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Effects of passive immunization of growth hormone-releasing hormone and somatostatin on growth hormone secretion under conditions of high somatostatin tone
Life Sciences, Vol. 50, pp. 951-958
Pergamon Press
Printed in the USA
EFFECTS OF PASSIVE IMMUNIZATION OF GROWTH H O R M O N E - R E L E A S I N G HORMONE AND SOMATOSTATIN ON GROWTH HORMONE SECRETION U N D E R CONDITIONS OF HIGH S O M A T O S T A T I N TONE Bethany A. Janowski and W i l l i a m B. W e h r e n b e r g D e p a r t m e n t s of Health Sciences and Biological Sciences, U n i v e r s i t y of Wisconsin-Milwaukee, Milwaukee, WI (Received in final form January 22, 1992) Summary Pulsatile GH secretion decreases during foodd e p r i v a t i o n in the rat. It has been h y p o t h e s i z e d that this decrease is d u e to e l e v a t e d hypothalamic somatostatin secretion. This is b a s e d on the o b s e r v a t i o n that GH i n c r e a s e s in f o o d - d e p r i v e d rats f o l l o w i n g r e m o v a l of e n d o g e n o u s s o m a t o s t a t i n u s i n g passive immunization techniques. C o g n i z a n t of the important stimulatory e f f e c t s of g r o w t h h o r m o n e r e l e a s i n g hormone (GHRH) on GH secretion, we sought to determine if t h i s n e u r o p e p t i d e p l a y s a n y r o l e in m e d i a t i n g GH s e c r e t i o n in f o o d - d e p r i v e d rats. Male rats w e r e p r e p a r e d w i t h i n d w e l l i n g v e n o u s c a t h e t e r s using sodium pentobarbital anesthesia seven days prior to experimentation. Animals were food-deprived for 72 h, a f t e r w h i c h control blood samples were drawn from -60 to 0 min. One group was then treated with normal r a b b i t s e r u m (NRS), while a second group was t r e a t e d with GHRH antiserum (GHRHab). At 55 m i n all a n i m a l s r e c e i v e d s o m a t o s t a t i n a n t i s e r u m (SSab). No animal e x h i b i t e d any spontaneous GH peak during the one hour c o n t r o l p e r i o d or in the s u b s e q u e n t one h o u r p e r i o d following the administration of G H R H a b or N R S . A b s e n c e of GH p u l s a t i l i t y d u r i n g f o o d - d e p r i v a t i o n , coupled with no decrease in GH levels in f o o d - d e p r i v e d rats treated with GHRHab suggest that d i m i n i s h e d GHRH pulsatility is l i k e l y during food-deprivation. Subsequent treatment of t h e s e a n i m a l s w i t h S S a b r e s u l t e d in an i d e n t i c a l 2.5 f o l d i n c r e a s e in GH concentrations. This result suggests that GHRH is not i n v o l v e d in the GH r e b o u n d f o l l o w i n g s o m a t o s t a t i n w i t h d r a w a l in food-deprived rats. Pituitary growth hormone (GH) s e c r e t i o n in the rat is c h a r a c t e r i z e d by an u l t r a d i a n rhythm, with intermittent peaks and troughs at a p p r o x i m a t e l y three hour intervals (i). The p u l s a t i l e p a t t e r n of s e c r e t i o n r e s u l t s from the i n t e r a c t i o n b e t w e e n the s t i m u l a t o r y e f f e c t s of g r o w t h h o r m o n e - r e l e a s i n g h o r m o n e (GHRH) and the inhibitory effects of somatostatin (2-10). H i g h levels of s o m a t o s t a t i n m a i n t a i n low GH c o n c e n t r a t i o n s d u r i n g t r o u g h 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc All rights reserved.
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periods since passive immunization with somatostatin antiserum (SSab) increases basal GH c o n c e n t r a t i o n s but does not a l t e r the p u l s a t i l e p a t t e r n of GH secretion (8, 9). High c o n c e n t r a t i o n s of G H R H m a i n t a i n e l e v a t e d GH c o n c e n t r a t i o n s d u r i n g p e a k p e r i o d s since administration of G H R H a n t i s e r u m (GHRHab) completely i n h i b i t s the p u l s a t i l e s e c r e t i o n of GH (i0). That GHRH and s o m a t o s t a t i n are released in a p u l s a t i l e fashion is s u g g e s t e d by the f a c t t h a t at t h e t i m e of an a n t i c i p a t e d GH p u l s e , G H R H c o n c e n t r a t i o n s are e l e v a t e d and s o m a t o s t a t i n c o n c e n t r a t i o n s are d e p r e s s e d in h y p o t h a l a m i c - h y p o p h y s e a l portal blood (ii). GH pulses are depressed in r a t s u n d e r c o n d i t i o n s of f o o d - d e p r i v a t i o n or m a l n u t r i t i o n (12-14) , and plasma s o m a t o s t a t i n c o n c e n t r a t i o n s are increased (15). Indeed, a GH rebound has been r e p o r t e d in 72 h f o o d - d e p r i v e d rats u p o n the a d m i n i s t r a t i o n of somatostatin antiserum (16). These findings suggest that s o m a t o s t a t i n m e d i a t e s the s u p p r e s s i o n of GH s e c r e t i o n d u r i n g food-deprivation. It is possible, however, that s u p p r e s s e d GH is a l s o d u e to d e c r e a s e d G H R H s e c r e t i o n , a h y p o t h e s i s yet to be tested. S u p p o r t i n g this h y p o t h e s i s is an e a r l i e r s t u d y w h i c h s u g g e s t e d that h y p o t h a l a m i c GHRH c o n c e n t r a t i o n s d e c r e a s e during f o o d - d e p r i v a t i o n (17). In an effort to further c h a r a c t e r i z e the neuroregulation of GH s e c r e t i o n d u r i n g f o o d - d e p r i v a t i o n , we examined the contribution of G H R H in r e g u l a t i n g basal GH s e c r e t i o n and in c o n t r o l l i n g the m a g n i t u d e of the GH r e b o u n d following immunoneutralization of s o m a t o s t a t i n in 72 h f o o d - d e p r i v e d rats. Methods Animals: A l l a n i m a l s in t h e s e s t u d i e s w e r e a c q u i r e d , maintained, a n d u s e d in a c c o r d a n c e with the guidelines e s t a b l i s h e d by the N a t i o n