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Effects of water restriction on circadian rhythms of corticosterone, growth hormone and thyroid stimulating hormone in adult male rats
Physiology & Behavior, Vol. 38, pp. 327-330. Copyright©PergamonPress Ltd., 1986. Printedin the U.S.A.
0031-9384/86$3.00 + .00
Effects of Water Restriction on Circadian Rhythms of Corticosterone, Growth Hormone and Thyroid Stimulating Hormone in Adult Male Rats A. A R M A R I O * A N D T. J O L I N t
*Departmento Fisiolog(a Animal, Fac. Ciencias, Univ. A u t r n o m a Barcelona and t D e p a r t m e n t o Endocrinolog(a Experimental, Inst. Investigaciones Biom~dicas CSIC, Madrid, Spain R e c e i v e d 30 O c t o b e r 1985 ARMARIO, A. AND T. JOLIN. Effects of water restriction on circadian rhythms of corticosterone, growth hormone and thyroid stimulating hormone in adult male rats. PHYSIOL BEHAV 38(3) 327-330, 1986.--The effects of acute and chronic water restriction on the circadian rhythms of corticosterone, growth hormone (GH), and thyroid stimulating hormone (TSH) were studied in adult male rats. Water restricted rats were allowed to drink between 9.00 and 9.30 a,m. only. Chronic water restriction but not one day of such treatment altered the circadian pattern of corticosterone so that a peak of this hormone appeared before water presentation. In contrast, neither acute nor chronic water restriction altered the qualitative patterns of circadian GH and TSH rhythms although both treatments depressed the secretion of the two hormones, the depression being greater in chronic than in acute water restricted rats. These results indicate that water restriction did not resynchronize the GH and TSH circadian rhythms. It appears that reduced secretion of GH and TSH observed in water restricted rats would be due to the concomitant reduction in food intake. Water restriction
Corticosterone
Growth hormone
A L T H O U G H light appears to be the most important synchronizer of circadian biological rhythms [24], it has been recently reported that nutrient availability might act as a synchronizer. Thus, restriction of water or food availability to a short period of time in the morning can shift the circadian rhythm of corticosteroids in the rat so that a peak of corticosteroids appears just before drinking or eating in addition to or instead of the normal peak at lights off [6, 8, 10, 15, 22, 25]. It should be taken into account that water or food restricted rats eat in the morning while control rats eat mainly in the dark phase of the fighting cycle [20,21]. It appears that food rather than water consumption is the determinant factor in the entrainment of circadian corticosteroid rhythm since water restricted rats eat immediately after drinking and the effects of food restriction are very similar to those of water restriction. Nevertheless, the latter regimen appears to be an interesting tool to study the influence of food availability on circadian rhythms as water restricted rats eat a great amount of food within the hour following daily water presentation. The present work aims at the possibility that daily water restriction could resynchronize the circadian rhythms of growth hormone and thyroid stimulating hormone. The effect of one day of previous water restriction was also studied to differentiate between the acute and chronic effects of this treatment. Moreover, it should be expected that one day of
327
Thyroid stimulating hormone
Circadian rhythms
water restriction would have no effect on the circadian pattern of secretion of these hormones. METHOD Male Spague-Dawley rats 50__-2 days old upon arrival at the laboratory were used. They were housed in groups of four per cage in a controlled environment (temperature 22°C, light on from 06.00 to 18.00) throughout all the experimental period. Food and water were available ad lib unless otherwise stated. The rats were allowed to become accustomed to our laboratory for ten days before the beginning of the experiment. Then they were assigned to three experimental groups: (a) Thirty-one control rats maintained in standard conditions. (b) Twenty-five rats subjected to one day of water restriction (WR1). They were maintained as control rats but the bottles of water were removed the day before the sacrifice at 9.30 a.m. (c) Thirty-two rats subjected to chronic water restriction (CWR) for 34 days so that water was available daily between 9 and 9.30 a.m. On day 35, rats from the three groups were killed at 6 hour intervals over a day. The number of rats at each time point was 6-7 in control and CWR groups, and 5 in WR1 group. Both WR1 and CWR rats were provided with water between 9 and 9.30 a.m. in the day of sacrifice. In order to study the influence of water and food ingestion on circulating levels of
328
ARMARIO AND JOLIN TABLE 1 PATTERN OF FOOD I N T A K E IN CONTROL AND WATER RESTRICTED RATS
,.°I
distribution of food intake over a day ~
f
t
Food intake g/rat/day
09-10
Control
25.5 + 0.2
--
Water restriction
18.1 _+ 0.7
Group*
09-13
0%19
4.9 _+ 1.7 10.6 _+ 3.4
28.8 _+ 2.8 46.9 +_ 1.7 54.3 _+ 0.9
*Number of cages controlled per group n = 5. Means _+ SEM are represented. tit is represented cumulative percent of total daily food intake. *p<0.001. 0
I
I
I
I
10
20
30
40
Days
FIG. 1. Body weight gain in control and water restricted rats. Means and SEM (n=8) are represented. Open squares represent controls and closed squares water restricted rats. The differences between the two experimental groups were always significant (p<0.01).
