOMAS, D. K., L. H. STORLIEN, W. P. BELLINGHAM AND K. GILLETTE. Ovarian hormone effects on activity, glucoregulation and thyroid hormones in the rat. PHYSIOL BEHAV 36(3) 567-573, 1986.--Ovarian hormonal influences on the range of physiological and behavioral variables which combine to affect overall energy balance are poorly delineated. In the present study 4 groups of virgin, female rats (intact, ovariectomized, ovariectomized with estrogen replacement and ovariectomized with estrogen plus progesterone) were allowed access to running wheels and activity; food intake and weight gain were measured initially under food restricted, then under ad lib conditions. Serum insulin, glucose, thyroxine (T4) and triiodothyronine (T:~)were determined on trunk blood samples obtained at the end of the experiment. Ovariectomy resulted in an increased rate of weight gain through reduced activity and T:~but food intake was unchanged. Insulin levels were greatly reduced. Estrogen replacement restored activity to the intact group's level and normalized weight gain. Insulin and T:~ were also raised to control levels but "1~4was reduced as was serum glucose. Estrogen plus progesterone replacement reduced weight gain markedly and increased T:~with normal T4. Despite the lower body weight this group was hyperglycemic and hyperinsulinemic suggesting insulin resistance. The results have important implications for the giucoregulatory and energy balance perturbations of ovarian hormone fluctuations and focus particularly on progesterone. Estrogen

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VARIATIONS in ovarian hormones, both over the estrus cycle and following ovariectomy, with and without exogenous replacement, are associated with major perturbations in energy balance. Thus body weight [15, 26, 27], food intake [15, 26, 27], activity [7, 15, 28], insulin levels [2,23], core temperature [7, 14, 19] and metabolic rate [10,18] have been reported to change significantly with ovarian hormone fluctuations. The interrelationships of the various behavioral and physiological aspects of energy balance regulation are poorly understood and somewhat paradoxical. Proestrus-estrus, when first estrogen and then progesterone peak, is associated with reduced food intake, elevated metabolic rate and body weight decline; all of which are consistent with an active regulation to a lower body weight. However, proestrus-estrus is also associated with increased insulin levels [2,23], which would counter lipolysis/proteolysis, and with increased activity [28] which is normally seen in both male and female animals suffering nutrient deprivation. Some metabolic parameters are then those of animals in energy surplus and others of animals in a state of depleted energy reserves. During diestrus, when both estrogen and progesterone levels are low, the opposite is seen. Increased food intake [26] and decreased metabolic rate [18] lead to increased body

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weight. Unlike a normal accumulation of weight, these changes are accompanied by reduced levels of the major anabolic hormone, insulin, [2,23] and also a decrease in activity [28] which normally reflects a current energy surplus. Thus, at the hormone extremes of the estrus cycle there is a curious anomaly within both behavioral (food intake and activity) and physiological (metabolic rate and insulin levels) mechanisms that regulate energy balance. The present study was aimed at further exploring the effects of ovarian hormones on energy balance by measuring food intake, body weight, activity, insulin, glucose and thyroid hormones in groups of intact, ovadectomized, ovariectomized-estrogen replaced and ovariectomizedestrogen and progesterone replaced female rats given access to activity wheels. METHOD Subjects were thirty-two, virgin, female, outbred Wistar hooded rats aged 98-105 days at the start of the experiment and ranging in weight from 205 to 250 grams. A 12-12 lightdark cycle (lights on 0830 hr) was imposed throughout the experiment with room temperature maintained at 21-23°C. Sixteen custom-built running wheels were used (diameter of 35 cm, track width 8.5 cm). The wheels opened via a

~Requests for reprints should be addressed to Dr. L. H. Storlien, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney. 2010, Australia.