a l I n s t i t u t e s of H e a l t h (18). Male S p r a g u e - D a w l e y rats (225-350 g) were m a i n t a i n e d in a t e m p e r a t u r e and h u m i d i t y - c o n t r o l l e d e n v i r o n m e n t and exposed to a 14 h light, I0 h dark lighting schedule (lights on at 0600 h). Surqical Preparation: Animals anesthetized with sodium p e n t o b a r b i t a l (NaPb, 40 mg/kg, ip) were o u t f i t t e d with a chronic indwelling venous c a t h e t e r one week prior to e x p e r i m e n t a t i o n (3). The c a t h e t e r was c o n n e c t e d to a single channel, fluid swivel to allow free m o v e m e n t of the rat following surgery. The rats were p l a c e d in isolation cages and received a constant infusion (0.08 ml/h) of h e p a r i n i z e d s a l i n e (i0 IU/ml) u n t i l t h e m o r n i n g of experimentation. Experimentation: F o o d - d e p r i v a t i o n was initiated four days after surgery and was continued for 72 h. B e g i n n i n g at 1200 h on t h e d a y of e x p e r i m e n t a t i o n , three control blood samples were d r a w n (0.35 ml), f o l l o w e d by an iv i n j e c t i o n of e i t h e r 0.5 ml n o r m a l r a b b i t s e r u m (NRS, n=7) or G H R H a b (n=ll). Subsequent b l o o d samples were drawn i0, 25 and 55 min following t r e a t m e n t . All animals then received 0.5 ml of SSab, iv, and blood samples w e r e d r a w n 5, i0, 15, 30 and 60 m i n f o l l o w i n g the i n j e c t i o n . Blood samples were centrifuged, plasma separated and then frozen for d e t e r m i n a t i o n of GH by RIA. The red blood cells were r e s u s p e n d e d in normal saline and returned to the animals.
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Antibody Preparation: Antiserum a g a i n s t rat G H R H w a s p r e p a r e d by i m m u n i z i n g r a b b i t s w i t h a m i x t u r e of s y n t h e t i c rat GHRH and methylated BSA emulsified in F r e u n d ' s a d j u v a n t as d e s c r i b e d p r e v i o u s l y (19). This a n t i s e r u m is s p e c i f i c for rat GHRH. It does not cross-react with GHRH from other species nor with p e p t i d e s that have considerable sequence h o m o l o g y with GHRH s u c h as s e c r e t i n , v a s o a c t i v e i n t e s t i n a l p e p t i d e and g l u c a g o n . The antibody d i l u t i o n required to bind a p p r o x i m a t e l y 50% of a rat GHRH-II25 labeled trace was 1:80,000. The dose a d m i n i s t e r e d was based on our observations that 0.5 ml of GHRHab is e f f e c t i v e in neutralizing e n d o g e n o u s G H R H as e v i d e n c e d by an a b s e n c e of p u l s a t i l e GH secretion (20). The SSab was p r e p a r e d by i m m u n i z i n g sheep w i t h s y n t h e t i c somatostatin. C o m p l e t e c h a r a c t e r i z a t i o n of t h e s o m a t o s t a t i n a n t i b o d y has b e e n p u b l i s h e d (21). Briefly, the antibody was partially purified by ammonium sulfate purification. It is d i r e c t e d toward the m i d - p o r t i o n of somatostatin. RIA s e n s i t i v i t y w a s 14 p g / t u b e at a f i n a l d i l u t i o n of 1 / 2 5 , 0 0 0 . The dose a d m i n i s t e r e d was based on our observations that 0.5 ml of SSab is effective in n e u t r a l i z i n g endogenous s o m a t o s t a t i n (22). R a d i o i m m u n o a s s a y Procedures: Plasma GH c o n c e n t r a t i o n s were d e t e r m i n e d in d u p l i c a t e by a d o u b l e a n t i b o d y p r o c e d u r e u s i n g materials s u p p l i e d by t h e N a t i o n a l P i t u i t a r y A g e n c y of t h e N a t i o n a l I n s t i t u t e s of Health. Blood s a m p l e s w e r e a s s a y e d in a l i q u o t s of e i t h e r i0 or 50 ~i. G r o w t h hormone c o n c e n t r a t i o n s are e x p r e s s e d in t e r m s of t h e r a t G H - R P - 2 s t a n d a r d . Assay sensitivity was approximately 0.08 ng/tube. Withinand b e t w e e n - a s s a y v a r i a t i o n s averaged less than 10%. Statistical Analyses: Significant t r e a t m e n t effects on GH c o n c e n t r a t i o n s were detected by analysis of v a r i a n c e for repeated m e a s u r e s (23). Since the variances of the mean GH c o n c e n t r a t i o n s were not homogenous, t h e d a t a w e r e f i r s t s u b j e c t e d to log transformation. E v a l u a t i o n of the overall GH response during the e x p e r i m e n t involved analyzing the area under the GH curve (AUC). The e x p e r i m e n t w a s s e p a r a t e d i n t o t h r e e d i f f e r e n t t r e a t m e n t periods. The control period was designated as -60 to 0 min, the N R S / G H R H a b period from 0 to 55 min, and SSab p e r i o d from 55 to 115 min. Less than 1% of the total number of blood samples to be drawn were missed due to technical problems during the experiment. In t h e s e i n s t a n c e s the GH v a l u e was e s t i m a t e d by c a l c u l a t i n g the average GH concentration from the p r e c e d i n g and following blood sample. All data are expressed as mean + SEM. Results Effects of F o o d - D e p r i v a t i o n : Seventy-two h o u r s of f o o d - d e p r i v a t i o n resulted in a 14-17% loss in total body weight. Pulsatile GH secretion was inhibited and baseline GH c o n c e n t r a t i o n s were elevated during the 60 min control p e r i o d in all animals (Fig. i). Although the control p e r i o d was too short to c o m p l e t e l y e v a l u a t e GH p u l s a t i l i t y , it has p r e v i o u s l y b e e n established that GH pulses are absent and baseline GH c o n c e n t r a t i o n s are elevated in food-deprived animals as c o m p a r e d to n o r m a l ones (3, I0, 13, 24). In addition to a l t e r i n g these e n d o c r i n e p a r a m e t e r s , f o o d - d e p r i v a t i o n a l s o m a d e the a n i m a l s agitated, restless and aggressive.
