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Robust Growth Hormone (GH) secretion in aged female rats co-administered GH-releasing hexapeptide (GHRP-6) and GH-releasing hormone (GHRH)
Life Sciences, Vol. 49, pp. 1499-1504 Printed in the U.S.A.
Pergamon Press
R O B U S T GROWTH HORMONE (GH) SECRETION IN AGED FEMALE RATS C O - A D M I N I S T E R E D G H - R E L E A S I N G H E X A P E P T I D E (GHRP-6) AND G H - R E L E A S I N G H O R M O N E (GHRH)
R.F. Walker,
S-W. Yang and B.B. Bercu
D i v i s i o n of Endocrinology, Department of P e d i a t r i c s , U n i v e r s i t y of South Florida College of Medicine,Tampa, Florida 33612 and All Children's Hospital,St. Petersburg, Florida 33701 (Received in final form September 12, 1991) Summary Aging is associated with a blunted growth h o r m o n e (GH) secretory response to G H - r e l e a s i n g h o r m o n e (GHRH), in vivo. The o b j e c t i v e of the present study was to assess the effects of aging on the GH secretory response to G H - r e l e a s i n g h e x a p e p t i d e (GHRP-6), a synthetic GH secretagogue. GHRP-6 (30 Bg/kg) was a d m i n i s t e r e d alone or in c o m b i n a t i o n with GHRH (2 ~g/kg) to a n e s t h e t i z e d female Fischer 344 rats, 3 or 19 months of age. The peptides were c o - a d m i n i s t e r e d to d e t e r m i n e the effect of aging upon the p o t e n t i a t i n g effect of GHRP-6 on GHRH activity. The increase in plasma GH as a function of time following a d m i n i s t r a t i o n of GHRP-6 was lower (p < 0.001) in old rats than in young rats; w h e r e a s the increase in plasma GH secretion as a function of time following c o - a d m i n i s t r a t i o n of GHRP-6 and GHPd4was h i g h e r (p < 0.001) in old rats than in young rats (mean Cmax = 8539 ± 790.6 ~g/l vs. 2970 ± 866 ~g/l, respectively; p <0.01). since p i t u i t a r y GH concentrations in old rats were lower than in young rats (257.0 ± 59.8 ~g/mg wet wt. vs. 639.7 ± 149.2 ~g/mg wet wt., respectively; p < 0.03), the results s u g g e s t e d that GH functional reserve in old female rats was not linked to p i t u i t a r y GH concentration. The d i f f e r e n t i a l r e s p o n s e s of old rats to individually a d m i n i s t e r e d and c o - a d m i n i s t e r e d GHRP-6 are important because they d e m o n s t r a t e that robust and immediate GH secretion can occur in old rats that are a p p r o p r i a t e l y stimulated. The data further suggest that the cellular p r o c e s s e s subserving GH secretion are intact in old rats, and that a g e - r e l a t e d decrements in GH s e c r e t i o n result from inadequate stimulation, rather than to m a l a d a p t i v e changes in the m e c h a n i s m of GH release. The d e c l i n e in serum growth hormone (GH) c o n c e n t r a t i o n s during aging is a s s o c i a t e d with a p r o g r e s s i v e i n s e n s i t i v i t y of the p i t u i t a r y gland to G H - r e l e a s i n g hormone (GHRH), in vivo. GH secretion following GHRH a d m i n i s t r a t i o n was inversely c o r r e l a t e d with age in men and women between the ages of 18 and 95 years (i4), and in a n e s t h e t i z e d male rats, between the ages of 3 and 21 m o n t h s (5,6). 0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc
1500
GHRP-6 in Aged Rats
Vol. 49, No. 20, 1991
During the past decade, a synthetic peptide, H i s - D - T r p - A l a - T r p - D P h e - L y s - N H z (growth h o r m o n e r e l e a s i n g hexapeptide; GHRP-6) was identified as a specific GH s e c r e t a g o g u e in vivo and in vitro (see 7). GHRP-6 has d i f f e r e n t b i n d i n g c h a r a c t e r i s t i c s and employs different intracellular messengers from GHRH (8-10), and it potentiates GHRH activity (i0,ii). Since GHRP-6 can act i n d e p e n d e n t l y of GHRH, it was of interest to d e t e r m i n e whether aging also d i m i n i s h e s the hexapeptides' G H - r e l e a s i n g activity. GHRP-6 and GHRH were also co-administered, so as to e v a l u a t e ager e l a t e d changes in the functional i n t e r a c t i o n of the two GH secretagogues. M a t e r i a l s and M e t h o d s
Animal Husbandry:
Fischer 344 female rats (2 and 18 months of age) were purchased from the NIA colony at Harlan Industries (Indianapolis, Ind.) and a c c l i m a t e d to local c o n d i t i o n s of light (0600h - 1800h), t e m p e r a t u r e (72 ± 2 °F) and h u m i d i t y (60 ± 5 %) for 30 days before being used in the present study. The animal facility at All Children's Hospital is fully a p p r o v e d for use by the A A A L A C and complies with federal statues and r e g u l a t i o n s r e l a t i n g to animal use in research.