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FIG. 1. Mean 24 hr activity (for Intact, Ovx, E-rep and E+P-rep groups (all n=8) during Phase I (food restriction) and Phase It (ad lib). A and C on the Phase 1 abscissa refer, respectively, to the first day in the wheel and the day on which the criterion of 5,000 revolutions per 24 hr period was reached. sliding metal door into a standard Wahmann living cage (20x 15x 12.5 cm high). The wheels were adjusted by a clutch mechanism such that with a 50 g weight attached at 90° to the vertical they revolved no more than one quarter of a revolution when released. The experiment was conducted in two partial replications with 16 animals in each. On the first day of the experiment animals were isolated in individual laboratory cages. Food and water were available at all times. At 1200-1230 hr every day animals were smeared by means of a small saline-wetted cotton bud inserted into the vagina. The smears were transferred to clean slides, allowed to dry, stained with methylene blue and examined under a microscope. After ten days, the animals were divided into four groups. These were: ovariectomized with estrogen replacement (E-rep, n = 8 , 1 /xg estradiol 17/3 in oil given SC daily); ovariectomized with estrogen and progesterone replacement (E+P-rep, n=8, estrogen as above plus 1 /zg progesterone again given SC daily); ovariectomized (Ovx, n=8) and sham ovariectomized (Intact, n=8). Animals were assigned to groups equated on the basis of their body weight. The dosages of progesterone and estrogen were chosen on the basis of observed changes in activity and body weight in pilot experiments and represent in both cases a daily injection of 4 to 5 ~g/kg. Bilateral ovariectomies were carried out under Nembutal anesthetic (60 mg/kg). Sham ovariectomies were also performed on intact animals by making an incision in each flank, extricating the ovary, removing a small piece of fat and returning the ovary to position unharmed. Animals were allowed at least one week to recover before hormone re-

placement was begun. Vaginal smears were taken throughout. After two days of stabilization under the hormone replacement conditions, all animals were placed in the running wheels with access to food for only 1 hr (1200-1300 hr) per 24 hr period. Water was continuously available. The deprivation schedule was maintained for each rat until that individual reached an arbitrary criterion of 5000 revolutions or more in any one 24 hour period after which food was reslored ad lib. The daily schedule was as follows. At 0830 the revolutions were recorded for the preceding dark period. At 1200 the rats were weighed and, if under deprivation conditions, were given a measured quantity of food (approximately 20 grams) for one hour. The food was placed on the floor of the cage. During this period access to the running wheel was blocked. At the end of the feeding period the food was removed and placed on the tray beneath the cage. 3"he relevant injections were then administered and the vaginal smears taken, after which remaining food was weighed along with the crumbs of food which had fallen through the mesh. Under ad lib food conditions, the food was removed after the animals were weighed. Access to the wheel was prevented while injections were administered and smears were taken; after which access was re-allowed. The remaining food was then weighed and approximately 50 grams of fresh food was given, again on the floor of the living cage. Food remained in the cage for 24 hours. At 2030 the revolutions were recorded for the preceding light period. On the final day of the experiment data were recorded as usual at 0830. Food was then removed and the animals locked from the wheels until 1230 when they were killed by decapitation. The trunk blood was collected on ice, allowed to clot and then separated. The serum was stored at --20°C for later analysis. Serum glucose was determined on a YSI 23A (Yellow Springs) glucose analyser. Insulin was determined by a radioimmunoassay using a rat insulin standard and human ~:'l-labelled insulin as a tracer. Triiodothyronine (T:3 and thyroxine (T,) analyses were carried out by radioimmunoassay: the T:~assay employed a second antibody separation and the T~ assay employed a methyl cellulose-charcoal separation of antibody-bound and free tracer. The experiment was divided into two sections for the purpose of analysis. The phases were: I. Deprivation, which remained in force until the animal reached a criterion of 5,000 revolutions in any one 24 hour period and II. Ad lib, which continued on from Phase I until the experiment was concluded. Since animals reached criterion after varying periods in Phase I, the time scale of the experiment was adjusted such that individual animals were aligned for deprivation days up to and including criterion and similarly for days after criterion. Some animals reached criterion rapidly (4 days), hence statistical analysis was carried out on the measures for the first day in the wheel, for the three days preceding criterion, the criterion day and 22 post-criterion days. For measures of food consumption there are only two days preceding criterion because of the change in feeding regime which occurred on the criterion day. There was no statistical difference between groups in the number of days to reach criterion. Analysis for the first day's measures of total activity, activity during the light period (a.m. activity), food intake and weight loss was by means of a one-way analysis of variance. All of the above measures were recorded during Phase I and Phase II and were analysed separately using a trend-oftrial-means technique [12].