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Time (min) FIG. 1 Mean plasma GH concentrations in 72 h f o o d - d e p r i v e d rats. NRS (n=7) o r G H R H a b (n=ll) w a s a d m i n i s t e r e d after the 0 min blood sample. All animals received SSab after t h e 55 m i n b l o o d sample• Baseline GH concentrations were elevated compared to previously e s t a b l i s h e d v a l u e s for n o r m a l r a t s ( c r o s s - h a t c h e d area, r e f s 3, i0, 24). GH concentrations did not change following NRS or GHRHab treatment• GH concentrations in b o t h N R S - a n d G H R H a b - t r e a t e d r a t s w e r e s i g n i f i c a n t l y e l e v a t e d a f t e r S S a b t r e a t m e n t (p<0.01).
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PERIOD FIG. 2 T h e a r e a u n d e r t h e G H c u r v e (AUC) in 72 h f o o d - d e p r i v e d rats. T h e G H A U C w a s s i g n i f i c a n t l y i n c r e a s e d (p<0.01) following SSab treatment• The GH AUCs between NRS- and GHRHab-treated rats were not different within periods•
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Control Period: In our laboratory GH trough c o n c e n t r a t i o n s in normal male rats are routinely less than 5 ng/ml (3). These v a l u e s are illustrated as the c r o s s - h a t c h e d area on Fig. i. In contrast, the GH values in food-deprived rats averaged b e t w e e n i0 and 20 ng/ml during the control period (Fig.l). E f f e c t s of N R S / G H R H a b : No s i g n i f i c a n t c h a n g e s in m e a n p l a s m a GH c o n c e n t r a t i o n s w e r e o b s e r v e d i m m e d i a t e l y f o l l o w i n g a d m i n i s t r a t i o n of GHRHab when compared to values o b s e r v e d during the control period or to values observed in N R S - t r e a t e d animals. T h e s e r e s u l t s are f u r t h e r s u p p o r t e d by c o m p a r i n g GH AUC. As illustrated in Figure 2, GH AUC was not a l t e r e d by G H R H a b w h e n c o m p a r e d to c o n t r o l v a l u e s or to the v a l u e of the N R S - t r e a t e d rats during the N R S / G H R H a b treatment period. E f f e c t s of S S a b : Administration of S S a b to N R S - or G H R H a b - t r e a t e d animals resulted in an identical GH rebound, with c o n c e n t r a t i o n s increasing a p p r o x i m a t e l y 2.5 fold (Fig. i). After t h e i n i t i a l r e b o u n d , GH c o n c e n t r a t i o n s r e m a i n e d e l e v a t e d in N R S - t r e a t e d animals, but steadily decreased in those t r e a t e d with GHRHab. This decline, however, was not s t a t i s t i c a l l y s i g n i f i c a n t when e v a l u a t e d by absolute GH concentrations (Fig. i) or GH A U C (Fig. 2). Discussion Our novel o b s e r v a t i o n s show that G H R H is not involved in the m a i n t e n a n c e of the e l e v a t e d basal GH c o n c e n t r a t i o n s , or in the acute GH rebound following somatostatin withdrawal in f o o d - d e p r i v e d rats. This is d e m o n s t r a t e d by the o b s e r v a t i o n that basal and r e b o u n d GH c o n c e n t r a t i o n s w e r e not d i f f e r e n t b e t w e e n NRS- and G H R H a b - t r e a t e d rats. These results suggest that the lack of p u l s a t i l e GH s e c r e t i o n in the f o o d - d e p r i v e d rat may be due to d e c r e a s e d GHRH secretion rather than elevated s o m a t o s t a t i n s e c r e t i o n (see next paragraph). Supporting this c o n c l u s i o n are the o b s e r v a t i o n s of Meites and Fiel (17) that GHRH c o n c e n t r a t i o n s are d e c r e a s e d following food-deprivation (bioassay) and GHRH m R N A decreases during food-deprivation (25). Even t h o u g h it a p p e a r s t h a t G H R H is not directly involved in c o n t r o l l i n g the m a g n i t u d e of the GH rebound, GHRH may play a role in m a i n t a i n i n g e l e v a t e d GH c o n c e n t r a t i o n s afterward. This is b a s e d on our o b s e r v a t i o n t h a t f o l l o w i n g s o m a t o s t a t i n r e m o v a l , GH r e m a i n e d e l e v a t e d in N R S - t r e a t e d a n i m a l s but d e c l i n e d in t h o s e t r e a t e d w i t h GHRHab. However, these results are not statistically different and studies of longer duration will be required to p r o p e r l y evaluate this possibility. B a s e d on GH c o n c e n t r a t i o n s , it may be i n f e r r e d t h a t the rhythmicity of G H R H a n d s o m a t o s t a t i n s e c r e t i o n is d i s r u p t e d during food-deprivation. R e s u l t s from the p r e s e n t e x p e r i m e n t s h o w an a b s e n c e of n o r m a l GH p u l s a t i l i t y and an e l e v a t e d GH b a s e l i n e in 72 h f o o d - d e p r i v e d rats. This s t u d y c o r r o b o r a t e s e a r l i e r o b s e r v a t i o n s t h a t s u p p r e s s i o n of n o r m a l p u l s a t i l e GH release following 72 h of food-deprivation is mediated, in part, by hypothalamic somatostatin secretion (12-16). It is of interest, however, that although peripheral somatostatin c o n c e n t r a t i o n s are reported to be elevated in the f o o d - d e p r i v e d rat (15), basal GH concentrations are elevated. In c o n t r a s t to a l m o s t u n d e t e c t a b l e b a s e l i n e GH c o n c e n t r a t i o n s in n o r m a l rats
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(GH
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T h e r e is no q u e s t i o n t h a t f o o d - d e p r i v e d r a t s (16). and t h o s e of o t h e r s (24), be diminished. Thus, t h e rats most likely reflects
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s o m a t o s t a t i n is f u n c t i o n a l l y a c t i v e in Yet, b a s e d on t h e p r e s e n t o b s e r v a t i o n s t h e c o n c e n t r a t i o n s of s o m a t o s t a t i n m a y a b s e n c e of GH p u l s e s in f o o d - d e p r i v e d an a b s e n c e of GHRH.
