Stimulatory Effect of Glycine on Human Growth Hormone Secretion Kikuo Kasai, Masami Glycine
(250
ministered subjects
0.3 to
and
trectomy. and
ml
orally In the
and blood fected more
normal
insulin
sugar
after
(BS)
pronounced
and
value
subjects
with
in serum
normal normal
gas-
a clear
that
human
tained
subiects.
of the in
drug
gastrectomied
normals
The facts demonstrated of
not
significant
tion,
afA
increase
found
than
stimulatory
pituitary
(PRL),
were
the
in the in the
to
of serum
intraduodenal
tine,
was
observed
ad-
that
secrete diabetics, hGH
in
was
to ob-
administration.
agents
in nonobese
increase the
gland
we
rise of hGH group
by this
im-
Thus
intraduodenally
The similar
ob-
administration.
gastrectomy
( p < 0.001).
the
was
prolactin
was
Shimoda
serum
level
levels
controls
ministered
subjects,
(IRI),
drug
of hGH
partial
whereas
the
ad-
normal
and Shin-lchi
of serum
(hGH)
( p < O.OOl),
munoreactive
with
increase
hormone
sewed
was
19 nonobese
12 subjects
significant
growth
M glycine)
Kobayashi,
glycine
is one
inducing hGH.
In
the addi-
no significant
level,
administration in the present
even
after
of
gly-
study.
I
T HAS BEEN REPORTED that various amino acids stimulate human growth hormone (hGH) secretion from the pituitary gland in man. These amino acids have been divided into two groups according to whether or not they may be contained in proteins. The amino acids contained in proteins are valine, leucine, threonine, arginine, lysine, histidine, and phenylalanine;’ the group of amino acids not contained in proteins is 5hydroxytryptophan’ and L-3,4_dihydroxyphenylalanine (L-dopa).3 Arginine, histidine, and lysine, which are the basic amino acids in the former group, are the well-known stimulators of the secretion of hGH. L-dopa and 5hydroxytryptophan also have a potent effect of hGH secretion. However, it has never been reported that the simplest amino acid, glycine, affects pituitary hGH secretion. From both neurochemical and neurophysiologic studies, glycine might be a neurotransmitter in the mamwe studied the effects of malian central nervous system (CNS). 4v5Therefore, glycine on hGH and prolactin (PRL) secretion from the pituitary gland in man. MATERIALS
AND
METHODS
In the first study, 19 nonobese normal subjects, 20-70 yr old (12 males and 7 females), and 12 partially gastrectomied patients with gastroduodenal anastomosis (Billroth I method), 29-67 yr old (7 males and 5 females), were employed to ascertain whether or not serum concentration of hGH might be increased by oral administration of 250 ml 0.3 M glycine (glycine: special grade, Kanto Chemical Co., Tokyo, Japan). In the second study, 7 nonobese normal subjects (5 males and 2 females, 25-63 yr old) and 8 nonobese diabetics (3 males and 5 females, 20-70 yr old) were employed. The glycine was administered intraduodenally in order to compare the hGH response patterns between these two groups. It should be noticed that the 7 normal subjects used in the second study were chosen from the 19 normal persons in the first study, and all diabetic patients
From the Department of Endocrinology, Internal Medicine, Dokkyo University, School of Medicine, Mibu. Tochigi. Japan. Received for publication April 4, 1977. Reprinr requests should be addressed to Kikuo Kasai, Department of Endocrinology, Internal Medicine, Dokkyo University, School of Medicine, Mibu. Tochigi 321-02. Japan. 1 0 1978 by Grune & Stratton, Inc. 0026~495/78/2702-0008%01.00/0
Metabolism,
Vol.