the hormones under investigation several rats from each experimental group were killed one hour after water presentation to water restricted rats (10.00 a.m.). The animals were killed by decapitation within 30 sec after they had been taken from the animal house. Care was taken to minimize disturbances associated with killing procedure. The trunk blood was collected in plastic tubes and centrifuged at 4000 rpm for 10 min. The serum was stored frozen at -20°C. Serum corticosterone was determined by a direct radioimmunoassay (RIA) as detailed elsewhere [1]. GH and TSH were determined by double antibody RIAs using reagents kindly provided by Dr. Parlow through the Rat Pituitary Hormone Program of the N I A D D K (NIH, Bethesda, MD). All samples were processed in the same assay to avoid inter-assay variations. Intra-assay coefficients of variations were below 10%. The statistical significance of the results was evaluated with one-way A N O V A . Two comparisons were programmed: One with time of day as main factor which was done for each experimental group; another with previous chronic treatment (control, WR1, CWR) as main factor which was done for each time of day. Where appropriate individual comparisons were done with Duncan's procedure. Data were log transformed to achieve homogeneity of variances.
2t~
15
_b\
LIM/!~F FIG. 2. Effects of acute and chronic water restriction on the circadian rhythm of corticosterone in male rats. Rats subjected to one day (-- - - --) or 34 days (. . . . . ) of water restriction so that water was daily available between 09 and 09.30 a.m. only, were killed jointly with control animals ( ) over a 24-hr period. Arrow indicates the time of water presentation. Groups labelled with different letters are significantly different from the others at the same time point of the circadian cycle. Means (n=5-7 per group) are represented.
RESULTS
Figure 1 shows body weight gain in control and CWR rats. CWR rats showed diminished body weight gain as compared with control rats at all the observed periods (p <0.001). Likewise, CWR rats consumed less food than control rats (p<0.001). Table 1 shows the daily pattern of food intake in the two experimental groups. This pattern was very different in control and CWR rats. Figure 2 depicts circadian rhythmicity of corticosterone in control and water restricted rats. The effect of time of day was significant for the three experimental groups (p <0.001). Control rats showed the well-known circadian pattern of
corticosterone characterized by low levels at lights on and high levels at lights off. WR1 rats showed the same pattern. In contrast, CWR rats showed higher corticosterone levels at 08.00 a.m. than the other two groups as revealed by Duncan's test. A dramatic fall in serum corticosterone levels was observed after water availability so that corticosterone levels were similar as those of the other two groups at 10.00 a.m. Corticosterone levels were similar in the three groups at lights off. The A N O V A revealed the existence of circadian GH rhythms in control (p<0.001), WR1 (/9<0.001) and CWR
W A T E R RESTRICTION A N D C I R C A D I A N R H Y T H M S
q
329
c--o F ,~
2b
o'2
1~
~
o~
FIG. 3. Effects of acute and chronic water restriction on circadian rhythm of GH in male rats. For details see legend of Fig. 2.
FIG. 4. Effects of acute and chronic water restriction on circadian rhythm of TSH in male rats. For details see legend of Fig. 2.