Acknowledqments We thank L. S t a g g , A. P i e r i n g a n d D. V o l t z for their technical assistance. T h i s w o r k w a s s u p p o r t e d b y t h e Univ. of WI Graduate School and the NIH Grants R01-DK-38324 and K04-DK-01874.
REFERENCES i. 2. 3.
4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22.
G.S. T A N N E N B A U M a n d J.B. M A R T I N , Endocrinology 9_88 5 6 2 - 5 6 9 (1976). R. G U I L L E M I N , P. B R A Z E A U , P. B O H L E N , F. E S C H , N. L I N G a n d W.B. W E H R E N B E R G , S c i e n c e 218 5 8 5 - 5 8 7 (1982). W.B. W E H R E N B E R G , N. LING, P. BOHLEN, F. ESCH, P. B R A Z E A U a n d R. G U I L L E M I N , Biochem. Biophys. Res. Commun. 109 5 6 2 - 5 6 7 (1982) . J.O. J A N S S O N , S. E D E N a n d O. ISAKSSON, Endocr. Rev. 6 1 2 8 - 1 5 0 (1985) . M. S A T O , J. T A K A H A R A , Y. F U J I O K A , M. N I I M I a n d S. IRINO, Endocrinology 123 1 9 2 8 - 1 9 3 3 (1988). P. B R A Z E A U , W. V A L E , R. B U R G U S , N. L I N G , M. B U T C H E R , J. R I V I E R a n d R. G U I L L E M I N , S c i e n c e 179 77-79 (1973). N.W. K A S T I N G , J.B. M A R T I N and M.A. ARNOLD, E n d o c r i n o l o g y 109 1 7 3 9 - 1 7 4 5 (1981). L. FERLAND, F. LABRIE, M. JOBIN, A. A R I M U R A a n d A.V. SCHALLY, B i o c h e m . Biophys. Res. Commun. 6_88 1 4 9 - 1 5 6 (1976). N. J O C O V I D O U and Y.C. PATEL, Endocrinology 121 782-785 (1987). W.B. WEHRENBERG, P. B R A Z E A U , R. L U B E N , P. B O H L E N a n d R. G U I L L E M I N , E n d o c r i n o l o g y iii 2 1 4 7 - 2 1 4 8 (1982). P.M. P L O T S K Y a n d W. VALE, S c i e n c e 230 4 6 1 - 4 6 3 (1985). E. D I C K E R M A N , A. N E G R O - V I L A R and J. MEITES, E n d o c r i n o l o g y 8_44 8 1 4 - 8 1 9 (1969). T A N N E N B A U M G. S., E P E L B A U M J. and P. BRAZEAU, Fed. Proc. 3_66 3 2 3 - 3 2 8 (1977). G.A. CAMPBELL, M. K U R C Z , S. M A R S H A L L a n d J. M E I T E S , Endocrinology i00 580-587 (1977). G.S. T A N N E N B A U M , O. R O R S T A D and P. BRAZEAU, E n d o c r i n o l o g y 104 1 7 3 3 - 1 7 3 8 (1979). G.S. T A N N E N B A U M , J. E P E L B A U M , E. COLLE, P. B R A Z E A U a n d J.B. M A R T I N , E n d o c r i n o l o g y 102 1 9 0 9 - 1 9 1 4 (1978). J. M E I T E S a n d N.J. FIEL, E n d o c r i n o l o g y 7_/7 4 5 5 - 4 6 0 (1965). G u i d e for t h e C a r e a n d U s e of L a b o r a t o r y A n i m a l s , (Department of Health, E d u c a t i o n and W e l f a r e , Bethesda, MD, 1978). R. B E N O I T , P. B O H L E N , N. L I N G , A. B R I S K E N , F. E S C H , P. B R A Z E A U , S.Y. Y I N G a n d R. G U I L L E M I N , Proc. Nat. Acad. Sci. 7_99 9 1 7 - 9 2 1 (1982). W.B. W E H R E N B E R G , E n d o c r i n o l o g y 118 4 8 9 - 4 9 4 (1986). O.P. RORSTAD, J. EPELBAUM and P.M., J.B. BRAZEAU, Endocrinology 105 1 0 8 3 - 1 0 9 2 (1979). W.B. W E H R E N B E R G , J A N O W S K I B. A., A.W. PIERING, F. C U L L E R a n d JONES K. L. , E n d o c r i n o l o g y 126 3200-3203 (1990) .