27, No. 2 (February),
1978
201
202
KASAI,
KOBAYASHI,
AND SHIMODA
employed in the present study did not receive any therapeutic management before or during the course of the study as they had relatively mild diabetes mellitus. After overnight fasting. 250 ml 0.3 M glycine was administered orally or intraduodenally for 2-3 min to each subject immediately after the blood sample was taken. Venous blood samples were again withdrawn at 5. 15. 30. 45. 60, 90, 120, 150, and I80 min after starting the administration. Aliquots of the serum separated from each blood sample were applied to measure serum immunoreactive insulin (IRI) and hGH levels with the radioimmunoassay kits supplied by Dainabot RI Lab, Tokyo, Japan. Blood sugar (BS) values were also determined by the method of Somogyi’ in all blood samples. Moreover, in some subjects, serum PRL levels were determined by the CEAIRE-SORIN kit, Midoryujt. Japan, and plasma levels of glycine were also measured by anionexchange amino acid autoanalyzer (Nippon Denshi). For the statistical analysis of the data obtained in the present study, Student’s t test was employed. RESULTS
Oral Administration of Glycine in Normal Subjects and Partially Gastrectomied Patients The administration of 250 ml 0.3 M glycine resulted in a significant increase of serum hGH with a mean (& SE) peak of 5.5 + 0.9 ng/ml in 12 male and 6.2 + 2.4 in 7 female normal controls (p < 0.001 and p < 0.05, respectively, versus each basal level), although serum IRI, PRL, and BS levels were not significantly affected (Table 1). When the same dose of the agent was given orally to subjects with gastrectomy, serum hGH response was greater, with a peak of 13.4 + 2.4 in 7 males and 11.1 f 2.9 ng/ml in 3 females (p < 0.001 and p < 0.05, respectively, versus each basal level), than that in normal subjects, whereas BS and serum IRI levels were not changed (Table 2). The difference in hGH response was found only 150 min after oral administration of the agent in both male and female normal subjects (p < 0.05). However, no significant difference of hGH response in both sexes was obtained in the subjects with gastrectomy. The mean + SE values in all 19 normals and the 12 subjects with gastrectomy are shown in Fig. I. The maximum rise of hGH in serum was 5.3 + 0.8 ng/ml in the normals and 12.5 f 1.8 ng/ml in the gastrectomied subjects (p < 0.001 and p < 0.001, respectively, versus each basal level). The significantly higher response of hGH in the latter group than in the former was obtained at 60 (p < 0.01) 90 (p < 0.001) and 120 min (p < 0.05) after the administration of glycine. Intraduodenal Administration of‘Glycine in Normal Subjects A clear rise of hGH in serum was observed by intraduodenal administration of glycine in normal subjects with a mean peak of 13.3 * 4.6 ng/ml (p < 0.05 versus basal level). This maximum level of hGH in the intraduodenal group was significantly higher (p < 0.02) than in the oral group, as shown in Fig. 2. In this study, no significant change in the levels of BS, IRI, and PRL was found (Table 3). This hGH response was similar to that obtained in the gastrectomied group; no significant difference of hGH level was observed between the normals (intraduodenal administration) and the subjects with gastrectomy (oral administration). Intraduodenal Administration of Glycine in Nonobese Diabetics Intraduodenal administration of the same dose of glycine had no effect on serum IRI and BS levels in diabetic patients either. Serum hGH level slightly
18.3 f 3.0
IRI(pU/ml)
gp < 0.001.
sp < 0.05.
difference
3.7 f 1.0 92.4 f 3.6 26.1 f 5.3
3.6 zt 0.7 91.2 f 2.9 21.5 f 4.0
4.6 zt 1.5 2.1 f 0.5
3.2 + 0.7
5
4.1 f 1.5
I5
from respective basal level, p < 0.02.
M+F(12)
2.5 z!z 0.4
2.7 f 0.7
2.1 zt 0.4
F(5)
0
M(7)
Sex(N)
91.4 f 3.0
tp < 0.01.
8.3 zt 3.5
82.6 * 1.6 23.7 zt 1.8
0.81
9.3 f 3.1
85.9 k 2.0 21.9 + 1.7
3.5 f
3.7 f 1.3 3.2 f 0.8
3.2 zt 1.0 3.4 f 0.5 3.3 f 0.61
45
82.6 i 2.2 22.0 + 2.2 a.2 f 2.8
4.2 f 1.01
18.1 i 2.8 7.0 f 2.3
19.3 f 2.4 a.8 f 3.3
la.4 f 2.5 7.2 f 3.3
3.3 f 0.6t 83.0 zt 1.7
5.1 f 1.0*
2.5 f 0.4t 5.0 f 1.3
4.4 * 0.7* 6.2 f 2.45 81.9 i 1.9
o.a*
150
120
83.3 i 1.7
5.3 f
5.5 f 0.9* 5.0 f 1.7
90
4.7 f 1.5
(min)
3.6 * 0.9
60
Time After Administration 30
20.6 f 2.9
90.0 f 4.1
5.4 f 1.7
3.6 f 1.7
6.7 f 2.7
30
22.4 f 4.3
93.1 f 2.8
6.4 f 1.9
3.2 f 1.6
a.2 f 2.6
45
Imin1
13.4 f 2.47
90
21.1 f 3.2
91.5 f 2.5
18.4 f 3.6
91.4 f 2.8
10.4 & 1.85 12.5 f 1.84
7.8 f 1.3' 11.1 f 2.91
12.2 f 2.a*
60
Time After Adminirtrotion
21.9 f 5.4
17.7 f 3.8
4.6 f 0.6t 92.6 f 2.7
9.3 zt 1.67 90.9 f 2.5
4.7 f 0.9* 4.3 f 0.5
7.7 f 2.1
150
10.3 f 2.2*
120
Table 2. Effect of Glycine Administered Orally on Serum hGH, IRI, and BS Levels (Mean f SE) in Subjects With Partial Gastrectomy
M+F(12)
*Significant
8.0 f 2.7
7.8 f 2.2
2.7 f 0.35
2.7 + 0.39 82.1 i 1.7 25.9 zt 2.8
2.3 f 0.4 3.4 f 0.5
2.5 f 0.5 2.9 f 0.4 83.1 i 2.8 25.7 zt 2.9
15
5
Administered Orally on Serum hGH, PRL, IRI, and BS Levels (Mean + SE) in Normal Subjects
from respective borolvalue,p < 0.001.