(p<0.05) rats. Circadian G H rhythm in control rats was characterized by a bimodal pattern with two peaks at 10.00 and 20.00 hours (Fig. 3). WR1 rats had significant lower levels of G H than control rats at 10.00 and 14.00 hours, but they showed the same circadian pattern. Chronic water restriction almost abolished circadian G H rhythm by markedly depressing GH peaks at 10.00 and 20.00 hours. The differences between WR1 and CWR rats were significant at 10.00, 20.00 and 02.00 hours. Circadian TSH rhythmicity (Fig. 4) was observed in the three experimental groups (p<0.001). These rhythms were characterized by the presence o f a peak at 10.00 in all groups. WR1 rats had lower TSH levels than control rats at I0.00 and 14.00 hours. Chronic water restriction resulted in even lower TSH levels so that these latter rats showed reduced TSH levels at 08.00, 10.00, 14.00 and 02.00 hours as compared with rats subjected to one day of water restriction.
11, 24]. In normal conditions the two synchronizers would be in phase as rats eat most food at lights off. The effects of water restriction on GH and TSH secretion were similar in the two cases. Thus, WR1 rats showed lower GH and TSH levels than control rats at some points of the circadian cycle especially when control rats showed the peaks of secretion. Chronic water restriction resulted in even more marked depression of GH and TSH levels. Nevertheless, the qualitative pattern of circadian rhythmicity was roughly similar in the three experimental groups. It should be concluded that water and food availability did not act as a zeitgeber for the entrainment of GH and TSH rhythms. This is in accordance with the results of Moberg et al. [14] who found that restriction of food availability to 2 hours in the morning induced a transient decrease in GH secretion but did not alter the pattern of circadian GH rhythm. In contrast, it has been found that inversion of the photoperiod reversed circadian TSH rhythm [17] so that light appears to be the most important synchronizer of TSH rhythms and perhaps GH rhythms. Reduced GH and TSH secretion induced by water restriction was likely due to a diminished food intake since water restricted rats eat approximately 60-70 percent of food consumed by control rats ([2], present data) and chronic food restriction was found to depress GH and TSH levels [16]. GH secretion is under the control of two hypothalamic factors: GH-releasing hormone (GH-RH) and somatostatin (GH-release inhibiting hormone). It seems possible that a decrease of G H - R H release might mediate the inhibition of G H secretion observed in water restricted rats; however, somatostatin might be likely involved since reduced GH levels caused by food deprivation were prevented by passive immunization of the rats with somatostatin antiserum [23]. TSH secretion is also under the inhibitory action of somatostatin [3] and increased somatostatinergic activity might, at least in part, contribute to the impairment of TSH release observed in water restricted rats. In sum, acute and chronic water restriction resulted in diminished G H and TSH secretion, but these treatments did not resynchronize the circadian rhythms of the two hormones. In contrast, chronic water restriction alters the circadian rhythm o f corticosterone, inducing the appearance of a peak before water presentation.
DISCUSSION
The circadian pattern of circulating hormone levels observed in the present study is in good agreement with those obtained by other authors [4, 7, 12, 18, 19]. The present results indicate that one day of previous water restriction did not alter the circadian pattern o f corticosterone. On the contrary, chronic water restriction induced the appearance of a corticosterone peak before water presentation in keeping with previous reports [6,8]. It appears that food rather than water intake w o u l d b e responsible for the appearance of such a peak. Firstly, restriction of food availability with water ad lib induced the same effects as water restriction [•0, 15, 25]. Secondly, water restricted rats eat a great amount of their total food intake within the hour following water presentation (see the Results section). Last of all, water availability per se appears to be a poor zeitgeber at least with regard to locomotor activity [13]. CWR rats showed a similar peak of corticosterone as control rats at lights off. This might be due to the fact that CWR rats consumed almost 50 percent of their total food intake at lights off. However, a similar peak has been frequently observed in food-restricted rats without access to food at lights off [5,14]. Therefore, it appears that both light and food availability can act as zeitgebers for the circadian corticosteroid rhythms likely acting on different neural centers [9,