Peptides
and Experimental Design: GHRP-6 and rat GHRH were purchased from Peninsula Laboratories (Belmont, CA) and a d m i n i s t e r e d as saline solutions in doses of 30 Bg/kg and 2 ~g/kg, respectively. In a p r e l i m i n a r y d o s e - r a n g e study, 30 ~g/kg GHRP-6 was the EDs^ in anesthetized, 3 month old female rats. The dose of GHRH (2 ~g/~g) was selected from p u b l i s h e d data showing d i f f e r e n c e s in GH s e c r e t o r y responses in 3 and 18 month old rats (5). On the day of testing, rats were a n e s t h e t i z e d w i t h k e t a m i n e (60 mg/kg i.m.) and p e n t o b a r b i t a l (35 mg/kg i.p.) and the right femoral artery was cannulated. Peptides were administered with c o n s i d e r a t i o n of time from surgery so that all animals were tested under the same duration of anesthesia. Blood samples (approximately 250 ~i) were c o l l e c t e d in h e p a r i n i z e d tubes before and at selected intervals after administration of the GH secretagogues. The rats were e x s a n g u i n a t e d at the last blood sample and their p i t u i t a r y glands were rapidly removed, w e i g h e d and frozen on dry ice. No adenomas were evident upon m a c r o s c o p i c e x a m i n a t i o n of the pituitaries. Radioimmunoassay:
Blood samples were c e n t r i f u g e d and plasma was c o l l e c t e d for r a d i o i m m u n o a s s a y (RIA) analysis of GH concentrations. P i t u i t a r i e s were h o m o g e n i z e d in 0.1 N NaOH/saline, d i l u t e d and aliquots were analyzed by RIA for GH concentrations. RIA reagents were s u p p l i e d by Dr. A. Parlow (Harbor-UCLA M e d i c a l Center, Torrence, CA) and the NIADD. GH was e x p r e s s e d in terms of rGH-RP2. I n t r a a s s a y and interassay c o e f f i c i e n t s of v a r i a t i o n were <5 % and <8 %, respectively.
Data Analysis:
P i t u i t a r y GH c o n c e n t r a t i o n s and peak plasma GH c o n c e n t r a t i o n s (Cm ) were compared among the groups of y o u n g and old rats using Student's t test. Changes in p l a s m a GH s e c r e t i o n over time w e r e c o m p a r e d using two way A N O V A w i t h r e p e a t e d m e a s u r e s analysis. R e s u l t s were e x p r e s s e d as means ± S.E.M. Plasma and p i t u i t a r y GH c o n c e n t r a t i o n s were e x p r e s s e d as ~g/l and ~g/mg wet weight, respectively.
Vol. 49, NO. 20, 1991
GHRP-6 in Aged Rats
1501
Results The data p r e s e n t e d in Figure 1 show that the increase in GH s e c r e t i o n as a function of time following GHRP-6 a d m i n i s t r a t i o n was lower (p <0.001) in old rats than in young rats. M e a n peak plasma GH values were lower in old rats than in young rats (179.8 ± 64.9 ~g/l vs. 356.4 ± 85.0 ~g/l, respectively); h o w e v e r the d i f f e r e n c e s were not s t a t i s t i c a l l y significant. P o t e n t i a t e d GH secretion occurred in rats of both ages following c o - a d m i n i s t r a t i o n of GHRP-6 and GHRH. However, the increase in plasma GH c o n c e n t r a t i o n s as a function of time (p <0.001), and peak plasma GH c o n c e n t r a t i o n s (p < 0.01) were s i g n i f i c a n t l y greater in old rats than in y o u n g rats (Cmx = 8539 ± 790.6 ~g/l vs. 2970 ± 866 ~g/l, respectively). 60O
............ OLD RATS •
Y O U N G RATS
~,3oo
"1-
O
2oo
100
0 0
I 10
I 20
I 30
I 40
I 60
6O
MINUTES Fiq. 1
GH s e c r e t i o n in young (3 months old) and old (19 m o n t h s old) female, Fischer 344 rats following a d m i n i s t r a t i o n of GHRP-6 (30 ~g/kg). Values are expressed as means + SEM; N = 4 r a t s / a g e group.