7.5 + 2.3
BS(mg/lOO ml) M+F(12)
hGH(ng/ml)
pp < 0.05.
$p < 0.02.
tp < 0.01.
*Significant difference
M(2)+F(l)
PRL(ng/ml)
84.6 i 3.5 21.1 f 1.7
1.6 f 0.2
M+F(19)
M+F(19) M+F(19)
1.5 f 0.3 1.7 f 0.3
0
Effectof Glycine
M(12) F(7)
BS(mg/lOOml) IRI(@U/ml)
hGH (ng/ml)
Sex(N)
Table 1.
18.7 f 3.6
92.1 f 1.9
2.9 f 0.4
2.4 f 0.4
3.3 * 0.7
180
17.0 f 2.2 6.1 f 2.9
83.4 i 1.9
2.8 f 0.5t
3.0 zto.7
2.4 &0.5x
180
P
5
x 0
P
3 ;:
F
3
2
204
KASAI,
KOBAYASHI,
AND SHlMODA
hG!$l,Wtl 1-3
0
30
60
90
120
mm
Fig. 1. Comparison of hGH responses to oral administration of glycine in 19 normals and 12 subjects with gastrectomy. All values ore the mean f SE. *p < 0.05, l*p < 0.02, l**p < 0.01, +‘**p < 0.001 (versus each basal level). lp < 0.05, ***p < 0.01, l**‘p < 0.001 (partial gostrectomy subjects versus normal controls at the given time).
180
increased, but not significantly, 30-90 min after the infusion of the drug (Table 4). Significantly higher hGH responses (p < 0.05) were obtained in the normals than in the diabetics 120 min after the administration of glycine (Fig. 3). Plasma Glycine Levels in Normal Subjects and Subjects With Gastrect0m.v After Glycine Administration Although the plasma levels of glycine within 60 min were not cause of the paucity of the specimens, the amino acid levels in determined by autoanalyzer at fasting, and 60, 90, 120, 180 min administration in some subjects. These results are presented in
Fig. 2. Comparison of hGH responses to oral and intraduodenal administrotion of glycine in normal subjects. All values are the mean f SE. l*p < 0.02 (oral group versus intraduodenal group 120 min after drug administration).
O
0
30
60
9oz%--
measured beplasma were after glycine Table 5. No
men 180
M+F(7) M+F(7) M(3)+F(2)
Bs(mg/loo ml) IRI(pU/ml) PRL(ng/ml)
92.2 f 5.1 19.8 f 3.5 5.5 f 0.9
2.2 f 0.5
0
89.1 f 4.8 24.2 f 6.4 5.7 f 1.1
2.6 f 0.4
5 3.1
8.8 f 1.4
9.4 f 1.2
Nosignificontdifferencefrom each basal value.
M+F/8)
IRI(uU/ml~
146*31
3.2 f 0.7
2.5 f 0.8
2.0 f 0.8
2.9 f 0.9
F(5)
M+F(8)
5
4.4 -f 1.4
0
4.4 f 2.0
M(3)
Sex(N)
149 +32
_~~~
87.6 f 5.0 23.2 f 3.2 4.6 f 1.2
3.6 i 0.9
30
87.6 f 5.0 18.0 f 3.8 5.5 f 1.5
5.6 * 2.1
45
82.6 f 3.4 18.4 f 3.3 4.5 f 1.1
6.6 f 2.7
60
(min)
89.4 f 4.0 18.0 f 3.3 4.1 zt 0.6
9.3 i 2.5*
90
89.3 f 3.6 18.0 zt3.6 4.7 * 1.1
13.3 zt 4.67
120
1.8t
90.0 f 3.7 16.8 zt 3.5 4.0 f 1.0
5.7 i
150
14.0 f 3.1
144+31
3.1 f 0.8
2.3 f 0.8
4.4 f 1.7
15
13.4 f 3.0
148 =t29
4.8 + 1.9
3.0 f 0.8
7.8 l 5.1
30
11.0 z+ 2.4
145 zt29
5.1 * 2.2
3.7 f 2.1
7.4 f 5.3
45
(min)
11.8 f 1.8
146zt29
5.5 f 2.1
5.8 f 3.1
4.8 z!z 2.9
60
Time After Administration
10.4 f- 1.5
142 ~331
4.1 + 1.4
4.7 f 2.2
3.2 f 1.0
90
13.2 zk 3.1
140 a30
3.3 f 0.9
3.2 f 1.3
3.5 f 1.5
120
11.0 f 2.6
135 &29
2.6 f 0.4
2.6 + 0.6
2.7 f 0.5
150
Table 4. Effect of Glycine Administered lntraduodenally on Serum hGH, IRI, and BS levels (Mean f SE) in Nonobese Diabetics
BS(mg/lOO ml) M+F(8)
hGH(ng/ml)
tp < 0.05.
iO.8
15
87.1 f 4.9 25.8 f 7.5 6.2 f 1.3
*Significant difference from respective basal level,p < 0.02).