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RESULTS

Phase I: Deprivation Total activity. Figure 1 depicts the mean 24 hour activity for all groups during Phase I. There was a significant difference in activity between groups on Day 1, F(3,28)=6.12, p<0.01, and this appears to be due to the fact that intact animals ran more than other groups on the first day in the wheel with 3 of the 8 intact animals in estrus at this time. However, there was no significant difference between groups after Day 1 and up to criterion, F(3,28)=0.37, p<0.05. As for daytime activity (Fig. 2), there was a significant effect due to activity increase under conditions of deprivation, F(3,84)= 141.11, p<0.01, but again no difference in the rate of activity increase between groups, F(9,84)=0.67, p>0.05. Food intake. The mean food intake for all groups during Phase I is shown in Fig. 3. There was a significant difference in food intake between groups on Day 1, F(3,28)=5.32, p<0.01. A post hoc comparison using Tukey's method revealed that the mean food intake for the intact group was significantly lower than the mean intake averaged over all other groups, F(4,28)= 14.37, p<0.01. However, this effect was only seen on the first day as there was no further difference in intake up to criterion, F(3,28)=0.26, p>0.05. There was a slight increase in food consumption for all groups over time, F(2,56)=4.91, p<0.05, but no significant difference in the rate of increase of food intake between groups, F(6,56) =0.29, p >0.05. Weight loss. Measures of body weight were expressed as a percentage of each animal's body weight on the day prior to being placed in activity wheels. The mean percentage loss

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during Phase I is depicted in Fig. 4. After 24 hours of deprivation there was no significant decrease in percentage weight loss between groups, F(3,28) =2.48, p >0.05. However, there was a significant difference in the weight lost between groups on the days leading up to criterion, F(3,28)=4.13, p<0.05, and weight loss was seen to increase with the degree of deprivation, F(3,84)=173.13, p<0.01. A set of orthogonal planned comparisons revealed that there was a significant difference between the E + P - r e p animals and all other groups in the amount of weight lost, t(28)=3.29, p<0.01. No other comparisons were significant.

Phase H: Ad Lib Total activity. Mean 24 hour activity for all groups during Phase II is represented in Fig. 1. The overall analysis revealed that there was a significant difference between groups, F(3,28)=4.15, p<0.05, and a significant increase in activity over days, F(21,588)= 17.30, p <0.01. There was also a significant difference in rate of activity increase between groups, F(63,588)=2.51, p<0.01. A set of planned orthogonal comparisons demonstrated that there was a significant difference in the activity of the Ovx animals when compared with all the other groups, t(28)=3.5, p<0.01. However, no other significant differences were found. Food intake. Figure 3 depicts the food intake for all groups during Phase II. The initial trend-of-trial-means analysis revealed no significant differences between groups, F(3,28)=2.42, p>0.05, indicating that all animals consumed approximately the same amounts during Phase II. Weight gain. The mean percentage weight gain for all animals during Phase II is represented in Fig. 4. The initial

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FIG. 5. Mean efficiency of weight gain scores (weight gain in grams/food intake in grams) plus one standard error of the mean, averaged over Days 13 to 22 of the ad lib phase for Intact, Ovx, E-rep and E+P-rep groups (all n=8).