As seen in Figure 2, the temporal profiles for GH secretion following c o - a d m i n i s t r a t i o n of GHRP-6 and GHRH were the same in the two age groups, except that plasma GH c o n c e n t r a t i o n s were up to three times higher in the old rats. P i t u i t a r y c o n c e n t r a t i o n s of GH in all y o u n g rats (639.7 ± 149.2 ~g/mg) were s i g n i f i c a n t l y higher (p < 0.03) than in all old rats (257.5 ± 59.8 ~g/mg). However, the data p r e s e n t e d in Table 1 show that the individual t r e a t m e n t s were a s s o c i a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s of p i t u i t a r y GH, p r o b a b l y reflecting the GH releasing efficacy of the individually a d m i n i s t e r e d or c o - a d m i n i s t e r e d peptides. C o - a d m i n i s t r a t i o n of GHRP-6 and GHRH was a s s o c i a t e d with the g r e a t e s t r e d u c t i o n in p i t u i t a r y GH c o n c e n t r a t i o n s in both age groups.
1502
GHRP-6
in A g e d R a t s
Vol.
49, No.
20,
1991
9,OOO T
I - - - = - ....
I
8,ooo
i I
i
•
OLD RATS YOUNG
RATS
7,0O0 I / I / I / I /
6,0O0
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v
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4,000
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MINUTES
Fig.
2
GH secretion in young and old female rats following coadministration of GHRP-6 and GHRH (30 ~g/kg and 2 ~g/kg, respectively). V a l u e s a r e e x p r e s s e d as m e a n ± SEM; N = 4 rats/age group. N o t e s c a l e d i f f e r e n c e o n t h e o r d i n a t e of t h i s g r a p h a n d t h a t in F i g u r e i.
TABLE
PITUITARY RATS AFTER
1
G H C O N C E N T R A T I O N S IN Y O U N G A N D O L D ADMINISTRATION OF GHRP-6 AND/OR GHRH PITUITARY GH CONCENTRATIONS Young Old
(~g/mg)
TREATMENT
N
GHRP-6
4
899
± 321
255
± 113"
GHRH
4
735
± 275
434
± 115"
GHRP-6 + GHRH
4
363 ± 122
170
±
p < 0.03;
o l d vs.
young
33*
Vol. 49, No. 20, 1991
GHRP-6 in Aged Rats
1503
Discussion The results of this study show that GH s e c r e t i o n following a d m i n i s t r a t i o n of GHRP-6 declines during aging in female rats. Similar r e s p o n s e s following a d m i n i s t r a t i o n of GHRH have been r e p o r t e d in rats and humans (i - 6). Despite these a g e - r e l a t e d d e c r e m e n t s in response to GHRP-6 or GHRH, the r e s p o n s e to coa d m i n i s t e r e d GHRP-6 and GHRH was higher in old rats than in young rats. This effect was inversely related to p i t u i t a r y GH concentrations, which were higher in the y o u n g rats. Thus, even t h o u g h p i t u i t a r y GH c o n c e n t r a t i o n s were reduced, the old rats e x p r e s s e d a g r e a t e r functional GH reserve than the young rats when GHRP-6 and G H R H w e r e co-administered. The d i f f e r e n t i a l effects of GHRP-6 on GH secretion when a d m i n i s t e r e d alone or in c o m b i n a t i o n with GHRH are very important because they suggest that a g e - r e l a t e d d e c r e m e n t s in GH secretion are not due to intrinsic p i t u i t a r y defects. A p p a r e n t l y the cellular processes s u b s e r v i n g GH secretion were intact in old rats used in this study, since they were capable of i m m e d i a t e l y releasing as much or more GH as y o u n