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B.J. W I N E R , S t a t i s t i c a l Principles in E x p e r i m e n t a l Desian , ( M c G r a w - Hill, N e w York, 1971), pp. 599. G.S. TANNENBAUM, J.C. PAINSON, A.M. J. L E N G Y E L a n d P. BRAZEAU, Endocrinology 124 1 3 8 0 - 1 3 8 8 (1989). J.F. BRUNO, D. O L C H O V S K Y , J.D. WHITE, J.W. LEIDY, J. S O N G a n d M. B E R E L O W I T Z , E n d o c r i n o l o g y 127 2 1 1 1 - 2 1 1 6 (1990). N. M I K I , M. O N O a n d K. S H I Z U M E , J. E n d o c r i n o l . 117 2 4 5 - 2 5 2 (1988). R.G. CLARK, L.M. S. C A R L S S O N , B. R A F F E R T Y a n d I.C. A. F. R O B I N S O N , J. E n d o c r i n o l . 119 3 9 7 - 4 0 4 (1988). K . Y . HO, J . D . V E L D H U I S , M.L. J O H N S O N , R. F U R L A N E T T O , W.S. EVANS, K.G. M. M. A L B E R T I a n d M.O. T H O R N E R , J. clin. Invest. 8_~i 9 6 8 - 9 7 5 (1988). T.J. M E R I M E E a n d S.E. F I N E B E R G , J. Clin. E n d o c r i n o l . Metab. 3 9 3 8 5 - 3 8 6 (1974). M.M. FICHTER and K.M. PIRKE, Psychneuroendocrinolcgy ii 2 9 5 - 3 0 5 (1986). I.F. S M I T H , M.C. L A T H A M , J.A. A Z U B U I K E , W.R. BUTLER, L.S. PHILLIPS, W.G. P O N D a n d C.O. ENWONWU, Proc. Soc. Exp. Biol. Med. 167 6 0 7 - 6 1 1 (1981). F. B R A M B I L L A , M. L A M P E R T I C O , L. S A L I , F. C A V A G N I N I , C. INVITTI, M. M A G G I O N I , C. C A N D O L F I and A.E. PANERAI, P s y c h i a t r y Res. 20 19-31 (1987). D.J. BECKER, Ann. Rev. Nutr. ~ 1 8 7 - 2 1 2 (1983). M.M. FICHTER, K.M. P I R K E a n d F. H O L S B O E R , P s y c h i a t r y Res. 1_/7 61-72 (1986). G.S. T A N N E N B A U M , J.B. M A R T I N and E. COLLE, E n d o c r i n o l o g y 9_99 7 2 0 - 7 2 6 (1976). L. DE M A R I N I S , G. FOLLI, C. D'AMICO, A. M A N C I N I , P. SAMBO, A. TOFANI, A. O R A D E I a n d A. B A R B A R I N O , E n d o c r i n o l o g y 6_66 5 9 8 - 6 0 1 (1988). P.S. SHARP, K. F O L E Y , P. C H A H A L and E.M. KOHNER, Clin. Endocrinol (Oxf) 2-0 4 9 7 - 5 0 1 (1984). T. IMAKI, T. S H I B A S A K I , K. SHIZUME, A. M A S U D A , M. H O T T A , Y. KIYOSAWA, K. J I B I K I a n d H. D E M U R A , J. C l i n . Endocrinol. Metab. 60 2 9 0 - 2 9 3 (1985). JANOWSKI. B.A., M.S. Thesis: University of WisconsinMilwaukee (January, 1991).
Pergamon Press
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EFFECTS OF PASSIVE IMMUNIZATION OF GROWTH H O R M O N E - R E L E A S I N G HORMONE AND SOMATOSTATIN ON GROWTH HORMONE SECRETION U N D E R CONDITIONS OF HIGH S O M A T O S T A T I N TONE Bethany A. Janowski and W i l l i a m B. W e h r e n b e r g D e p a r t m e n t s of Health Sciences and Biological Sciences, U n i v e r s i t y of Wisconsin-Milwaukee, Milwaukee, WI (Received in final form January 22, 1992) Summary Pulsatile GH secretion decreases during foodd e p r i v a t i o n in the rat. It has been h y p o t h e s i z e d that this decrease is d u e to e l e v a t e d hypothalamic somatostatin secretion. This is b a s e d on the o b s e r v a t i o n that GH i n c r e a s e s in f o o d - d e p r i v e d rats f o l l o w i n g r e m o v a l of e n d o g e n o u s s o m a t o s t a t i n u s i n g passive immunization techniques. C o g n i z a n t of the important stimulatory e f f e c t s of g r o w t h h o r m o n e r e l e a s i n g hormone (GHRH) on GH secretion, we sought to determine if t h i s n e u r o p e p t i d e p l a y s a n y r o l e in m e d i a t i n g GH s e c r e t i o n in f o o d - d e p r i v e d rats. Male rats w e r e p r e p a r e d w i t h i n d w e l l i n g v e n o u s c a t h e t e r s using sodium pentobarbital anesthesia seven days prior to experimentation. Animals were food-deprived for 72 h, a f t e r w h i c h control blood samples were drawn from -60 to 0 min. One group was then treated with normal r a b b i t s e r u m (NRS), while a second group was t r e a t e d with GHRH antiserum (GHRHab). At 55 m i n all a n i m a l s r e c e i v e d s o m a t o s t a t i n a n t i s e r u m (SSab). No animal e x h i b i t e d any spontaneous GH peak during the one hour c o n t r o l p e r i o d or in the s u b s e q u e n t one h o u r p e r i o d following the administration of G H R H a b or N R S . A b s e n c e of GH p u l s a t i l i t y d u r i n g f o o d - d e p r i v a t i o n , coupled with no decrease in GH levels in f o o d - d e p r i v e d rats treated with GHRHab suggest that d i m i n i s h e d GHRH pulsatility is l i k e l y during food-deprivation. Subsequent treatment of t h e s e a n i m a l s w i t h S S a b r e s u l t e d in an i d e n t i c a l 2.5 f o l d i n c r e a s e in GH concentrations. This result suggests that GHRH is not i n v o l v e d in the GH r e b o u n d f o l l o w i n g s o m a t o s t a t i n w i t h d r a w a l in food-deprived rats. Pituitary growth hormone (GH) s e c r e t i o n in the rat is c h a r a c t e r i z e d by an u l t r a d i a n rhythm, with intermittent peaks and troughs at a p p r o x i m a t e l y three hour intervals (i). The p u l s a t i l e p a t t e r n of s e c r e t i o n r e s u l t s from the i n t e r a c t i o n b e t w e e n the s t i m u l a t o r y e f f e c t s of g r o w t h h o r m o n e - r e l e a s i n g h o r m o n e (GHRH) and the inhibitory effects of somatostatin (2-10). H i g h levels of s o m a t o s t a t i n m a i n t a i n low GH c o n c e n t r a t i o n s d u r i n g t r o u g h 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc All rights reserved.