analysis revealed that there was a significant difference between groups, F(3,28)= 15.79, p<0.01, and that there was a significant increase in weight over days, F(21,588)= 128.04, p<0.01. Also, the rate of weight gain was different for the different hormonal conditions, F(63,588)=2.92, p<0.01. Planned orthogonal comparisons revealed that the mean percentage weight gain for the E+P-rep group was significantly different from that of all other groups over all 22 postdeprivation days, t(28)=6.82, p<0.01. No significant differences in weight gain were found between intact animals and E-rep animals, t(28)=0.12, p>0.05, or between these animals and ovariectomized animals. However, as can be seen in Fig. 4, the ovariectomized animals appeared to be gaining at a somewhat higher rate and the results of an analysis of efficiency of weight gain are next reported. Efficiency o f weight gain score. Efficiency scores (weight gain/food intake) were calculated by averaging over postdeprivation days 13-22 for each animal and are represented in Fig. 5. A one-way analysis of variance revealed a significant difference between groups, F(3,28)=9.47, p<0.01. A set of orthogonal planned contrasts revealed that the ovariectomized group had the highest efficiency score when compared with the average score of those groups with hormonal replacement, t(28)=5.33, p<0.01. No other planned comparisons were significant. Vaginal smears. For individual animals in the intact group it was found that peak activity occurred on the night of

estrus. Activity when declined during periods of diestrus increasing again at proestrus. Figure 2 clearly depicts the effects of the estrus cycle on activity for the intact group; however, the 4 day cycle is not visible due to the fact that the intact animals were not all in synchrony during their estrus cycle. The smear pattern for E-rep and E+P-rep groups was that of permanent estrus. However, occasional and transitory lapses into a pattern most closely resembling that of proestrus were observed in all animals in both groups. There was no evidence of cyclical activity in either the smear patterns or in running behaviour. The smear picture for Ovx animals was that of anestrus, also, with no evidence of cyclicity. With only a transitory exception in one animal, deprivation did not affect the smear patterns for any of the groups. On the first day in the wheels, vaginal smears from all animals exhibited a high proportion of leucocytes with respect to all other cell types. This pattern may well be attributed to stress. Blood Analysis Five samples were lost through tube breakage and blood analysis was finally performed on 6 each from the Intact and Ovx groups, 7 from the E+P-group and 8 from the E-rep group. Statistical analysis was done by one-way ANOVA followed by Tukey between-group comparisons. Glucose and insulin. Serum glucose and insulin values for

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the four groups are shown in Fig. 6. Overall the groups differed in glucose levels, F(3,23)=26.94,p<0.01. Compared to the intact group the Ovx and E-rep groups had lower (p <0.05 and p<0.01 respectively), and the E+P-rep group higher (p<0.01), glucose levels. A similar pattern was evident in insulin, F(3,23)=8.58, p<0.01, with the Ovx group lower (p<0.01) and the E+P-rep group higher, than intact animals. The insulin/glucose ratios were: Intact 3.88__.1.03, Ovx 1.49__.0.20; E-rep 3.59-+0.65; E+P-rep 5.31-+0.64. Thyroid hormones. T4 and T3 values for the four groups are shown in Fig. 7. T4 differed between groups, F(3,28)ffi3.41, p<0.05, with the E-rep group lower than the other three (p<0.05 in each case). T.~ levels differed, F(3,23)=5.27, p<0.01, and, as might be expected, were the reciprocal of the efficiency scores (correlation r=-0.51). The differences between groups were only marginally significant (Intact versus Ovx, p<0.05; Intact versus E+P-rep, p =0.08).

to food (Phase II) weight gain was reinstated in all groups and all groups ate the same amount but the dual hormone replaced group gained weight at a slower rate than the other three groups which again did not differ (Figs. 3 and 4). Efficiency of weight gain (weight gained/food eaten) showed the ovariectomized animals to be more efficient than the other three groups (Fig. 5). During Phase II the ovariectomized group was less active than the other three groups which did not differ (Fig. 1). Among the plasma variables and compared to intact controls, glucose was reduced by ovariectomy and estrogen replacement but increased by ovariectomy and estrogen plus progesterone replacement (Fig. 6). Insulin was reduced by ovariectomy, normalized by estrogen replacement and increased by estrogen plus progesterone replacement (Fig. 6). T3 concentration precisely mirrored insulin being reduced by ovariectomy, normalized by estrogen replacement and increased by dual estrogen/progesterone administration (Fig. 7). T4 was affected only by ovariectomy plus estrogen replacement being reduced (Fig. 7).

Intact (n=6), Ovx (n=6), E-rep (n=8) and E+P-rep (n=7) groups.