330
ARMARIO AND JOLIN REFERENCES
1. Armario, A. and J. M. Castellanos. A simple procedure for direct corticosterone radioimmunoassay in the rat. Roy Esp t~7siol 40: 437-442, 1984. 2. Armario, A., J. M. Castellanos and J. Balasch. Chronic noise and water restriction as stress models in relation to food and water intake and hormonal profiles in adult male rats. Nutr Rep lnt 28: 1333-1339, 1983. 3. Drouin, J., A. De Lean, D. Rainville. R. Lachance and F. Labile. Characteristics of the interaction between thyrotropin releasing hormone and somatostatin for thyrotropin and prolactin release. Endocrinology 98: 514-521, 1976. 4. Guillemin, R., W. E. Dear and R. A. Liebelt. Nycthemeral variations in plasma free corticosteroid levels of the rat. Proe Soc Exp Bh)l Med 101: 394-399, 1959. 5. Honma, K-I., S. Honma and T. Hiroshige. Critical role of food amount for prefeeding corticosterone peak in rats. Arn J Physiol 245: R33%R344, 1983. 6. Johnson, J. T. and S. Levine. Influence of water deprivation on adrenocortical rhythms. Neuroendocrinology 11: 268--273, 1973. 7. Jordan, D., B. Rousset, F. Perrin, M. Fournier and J. Orgiazzi. Evidence for circadian variations in serum thyrotropin, 3,5,Ytriiodothironine and thyroxine in the rat. Endocrinology 107: 1245-1248, 1980. 8. Krieger, D. T. Food and water restriction shifts corticosterone, temperature, activity and brain amines periodicity. Endocrinology 95: 1195-1201, 1974. 9. Krieger, D. T. Ventromedial hypothalamic lesions abolish food-shifted circadian adrenal and temperature rhythmicity. Endocrinology 106: 649-654, 1980. 10. Krieger, D. T. and H. Herbert. Comparison of synchronization of circadian corticosteroid rhythms by photoperiod and food. Proc Natl Acad Sci USA 75: 1577-1681, 1978. 11. Krieger, D. T., H. Hauser and L. C. Krey. Suprachiasmatic nuclear lesions do not abolish food-shifted circadian adrenal and temperature rhythmicity. Science 197: 398-399, 1977. 12. Martin, J. B., L. P. Renaud and P. Brazeau. Pulsatile growth hormone secretion:suppression by hypothalamic ventromedial lesions and by long-acting somatostatin. Science 186: 538-540, 1974. 13. Mistlberger, R. E. and A. Rechtschaffen. Periodic water availability is not a potent zeitgeber for entrainment of circadian locomotor rhythms in rats. Physiol Behav 34: 17-22, 1985.
14. Moberg, G. P,, L. L. Bellinger and V. E. Mendel. Effect of meal feeding on daily rhythms of plasma corticosterone and growth hormone in the rat. Neuroendocrinology 19: 160-169, 1975. 15. Morimoto, Y., K. Arisue and Y. Yamamura. Relationship between circadian rhythm of food intake and that of plasma corticosterone and effect of food restriction on circadian adrenocortical rhythm in the rat. Neuroendocrinology 23:212-222, 1977. 16. Ortiz-Caro, J., C. Gonzalez and T. Jolin. Diurnal variations of plasma growth hormone, thyrotropin, thyroxine and triiodothyronine in streptozotocin-diabetic and food-restricted rats. Endocrinology !15: 2227-2232, 1984. 17. Ottenweller, J. E. and G. A. Hedge. Diurnal variations of plasma thyrotropin, thyroxine, and triiodothyronine in female rats are shifted after inversion of the photoperiod, l;'ndo~rinolo k,y 111: 50%514, 1982. 18. Retiene, K., E. Zimmerman, W. J. Schindler, J. Neuenschwander and H. S. Lipscomb. A correlative study of endocrine rhythms in rats. Acta Endocrinol (Copenh) 57: 613-623, 1968. 19. Rookh, H. V., M. Azukizawa, J. J. Distefano, III, Y. Ogihara and J. M. Hersman. Pituitary-thyroid hormone periodicities in serially sampled plasma of unanesthetized rats. End, crinology 104': 851-856, 1979. 20. Siegel, P. S. Food intake in the rat in relation to the light-dark cycle. J Comp Physiol Psychol 54: 294-301, 1961. 21. Spiteri, N. J. Circadian patterning of feeding, drinking and activity during diurnal food access in rats. Physiol Behav 28: 139147, 1982. 22. Takahashi, K., K. Inoue and Y. Takahashi. Parallel shift in circadian rhythms of adrenocortical activity and food intake in blinded and intact rats exposed to continuous illumination. Endoerinology 100: 1097-1107, 1977. 23. Tannenbaum, G. S., J. Epelbaum, E. Colle, P. Brazeau and J. B. Martin. Antiserum to somatostatin reverses starvationinduced inhibition of growth hormone but not insulin secretion. Endocrinology 102: 1909--1914, 1978. 24. Turek, F. W. Circadian neural rhythms in mammals. Annu Rev Physiol 47: 49-64, 1985. 25. Wilkinson, C. W., J. Shinsako and M. F. Dallman. Daily rhythms in adrenal responsiveness to adrenocorticotropin are determined primarily by the time of feeding in the rat. Endocrinolo~,y 104: 350-359, 1979.