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periods since passive immunization with somatostatin antiserum (SSab) increases basal GH c o n c e n t r a t i o n s but does not a l t e r the p u l s a t i l e p a t t e r n of GH secretion (8, 9). High c o n c e n t r a t i o n s of G H R H m a i n t a i n e l e v a t e d GH c o n c e n t r a t i o n s d u r i n g p e a k p e r i o d s since administration of G H R H a n t i s e r u m (GHRHab) completely i n h i b i t s the p u l s a t i l e s e c r e t i o n of GH (i0). That GHRH and s o m a t o s t a t i n are released in a p u l s a t i l e fashion is s u g g e s t e d by the f a c t t h a t at t h e t i m e of an a n t i c i p a t e d GH p u l s e , G H R H c o n c e n t r a t i o n s are e l e v a t e d and s o m a t o s t a t i n c o n c e n t r a t i o n s are d e p r e s s e d in h y p o t h a l a m i c - h y p o p h y s e a l portal blood (ii). GH pulses are depressed in r a t s u n d e r c o n d i t i o n s of f o o d - d e p r i v a t i o n or m a l n u t r i t i o n (12-14) , and plasma s o m a t o s t a t i n c o n c e n t r a t i o n s are increased (15). Indeed, a GH rebound has been r e p o r t e d in 72 h f o o d - d e p r i v e d rats u p o n the a d m i n i s t r a t i o n of somatostatin antiserum (16). These findings suggest that s o m a t o s t a t i n m e d i a t e s the s u p p r e s s i o n of GH s e c r e t i o n d u r i n g food-deprivation. It is possible, however, that s u p p r e s s e d GH is a l s o d u e to d e c r e a s e d G H R H s e c r e t i o n , a h y p o t h e s i s yet to be tested. S u p p o r t i n g this h y p o t h e s i s is an e a r l i e r s t u d y w h i c h s u g g e s t e d that h y p o t h a l a m i c GHRH c o n c e n t r a t i o n s d e c r e a s e during f o o d - d e p r i v a t i o n (17). In an effort to further c h a r a c t e r i z e the neuroregulation of GH s e c r e t i o n d u r i n g f o o d - d e p r i v a t i o n , we examined the contribution of G H R H in r e g u l a t i n g basal GH s e c r e t i o n and in c o n t r o l l i n g the m a g n i t u d e of the GH r e b o u n d following immunoneutralization of s o m a t o s t a t i n in 72 h f o o d - d e p r i v e d rats. Methods Animals: A l l a n i m a l s in t h e s e s t u d i e s w e r e a c q u i r e d , maintained, a n d u s e d in a c c o r d a n c e with the guidelines e s t a b l i s h e d by the N a t i o n a l I n s t i t u t e s of H e a l t h (18). Male S p r a g u e - D a w l e y rats (225-350 g) were m a i n t a i n e d in a t e m p e r a t u r e and h u m i d i t y - c o n t r o l l e d e n v i r o n m e n t and exposed to a 14 h light, I0 h dark lighting schedule (lights on at 0600 h). Surqical Preparation: Animals anesthetized with sodium p e n t o b a r b i t a l (NaPb, 40 mg/kg, ip) were o u t f i t t e d with a chronic indwelling venous c a t h e t e r one week prior to e x p e r i m e n t a t i o n (3). The c a t h e t e r was c o n n e c t e d to a single channel, fluid swivel to allow free m o v e m e n t of the rat following surgery. The rats were p l a c e d in isolation cages and received a constant infusion (0.08 ml/h) of h e p a r i n i z e d s a l i n e (i0 IU/ml) u n t i l t h e m o r n i n g of experimentation. Experimentation: F o o d - d e p r i v a t i o n was initiated four days after surgery and was continued for 72 h. B e g i n n i n g at 1200 h on t h e d a y of e x p e r i m e n t a t i o n , three control blood samples were d r a w n (0.35 ml), f o l l o w e d by an iv i n j e c t i o n of e i t h e r 0.5 ml n o r m a l r a b b i t s e r u m (NRS, n=7) or G H R H a b (n=ll). Subsequent b l o o d samples were drawn i0, 25 and 55 min following t r e a t m e n t . All animals then received 0.5 ml of SSab, iv, and blood samples w e r e d r a w n 5, i0, 15, 30 and 60 m i n f o l l o w i n g the i n j e c t i o n . Blood samples were centrifuged, plasma separated and then frozen for d e t e r m i n a t i o n of GH by RIA. The red blood cells were r e s u s p e n d e d in normal saline and returned to the animals.