Summary of Results During the period of restricted access to food (Phase I) the activity of all animals increased progressively with no significant difference in rate between groups. All groups lost weight, with the estrogen plus progesterone-replaced group losing at a greater rate than the other three groups, among which there were no differences. Given unrestricted access

DISCUSSION

We [23] and others [2] have previously demonstrated, in rats, a significant difference in insulin levels over the estrus cycle. Basal, and glucose-stimulated, insulin secretion is depressed during diestrus, a period of low estrogen and progesterone, but elevated during proestrus/estrus when

s72 these hormones are elevated. Glucose levels, however, were well regulated and did not vary over the estrus cycle. In the present experiment the insulin levels were consistent with the above observations, being low following ovariectomy and elevated by estrogen plus progesterone replacement. The pattern of hyperinsulinemia/insulinresistance following progesterone administration has been demonstrated previously [I, 11, 24] but, as in the normal estrus cycle, no increase in blood glucose was observed [I, 10]. What is significant in the present results is that despite elevated insulin (and insulin/glucose ratio) in the E+P-rep group, glucose levels were significantly higher than the intact control group and over 2 retools per liter higher than that observed for the estrogen-replaced group. Further to the above point, the diabetogenic aspects of human pregnancy are thought to be largely a function of increases in placental lactogen production [3]. However a cellular basis of a direct and significant anti-insulin role for progesterone has certainly been established [1, I1, 24, 25]. One problem is that it has been difficult previously to separate in vivo the direct effects of progesterone from the usually found increases in body weight and food intake. Such a separation was achieved in the present study. If elevated insulin/glucose ratios are taken as an index of insulin in resistance then the present results confirm that progesterone has a primary diabetogenic action. In fact, given the marked reduction in body weight in the E+P-rep group, and the usual finding that basal insulin levels fall and insulin sensitivity increases with weight loss. the hyperinsulinemia/insulin resistance with estrogen plus progesterone replacement is particularly striking. The results then have implications for glucoregulatory stresses in a diversity of conditions in the female where ovarian hormone levels fluctuate (pregnancy, menstrual cycle, birth control via estrogen/progesterone pills, menopause and hormone replacement following menopause) and suggest progesterone is of prime importance in this regard. The reduced T:+ level of the Ovx group is interesting. Hyperphagia and approximately a 20a/~ increase in body weight compared to intact controls is usually seen following ovariectomy [26,27]. In the present study where ovariectomy was combined with access to a running wheel food intake was suppressed to control levels. However, to achieve a higher body weight an animal can either increase food intake or reduce expenditure. Under the present circumstances it is apparent that the ovariectomized animals employed the latter means. The reduced expenditure can be seen both from the low activity (Fig. 1) and from the reduced T:~ (Fig. 7) as an indicator of metabolic rate. It would seem that the Ovx group are demonstrating the expected drive to higher body weight, but by reducing energy expenditure rather than increasing intake, and the rate of accumulation of excess weight is at a much slower rate than would normally be expected (see [26] for example). Why access to the running wheel prevented the expected hyperphagia following ovariectomy is not clear but the effect may be an important one for our understanding of the central determinants of food intake regulation. It should also be pointed out that the thyroid hormone pattern of low TJnormal T, is characteristic of a +'sick, euthyroid" patient where the depressed T:+ is also thought to reflect an adaptation to reduce metabolic rate and conserve body protein and energy reserves [8,9]. Little is known of the mechanism of this adaptation. In this respect it is also significant that estrogen replacement reduced T+ levels while T:~