M(5)+ F(2)
hGH(ng/ml)
Sex(N)
Time After Administration
Table 3. Effect of Glycine Administered Intraduodenally on Serum hGH, PRL, IRI, and BS levels (Meon + SE) in Normal Subjects
0.8
10.2 f 2.2
131 k226
2.2 f 0.3
2.3 f 0.4
2.0 f 0.1
180
88.2 f 4.6 19.0 zt 5.8 4.0 zk0.5
2.8 i
180
!?
?A
z
?I
3
5 5
KASAI,
206
KOBAYASHI,
AND SHIMODA
hGH fig/ml
15.
Glycine 4 Udr IO-
I/
%
6
$6
i$j
;-I”
Comparison of hGH responses to inFig. 3. traduodenal administration of glycine in 7 normal subjects and 8 diabetics. All values are the mean f SE. lp < 0.05 (diabetics versus controls ot 120 min).
GO
significant difference of plasma glycine levels between jects with gastrectomy was found at these times.
the normals
and the sub-
DISCUSSION It is well known that various amino acids and their metabolites modulate hypothalamohypophyseal functions. There is abundant evidence that monoaminergic neuron systems in the hypothalamus and preoptic area play important roles in regulating neuroendocrine functions.7,8 In man, it has been reported that alpha-adrenergic,’ dopaminergic3 and serotoninergic? neuron systems have stimulatory effects on hGH release from the pituitary gland. Moreover, the dopaminergic system inhibits PRL release” and the serotoninergic stimulates secretion of PRL.” On the other hand, amino acids contained in proteins (valine, leucine, threonine, arginine, lysine, histidine, and phenylalanine) stimulate hGH secretion. ’ Among these amino acids, arginine, a basic amino acid, is especially well-known to stimulate secretion of hGH in man. However, there has been no report that glycine, which is the simplest amino acid, might modulate hypothalamohypophyseal functions. From both neurochemical and neurophysiologic studies, 4s it has been suggested that glycine Table 5. Plasma levels of Glycine (fig/ml, Subjects With Gastrectomy
Time Sex(N)
Meun * SE) in Normal Subiects and
Before and After Glycine Administration After Administration
0
(min) 90
60
120
180
M(l)+F(2)
25.2 + 5.1
76.0 f
16.8
62.6 zt 24.3
61.9 + 31.4
38.1 + 13.1
Gastr. (p.0.)
M(3)+F(2)
24.4 f
66.4 f
19.1
79.6 f 22.5
46.9 f 4.1
47.4 zt 5.1
Normal (i.d.)t
M(l)+F(l)
27.1
67.4
41.4
30.2
Normal
(p.o.)*
2.7
50.9
No significant difference in glycine level among these groups. ‘Oral administration. tlntroduodenal administration.
GLYCINE-STIMULATED
hGH SECRETION
207
may be a neurotransmitter in mammalian CNS, in addition to the amines mentioned above and gamma-aminobutyric acid, etc. The present study was undertaken to determine whether or not glycine stimulates the pituitary gland to secrete hGH. At present this agent is commonly used in clinical provocative tests to release gastrin in the stomach.12 The results in the first study indicated that the drug was able to stimulate secretion of hGH from the gland as well as the other amino acids and that serum hGH response was greater in the subjects with gastrectomy than in the normal controls. Because plasma glycine levels were not significantly different between the normals and the subjects with gastrectomy (Table 5), it was suggested that unknown factors might facilitate the hGH release by glycine in the intestine. However, there is also the possibility that glycine might be absorbed rapidly and adequately within 60 min, as is alanine,13 and that it might facilitate hGH release more pronouncedly in the subjects with gastrectomy than in the normals (oral administration). Since glycine is a putative neurotransmitter in CNS, the hypothesis would be attractive that the mechanism of hGH release by glycine might be mediated through the growth hormone-releasing hormoneI or somatostatin” in the hypothalamus and/or stimulate the gland directly. However, understanding of the exact mechanism of glycine stimulation of hGH secretion from the gland must await further investigation. Although it is known that growth hormone may have a diabetogenic action 3‘6~17 the exact role of hGH in the development of diabetes mellitus and its vascular complications is still uncertain. Recently we reported that the effectiver8,” stimulation of secretion of hGH ness of L-dopa, arginine, and tolbutamide from the pituitary was less in diabetics than in normals; we thus hypothesized that the secretory ability of hGH in diabetics might be retarded. Therefore, the present study extended to diabetics to confirm our previous reports. In nonobese diabetics, glycine was administered intraduodenally, but no pronounced rise of hGH was found (Table 4 and Fig. 3). Although there were several problems in this new stimulation of hGH by glycine, such as absorption rate in the intestine, it may be concluded that glycine given intraduodenally to stimulate release of hGH is less effective in diabetics than in the normal controls.