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Antibody Preparation: Antiserum a g a i n s t rat G H R H w a s p r e p a r e d by i m m u n i z i n g r a b b i t s w i t h a m i x t u r e of s y n t h e t i c rat GHRH and methylated BSA emulsified in F r e u n d ' s a d j u v a n t as d e s c r i b e d p r e v i o u s l y (19). This a n t i s e r u m is s p e c i f i c for rat GHRH. It does not cross-react with GHRH from other species nor with p e p t i d e s that have considerable sequence h o m o l o g y with GHRH s u c h as s e c r e t i n , v a s o a c t i v e i n t e s t i n a l p e p t i d e and g l u c a g o n . The antibody d i l u t i o n required to bind a p p r o x i m a t e l y 50% of a rat GHRH-II25 labeled trace was 1:80,000. The dose a d m i n i s t e r e d was based on our observations that 0.5 ml of GHRHab is e f f e c t i v e in neutralizing e n d o g e n o u s G H R H as e v i d e n c e d by an a b s e n c e of p u l s a t i l e GH secretion (20). The SSab was p r e p a r e d by i m m u n i z i n g sheep w i t h s y n t h e t i c somatostatin. C o m p l e t e c h a r a c t e r i z a t i o n of t h e s o m a t o s t a t i n a n t i b o d y has b e e n p u b l i s h e d (21). Briefly, the antibody was partially purified by ammonium sulfate purification. It is d i r e c t e d toward the m i d - p o r t i o n of somatostatin. RIA s e n s i t i v i t y w a s 14 p g / t u b e at a f i n a l d i l u t i o n of 1 / 2 5 , 0 0 0 . The dose a d m i n i s t e r e d was based on our observations that 0.5 ml of SSab is effective in n e u t r a l i z i n g endogenous s o m a t o s t a t i n (22). R a d i o i m m u n o a s s a y Procedures: Plasma GH c o n c e n t r a t i o n s were d e t e r m i n e d in d u p l i c a t e by a d o u b l e a n t i b o d y p r o c e d u r e u s i n g materials s u p p l i e d by t h e N a t i o n a l P i t u i t a r y A g e n c y of t h e N a t i o n a l I n s t i t u t e s of Health. Blood s a m p l e s w e r e a s s a y e d in a l i q u o t s of e i t h e r i0 or 50 ~i. G r o w t h hormone c o n c e n t r a t i o n s are e x p r e s s e d in t e r m s of t h e r a t G H - R P - 2 s t a n d a r d . Assay sensitivity was approximately 0.08 ng/tube. Withinand b e t w e e n - a s s a y v a r i a t i o n s averaged less than 10%. Statistical Analyses: Significant t r e a t m e n t effects on GH c o n c e n t r a t i o n s were detected by analysis of v a r i a n c e for repeated m e a s u r e s (23). Since the variances of the mean GH c o n c e n t r a t i o n s were not homogenous, t h e d a t a w e r e f i r s t s u b j e c t e d to log transformation. E v a l u a t i o n of the overall GH response during the e x p e r i m e n t involved analyzing the area under the GH curve (AUC). The e x p e r i m e n t w a s s e p a r a t e d i n t o t h r e e d i f f e r e n t t r e a t m e n t periods. The control period was designated as -60 to 0 min, the N R S / G H R H a b period from 0 to 55 min, and SSab p e r i o d from 55 to 115 min. Less than 1% of the total number of blood samples to be drawn were missed due to technical problems during the experiment. In t h e s e i n s t a n c e s the GH v a l u e was e s t i m a t e d by c a l c u l a t i n g the average GH concentration from the p r e c e d i n g and following blood sample. All data are expressed as mean + SEM. Results Effects of F o o d - D e p r i v a t i o n : Seventy-two h o u r s of f o o d - d e p r i v a t i o n resulted in a 14-17% loss in total body weight. Pulsatile GH secretion was inhibited and baseline GH c o n c e n t r a t i o n s were elevated during the 60 min control p e r i o d in all animals (Fig. i). Although the control p e r i o d was too short to c o m p l e t e l y e v a l u a t e GH p u l s a t i l i t y , it has p r e v i o u s l y b e e n established that GH pulses are absent and baseline GH c o n c e n t r a t i o n s are elevated in food-deprived animals as c o m p a r e d to n o r m a l ones (3, I0, 13, 24). In addition to a l t e r i n g these e n d o c r i n e p a r a m e t e r s , f o o d - d e p r i v a t i o n a l s o m a d e the a n i m a l s agitated, restless and aggressive.
954
Neuroregulation of GH in Food-deprived Rats
60 ~"
50
0 NRS • GHRHab
40 I:~ C"
All Receive SSab
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:kT-I
30
~
20
~
100
-60-30
0
30
60
90 120
Time (min) FIG. 1 Mean plasma GH concentrations in 72 h f o o d - d e p r i v e d rats. NRS (n=7) o r G H R H a b (n=ll) w a s a d m i n i s t e r e d after the 0 min blood sample. All animals received SSab after t h e 55 m i n b l o o d sample• Baseline GH concentrations were elevated compared to previously e s t a b l i s h e d v a l u e s for n o r m a l r a t s ( c r o s s - h a t c h e d area, r e f s 3, i0, 24). GH concentrations did not change following NRS or GHRHab treatment• GH concentrations in b o t h N R S - a n d G H R H a b - t r e a t e d r a t s w e r e s i g n i f i c a n t l y e l e v a t e d a f t e r S S a b t r e a t m e n t (p<0.01).