g rats when a p p r o p r i a t e l y stimulated. These findings are c o n s i s t e n t with data from in vitro studies showing that GH secretion from p i t u i t a r y slices is not d i m i n i s h e d with advancing age (6). The p a r a d o x i c a l response of old rats to c o - a d m i n i s t e r e d GHRH and GHRP-6 is quite interesting. An attenuated r e s p o n s e to the coa d m i n i s t e r e d peptides would be consistent with blunted r e s p o n s e s to individually administered GHRH or GHRP-6. Instead, coa d m i n i s t r a t i o n of GHRP-6 and GHRH stimulated more GH secretion in old rats than in young rats, as if the former group were h y p e r s e n s i t i v e rather than h y p o s e n s i t i v e to the GH secretagogues. One p o s s i b l e i n t e r p r e t a t i o n of these data is that in addition to r e d u c e d GHRH secretion in old rats (12, 13), c o n c e n t r a t i o n s of an a n c i l l a r y factor that facilitates G H R H a c t i v i t y are also d i m i n i s h e d during aging. With a progressive decrease in this p u t a t i v e a n c i l l a r y factor during aging, GHRH activity w o u l d become blunted. A l t h o u g h hypothetical, the existence of a yet unidentified, endogenous analog of GHRP-6 as proposed by Bowers et al. (7), is supported by the consistent, potent and specific GH r e l e a s i n g a c t i v i t y of the hexapeptide. The reduced s e n s i t i v i t y of old rats to i n d i v i d u a l l y but not c o - a d m i n i s t e r e d GHRH and GHRP-6 suggests that the concentrations of both functionally interdependent m o l e c u l e s are d e p l e t e d during aging. REFERENCES i.
2. 3. 4.
5.
E.
GHIGO,
S. GOFFI,
E. ARVAT,
M.
NICOLOSI,
M.
PROCOPIO,
J.
BELLONE, E. IMPERIALE, E. MAZZA, G. BARACCHI, and F. CAMANNI, Acta Endocrinol (Copenh) 123:160-174 (1990). I. LANG, G. SCHERNTHANER, P. PIETSCHMANN, R. KURZ, J.M. STEPHENSON, and H. TEMPL, J Clin Endocrinol M e t a b 6--5:535-540 (1987). I. LANG, R.KURZ, G. GEYER, and K.H. TRAGL, H o r m M e t a b o l Res 2--0:574-578 (1988). T. SHIBASAKI, K. SHIZUME, M. NAKAHAR, A. MASUDA, K. JIBIKI, H. DEMURA, I. WAKABAYASHI, and N. LING, J Clin E n d o c r i n o l M e t a b 5--8:212-214 (1984). G.P. CEDA, G. VALENTI, U. BUTTURINI, and A.R. HOFFMAN, E n d o c r i n o l o q y 118:2109-2114 (1986).
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6. 7. 8. 9. i0. ii.
GHRP-6 in Aged Rats
Vol. 49, No. 20, 1991
W.E. SONNTAG, V.W. HYLKA, and J. MEITES, Endocrinology 113:2305-2307 (1983). C.Y. BOWERS, A.O. SARTOR, G.A. REYNOLDS, and T.M. BADGER, E n d o c r i n o l o g y 128:2027-2035 (1991). E.E. CODD, A.Y.L. SHU, and R.F. WALKER, N e u r o p h a r m a c o l o g y 2--8:1139-1144 (1989). A.D. BLAKE, and R.G. SMITH, J Endocrinol 129:11-19 (1991). K. CHENG, W.W-S. CHAN, A. BARRETO, D.M. CONVEY, and R.G. SMITH, E n d o c r i n o l o g y 124:2791-2798 (1989). C.Y. BOWERS, G.A. REYNOLDS, D. DURHAM, C.M. BARRERA, S.S. PEZZOLI, and M.O. THORNER, J clin E n d o c r i n o l M e t a b 7--0:975-982
(1990). 12.
V. DE G E N N A R O COLLONA, M. ZOLI, D. COCCHI, A. MARRAMA, L.F. AGNATI and E.E. MULLER, Peptides
MAGGI, P. 1-0:705-708
13.
F. GE, S. TSAGARAKIS, L.H. REES, G.M. BESSER, and A. GROSSMAN, J E n d o c r i n o l 123:53-58 (1989)
(1989).