FH()MAS t:7 ,t1.. returned to control levels. This may indicate a direct inhibitory effect of estrogen on thyroid function and under conditions where the thyroid is suppressed it has been demonstrated that a higher percentage of the thyroid hormone output can be as T:~ [17]. However. estrogen replacemenl in ovariectomized animals usually results in both physiological and behavioral drive to a lower body weight. It is then adaptNe for the estrogen-treated animals to maintain a high metabolic rate. Thus the observation in the E-rep group of normal T j l o w T, could reflect a higher rate of deiodination. This pattern is the opposite of that seen in the Ovx group. Both the Ovx and E-rep treatments of the present paradigm may then provide important models for the study of factors controlling the rate of peripheral deiodination of T i+ The effects of estrogen plus progesterone replacement on activity and body weight regulation are notable. Contrary to previous findings ]15,211, progesterone did not inhibit the facilitory effect of estrogen on activity. The dosage, however. in these studies was 1000 times that used here and of the order of magnitude of injections of progesterone used to induce ovulation in estrogen-primed rats. Such was not the intention here and the dosage of 1 p.g/day was about one-tenth of that used by Krotkiewski und Bjorntorp II61 to induce changes in adipose tissue weight in intact female rats. With the level of progesterone replacement in the present experiment, activity and food intake are not affected but body weight is suppressed (Fig. 4) and T:~ elevated (Fig. 7). This latter result is consistent with previously reported calorigenic effects of progesterone [14,191. Thus we have the situation where animals are quite severely depleted (to some 86¢~ of control body weight on the last day of the experiment) but are making no behavioral (i.e., increasing food intake or decreasing activity) or physiological compensation to restore body weight to control levels. In fact T:=. as an index of metabolic rate, is elevated. This body weight suppressing effect of progesterone has not been reported previously. Despite the above-mentioned calorigenic effects of progesterone [14,19], either no effect or even a slight increase in body weight has been found with very large amounts of progesterone in sedentary rats [21,26]. It is not clear whether the low dose of progesterone or the availability of the activity wheel or both conditions are necessary to reveal the weight reducing effects of progesterone. In this regard it is interesting that recently excessive activity has been emphasised in the etiology of anorexia nervosa [5,13]. It may be that the present pardigm will provide the basis for a suitable animal model for investigating this poorly understood pathophysiological condition. Hormone replacement has a marked effect on the diurnal rhythm of wheel running. As is well known, rats run mostly in the dark period of the light-dark cycle. This was confirmed for the Intact group where only 16.6% of revolutions were completed during the light (see Figs. 1 and 2). What is interesting is that ovariectomy and ovariectomy with estrogen replacement increases the percentage of activity in the light period to 29 and 32% respectively which are both significantly higher than the Intact group (/)<0.05). The further addition of progesterone reverses this effect and the E+Prep group run very little in the light even though their total 24-hour activity does not differ from the intact and estrogenreplaced groups. As a percentage of total activity, the E + P-rep group run only 7.3% in the light. It may be that giving our replacement injections at noon has influenced these results; however, we would anticipate a relatively stable level of hormone over the 24 hour period with our methodology.

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573

W e are n o w p u r s u i n g this issue u s i n g silastic i m p l a n t s containing estrogen or progesterone. Finally, t h e r e is a m p l e e v i d e n c e t h a t o v a r i a n h o r m o n e s act directly o n n e r v e cells, p a r t i c u l a r l y in t h e medial b a s a l h y p o t h a l a m u s , to e i t h e r directly a l t e r electrical activity or m o d i f y r e s p o n s i v e n e s s to o t h e r stimuli (see [20] for a r e c e n t review). T h e r e is also n o w a c o m p r e h e n s i v e b o d y o f literature w h i c h strongly suggests t h a t the v e n t r o m e d i a l h y p o t h a l a m u s e x e r t s its p o w e r f u l effects o n e n e r g y b a l a n c e via autonomic nervous system control of visceral glucoregulatory o r g a n s [4, 6, 22]. It is likely t h a t the o v a r i a n h o r m o n e man i p u l a t i o n s o f the p r e s e n t s t u d y r e s u l t e d in m o d u l a t i o n o f

medial basal h y p o t h a l a m i c n e u r o n a l activity. T h a t modulation m a y b e r e s p o n s i b l e for the o b s e r v e d c h a n g e s in g l u c o r e g u l a t i o n a n d e n e r g y b a l a n c e via i n f l u e n c e o n the autonomic nervous system.

ACKNOWLEDGEMENTS The authors thank A. Jenkins and R. Symons for providing the insulin and thyroid assays. This research was supported in part by an NH & MRC (Australia) Program Grant to the Garvan Institute. Our friend and colleague William P. Bellingham is deceased.

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