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Hypothalamic regulation of growth hormone secretion during food deprivation in the rat
Life Sciences, Vol. Printed in the USA
52, pp.
981-987
Pergamon
Press
HYPOTHALAMIC R E G U L A T I O N OF G R O W T H H O R M O N E SECRETION D U R I N G FOOD DEPRIVATION IN T H E RAT Bethany A. Janowski, Nicholas C. Ling*, Andrea Giustina**, William B. Wehrenberg Depts. of Health Sciences and Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211; *Whittier Institute, San Diego, CA 92037; ** Clinica Medica, University of Brescia, 25125 Brescia, Italy. (Received
in final
form December
30,
1992)
Summary Suppressed pulsatile GH secretion in food-deprived rats has been hypothesized to be due to an increase in hypothalamic somatostatin secretion. We investigated this hypothesis and the role of GHRH in regulating GH secretion during food deprivation using two different models. In experiment one, rats were food deprived for 72h during which time they received a saline infusion (n = 5). At the same time rats were normal fed for 72h during which time they received a somatostatin infusion (5/~g/h, n=7). After the 72h infusion period, all rats received two iv injections of GHRH (1/~g/rat) at 2h intervals. GH concentrations in food-deprived rats rose from approximately 10 ng/ml to 400-800 ng/ml in response to both GHRH injections. This increase was significantly greater (p<0.01) than the GH response (100-400 ng/ml) observed in somatostatin-infused animals. The significantly higher GH response observed in food-deprived rats as compared to somatostatin-infused, normal-fed rats suggests that somatostatin concentrations may decrease during food deprivation. In experiment two, rats were infused for 5h with either saline (n=6) or GHRH (10 #g/h, n=9) at the end of a 72h fast. GH concentrations did not change in saline-infused animals. In contrast, GH concentrations significantly increased (p<0.01) upon initiation of the continuous GHRH infusion. Yet, this release of GH was pulsatile in nature. Pulsatile GH secretion in the presence of a constant GHRH infusion suggests that pulsatile somatostatin release from the hypothalamus is maintained during food deprivation. These studies suggest that during food deprivation in the rat 1) absolute concentrations of somatostatin decrease, but its pattern of secretion remains pulsatile, and 2) decreased GHRH release may be responsible for the absence of spontaneous GH pulses. GH secretion in the rat is pulsatile in nature. It is regulated by interactions between growth hormone-releasing hormone (GHRH) and somatostatin (1-5). Pulsatile GH secretion during a continuous GHRH infusion in normal humans (6, 7) and in normal, conscious rats (8) suggests intermittent or pulsatile somatostatin secretion since somatostatin is known to inhibit GHRH-induced GH release from the anterior pituitary both in vivo (9) andin vitro (10-13). Endogenous pulsatile somatostatin secretion is further suggested by results obtained in normal rats passively immunized against somatostatin. In this model, a constant GHRH infusion results in elevated and continuous GH release (14). Furthermore, direct measurement of GHRH and somatostatin in hypophyseal portal blood suggests their intermittent release (15, 16). During food deprivation in rats, pulsatile GH secretion is suppressed (17-19). It has been previously proposed that this is a result of increased somatostatin tone since immunoneutralization of somatostatin increases baseline GH levels in these animals (20). However, there is now evidence to suggest that both GHRH and somatostatin are involved in regulating altered GH secretion during food deprivation. Specifically, the absence of GH pulses could result from a GHRH decrease (5) as well as from a somatostatin increase. Baseline serum GH concentrations are elevated in fasted rats with respect to normal-fed animals (21). And finally, an acute GHRH injection elicits GH release in food-deprived rats (1, 22), yet it is recognized the GHRH-induced GH secretion is significantly inhibited under conditions of high somatostatin tone (1). The aim of our study was to investigate the role of GHRH and somatostatin in regulating the altered GH secretory patterns observed in food-deprived animals. Our findings suggest that during food deprivation Copyright
0024-3205/93 $6.00 + .00 © 1993 Pergamon Press Ltd All rights reserved.
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hypothalamic somatostatin secretion is decreased but that its pattern of release remains pulsatile. This would suggest that decreased hypothalamic GHRH release is responsible for the absence of spontaneous GH peaks. Materials and Methods Animals and Surgical Preparation: All animals used in this study were acquired, maintained, and handled accordin~ to the guidelines established by the National Institutes of Health (23). Animals were housed in a temperature- and humidity-controlled environment under a 14h light, 10h dark lighting schedule. Male rats (350-450 g) were outfitted with two indwelling venous catheters under sodium pentobarbital anesthesia (45 mg/kg, ip) seven or eight days prior to experimentation. Details of the construction of the catheters and the surgical preparation of the animals are described elsewhere (14, 24). Following surgery, the animals were housed in individual isolation cages. One catheter was connected to a single channel swivel through which a constant infusion (0.08 ml/h) of heparinized saline (10 IU/ml) was administered. The second catheter was filled with heparinized saline (50 IU/ml) and plugged. This catheter was flushed twice a day (0.25 ml) until the morning of experimentation. Experiment one: Five days after surgery, a constant 72h saline infusion (0.8 ml/h) and 72h food deprivation was initiated in one group of rats (n=5). At the same time a constant 72h somatostatin infusion (5 #g/h) was initiated in normal-fed rats (n=7). This dose was selected since it is known to approach the minimum dose needed to suppress endogenous GH secretion (25, 26). Since the biological end point, pulsatile GH secretion, was the same in both the food-deprived and somatostatin-infused rats, one can hypothesize that the pituitary should be exposed to similar concentrations of somatostatin in both conditions. After 72h of food deprivation or somatostatin infusion, three control blood samples were drawn (0.35 ml) from all animals at 30 min intervals. Following the third sample, animals received an iv injection of GHRH (1 ug). Blood samples were drawn 5, 10, 15, 30, 45, 60, 90 and 120 min after administration of GHRH. Immediately following the 120 min blood sample all animals received a second GHRH injection. Subsequent samples were drawn at the times noted. The samples were centrifuged, and the plasma was frozen until measurement of GH by RIA. Red blood cells were