0031-9384/86$3.00 + .00
Effects of Water Restriction on Circadian Rhythms of Corticosterone, Growth Hormone and Thyroid Stimulating Hormone in Adult Male Rats A. A R M A R I O * A N D T. J O L I N t
*Departmento Fisiolog(a Animal, Fac. Ciencias, Univ. A u t r n o m a Barcelona and t D e p a r t m e n t o Endocrinolog(a Experimental, Inst. Investigaciones Biom~dicas CSIC, Madrid, Spain R e c e i v e d 30 O c t o b e r 1985 ARMARIO, A. AND T. JOLIN. Effects of water restriction on circadian rhythms of corticosterone, growth hormone and thyroid stimulating hormone in adult male rats. PHYSIOL BEHAV 38(3) 327-330, 1986.--The effects of acute and chronic water restriction on the circadian rhythms of corticosterone, growth hormone (GH), and thyroid stimulating hormone (TSH) were studied in adult male rats. Water restricted rats were allowed to drink between 9.00 and 9.30 a,m. only. Chronic water restriction but not one day of such treatment altered the circadian pattern of corticosterone so that a peak of this hormone appeared before water presentation. In contrast, neither acute nor chronic water restriction altered the qualitative patterns of circadian GH and TSH rhythms although both treatments depressed the secretion of the two hormones, the depression being greater in chronic than in acute water restricted rats. These results indicate that water restriction did not resynchronize the GH and TSH circadian rhythms. It appears that reduced secretion of GH and TSH observed in water restricted rats would be due to the concomitant reduction in food intake. Water restriction
Corticosterone
Growth hormone
A L T H O U G H light appears to be the most important synchronizer of circadian biological rhythms [24], it has been recently reported that nutrient availability might act as a synchronizer. Thus, restriction of water or food availability to a short period of time in the morning can shift the circadian rhythm of corticosteroids in the rat so that a peak of corticosteroids appears just before drinking or eating in addition to or instead of the normal peak at lights off [6, 8, 10, 15, 22, 25]. It should be taken into account that water or food restricted rats eat in the morning while control rats eat mainly in the dark phase of the fighting cycle [20,21]. It appears that food rather than water consumption is the determinant factor in the entrainment of circadian corticosteroid rhythm since water restricted rats eat immediately after drinking and the effects of food restriction are very similar to those of water restriction. Nevertheless, the latter regimen appears to be an interesting tool to study the influence of food availability on circadian rhythms as water restricted rats eat a great amount of food within the hour following daily water presentation. The present work aims at the possibility that daily water restriction could resynchronize the circadian rhythms of growth hormone and thyroid stimulating hormone. The effect of one day of previous water restriction was also studied to differentiate between the acute and chronic effects of this treatment. Moreover, it should be expected that one day of
327
Thyroid stimulating hormone
Circadian rhythms
water restriction would have no effect on the circadian pattern of secretion of these hormones. METHOD Male Spague-Dawley rats 50__-2 days old upon arrival at the laboratory were used. They were housed in groups of four per cage in a controlled environment (temperature 22°C, light on from 06.00 to 18.00) throughout all the experimental period. Food and water were available ad lib unless otherwise stated. The rats were allowed to become accustomed to our laboratory for ten days before the beginning of the experiment. Then they were assigned to three experimental groups: (a) Thirty-one control rats maintained in standard conditions. (b) Twenty-five rats subjected to one day of water restriction (WR1). They were maintained as control rats but the bottles of water were removed the day before the sacrifice at 9.30 a.m. (c) Thirty-two rats subjected to chronic water restriction (CWR) for 34 days so that water was available daily between 9 and 9.30 a.m. On day 35, rats from the three groups were killed at 6 hour intervals over a day. The number of rats at each time point was 6-7 in control and CWR groups, and 5 in WR1 group. Both WR1 and CWR rats were provided with water between 9 and 9.30 a.m. in the day of sacrifice. In order to study the influence of water and food ingestion on circulating levels of