REFERENCES 1. Knopf RF, Conn JW, Fajans SS, et al: Plasma growth hormone response to intravenous administration of amino acids. J Clin Endocrinol25: I 140, 1965 2. Imura H, Nakai Y, Yoshimi T: Effect of S-hydroxytryptophan (5HTP) on growth hormone and ACTH release in man. J Clin Endocrinol36:204, 1973 3. Boyd AE, Levovitz HE, Pfeiffer JB: Stimulation of human growth hormone by L-dopa. N Engl J Med 2381425, 1970 4. Cutler RWP, Hammerstad JP, Cornick LR, et al: Efflux of amino acid neurotransmitters from rat spinal cord slices. 1. Factors
influencing the spontaneous efflux of (14C) glytine and ‘H-GABA. Brain Res 35:337, 1971 5. Bruin WJ, Frantz BM, Sallach HJ: The occurrence of a glycine cleavage system in mammalian brain. J Neurochem 20:1649, 1973 6. Somogyi M: Notes on sugar determination. J Biol Chem 195:19, 1952 7. Arimura A. Davis TS, Nishi N, et al: Catecholamines and the pituitary, in Charro Salgado AL, et al (eds): Basic Applications and Clinical Uses of Hypothalmic Hormones. Amsterdam, Excerpta Medica, 1976, p 200 8. Mtiller EE, Gil-AD I, Panerai AE, et al: Involvement of brain monoamines in the con-
208
trol of growth hormone secretion, in Charro Salgado AL, et al (eds): Basic Applications and Clinical Uses of Hypothalmic Hormones. Amsterdam, Excerpta Medica, 1976, p 2 I 1 9. Imura H, Kato Y. lkeda M, et al: ElTect of adrenergic-blocking or -stimulating agents on plasma growth hormone, immunoreactive insulin, and blood free fatty acid levels in man. J Clin Invest 50:1069, 1971 10. Kleinberg DL, Noel GL, Frantz AG: Chrolpromazine stimulation and L-dopa suppression of plasma prolactin in man. J Clin Endocrinol 33:873, 1971 Il. Kato Y, Nakai Y, Imura H, et al: Effect of 5hydroxytryptophan (5HTP) on plasma prolactin levels in man. J Clin Endocrinol 38:695, 1974 12. McGuian JE, Trudeau WL: Studies with antibodies to gastrin: Radioimmunoassay in human serum and physiological studies. Gastroenterology 58: 139, 1970 13. Rossini AA, Aoki TT, Ganda OP, et al: Alanine-induced amino acid interrelationships. Metabolism 24: 1185, 1975 14. Schally AV. Baba Y, Nair PMG, et al:
KASAI.
KOBAYASHI,
AND SHIMODA
The amino acid sequence of a peptide with isolated growth hormoneereleasing activity from porcine hypothalamus. J Biol Chem 246: 6647, 1971 15. Brazeau P, Vale W, Burgus R, et al: Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:77, 1973 16. Houssay BA, Anderson E: Diabetogenic action of purified anterior pituitary hormones. Endocrinology 45:627, 1949 17. Luft R, Guillemin R: Growth hormone and diabetes in man: Old concepts-New implications. Diabetes 23:783, 1974 18. Shimoda S, Takemura Y, Ieiri T, et al: A low response of pituitary growth hormone secretion in the patient with diabetes mellitus after intravenous administration of L-dopa. Folia Endocrinol Jpn 51:9, 1975 (in Japanese) 19. Kasai K. Kikuchi T. Shimoda S: Growth hormone secretion from the pituitary after a single dose of tolbutamidr injection in normal subjects, hyperthyroidism, hypothyroidism, diabetes mellitus and nephrotic syndrome. Folia Endocrinol Jpn 50: 1353. 1974 (in Japanese)
(250
ministered subjects
0.3 to
and
trectomy. and
ml
orally In the
and blood fected more
normal
insulin
sugar
after
(BS)
pronounced
and
value
subjects
with
in serum
normal normal
gas-
a clear
that
human
tained
subiects.
of the in
drug
gastrectomied
normals
The facts demonstrated of
not
significant
tion,
afA
increase
found
than
stimulatory
pituitary
(PRL),
were
the
in the in the
to
of serum
intraduodenal
tine,
was
observed
ad-
that
secrete diabetics, hGH
in
was
to ob-
administration.
agents
in nonobese
increase the
gland
we
rise of hGH group
by this
im-
Thus
intraduodenally
The similar
ob-
administration.
gastrectomy
( p < 0.001).
the
was
prolactin
was
Shimoda
serum
level
levels
controls
ministered
subjects,
(IRI),
drug
of hGH
partial
whereas
the
ad-
normal
and Shin-lchi
of serum
(hGH)
( p < O.OOl),
munoreactive
with
increase
hormone
sewed
was
19 nonobese
12 subjects
significant
growth
M glycine)
Kobayashi,
glycine
is one
inducing hGH.
In
the addi-
no significant
level,
administration in the present
even
after
of
gly-
study.