resuspended in saline and returned to the animals. All animals were immediately decapitated after the experiment. The pituitaries were removed and frozen until assayed for GH content. Experiment two: All rats were food deprived for 72h beginning four days after surgery. After 72h of food deprivation, three control blood samples (0.35 ml) were drawn at 20 min intervals, starting at 10:00 h. Immediately after the third blood sample a 5h constant iv infusion of either GHRH (10 #g/h, n=9) or saline (0.5 ml/h, n=6) was initiated. Blood samples were drawn every 20 minutes for the duration of the infusion. Samples and pituitaries were handled as in the first experiment. Radioimmunoassay: GH concentrations were determined in duplicate by RIA using a double antibody method. Reagents were provided by the National Pituitary Agency of the National Institutes of Health. Blood samples were assayed in aliquots of 10 and 50 #1. GH concentrations are expressed in terms of the NIH-RP-2 standard. The minimum sensitivity (90% bound) was approximately 0.04 ng/tube. Within- and between-assay variation averaged less than 10%. Some samples drawn following the initiation of the GHRH infusion were re-assayed in duplicate since GH concentrations exceeded the maximum sensitivity of the assay. These samples were diluted 1:10 in phosphate-buffered saline and assayed in 20 #1 aliquots. The pituitaries were thawed and placed in 1 ml of saline. The tissues were sonicated at 4°C for approximately five min then refrozen. Tissue homogenates were thawed and centrifuged for 15 min. Aliquots of the supernatant were diluted as necessary for RIA of GH. Peptides: Somatostatin was first dissolved in 0.1 M acetic acid and then diluted to 320 #g/ml in saline containing 0.1% bovine serum albumin. The diluted somatostatin was snap frozen and stored at -20°C until used. On the day of use, the peptide was diluted to 6.4 #g/ml in sterile saline. The somatostatin solution was changed in the syringes every 12h over the 72h infusion period. Rat GHRH was diluted to 1 mg/ml in 0.01 M acetic acid, snap frozen and stored at -20°C until used. On the morning of experimentation the peptide was diluted to 20/~g/ml in sterile saline. Data Analysis: Data are expressed as mean + SEM. Since the variances of the mean GH values were not homogenous, the data were subjected to log transformation prior to statistical analysis. Determination of the overall GH response involved determining the area under the GH curve (AUC) by trapezoid analysis. Significant differences in GH concentrations, AUC and pituitary GH content were determined by one way analysis of variance (27). Pulsatile GH secretion in experiment two was identified with the use of the Pulsar algorithm (28).
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Results Experiment 1: G H concentrations in 72h food-deprived rats significantly increased (p<0.01) following both G H R H injections (Fig. 1A). Individual examples of the GH profiles from two food-deprived animals demonstrated that, following G H R H administration, GH concentrations increased from approximately 10 to 400800 ng/ml (Fig. 1B). GH concentrations also rose following GHRH injection in somatostatin-infused rats, but the magnitude of the increase was significantly lower than the increase observed in food-deprived rats (Fig. 1A). Individual profiles show that GH concentrations increased from approximately 10 to only 100-400 ng/ml in somatostatin-infused rats following administration of GHRH (Fig. 1B). GH AUC was significantly higher (p < 0.01) in food-deprived (0.44+0.08 #g/ml/5h) than in somatostatin-infused rats (0.08+0.01 #g/ml/5h). Pituitary GH content was significantly higher (p<0.05) in somatostatin-infused rats (693+79 #g/pit) as compared to fooddeprived rats (377+90/~g/pit) suggesting that GH is synthesized but the amount released is decreased in response to the constant influence of exogenous somatostatin.
B
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FIG. 1 Panel A - The mean plasma GH response following two iv injections of GHRH (1 #g, indicated by arrows) in 72h food-deprived, saline-infused rats (e e, 0.8 ml/h, n=5) or in normal-fed, 72h somatostatin-infused rats ((3 .... o, 5 #g/h, n=7). GH concentrations are expressed as mean + SEM. GH concentrations were significantly higher (**, p<0.01) in food-deprived animals as compared to the somatostatin-infused rats. Panel B - Plasma GH concentrations in two individual 72h food-deprived (FD), saline-infused (SAL) rats and two individual 72h normal-fed (FED), somatostatin-infused (SS) rats receiving two iv injections of GHRH. Experiment 2: Plasma GH concentrations averaged 10-20 ng/ml in both food-deprived groups prior to the onset of the saline or G H R H infusion. GH concentrations in saline-infused rats did not change from control values during the 5h infusion period (Fig. 2A) and were virtually devoid of any GH pulses as identified by the Pulsar algorithm. In contrast, GH concentrations increased approximately 30 fold above control values upon initiation of the G H R H infusion. These elevated GH concentrations demonstrate an underlying pulsatile pattern of GH secretion. Representative GHRH-infused and saline-infused animals are illustrated in Figure 2B. No pulses of GH were observed in five of the seven saline-infused animals. One saline-infused rat demonstrated one GH pulse (Fig. 2B) with an amplitude of 13 ng/ml and the other saline-infused rat demonstrated two pulses with a mean amplitude of 28 ng/ml and an inter-peak interval of two hours. In contrast, pulsatile GH secretion was evident in animals receiving a constant GHRH infusion. There was an immediate peak in GH concentrations upon the initiation of the G H R H infusion with GH values rising to greater than 400 ng/ml. Over the next two hours, GH concentrations declined to approximately 100 ng/ml. Over the last two hours of the infusion, GH concentrations again rose to greater than 400 ng/ml.
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B GHRH
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Saline Infusion
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FIG. 2 Panel A - Mean plasma GH concentrations in 72h food-deprived rats during a constant 5h infusion (represented by - at the top of the graph) of saline (0.5 ml/h, n=6) or GHRH (10/~g/h, n=9). GH concentrations are expressed as mean + SEM. GH concentrations did not change in saline-infused animals. GH concentrations significantly increased (**, p < 0.01) upon initiation of the GHRH infusion and remained significantly elevated (indicated by arrow), though pulsatile in nature, for the duration of the experiment. Panel B - Plasma GH concentrations in individual 72h food-deprived rats infused with either saline or GHRH for 5 h. [ ] represents pulses of GH as identified by the pulse detection algorithm Pulsar (28).