Pergamon Press
R O B U S T GROWTH HORMONE (GH) SECRETION IN AGED FEMALE RATS C O - A D M I N I S T E R E D G H - R E L E A S I N G H E X A P E P T I D E (GHRP-6) AND G H - R E L E A S I N G H O R M O N E (GHRH)
R.F. Walker,
S-W. Yang and B.B. Bercu
D i v i s i o n of Endocrinology, Department of P e d i a t r i c s , U n i v e r s i t y of South Florida College of Medicine,Tampa, Florida 33612 and All Children's Hospital,St. Petersburg, Florida 33701 (Received in final form September 12, 1991) Summary Aging is associated with a blunted growth h o r m o n e (GH) secretory response to G H - r e l e a s i n g h o r m o n e (GHRH), in vivo. The o b j e c t i v e of the present study was to assess the effects of aging on the GH secretory response to G H - r e l e a s i n g h e x a p e p t i d e (GHRP-6), a synthetic GH secretagogue. GHRP-6 (30 Bg/kg) was a d m i n i s t e r e d alone or in c o m b i n a t i o n with GHRH (2 ~g/kg) to a n e s t h e t i z e d female Fischer 344 rats, 3 or 19 months of age. The peptides were c o - a d m i n i s t e r e d to d e t e r m i n e the effect of aging upon the p o t e n t i a t i n g effect of GHRP-6 on GHRH activity. The increase in plasma GH as a function of time following a d m i n i s t r a t i o n of GHRP-6 was lower (p < 0.001) in old rats than in young rats; w h e r e a s the increase in plasma GH secretion as a function of time following c o - a d m i n i s t r a t i o n of GHRP-6 and GHPd4was h i g h e r (p < 0.001) in old rats than in young rats (mean Cmax = 8539 ± 790.6 ~g/l vs. 2970 ± 866 ~g/l, respectively; p <0.01). since p i t u i t a r y GH concentrations in old rats were lower than in young rats (257.0 ± 59.8 ~g/mg wet wt. vs. 639.7 ± 149.2 ~g/mg wet wt., respectively; p < 0.03), the results s u g g e s t e d that GH functional reserve in old female rats was not linked to p i t u i t a r y GH concentration. The d i f f e r e n t i a l r e s p o n s e s of old rats to individually a d m i n i s t e r e d and c o - a d m i n i s t e r e d GHRP-6 are important because they d e m o n s t r a t e that robust and immediate GH secretion can occur in old rats that are a p p r o p r i a t e l y stimulated. The data further suggest that the cellular p r o c e s s e s subserving GH secretion are intact in old rats, and that a g e - r e l a t e d decrements in GH s e c r e t i o n result from inadequate stimulation, rather than to m a l a d a p t i v e changes in the m e c h a n i s m of GH release. The d e c l i n e in serum growth hormone (GH) c o n c e n t r a t i o n s during aging is a s s o c i a t e d with a p r o g r e s s i v e i n s e n s i t i v i t y of the p i t u i t a r y gland to G H - r e l e a s i n g hormone (GHRH), in vivo. GH secretion following GHRH a d m i n i s t r a t i o n was inversely c o r r e l a t e d with age in men and women between the ages of 18 and 95 years (i4), and in a n e s t h e t i z e d male rats, between the ages of 3 and 21 m o n t h s (5,6). 0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc
1500
GHRP-6 in Aged Rats
Vol. 49, No. 20, 1991
During the past decade, a synthetic peptide, H i s - D - T r p - A l a - T r p - D P h e - L y s - N H z (growth h o r m o n e r e l e a s i n g hexapeptide; GHRP-6) was identified as a specific GH s e c r e t a g o g u e in vivo and in vitro (see 7). GHRP-6 has d i f f e r e n t b i n d i n g c h a r a c t e r i s t i c s and employs different intracellular messengers from GHRH (8-10), and it potentiates GHRH activity (i0,ii). Since GHRP-6 can act i n d e p e n d e n t l y of GHRH, it was of interest to d e t e r m i n e whether aging also d i m i n i s h e s the hexapeptides' G H - r e l e a s i n g activity. GHRP-6 and GHRH were also co-administered, so as to e v a l u a t e ager e l a t e d changes in the functional i n t e r a c t i o n of the two GH secretagogues. M a t e r i a l s and M e t h o d s
Animal Husbandry:
Fischer 344 female rats (2 and 18 months of age) were purchased from the NIA colony at Harlan Industries (Indianapolis, Ind.) and a c c l i m a t e d to local c o n d i t i o n s of light (0600h - 1800h), t e m p e r a t u r e (72 ± 2 °F) and h u m i d i t y (60 ± 5 %) for 30 days before being used in the present study. The animal facility at All Children's Hospital is fully a p p r o v e d for use by the A A A L A C and complies with federal statues and r e g u l a t i o n s r e l a t i n g to animal use in research.