I
T HAS BEEN REPORTED that various amino acids stimulate human growth hormone (hGH) secretion from the pituitary gland in man. These amino acids have been divided into two groups according to whether or not they may be contained in proteins. The amino acids contained in proteins are valine, leucine, threonine, arginine, lysine, histidine, and phenylalanine;’ the group of amino acids not contained in proteins is 5hydroxytryptophan’ and L-3,4_dihydroxyphenylalanine (L-dopa).3 Arginine, histidine, and lysine, which are the basic amino acids in the former group, are the well-known stimulators of the secretion of hGH. L-dopa and 5hydroxytryptophan also have a potent effect of hGH secretion. However, it has never been reported that the simplest amino acid, glycine, affects pituitary hGH secretion. From both neurochemical and neurophysiologic studies, glycine might be a neurotransmitter in the mamwe studied the effects of malian central nervous system (CNS). 4v5Therefore, glycine on hGH and prolactin (PRL) secretion from the pituitary gland in man. MATERIALS
AND
METHODS
In the first study, 19 nonobese normal subjects, 20-70 yr old (12 males and 7 females), and 12 partially gastrectomied patients with gastroduodenal anastomosis (Billroth I method), 29-67 yr old (7 males and 5 females), were employed to ascertain whether or not serum concentration of hGH might be increased by oral administration of 250 ml 0.3 M glycine (glycine: special grade, Kanto Chemical Co., Tokyo, Japan). In the second study, 7 nonobese normal subjects (5 males and 2 females, 25-63 yr old) and 8 nonobese diabetics (3 males and 5 females, 20-70 yr old) were employed. The glycine was administered intraduodenally in order to compare the hGH response patterns between these two groups. It should be noticed that the 7 normal subjects used in the second study were chosen from the 19 normal persons in the first study, and all diabetic patients
From the Department of Endocrinology, Internal Medicine, Dokkyo University, School of Medicine, Mibu. Tochigi. Japan. Received for publication April 4, 1977. Reprinr requests should be addressed to Kikuo Kasai, Department of Endocrinology, Internal Medicine, Dokkyo University, School of Medicine, Mibu. Tochigi 321-02. Japan. 1 0 1978 by Grune & Stratton, Inc. 0026~495/78/2702-0008%01.00/0
Metabolism,
Vol.
27, No. 2 (February),
1978
201
202
KASAI,
KOBAYASHI,
AND SHIMODA
employed in the present study did not receive any therapeutic management before or during the course of the study as they had relatively mild diabetes mellitus. After overnight fasting. 250 ml 0.3 M glycine was administered orally or intraduodenally for 2-3 min to each subject immediately after the blood sample was taken. Venous blood samples were again withdrawn at 5. 15. 30. 45. 60, 90, 120, 150, and I80 min after starting the administration. Aliquots of the serum separated from each blood sample were applied to measure serum immunoreactive insulin (IRI) and hGH levels with the radioimmunoassay kits supplied by Dainabot RI Lab, Tokyo, Japan. Blood sugar (BS) values were also determined by the method of Somogyi’ in all blood samples. Moreover, in some subjects, serum PRL levels were determined by the CEAIRE-SORIN kit, Midoryujt. Japan, and plasma levels of glycine were also measured by anionexchange amino acid autoanalyzer (Nippon Denshi). For the statistical analysis of the data obtained in the present study, Student’s t test was employed. RESULTS
Oral Administration of Glycine in Normal Subjects and Partially Gastrectomied Patients The administration of 250 ml 0.3 M glycine resulted in a significant increase of serum hGH with a mean (& SE) peak of 5.5 + 0.9 ng/ml in 12 male and 6.2 + 2.4 in 7 female normal controls (p < 0.001 and p < 0.05, respectively, versus each basal level), although serum IRI, PRL, and BS levels were not significantly affected (Table 1). When the same dose of the agent was given orally to subjects with gastrectomy, serum hGH response was greater, with a peak of 13.4 + 2.4 in 7 males and 11.1 f 2.9 ng/ml in 3 females (p < 0.001 and p < 0.05, respectively, versus each basal level), than that in normal subjects, whereas BS and serum IRI levels were not changed (Table 2). The difference in hGH response was found only 150 min after oral administration of the agent in both male and female normal subjects (p < 0.05). However, no significant difference of hGH response in both sexes was obtained in the subjects with gastrectomy. The mean + SE values in all 19 normals and the 12 subjects with gastrectomy are shown in Fig. I. The maximum rise of hGH in serum was 5.3 + 0.8 ng/ml in the normals and 12.5 f 1.8 ng/ml in the gastrectomied subjects (p < 0.001 and p < 0.001, respectively, versus each basal level). The significantly higher response of hGH in the latter group than in the former was obtained at 60 (p < 0.01) 90 (p < 0.001) and 120 min (p < 0.05) after the administration of glycine. Intraduodenal Administration of‘Glycine in Normal Subjects A clear rise of hGH in serum was observed by intraduodenal administration of glycine in normal subjects with a mean peak of 13.3 * 4.6 ng/ml (p < 0.05 versus basal level). This maximum level of hGH in the intraduodenal group was significantly higher (p < 0.02) than in the oral group, as shown in Fig. 2. In this study, no significant change in the levels of BS, IRI, and PRL was found (Table 3). This hGH response was similar to that obtained in the gastrectomied group; no significant difference of hGH level was observed between the normals (intraduodenal administration) and the subjects with gastrectomy (oral administration). Intraduodenal Administration of Glycine in Nonobese Diabetics Intraduodenal administration of the same dose of glycine had no effect on serum IRI and BS levels in diabetic patients either. Serum hGH level slightly
18.3 f 3.0
IRI(pU/ml)
gp < 0.001.
sp < 0.05.
difference
3.7 f 1.0 92.4 f 3.6 26.1 f 5.3
3.6 zt 0.7 91.2 f 2.9 21.5 f 4.0
4.6 zt 1.5 2.1 f 0.5
3.2 + 0.7
5
4.1 f 1.5
I5
from respective basal level, p < 0.02.