Parameters of pulsatile GH secretion as estimated by Pulsar are illustrated in Fig. 3. GH AUC was significantly higher (p<0.01) in GHRH-infused (1.52+0.27 #g/ml/5h) than in saline-infused rats (0.09+_0.03 #g/ml/5h). There was no difference in pituitary GH content between salineinfused animals (494+113 #g/pit) and GHRH-infused animals (333+28 ~zg/pit). Discussion
$* E_
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FIG. 3 The interactions between somatostatin and GHRH appear GH pulse frequency, amplitude, and to be altered during food deprivation since pulsatile GH interval in 72h food-deprived rats release is depressed following 72h of food deprivation (18receiving a 5h infusion of saline (open 21). Tannenbaum et al. (22) have suggested that increased bars) or GHRH (hatched bars). somatostatin concentrations may be the primary inhibitor Parameters were determined by the of GH following 72h of food deprivation. Indeed, direct pulse detection algorithm Pulsar (28). measurement of peripheral plasma somatostatin concentrations show an increase during food deprivation (29, 30) and passive immunization with somatostatin antiserum increases GH in food-deprived animals (20). If this is the case, then GHRH should have little or no effect in food-deprived animals since somatostatin is known to inhibit GHRH-indueed GH secretion (1, 9-13). However, it has been reported that GHRH increases GH
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secretion in food-deprived animals (1, 22). This leads to the conclusion that increased somatostatin tone may not explain decreased GH concentrations during food deprivation in the rat. It would be ideal to evaluate GHRH and somatostatin secretion in food-deprived rats by directly measuring portal blood concentrations. Unfortunately, this technique is only available in anesthetized rats and it is known that anesthesia affects GHRH and somatostatin secretion (31). Thus, we chose to evaluate the secretion of these two neuropeptides by measuring GH levels during the constant infusion of either G H R H or somatostatin. Our results suggest that the loss of pulsatile GH secretion reflects an absence of pulsatile GHRH secretion and a diminished yet still pulsatile release of somatostatin. The following observations support this hypothesis. First - the somatostatin infusion in normal-fed rats in experiment one was designed to mimic the same biological effect (i.e. inhibition of pulsatile GH secretion) observed during food deprivation. If somatostatin concentrations are indeed elevated during food deprivation, the GH response to exogenous GHRH in both food-deprived and somatostatin-infused rats should be similar. Our results show that this is not the case. GH concentrations following GHRH injection are significantly higher in food-deprived rats as compared to the somatostatin-infused rats. In fact, the GH response to GHRH in food-deprived rats was both higher and more consistent than that observed in normal rats (1). These results suggest that somatostatin concentrations decrease during food deprivation. In addition, pituitary GH content was significantly higher (p<0.01) in somatostatin-infused rats than in food-deprived rats, suggesting that GH is synthesized but not released during the constant somatostatin infusion. It is possible that the somatostatin infusion produced circulating concentrations higher than those observed during food deprivation. However, the role of peripheral concentrations of somatostatin in regulating GH secretion is unknown (29, 32). Furthermore, the infusion dose was the minimum dose of somatostatin required to suppress pulsatile GH secretion (25, 26) as observed in food-deprived rats. That is to say that the biological end point, pulsatile GH secretion, was identical in the two animal models. Second - baseline GH concentrations are elevated (10-20 ng/ml) in food-deprived rats, while they are virtually non-detectable ( < 2 ng/ml) during baseline or trough periods in normal rats (1, 8, 9). This suggests that somatostatin tone is decreased during food deprivation. Third - experiment two was designed to determine if the pattern of somatostatin release during food deprivation is pulsatile in nature. The initial GH response to the GHRH infusion was immediate and high in magnitude. Subsequent release was characterized by a gradual decrease in GH concentrations during the second hour of the infusion, followed by an increase during the third and fourth hours. Interestingly, the GH response to the infusion of GHRH in 72h food-deprived rats reflects a rhythm similar to that observed in fed, freely-moving rats during a constant GHRH infusion (8). In that study, as in this study, animals retained a pulsatile rhythm of GH secretion during a constant GHRH infusion. We attribute the phasic changes in the pituitary response to a constant G H R H stimulus to the presence of phasic endogenous somatostatin secretion. This is further supported by the observation that GH secretion is constant and non-pulsatile in nature in rats receiving a G H R H infusion and pre-treated with somatostatin antiserum (14). The present experiment suggests that episodic release of somatostatin, known, in part, to regulate the pulsatile rhythm of GH secretion, is still present but not eliminated following 72h of food deprivation. It is unlikely that the pulses in G H secretion reflect release of a secondary storage pool or de novo synthesis of GH for three reasons. One, pituitary GH content was not different between the two treatment groups, a fact which does not support increased synthesis. Two, we have previously demonstrated that the pituitary maintains its ability to respond to GHRH following repeated challenges with this neuropeptide (24). Three, pituitary GH depletion and decreased plasma GH concentrations begin to occur as early as four hours after the initiation of a continuous GHRH infusion when somatostatin is not present (14). Although we hypothesize that somatostatin is lower in food-deprived rats as compared to normal-fed rats, it is still biologically important as evidenced by the fact that somatostatin passive immunization is able to elicit a GH rebound in food-deprived rats (20) and GHRH administration to food-deprived rats which have been pretreated with somatostatin antibodies results in a larger GH response with respect to food-deprived animals which have not been pretreated (1). GH pulses are absent in food-deprived rats, even though it appears such animals have suppressed hypothalamic somatostatin tone. This leads to the conclusion that the absence of pulses is due to an absence of GHRH. This is consistent with the fact that G H R H is responsible for GH pulses in normal animals (5). In addition, it has been observed that during food deprivation, hypothalamic GHRH concentrations decrease (33). Bruno et al. (34) have recently reported that prepro-GHRH mRNA is reduced in 72h food-deprived rats. These observations further suggest that the suppression of pulsatile GH secretion during food deprivation is the result of decreased