Peptides
and Experimental Design: GHRP-6 and rat GHRH were purchased from Peninsula Laboratories (Belmont, CA) and a d m i n i s t e r e d as saline solutions in doses of 30 Bg/kg and 2 ~g/kg, respectively. In a p r e l i m i n a r y d o s e - r a n g e study, 30 ~g/kg GHRP-6 was the EDs^ in anesthetized, 3 month old female rats. The dose of GHRH (2 ~g/~g) was selected from p u b l i s h e d data showing d i f f e r e n c e s in GH s e c r e t o r y responses in 3 and 18 month old rats (5). On the day of testing, rats were a n e s t h e t i z e d w i t h k e t a m i n e (60 mg/kg i.m.) and p e n t o b a r b i t a l (35 mg/kg i.p.) and the right femoral artery was cannulated. Peptides were administered with c o n s i d e r a t i o n of time from surgery so that all animals were tested under the same duration of anesthesia. Blood samples (approximately 250 ~i) were c o l l e c t e d in h e p a r i n i z e d tubes before and at selected intervals after administration of the GH secretagogues. The rats were e x s a n g u i n a t e d at the last blood sample and their p i t u i t a r y glands were rapidly removed, w e i g h e d and frozen on dry ice. No adenomas were evident upon m a c r o s c o p i c e x a m i n a t i o n of the pituitaries. Radioimmunoassay:
Blood samples were c e n t r i f u g e d and plasma was c o l l e c t e d for r a d i o i m m u n o a s s a y (RIA) analysis of GH concentrations. P i t u i t a r i e s were h o m o g e n i z e d in 0.1 N NaOH/saline, d i l u t e d and aliquots were analyzed by RIA for GH concentrations. RIA reagents were s u p p l i e d by Dr. A. Parlow (Harbor-UCLA M e d i c a l Center, Torrence, CA) and the NIADD. GH was e x p r e s s e d in terms of rGH-RP2. I n t r a a s s a y and interassay c o e f f i c i e n t s of v a r i a t i o n were <5 % and <8 %, respectively.
Data Analysis:
P i t u i t a r y GH c o n c e n t r a t i o n s and peak plasma GH c o n c e n t r a t i o n s (Cm ) were compared among the groups of y o u n g and old rats using Student's t test. Changes in p l a s m a GH s e c r e t i o n over time w e r e c o m p a r e d using two way A N O V A w i t h r e p e a t e d m e a s u r e s analysis. R e s u l t s were e x p r e s s e d as means ± S.E.M. Plasma and p i t u i t a r y GH c o n c e n t r a t i o n s were e x p r e s s e d as ~g/l and ~g/mg wet weight, respectively.
Vol. 49, NO. 20, 1991
GHRP-6 in Aged Rats
1501
Results The data p r e s e n t e d in Figure 1 show that the increase in GH s e c r e t i o n as a function of time following GHRP-6 a d m i n i s t r a t i o n was lower (p <0.001) in old rats than in young rats. M e a n peak plasma GH values were lower in old rats than in young rats (179.8 ± 64.9 ~g/l vs. 356.4 ± 85.0 ~g/l, respectively); h o w e v e r the d i f f e r e n c e s were not s t a t i s t i c a l l y significant. P o t e n t i a t e d GH secretion occurred in rats of both ages following c o - a d m i n i s t r a t i o n of GHRP-6 and GHRH. However, the increase in plasma GH c o n c e n t r a t i o n s as a function of time (p <0.001), and peak plasma GH c o n c e n t r a t i o n s (p < 0.01) were s i g n i f i c a n t l y greater in old rats than in y o u n g rats (Cmx = 8539 ± 790.6 ~g/l vs. 2970 ± 866 ~g/l, respectively). 60O
............ OLD RATS •
Y O U N G RATS
~,3oo
"1-
O
2oo
100
0 0
I 10
I 20
I 30
I 40
I 60
6O
MINUTES Fiq. 1
GH s e c r e t i o n in young (3 months old) and old (19 m o n t h s old) female, Fischer 344 rats following a d m i n i s t r a t i o n of GHRP-6 (30 ~g/kg). Values are expressed as means + SEM; N = 4 r a t s / a g e group.