328
ARMARIO AND JOLIN TABLE 1 PATTERN OF FOOD I N T A K E IN CONTROL AND WATER RESTRICTED RATS
,.°I
distribution of food intake over a day ~
f
t
Food intake g/rat/day
09-10
Control
25.5 + 0.2
--
Water restriction
18.1 _+ 0.7
Group*
09-13
0%19
4.9 _+ 1.7 10.6 _+ 3.4
28.8 _+ 2.8 46.9 +_ 1.7 54.3 _+ 0.9
*Number of cages controlled per group n = 5. Means _+ SEM are represented. tit is represented cumulative percent of total daily food intake. *p<0.001. 0
I
I
I
I
10
20
30
40
Days
FIG. 1. Body weight gain in control and water restricted rats. Means and SEM (n=8) are represented. Open squares represent controls and closed squares water restricted rats. The differences between the two experimental groups were always significant (p<0.01).
the hormones under investigation several rats from each experimental group were killed one hour after water presentation to water restricted rats (10.00 a.m.). The animals were killed by decapitation within 30 sec after they had been taken from the animal house. Care was taken to minimize disturbances associated with killing procedure. The trunk blood was collected in plastic tubes and centrifuged at 4000 rpm for 10 min. The serum was stored frozen at -20°C. Serum corticosterone was determined by a direct radioimmunoassay (RIA) as detailed elsewhere [1]. GH and TSH were determined by double antibody RIAs using reagents kindly provided by Dr. Parlow through the Rat Pituitary Hormone Program of the N I A D D K (NIH, Bethesda, MD). All samples were processed in the same assay to avoid inter-assay variations. Intra-assay coefficients of variations were below 10%. The statistical significance of the results was evaluated with one-way A N O V A . Two comparisons were programmed: One with time of day as main factor which was done for each experimental group; another with previous chronic treatment (control, WR1, CWR) as main factor which was done for each time of day. Where appropriate individual comparisons were done with Duncan's procedure. Data were log transformed to achieve homogeneity of variances.
2t~
15
_b\
LIM/!~F FIG. 2. Effects of acute and chronic water restriction on the circadian rhythm of corticosterone in male rats. Rats subjected to one day (-- - - --) or 34 days (. . . . . ) of water restriction so that water was daily available between 09 and 09.30 a.m. only, were killed jointly with control animals ( ) over a 24-hr period. Arrow indicates the time of water presentation. Groups labelled with different letters are significantly different from the others at the same time point of the circadian cycle. Means (n=5-7 per group) are represented.
RESULTS
Figure 1 shows body weight gain in control and CWR rats. CWR rats showed diminished body weight gain as compared with control rats at all the observed periods (p <0.001). Likewise, CWR rats consumed less food than control rats (p<0.001). Table 1 shows the daily pattern of food intake in the two experimental groups. This pattern was very different in control and CWR rats. Figure 2 depicts circadian rhythmicity of corticosterone in control and water restricted rats. The effect of time of day was significant for the three experimental groups (p <0.001). Control rats showed the well-known circadian pattern of
corticosterone characterized by low levels at lights on and high levels at lights off. WR1 rats showed the same pattern. In contrast, CWR rats showed higher corticosterone levels at 08.00 a.m. than the other two groups as revealed by Duncan's test. A dramatic fall in serum corticosterone levels was observed after water availability so that corticosterone levels were similar as those of the other two groups at 10.00 a.m. Corticosterone levels were similar in the three groups at lights off. The A N O V A revealed the existence of circadian GH rhythms in control (p<0.001), WR1 (/9<0.001) and CWR
W A T E R RESTRICTION A N D C I R C A D I A N R H Y T H M S
q
329
c--o F ,~
2b
o'2
1~
~
o~
FIG. 3. Effects of acute and chronic water restriction on circadian rhythm of GH in male rats. For details see legend of Fig. 2.