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synthesis and/or release of GHRH. Consistent with this, we have demonstrated that GHRH is neither involved in the maintenance of basal GH concentrations nor in the GH rebound following withdrawal of somatostatin in 72h food-deprived rats (21). Short term somatostatin infusions of 5-50 #g/h have been shown to inhibit GHRH-induced GH release in the rat (26). Yet, in the present experiment, the 72h somatostatin infusion did not completely inhibit the response of GH to GHRH. This may be attributed to partial desensitization of pituitary somatotropes to somatostatin due to the long-term infusion. Numerous studies which demonstrate that constant exposure of the pituitary to almost any neuropeptide results in a desensitized response would support this point (35, 36). There are marked differences in the GH response to food deprivation between rats and humans. GH secretion decreases in the rat, while it increases in human. In part this may be due to the fact that in most human studies, food deprivation is for 24 to 72h and results in negligible weight changes while in the rat, the same treatment results in a 20 to 30% decrease in body weight. The present study which supports the concept that somatostatin concentrations decrease during food deprivation in the rat, yet maintain a pulsatile pattern of secretion, is consistent with the human data. However, due to the fact that 72h of food deprivation in the rat also results in decreased hypothalamic GHRH release yields results which are different than in the human. Acknowledgements We thank L. Stagg and D. Voltz for their technical assistance. This work was supported by the University of Wisconsin Graduate School and NIH Grants R01-DK-38324 and K04-DK-01874 and by the Centro Studi e Ricerche di Neuroendocrinologia, Brescia, Italy. References 1. W.B. WEHRENBERG, N. LING, P. BOHLEN, F. ESCH, P. BRAZEAU and R. GUILLEMIN, Biochem Biophys Res Commun 109 562-567 (1982). 2. M. SATO, J. TAKAHARA, Y. FUJIOKA, M. NIIMI and S. IRINO, Endocrinology 123 1928-1933 (1988). 3. N.W. KASTING, J.B. MARTIN and M.A. ARNOLD, Endocrinology 109 1739-1745 (1981). 4. L. FERLAND, F. LABRIE, M. JOBIN, A. ARIMURA and A.V. SCHALLY, Biochem Biophys Res Commun 6.__88149-156 (1976). 5. W.B. WEHRENBERG, P. BRAZEAU, R. LUBEN, P. BOHLEN and R. GUILLEMIN, Endocrinology 111 2147-2148 (1982). 6. C.B. WEBB, M.L. VANCE, M.O. THORNER, G. PERISUTTI, J. THOMINET, J. RIVIER, W. VALE and L.A. FROHMAN, J Clin Invest 7_4496-103 (1984). 7. M.L. VANCE, D.L. KAISER, W.S. EVANS, M.O. THORNER, R. FURLANETTO, J. RIVIER, W. VALE and G. PERISUTTI, J Clin Endocrinol Metab 6.__0370-375 (1985). 8. W.B. WEHRENBERG, Neuroendocrinology 4._!391-396 (1986). 9. W.B. WEHRENBERG, A. BAIRD, F. ZEYTIN, F. ESCH, P. BOHLEN, N. LING, S.Y. YING and R. GUILLEMIN, Ann Rev Pharmacol Toxieol 25 463-483 (1985). 10. L. KRULICH, A.P.S. DHARIWAL and S.M. MCCANN, Endocrinology 8__!783-790 (1968). 11. W. VALE, J. VAUGHAN, G. YAMAMOTO, J. SPIESS and J. RIVIER, Endocrinology 112 1553-1555 (1983). 12. R.S. RITTMASTER, D.L. LORIAUX and G.R. MERRIAM, Neuroendocrinology 4__55118-122 (1987). 13. M.E. STACHURA, J.M. TYLER and P.K. FARMER, Endocrinology 123 1476-1482 (1988). 14. W.B. WEHRENBERG, P. BRAZEAU, N. LING, G. TEXTOR and R. GUILLEMIN, Endocrinology 114 1613-1616 (1984). 15. P.M. PLOTSKY and W. VALE, Science 23___0_0461-463 (1985). 16. L.A. FROHMAN, T.R. DOWNS, I.J. CLARKE and G.B. THOMAS, J Clin Invest 8_.6617-24 (1990). 17. M. KJAER, Int J Sports Med 1__0.02-15 (1989). 18. G. TANNENBAUM, J. EPELBAUM and P. BRAZEAU, Fed Proc 3__6_6323- 328 (1977). 19. G.A. CAMPBELL, M. KURCZ, S. MARSHALL and J. MEITES, Endocrinology 100 580-587 (1977). 20. G. TANNENBAUM, J. EPELBAUM, E. COLLE, P. BRAZEAU and J.B. MARTIN, Endocrinology 10__._22 1909-1914 (1978). 21. B.A. JANOWSKI and W.B. WEHRENBERG, Life Sci 5__0951-958 (1992). 22. G. TANNENBAUM, J.C. PAINSON, A.M.J. LENGYEL and P. BRAZEAU, Endocrinology 124 1380-1388 (1989). 23. Guide for the Care and Use of Laboratory Animals, Dept. of Health, Education and Welfare, Bethsda, MD
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HYPOTHALAMIC R E G U L A T I O N OF G R O W T H H O R M O N E SECRETION D U R I N G FOOD DEPRIVATION IN T H E RAT Bethany A. Janowski, Nicholas C. Ling*, Andrea Giustina**, William B. Wehrenberg Depts. of Health Sciences and Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211; *Whittier Institute, San Diego, CA 92037; ** Clinica Medica, University of Brescia, 25125 Brescia, Italy. (Received
in final
form December
30,
1992)
Summary Suppressed pulsatile GH secretion in food-deprived rats has been hypothesized to be due to an increase in hypothalamic somatostatin secretion. We investigated this hypothesis and the role of GHRH in regulating GH secretion during food deprivation using two different models. In experiment one, rats were food deprived for 72h during which time they received a saline infusion (n = 5). At the same time rats were normal fed for 72h during which time they received a somatostatin infusion (5/~g/h, n=7). After the 72h infusion period, all rats received two iv injections of GHRH (1/~g/rat) at 2h intervals. GH concentrations in food-deprived rats rose from approximately 10 ng/ml to 400-800 ng/ml in response to both GHRH injections. This increase was significantly greater (p<0.01) than the GH response (100-400 ng/ml) observed in somatostatin-infused animals. The significantly higher GH response observed in food-deprived rats as compared to somatostatin-infused, normal-fed rats suggests that somatostatin concentrations may decrease during food deprivation. In experiment two, rats were infused for 5h with either saline (n=6) or GHRH (10 #g/h, n=9) at the end of a 72h fast. GH concentrations did not change in saline-infused animals. In contrast, GH concentrations significantly increased (p<0.01) upon initiation of the continuous GHRH infusion. Yet, this release of GH was pulsatile in nature. Pulsatile GH secretion in the presence of a constant GHRH infusion suggests that pulsatile somatostatin release from the hypothalamus is maintained during food deprivation. These studies suggest that during food deprivation in the rat 1) absolute concentrations of somatostatin decrease, but its pattern of secretion remains pulsatile, and 2) decreased GHRH release may be responsible for the absence of spontaneous GH pulses. GH secretion in the rat is pulsatile in nature. It is regulated by interactions between growth hormone-releasing hormone (GHRH) and somatostatin (1-5). Pulsatile GH secretion during a continuous GHRH infusion in normal humans (6, 7) and in normal, conscious rats (8) suggests intermittent or pulsatile somatostatin secretion since somatostatin is known to inhibit GHRH-induced GH release from the anterior pituitary both in vivo (9) andin vitro (10-13). Endogenous pulsatile somatostatin secretion is further suggested by results obtained in normal rats passively immunized against somatostatin. In this model, a constant GHRH infusion results in elevated and continuous GH release (14). Furthermore, direct measurement of GHRH and somatostatin in hypophyseal portal blood suggests their intermittent release (15, 16). During food deprivation in rats, pulsatile GH secretion is suppressed (17-19). It has been previously proposed that this is a result of increased somatostatin tone since immunoneutralization of somatostatin increases baseline GH levels in these animals (20). However, there is now evidence to suggest that both GHRH and somatostatin are involved in regulating altered GH secretion during food deprivation. Specifically, the absence of GH pulses could result from a GHRH decrease (5) as well as from a somatostatin increase. Baseline serum GH concentrations are elevated in fasted rats with respect to normal-fed animals (21). And finally, an acute GHRH injection elicits GH release in food-deprived rats (1, 22), yet it is recognized the GHRH-induced GH secretion is significantly inhibited under conditions of high somatostatin tone (1). The aim of our study was to investigate the role of GHRH and somatostatin in regulating the altered GH secretory patterns observed in food-deprived animals. Our findings suggest that during food deprivation Copyright
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hypothalamic somatostatin secretion is decreased but that its pattern of release remains pulsatile. This would suggest that decreased hypothalamic GHRH release is responsible for the absence of spontaneous GH peaks. Materials and Methods Animals and Surgical Preparation: All animals used in this study were acquired, maintained, and handled accordin~ to the guidelines established by the National Institutes of Health (23). Animals were housed in a temperature- and humidity-controlled environment under a 14h light, 10h dark lighting schedule. Male rats (350-450 g) were outfitted with two indwelling venous catheters under sodium pentobarbital anesthesia (45 mg/kg, ip) seven or eight days prior to experimentation. Details of the construction of the catheters and the surgical preparation of the animals are described elsewhere (14, 24). Following surgery, the animals were housed in individual isolation cages. One catheter was connected to a single channel swivel through which a constant infusion (0.08 ml/h) of heparinized saline (10 IU/ml) was administered. The second catheter was filled with heparinized saline (50 IU/ml) and plugged. This catheter was flushed twice a day (0.25 ml) until the morning of experimentation. Experiment one: Five days after surgery, a constant 72h saline infusion (0.8 ml/h) and 72h food deprivation was initiated in one group of rats (n=5). At the same time a constant 72h somatostatin infusion (5 #g/h) was initiated in normal-fed rats (n=7). This dose was selected since it is known to approach the minimum dose needed to suppress endogenous GH secretion (25, 26). Since the biological end point, pulsatile GH secretion, was the same in both the food-deprived and somatostatin-infused rats, one can hypothesize that the pituitary should be exposed to similar concentrations of somatostatin in both conditions. After 72h of food deprivation or somatostatin infusion, three control blood samples were drawn (0.35 ml) from all animals at 30 min intervals. Following the third sample, animals received an iv injection of GHRH (1 ug). Blood samples were drawn 5, 10, 15, 30, 45, 60, 90 and 120 min after administration of GHRH. Immediately following the 120 min blood sample all animals received a second GHRH injection. Subsequent samples were drawn at the times noted. The samples were centrifuged, and the plasma was frozen until measurement of GH by RIA. Red blood cells were resuspended in saline and returned to the animals. All animals were immediately decapitated after the experiment. The pituitaries were removed and frozen until assayed for GH content. Experiment two: All rats were food deprived for 72h beginning four days after surgery. After 72h of food deprivation, three control blood samples (0.35 ml) were drawn at 20 min intervals, starting at 10:00 h. Immediately after the third blood sample a 5h constant iv infusion of either GHRH (10 #g/h, n=9) or saline (0.5 ml/h, n=6) was initiated. Blood samples were drawn every 20 minutes for the duration of the infusion. Samples and pituitaries were handled as in the first experiment. Radioimmunoassay: GH concentrations were determined in duplicate by RIA using a double antibody method. Reagents were provided by the National Pituitary Agency of the National Institutes of Health. Blood samples were assayed in aliquots of 10 and 50 #1. GH concentrations are expressed in terms of the NIH-RP-2 standard. The minimum sensitivity (90% bound) was approximately 0.04 ng/tube. Within- and between-assay variation averaged less than 10%. Some samples drawn following the initiation of the GHRH infusion were re-assayed in duplicate since GH concentrations exceeded the maximum sensitivity of the assay. These samples were diluted 1:10 in phosphate-buffered saline and assayed in 20 #1 aliquots. The pituitaries were thawed and placed in 1 ml of saline. The tissues were sonicated at 4°C for approximately five min then refrozen. Tissue homogenates were thawed and centrifuged for 15 min. Aliquots of the supernatant were diluted as necessary for RIA of GH. Peptides: Somatostatin was first dissolved in 0.1 M acetic acid and then diluted to 320 #g/ml in saline containing 0.1% bovine serum albumin. The diluted somatostatin was snap frozen and stored at -20°C until used. On the day of use, the peptide was diluted to 6.4 #g/ml in sterile saline. The somatostatin solution was changed in the syringes every 12h over the 72h infusion period. Rat GHRH was diluted to 1 mg/ml in 0.01 M acetic acid, snap frozen and stored at -20°C until used. On the morning of experimentation the peptide was diluted to 20/~g/ml in sterile saline. Data Analysis: Data are expressed as mean + SEM. Since the variances of the mean GH values were not homogenous, the data were subjected to log transformation prior to statistical analysis. Determination of the overall GH response involved determining the area under the GH curve (AUC) by trapezoid analysis. Significant differences in GH concentrations, AUC and pituitary GH content were determined by one way analysis of variance (27). Pulsatile GH secretion in experiment two was identified with the use of the Pulsar algorithm (28).