As seen in Figure 2, the temporal profiles for GH secretion following c o - a d m i n i s t r a t i o n of GHRP-6 and GHRH were the same in the two age groups, except that plasma GH c o n c e n t r a t i o n s were up to three times higher in the old rats. P i t u i t a r y c o n c e n t r a t i o n s of GH in all y o u n g rats (639.7 ± 149.2 ~g/mg) were s i g n i f i c a n t l y higher (p < 0.03) than in all old rats (257.5 ± 59.8 ~g/mg). However, the data p r e s e n t e d in Table 1 show that the individual t r e a t m e n t s were a s s o c i a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s of p i t u i t a r y GH, p r o b a b l y reflecting the GH releasing efficacy of the individually a d m i n i s t e r e d or c o - a d m i n i s t e r e d peptides. C o - a d m i n i s t r a t i o n of GHRP-6 and GHRH was a s s o c i a t e d with the g r e a t e s t r e d u c t i o n in p i t u i t a r y GH c o n c e n t r a t i o n s in both age groups.
1502
GHRP-6
in A g e d R a t s
Vol.
49, No.
20,
1991
9,OOO T
I - - - = - ....
I
8,ooo
i I
i
•
OLD RATS YOUNG
RATS
7,0O0 I / I / I / I /
6,0O0
!/
G"
"Oh %oo0 i
v
"r
4,000
i
"M! i
i
3,OO0
/
2,OOO
/I
,
k
i
1,000 0 0
"\.
I 10
i 2O
I 30
1 4O
I 6O
6o
MINUTES
Fig.
2
GH secretion in young and old female rats following coadministration of GHRP-6 and GHRH (30 ~g/kg and 2 ~g/kg, respectively). V a l u e s a r e e x p r e s s e d as m e a n ± SEM; N = 4 rats/age group. N o t e s c a l e d i f f e r e n c e o n t h e o r d i n a t e of t h i s g r a p h a n d t h a t in F i g u r e i.
TABLE
PITUITARY RATS AFTER
1
G H C O N C E N T R A T I O N S IN Y O U N G A N D O L D ADMINISTRATION OF GHRP-6 AND/OR GHRH PITUITARY GH CONCENTRATIONS Young Old
(~g/mg)
TREATMENT
N
GHRP-6
4
899
± 321
255
± 113"
GHRH
4
735
± 275
434
± 115"
GHRP-6 + GHRH
4
363 ± 122
170
±
p < 0.03;
o l d vs.
young
33*
Vol. 49, No. 20, 1991
GHRP-6 in Aged Rats
1503
Discussion The results of this study show that GH s e c r e t i o n following a d m i n i s t r a t i o n of GHRP-6 declines during aging in female rats. Similar r e s p o n s e s following a d m i n i s t r a t i o n of GHRH have been r e p o r t e d in rats and humans (i - 6). Despite these a g e - r e l a t e d d e c r e m e n t s in response to GHRP-6 or GHRH, the r e s p o n s e to coa d m i n i s t e r e d GHRP-6 and GHRH was higher in old rats than in young rats. This effect was inversely related to p i t u i t a r y GH concentrations, which were higher in the y o u n g rats. Thus, even t h o u g h p i t u i t a r y GH c o n c e n t r a t i o n s were reduced, the old rats e x p r e s s e d a g r e a t e r functional GH reserve than the young rats when GHRP-6 and G H R H w e r e co-administered. The d i f f e r e n t i a l effects of GHRP-6 on GH secretion when a d m i n i s t e r e d alone or in c o m b i n a t i o n with GHRH are very important because they suggest that a g e - r e l a t e d d e c r e m e n t s in GH secretion are not due to intrinsic p i t u i t a r y defects. A p p a r e n t l y the cellular processes s u b s e r v i n g GH secretion were intact in old rats used in this study, since they were capable of i m m e d i a t e l y releasing as much or more GH as y o u n g rats when a p p r o p r i a t e l y stimulated. These findings are c o n s i s t e n t with data from