4 .¢0
~NRS ~---~---~"~GHRHab
0
<~
2
o~ 1 v
0
Control
NRS/GHRHab
SSab
PERIOD FIG. 2 T h e a r e a u n d e r t h e G H c u r v e (AUC) in 72 h f o o d - d e p r i v e d rats. T h e G H A U C w a s s i g n i f i c a n t l y i n c r e a s e d (p<0.01) following SSab treatment• The GH AUCs between NRS- and GHRHab-treated rats were not different within periods•
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Control Period: In our laboratory GH trough c o n c e n t r a t i o n s in normal male rats are routinely less than 5 ng/ml (3). These v a l u e s are illustrated as the c r o s s - h a t c h e d area on Fig. i. In contrast, the GH values in food-deprived rats averaged b e t w e e n i0 and 20 ng/ml during the control period (Fig.l). E f f e c t s of N R S / G H R H a b : No s i g n i f i c a n t c h a n g e s in m e a n p l a s m a GH c o n c e n t r a t i o n s w e r e o b s e r v e d i m m e d i a t e l y f o l l o w i n g a d m i n i s t r a t i o n of GHRHab when compared to values o b s e r v e d during the control period or to values observed in N R S - t r e a t e d animals. T h e s e r e s u l t s are f u r t h e r s u p p o r t e d by c o m p a r i n g GH AUC. As illustrated in Figure 2, GH AUC was not a l t e r e d by G H R H a b w h e n c o m p a r e d to c o n t r o l v a l u e s or to the v a l u e of the N R S - t r e a t e d rats during the N R S / G H R H a b treatment period. E f f e c t s of S S a b : Administration of S S a b to N R S - or G H R H a b - t r e a t e d animals resulted in an identical GH rebound, with c o n c e n t r a t i o n s increasing a p p r o x i m a t e l y 2.5 fold (Fig. i). After t h e i n i t i a l r e b o u n d , GH c o n c e n t r a t i o n s r e m a i n e d e l e v a t e d in N R S - t r e a t e d animals, but steadily decreased in those t r e a t e d with GHRHab. This decline, however, was not s t a t i s t i c a l l y s i g n i f i c a n t when e v a l u a t e d by absolute GH concentrations (Fig. i) or GH A U C (Fig. 2). Discussion Our novel o b s e r v a t i o n s show that G H R H is not involved in the m a i n t e n a n c e of the e l e v a t e d basal GH c o n c e n t r a t i o n s , or in the acute GH rebound following somatostatin withdrawal in f o o d - d e p r i v e d rats. This is d e m o n s t r a t e d by the o b s e r v a t i o n that basal and r e b o u n d GH c o n c e n t r a t i o n s w e r e not d i f f e r e n t b e t w e e n NRS- and G H R H a b - t r e a t e d rats. These results suggest that the lack of p u l s a t i l e GH s e c r e t i o n in the f o o d - d e p r i v e d rat may be due to d e c r e a s e d GHRH secretion rather than elevated s o m a t o s t a t i n s e c r e t i o n (see next paragraph). Supporting this c o n c l u s i o n are the o b s e r v a t i o n s of Meites and Fiel (17) that GHRH c o n c e n t r a t i o n s are d e c r e a s e d following food-deprivation (bioassay) and GHRH m R N A decreases during food-deprivation (25). Even t h o u g h it a p p e a r s t h a t G H R H is not directly involved in c o n t r o l l i n g the m a g n i t u d e of the GH rebound, GHRH may play a role in m a i n t a i n i n g e l e v a t e d GH c o n c e n t r a t i o n s afterward. This is b a s e d on our o b s e r v a t i o n t h a t f o l l o w i n g s o m a t o s t a t i n r e m o v a l , GH r e m a i n e d e l e v a t e d in N R S - t r e a t e d a n i m a l s but d e c l i n e d in t h o s e t r e a t e d w i t h GHRHab. However, these results are not statistically different and studies of longer duration will be required to p r o p e r l y evaluate this possibility. B a s e d on GH c o n c e n t r a t i o n s , it may be i n f e r r e d t h a t the rhythmicity of G H R H a n d s o m a t o s t a t i n s e c r e t i o n is d i s r u p t e d during food-deprivation. R e s u l t s from the p r e s e n t e x p e r i m e n t s h o w an a b s e n c e of n o r m a l GH p u l s a t i l i t y and an e l e v a t e d GH b a s e l i n e in 72 h f o o d - d e p r i v e d rats. This s t u d y c o r r o b o r a t e s e a r l i e r o b s e r v a t i o n s t h a t s u p p r e s s i o n of n o r m a l p u l s a t i l e GH release following 72 h of food-deprivation is mediated, in part, by hypothalamic somatostatin secretion (12-16). It is of interest, however, that although peripheral somatostatin c o n c e n t r a t i o n s are reported to be elevated in the f o o d - d e p r i v e d rat (15), basal GH concentrations are elevated. In c o n t r a s t to a l m o s t u n d e t e c t a b l e b a s e l i n e GH c o n c e n t r a t i o n s in n o r m a l rats
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(GH
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T h e r e is no q u e s t i o n t h a t f o o d - d e p r i v e d r a t s (16). and t h o s e of o t h e r s (24), be diminished. Thus, t h e rats most likely reflects
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s o m a t o s t a t i n is f u n c t i o n a l l y a c t i v e in Yet, b a s e d on t h e p r e s e n t o b s e r v a t i o n s t h e c o n c e n t r a t i o n s of s o m a t o s t a t i n m a y a b s e n c e of GH p u l s e s in f o o d - d e p r i v e d an a b s e n c e of GHRH.
Acknowledqments We thank L. S t a g g , A. P i e r i n g a n d D. V o l t z for their technical assistance. T h i s w o r k w a s s u p p o r t e d b y t h e Univ. of WI Graduate School and the NIH Grants R01-DK-38324 and K04-DK-01874.
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