FIG. 4. Effects of acute and chronic water restriction on circadian rhythm of TSH in male rats. For details see legend of Fig. 2.
(p<0.05) rats. Circadian G H rhythm in control rats was characterized by a bimodal pattern with two peaks at 10.00 and 20.00 hours (Fig. 3). WR1 rats had significant lower levels of G H than control rats at 10.00 and 14.00 hours, but they showed the same circadian pattern. Chronic water restriction almost abolished circadian G H rhythm by markedly depressing GH peaks at 10.00 and 20.00 hours. The differences between WR1 and CWR rats were significant at 10.00, 20.00 and 02.00 hours. Circadian TSH rhythmicity (Fig. 4) was observed in the three experimental groups (p<0.001). These rhythms were characterized by the presence o f a peak at 10.00 in all groups. WR1 rats had lower TSH levels than control rats at I0.00 and 14.00 hours. Chronic water restriction resulted in even lower TSH levels so that these latter rats showed reduced TSH levels at 08.00, 10.00, 14.00 and 02.00 hours as compared with rats subjected to one day of water restriction.
11, 24]. In normal conditions the two synchronizers would be in phase as rats eat most food at lights off. The effects of water restriction on GH and TSH secretion were similar in the two cases. Thus, WR1 rats showed lower GH and TSH levels than control rats at some points of the circadian cycle especially when control rats showed the peaks of secretion. Chronic water restriction resulted in even more marked depression of GH and TSH levels. Nevertheless, the qualitative pattern of circadian rhythmicity was roughly similar in the three experimental groups. It should be concluded that water and food availability did not act as a zeitgeber for the entrainment of GH and TSH rhythms. This is in accordance with the results of Moberg et al. [14] who found that restriction of food availability to 2 hours in the morning induced a transient decrease in GH secretion but did not alter the pattern of circadian GH rhythm. In contrast, it has been found that inversion of the photoperiod reversed circadian TSH rhythm [17] so that light appears to be the most important synchronizer of TSH rhythms and perhaps GH rhythms. Reduced GH and TSH secretion induced by water restriction was likely due to a diminished food intake since water restricted rats eat approximately 60-70 percent of food consumed by control rats ([2], present data) and chronic food restriction was found to depress GH and TSH levels [16]. GH secretion is under the control of two hypothalamic factors: GH-releasing hormone (GH-RH) and somatostatin (GH-release inhibiting hormone). It seems possible that a decrease of G H - R H release might mediate the inhibition of G H secretion observed in water restricted rats; however, somatostatin might be likely involved since reduced GH levels caused by food deprivation were prevented by passive immunization of the rats with somatostatin antiserum [23]. TSH secretion is also under the inhibitory action of somatostatin [3] and increased somatostatinergic activity might, at least in part, contribute to the impairment of TSH release observed in water restricted rats. In sum, acute and chronic water restriction resulted in diminished G H and TSH secretion, but these treatments did not resynchronize the circadian rhythms of the two hormones. In contrast, chronic water restriction alters the circadian rhythm o f corticosterone, inducing the appearance of a peak before water presentation.
DISCUSSION
The circadian pattern of circulating hormone levels observed in the present study is in good agreement with those obtained by other authors [4, 7, 12, 18, 19]. The present results indicate that one day of previous water restriction did not alter the circadian pattern o f corticosterone. On the contrary, chronic water restriction induced the appearance of a corticosterone peak before water presentation in keeping with previous reports [6,8]. It appears that food rather than water intake w o u l d b e responsible for the appearance of such a peak. Firstly, restriction of food availability with water ad lib induced the same effects as water restriction [•0, 15, 25]. Secondly, water restricted rats eat a great amount of their total food intake within the hour following water presentation (see the Results section). Last of all, water availability per se appears to be a poor zeitgeber at least with regard to locomotor activity [13]. CWR rats showed a similar peak of corticosterone as control rats at lights off. This might be due to the fact that CWR rats consumed almost 50 percent of their total food intake at lights off. However, a similar peak has been frequently observed in food-restricted rats without access to food at lights off [5,14]. Therefore, it appears that both light and food availability can act as zeitgebers for the circadian corticosteroid rhythms likely acting on different neural centers [9,
330
ARMARIO AND JOLIN REFERENCES
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