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Results Experiment 1: G H concentrations in 72h food-deprived rats significantly increased (p<0.01) following both G H R H injections (Fig. 1A). Individual examples of the GH profiles from two food-deprived animals demonstrated that, following G H R H administration, GH concentrations increased from approximately 10 to 400800 ng/ml (Fig. 1B). GH concentrations also rose following GHRH injection in somatostatin-infused rats, but the magnitude of the increase was significantly lower than the increase observed in food-deprived rats (Fig. 1A). Individual profiles show that GH concentrations increased from approximately 10 to only 100-400 ng/ml in somatostatin-infused rats following administration of GHRH (Fig. 1B). GH AUC was significantly higher (p < 0.01) in food-deprived (0.44+0.08 #g/ml/5h) than in somatostatin-infused rats (0.08+0.01 #g/ml/5h). Pituitary GH content was significantly higher (p<0.05) in somatostatin-infused rats (693+79 #g/pit) as compared to fooddeprived rats (377+90/~g/pit) suggesting that GH is synthesized but the amount released is decreased in response to the constant influence of exogenous somatostatin.
B
A
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FIG. 1 Panel A - The mean plasma GH response following two iv injections of GHRH (1 #g, indicated by arrows) in 72h food-deprived, saline-infused rats (e e, 0.8 ml/h, n=5) or in normal-fed, 72h somatostatin-infused rats ((3 .... o, 5 #g/h, n=7). GH concentrations are expressed as mean + SEM. GH concentrations were significantly higher (**, p<0.01) in food-deprived animals as compared to the somatostatin-infused rats. Panel B - Plasma GH concentrations in two individual 72h food-deprived (FD), saline-infused (SAL) rats and two individual 72h normal-fed (FED), somatostatin-infused (SS) rats receiving two iv injections of GHRH. Experiment 2: Plasma GH concentrations averaged 10-20 ng/ml in both food-deprived groups prior to the onset of the saline or G H R H infusion. GH concentrations in saline-infused rats did not change from control values during the 5h infusion period (Fig. 2A) and were virtually devoid of any GH pulses as identified by the Pulsar algorithm. In contrast, GH concentrations increased approximately 30 fold above control values upon initiation of the G H R H infusion. These elevated GH concentrations demonstrate an underlying pulsatile pattern of GH secretion. Representative GHRH-infused and saline-infused animals are illustrated in Figure 2B. No pulses of GH were observed in five of the seven saline-infused animals. One saline-infused rat demonstrated one GH pulse (Fig. 2B) with an amplitude of 13 ng/ml and the other saline-infused rat demonstrated two pulses with a mean amplitude of 28 ng/ml and an inter-peak interval of two hours. In contrast, pulsatile GH secretion was evident in animals receiving a constant GHRH infusion. There was an immediate peak in GH concentrations upon the initiation of the G H R H infusion with GH values rising to greater than 400 ng/ml. Over the next two hours, GH concentrations declined to approximately 100 ng/ml. Over the last two hours of the infusion, GH concentrations again rose to greater than 400 ng/ml.
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B GHRH
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o
Saline Infusion
_~ E
(10 /4g/h)
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FIG. 2 Panel A - Mean plasma GH concentrations in 72h food-deprived rats during a constant 5h infusion (represented by - at the top of the graph) of saline (0.5 ml/h, n=6) or GHRH (10/~g/h, n=9). GH concentrations are expressed as mean + SEM. GH concentrations did not change in saline-infused animals. GH concentrations significantly increased (**, p < 0.01) upon initiation of the GHRH infusion and remained significantly elevated (indicated by arrow), though pulsatile in nature, for the duration of the experiment. Panel B - Plasma GH concentrations in individual 72h food-deprived rats infused with either saline or GHRH for 5 h. [ ] represents pulses of GH as identified by the pulse detection algorithm Pulsar (28).