M+F(12)
2.5 z!z 0.4
2.7 f 0.7
2.1 zt 0.4
F(5)
0
M(7)
Sex(N)
91.4 f 3.0
tp < 0.01.
8.3 zt 3.5
82.6 * 1.6 23.7 zt 1.8
0.81
9.3 f 3.1
85.9 k 2.0 21.9 + 1.7
3.5 f
3.7 f 1.3 3.2 f 0.8
3.2 zt 1.0 3.4 f 0.5 3.3 f 0.61
45
82.6 i 2.2 22.0 + 2.2 a.2 f 2.8
4.2 f 1.01
18.1 i 2.8 7.0 f 2.3
19.3 f 2.4 a.8 f 3.3
la.4 f 2.5 7.2 f 3.3
3.3 f 0.6t 83.0 zt 1.7
5.1 f 1.0*
2.5 f 0.4t 5.0 f 1.3
4.4 * 0.7* 6.2 f 2.45 81.9 i 1.9
o.a*
150
120
83.3 i 1.7
5.3 f
5.5 f 0.9* 5.0 f 1.7
90
4.7 f 1.5
(min)
3.6 * 0.9
60
Time After Administration 30
20.6 f 2.9
90.0 f 4.1
5.4 f 1.7
3.6 f 1.7
6.7 f 2.7
30
22.4 f 4.3
93.1 f 2.8
6.4 f 1.9
3.2 f 1.6
a.2 f 2.6
45
Imin1
13.4 f 2.47
90
21.1 f 3.2
91.5 f 2.5
18.4 f 3.6
91.4 f 2.8
10.4 & 1.85 12.5 f 1.84
7.8 f 1.3' 11.1 f 2.91
12.2 f 2.a*
60
Time After Adminirtrotion
21.9 f 5.4
17.7 f 3.8
4.6 f 0.6t 92.6 f 2.7
9.3 zt 1.67 90.9 f 2.5
4.7 f 0.9* 4.3 f 0.5
7.7 f 2.1
150
10.3 f 2.2*
120
Table 2. Effect of Glycine Administered Orally on Serum hGH, IRI, and BS Levels (Mean f SE) in Subjects With Partial Gastrectomy
M+F(12)
*Significant
8.0 f 2.7
7.8 f 2.2
2.7 f 0.35
2.7 + 0.39 82.1 i 1.7 25.9 zt 2.8
2.3 f 0.4 3.4 f 0.5
2.5 f 0.5 2.9 f 0.4 83.1 i 2.8 25.7 zt 2.9
15
5
Administered Orally on Serum hGH, PRL, IRI, and BS Levels (Mean + SE) in Normal Subjects
from respective borolvalue,p < 0.001.
7.5 + 2.3
BS(mg/lOO ml) M+F(12)
hGH(ng/ml)
pp < 0.05.
$p < 0.02.
tp < 0.01.
*Significant difference
M(2)+F(l)
PRL(ng/ml)
84.6 i 3.5 21.1 f 1.7
1.6 f 0.2
M+F(19)
M+F(19) M+F(19)
1.5 f 0.3 1.7 f 0.3
0
Effectof Glycine
M(12) F(7)
BS(mg/lOOml) IRI(@U/ml)
hGH (ng/ml)
Sex(N)
Table 1.
18.7 f 3.6
92.1 f 1.9
2.9 f 0.4
2.4 f 0.4
3.3 * 0.7
180
17.0 f 2.2 6.1 f 2.9
83.4 i 1.9
2.8 f 0.5t
3.0 zto.7
2.4 &0.5x
180
P
5
x 0
P
3 ;:
F
3
2
204
KASAI,
KOBAYASHI,
AND SHlMODA
hG!$l,Wtl 1-3
0
30
60
90
120
mm
Fig. 1. Comparison of hGH responses to oral administration of glycine in 19 normals and 12 subjects with gastrectomy. All values ore the mean f SE. *p < 0.05, l*p < 0.02, l**p < 0.01, +‘**p < 0.001 (versus each basal level). lp < 0.05, ***p < 0.01, l**‘p < 0.001 (partial gostrectomy subjects versus normal controls at the given time).