in vitro studies showing that GH secretion from p i t u i t a r y slices is not d i m i n i s h e d with advancing age (6). The p a r a d o x i c a l response of old rats to c o - a d m i n i s t e r e d GHRH and GHRP-6 is quite interesting. An attenuated r e s p o n s e to the coa d m i n i s t e r e d peptides would be consistent with blunted r e s p o n s e s to individually administered GHRH or GHRP-6. Instead, coa d m i n i s t r a t i o n of GHRP-6 and GHRH stimulated more GH secretion in old rats than in young rats, as if the former group were h y p e r s e n s i t i v e rather than h y p o s e n s i t i v e to the GH secretagogues. One p o s s i b l e i n t e r p r e t a t i o n of these data is that in addition to r e d u c e d GHRH secretion in old rats (12, 13), c o n c e n t r a t i o n s of an a n c i l l a r y factor that facilitates G H R H a c t i v i t y are also d i m i n i s h e d during aging. With a progressive decrease in this p u t a t i v e a n c i l l a r y factor during aging, GHRH activity w o u l d become blunted. A l t h o u g h hypothetical, the existence of a yet unidentified, endogenous analog of GHRP-6 as proposed by Bowers et al. (7), is supported by the consistent, potent and specific GH r e l e a s i n g a c t i v i t y of the hexapeptide. The reduced s e n s i t i v i t y of old rats to i n d i v i d u a l l y but not c o - a d m i n i s t e r e d GHRH and GHRP-6 suggests that the concentrations of both functionally interdependent m o l e c u l e s are d e p l e t e d during aging. REFERENCES i.
2. 3. 4.
5.
E.
GHIGO,
S. GOFFI,
E. ARVAT,
M.
NICOLOSI,
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PROCOPIO,
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BELLONE, E. IMPERIALE, E. MAZZA, G. BARACCHI, and F. CAMANNI, Acta Endocrinol (Copenh) 123:160-174 (1990). I. LANG, G. SCHERNTHANER, P. PIETSCHMANN, R. KURZ, J.M. STEPHENSON, and H. TEMPL, J Clin Endocrinol M e t a b 6--5:535-540 (1987). I. LANG, R.KURZ, G. GEYER, and K.H. TRAGL, H o r m M e t a b o l Res 2--0:574-578 (1988). T. SHIBASAKI, K. SHIZUME, M. NAKAHAR, A. MASUDA, K. JIBIKI, H. DEMURA, I. WAKABAYASHI, and N. LING, J Clin E n d o c r i n o l M e t a b 5--8:212-214 (1984). G.P. CEDA, G. VALENTI, U. BUTTURINI, and A.R. HOFFMAN, E n d o c r i n o l o q y 118:2109-2114 (1986).
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6. 7. 8. 9. i0. ii.
GHRP-6 in Aged Rats
Vol. 49, No. 20, 1991
W.E. SONNTAG, V.W. HYLKA, and J. MEITES, Endocrinology 113:2305-2307 (1983). C.Y. BOWERS, A.O. SARTOR, G.A. REYNOLDS, and T.M. BADGER, E n d o c r i n o l o g y 128:2027-2035 (1991). E.E. CODD, A.Y.L. SHU, and R.F. WALKER, N e u r o p h a r m a c o l o g y 2--8:1139-1144 (1989). A.D. BLAKE, and R.G. SMITH, J Endocrinol 129:11-19 (1991). K. CHENG, W.W-S. CHAN, A. BARRETO, D.M. CONVEY, and R.G. SMITH, E n d o c r i n o l o g y 124:2791-2798 (1989). C.Y. BOWERS, G.A. REYNOLDS, D. DURHAM, C.M. BARRERA, S.S. PEZZOLI, and M.O. THORNER, J clin E n d o c r i n o l M e t a b 7--0:975-982
(1990). 12.
V. DE G E N N A R O COLLONA, M. ZOLI, D. COCCHI, A. MARRAMA, L.F. AGNATI and E.E. MULLER, Peptides
MAGGI, P. 1-0:705-708
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F. GE, S. TSAGARAKIS, L.H. REES, G.M. BESSER, and A. GROSSMAN, J E n d o c r i n o l 123:53-58 (1989)
(1989).