Parameters of pulsatile GH secretion as estimated by Pulsar are illustrated in Fig. 3. GH AUC was significantly higher (p<0.01) in GHRH-infused (1.52+0.27 #g/ml/5h) than in saline-infused rats (0.09+_0.03 #g/ml/5h). There was no difference in pituitary GH content between salineinfused animals (494+113 #g/pit) and GHRH-infused animals (333+28 ~zg/pit). Discussion
$* E_
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FIG. 3 The interactions between somatostatin and GHRH appear GH pulse frequency, amplitude, and to be altered during food deprivation since pulsatile GH interval in 72h food-deprived rats release is depressed following 72h of food deprivation (18receiving a 5h infusion of saline (open 21). Tannenbaum et al. (22) have suggested that increased bars) or GHRH (hatched bars). somatostatin concentrations may be the primary inhibitor Parameters were determined by the of GH following 72h of food deprivation. Indeed, direct pulse detection algorithm Pulsar (28). measurement of peripheral plasma somatostatin concentrations show an increase during food deprivation (29, 30) and passive immunization with somatostatin antiserum increases GH in food-deprived animals (20). If this is the case, then GHRH should have little or no effect in food-deprived animals since somatostatin is known to inhibit GHRH-indueed GH secretion (1, 9-13). However, it has been reported that GHRH increases GH
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secretion in food-deprived animals (1, 22). This leads to the conclusion that increased somatostatin tone may not explain decreased GH concentrations during food deprivation in the rat. It would be ideal to evaluate GHRH and somatostatin secretion in food-deprived rats by directly measuring portal blood concentrations. Unfortunately, this technique is only available in anesthetized rats and it is known that anesthesia affects GHRH and somatostatin secretion (31). Thus, we chose to evaluate the secretion of these two neuropeptides by measuring GH levels during the constant infusion of either G H R H or somatostatin. Our results suggest that the loss of pulsatile GH secretion reflects an absence of pulsatile GHRH secretion and a diminished yet still pulsatile release of somatostatin. The following observations support this hypothesis. First - the somatostatin infusion in normal-fed rats in experiment one was designed to mimic the same biological effect (i.e. inhibition of pulsatile GH secretion) observed during food deprivation. If somatostatin concentrations are indeed elevated during food deprivation, the GH response to exogenous GHRH in both food-deprived and somatostatin-infused rats should be similar. Our results show that this is not the case. GH concentrations following GHRH injection are significantly higher in food-deprived rats as compared to the somatostatin-infused rats. In fact, the GH response to GHRH in food-deprived rats was both higher and more consistent than that observed in normal rats (1). These results suggest that somatostatin concentrations decrease during food deprivation. In addition, pituitary GH content was significantly higher (p<0.01) in somatostatin-infused rats than in food-deprived rats, suggesting that GH is synthesized but not released during the constant somatostatin infusion. It is possible that the somatostatin infusion produced circulating concentrations higher than those observed during food deprivation. However, the role of peripheral concentrations of somatostatin in regulating GH secretion is unknown (29, 32). Furthermore, the infusion dose was the minimum dose of somatostatin required to suppress pulsatile GH secretion (25, 26) as observed in food-deprived rats. That is to say that the biological end point, pulsatile GH secretion, was identical in the two animal models. Second - baseline GH concentrations are elevated (10-20 ng/ml) in food-deprived rats, while they are virtually non-detectable ( < 2 ng/ml) during baseline or trough periods in normal rats (1, 8, 9). This suggests that somatostatin tone is decreased during food deprivation. Third - experiment two was designed to determine if the pattern of somatostatin release during food deprivation is pulsatile in nature. The initial GH response to the GHRH infusion was immediate and high in magnitude. Subsequent release was characterized by a gradual decrease in GH concentrations during the second hour of the infusion, followed by an increase during the third and fourth hours. Interestingly, the GH response to the infusion of GHRH in 72h food-deprived rats reflects a rhythm similar to that observed in fed, freely-moving rats during a constant GHRH infusion (8). In that study, as in this study, animals retained a pulsatile rhythm of GH secretion during a constant GHRH infusion. We attribute the phasic changes in the pituitary response to a constant G H R H stimulus to the presence of phasic endogenous somatostatin secretion. This is further supported by the observation that GH secretion is constant and non-pulsatile in nature in rats receiving a G H R H infusion and pre-treated with somatostatin antiserum (14). The present experiment suggests that episodic release of somatostatin, known, in part, to regulate the pulsatile rhythm of GH secretion, is still present but not eliminated following 72h of food deprivation. It is unlikely that the pulses in G H secretion reflect release of a secondary storage pool or de novo synthesis of GH for three reasons. One, pituitary GH content was not different between the two treatment groups, a fact which does not support increased synthesis. Two, we have previously demonstrated that the pituitary maintains its ability to respond to GHRH following repeated challenges with this neuropeptide (24). Three, pituitary GH depletion and decreased plasma GH concentrations begin to occur as early as four hours after the initiation of a continuous GHRH infusion when somatostatin is not present (14). Although we hypothesize that somatostatin is lower in food-deprived rats as compared to normal-fed rats, it is still biologically important as evidenced by the fact that somatostatin passive immunization is able to elicit a GH rebound in food-deprived rats (20) and GHRH administration to food-deprived rats which have been pretreated with somatostatin antibodies results in a larger GH response with respect to food-deprived animals which have not been pretreated (1). GH pulses are absent in food-deprived rats, even though it appears such animals have suppressed hypothalamic somatostatin tone. This leads to the conclusion that the absence of pulses is due to an absence of GHRH. This is consistent with the fact that G H R H is responsible for GH pulses in normal animals (5). In addition, it has been observed that during food deprivation, hypothalamic GHRH concentrations decrease (33). Bruno et al. (34) have recently reported that prepro-GHRH mRNA is reduced in 72h food-deprived rats. These observations further suggest that the suppression of pulsatile GH secretion during food deprivation is the result of decreased
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synthesis and/or release of GHRH. Consistent with this, we have demonstrated that GHRH is neither involved in the maintenance of basal GH concentrations nor in the GH rebound following withdrawal of somatostatin in 72h food-deprived rats (21). Short term somatostatin infusions of 5-50 #g/h have been shown to inhibit GHRH-induced GH release in the rat (26). Yet, in the present experiment, the 72h somatostatin infusion did not completely inhibit the response of GH to GHRH. This may be attributed to partial desensitization of pituitary somatotropes to somatostatin due to the long-term infusion. Numerous studies which demonstrate that constant exposure of the pituitary to almost any neuropeptide results in a desensitized response would support this point (35, 36). There are marked differences in the GH response to food deprivation between rats and humans. GH secretion decreases in the rat, while it increases in human. In part this may be due to the fact that in most human studies, food deprivation is for 24 to 72h and results in negligible weight changes while in the rat, the same treatment results in a 20 to 30% decrease in body weight. The present study which supports the concept that somatostatin concentrations decrease during food deprivation in the rat, yet maintain a pulsatile pattern of secretion, is consistent with the human data. However, due to the fact that 72h of food deprivation in the rat also results in decreased hypothalamic GHRH release yields results which are different than in the human. Acknowledgements We thank L. Stagg and D. Voltz for their technical assistance. This work was supported by the University of Wisconsin Graduate School and NIH Grants R01-DK-38324 and K04-DK-01874 and by the Centro Studi e Ricerche di Neuroendocrinologia, Brescia, Italy. References 1. W.B. WEHRENBERG, N. LING, P. BOHLEN, F. ESCH, P. BRAZEAU and R. GUILLEMIN, Biochem Biophys Res Commun 109 562-567 (1982). 2. M. SATO, J. TAKAHARA, Y. FUJIOKA, M. NIIMI and S. IRINO, Endocrinology 123 1928-1933 (1988). 3. N.W. KASTING, J.B. MARTIN and M.A. ARNOLD, Endocrinology 109 1739-1745 (1981). 4. L. FERLAND, F. LABRIE, M. JOBIN, A. ARIMURA and A.V. SCHALLY, Biochem Biophys Res Commun 6.__88149-156 (1976). 5. W.B. WEHRENBERG, P. BRAZEAU, R. LUBEN, P. BOHLEN and R. GUILLEMIN, Endocrinology 111 2147-2148 (1982). 6. C.B. WEBB, M.L. VANCE, M.O. THORNER, G. PERISUTTI, J. THOMINET, J. RIVIER, W. VALE and L.A. FROHMAN, J Clin Invest 7_4496-103 (1984). 7. M.L. VANCE, D.L. KAISER, W.S. EVANS, M.O. THORNER, R. FURLANETTO, J. RIVIER, W. VALE and G. PERISUTTI, J Clin Endocrinol Metab 6.__0370-375 (1985). 8. W.B. WEHRENBERG, Neuroendocrinology 4._!391-396 (1986). 9. W.B. WEHRENBERG, A. BAIRD, F. ZEYTIN, F. ESCH, P. BOHLEN, N. LING, S.Y. YING and R. GUILLEMIN, Ann Rev Pharmacol Toxieol 25 463-483 (1985). 10. L. KRULICH, A.P.S. DHARIWAL and S.M. MCCANN, Endocrinology 8__!783-790 (1968). 11. W. VALE, J. VAUGHAN, G. YAMAMOTO, J. SPIESS and J. RIVIER, Endocrinology 112 1553-1555 (1983). 12. R.S. RITTMASTER, D.L. LORIAUX and G.R. MERRIAM, Neuroendocrinology 4__55118-122 (1987). 13. M.E. STACHURA, J.M. TYLER and P.K. FARMER, Endocrinology 123 1476-1482 (1988). 14. W.B. WEHRENBERG, P. BRAZEAU, N. LING, G. TEXTOR and R. GUILLEMIN, Endocrinology 114 1613-1616 (1984). 15. P.M. PLOTSKY and W. VALE, Science 23___0_0461-463 (1985). 16. L.A. FROHMAN, T.R. DOWNS, I.J. CLARKE and G.B. THOMAS, J Clin Invest 8_.6617-24 (1990). 17. M. KJAER, Int J Sports Med 1__0.02-15 (1989). 18. G. TANNENBAUM, J. EPELBAUM and P. BRAZEAU, Fed Proc 3__6_6323- 328 (1977). 19. G.A. CAMPBELL, M. KURCZ, S. MARSHALL and J. MEITES, Endocrinology 100 580-587 (1977). 20. G. TANNENBAUM, J. EPELBAUM, E. COLLE, P. BRAZEAU and J.B. MARTIN, Endocrinology 10__._22 1909-1914 (1978). 21. B.A. JANOWSKI and W.B. WEHRENBERG, Life Sci 5__0951-958 (1992). 22. G. TANNENBAUM, J.C. PAINSON, A.M.J. LENGYEL and P. BRAZEAU, Endocrinology 124 1380-1388 (1989). 23. Guide for the Care and Use of Laboratory Animals, Dept. of Health, Education and Welfare, Bethsda, MD
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(1988). 26. R.G. CLARK and I.C.A.F. ROBINSON, Endocrinology 12__!2675-2682 (1988). 27. B. J. WINER, Statistical Principles in Experimental Design, Multifactorial experiments having repeated measures on the same elements, B. J. Winer, (McGraw-Hill, New York, 1971), pp. 603. 28. G.R. MERRIAM and K.W. WACHTER, Am J Physiol 24..__!E310-E318 (1982). 29. B. SHAPIRO, M. BERELOWlTZ, B.L. PIMSTONE, S. KRONHEIM and M. SHEPPARD, Diabetes 28 182-186 (1979). 30. G. TANNENBAUM, O. RORSTAD and P. BRAZEAU, Endocrinology 104 1733-1738 (1979). 31. J.B. MARTIN, Neuroendocrinology 1__/3339-350 (1973). 32. G.B. THOMAS, J.T. CUMMINS, H. FRANCIS, A.W. SUDBURY, P.I. MCCLOUD and I.J. CLARKE, Endocrinology 128 1151-1158 (1991). 33. J. MEITES and N.J. FIEL, Endocrinology 7_/7455-460 (1965). 34. J.F. BRUNO, D. OLCHOVSKY, J.D. WHITE, J.W. LEIDY, J.F. SONG and M. BERELOWITZ, Endocrinology 12__.!2111-2116 (1990). 35. W.B. WEHRENBERG, H. SEIFERT, L.M. BILEZIKJIAN and W. VALE, Neuroendocrinology 43 266-268 (1986). 36. T.M. BADGER, W.J. MILLARD, G.F. MCCORMICK, C.Y. BOWERS and J.B. MARTIN, Endocrinology 115 1432-1438 (1984).