180
increased, but not significantly, 30-90 min after the infusion of the drug (Table 4). Significantly higher hGH responses (p < 0.05) were obtained in the normals than in the diabetics 120 min after the administration of glycine (Fig. 3). Plasma Glycine Levels in Normal Subjects and Subjects With Gastrect0m.v After Glycine Administration Although the plasma levels of glycine within 60 min were not cause of the paucity of the specimens, the amino acid levels in determined by autoanalyzer at fasting, and 60, 90, 120, 180 min administration in some subjects. These results are presented in
Fig. 2. Comparison of hGH responses to oral and intraduodenal administrotion of glycine in normal subjects. All values are the mean f SE. l*p < 0.02 (oral group versus intraduodenal group 120 min after drug administration).
O
0
30
60
9oz%--
measured beplasma were after glycine Table 5. No
men 180
M+F(7) M+F(7) M(3)+F(2)
Bs(mg/loo ml) IRI(pU/ml) PRL(ng/ml)
92.2 f 5.1 19.8 f 3.5 5.5 f 0.9
2.2 f 0.5
0
89.1 f 4.8 24.2 f 6.4 5.7 f 1.1
2.6 f 0.4
5 3.1
8.8 f 1.4
9.4 f 1.2
Nosignificontdifferencefrom each basal value.
M+F/8)
IRI(uU/ml~
146*31
3.2 f 0.7
2.5 f 0.8
2.0 f 0.8
2.9 f 0.9
F(5)
M+F(8)
5
4.4 -f 1.4
0
4.4 f 2.0
M(3)
Sex(N)
149 +32
_~~~
87.6 f 5.0 23.2 f 3.2 4.6 f 1.2
3.6 i 0.9
30
87.6 f 5.0 18.0 f 3.8 5.5 f 1.5
5.6 * 2.1
45
82.6 f 3.4 18.4 f 3.3 4.5 f 1.1
6.6 f 2.7
60
(min)
89.4 f 4.0 18.0 f 3.3 4.1 zt 0.6
9.3 i 2.5*
90
89.3 f 3.6 18.0 zt3.6 4.7 * 1.1
13.3 zt 4.67
120
1.8t
90.0 f 3.7 16.8 zt 3.5 4.0 f 1.0
5.7 i
150
14.0 f 3.1
144+31
3.1 f 0.8
2.3 f 0.8
4.4 f 1.7
15
13.4 f 3.0
148 =t29
4.8 + 1.9
3.0 f 0.8
7.8 l 5.1
30
11.0 z+ 2.4
145 zt29
5.1 * 2.2
3.7 f 2.1
7.4 f 5.3
45
(min)
11.8 f 1.8
146zt29
5.5 f 2.1
5.8 f 3.1
4.8 z!z 2.9
60
Time After Administration
10.4 f- 1.5
142 ~331
4.1 + 1.4
4.7 f 2.2
3.2 f 1.0
90
13.2 zk 3.1
140 a30
3.3 f 0.9
3.2 f 1.3
3.5 f 1.5
120
11.0 f 2.6
135 &29
2.6 f 0.4
2.6 + 0.6
2.7 f 0.5
150
Table 4. Effect of Glycine Administered lntraduodenally on Serum hGH, IRI, and BS levels (Mean f SE) in Nonobese Diabetics
BS(mg/lOO ml) M+F(8)
hGH(ng/ml)
tp < 0.05.
iO.8
15
87.1 f 4.9 25.8 f 7.5 6.2 f 1.3
*Significant difference from respective basal level,p < 0.02).
M(5)+ F(2)
hGH(ng/ml)
Sex(N)
Time After Administration
Table 3. Effect of Glycine Administered Intraduodenally on Serum hGH, PRL, IRI, and BS levels (Meon + SE) in Normal Subjects
0.8
10.2 f 2.2
131 k226
2.2 f 0.3
2.3 f 0.4
2.0 f 0.1
180
88.2 f 4.6 19.0 zt 5.8 4.0 zk0.5
2.8 i
180
!?
?A
z
?I
3
5 5
KASAI,
206
KOBAYASHI,
AND SHIMODA
hGH fig/ml
15.
Glycine 4 Udr IO-
I/
%
6
$6
i$j
;-I”
Comparison of hGH responses to inFig. 3. traduodenal administration of glycine in 7 normal subjects and 8 diabetics. All values are the mean f SE. lp < 0.05 (diabetics versus controls ot 120 min).
GO
significant difference of plasma glycine levels between jects with gastrectomy was found at these times.
the normals
and the sub-
DISCUSSION It is well known that various amino acids and their metabolites modulate hypothalamohypophyseal functions. There is abundant evidence that monoaminergic neuron systems in the hypothalamus and preoptic area play important roles in regulating neuroendocrine functions.7,8 In man, it has been reported that alpha-adrenergic,’ dopaminergic3 and serotoninergic? neuron systems have stimulatory effects on hGH release from the pituitary gland. Moreover, the dopaminergic system inhibits PRL release” and the serotoninergic stimulates secretion of PRL.” On the other hand, amino acids contained in proteins (valine, leucine, threonine, arginine, lysine, histidine, and phenylalanine) stimulate hGH secretion. ’ Among these amino acids, arginine, a basic amino acid, is especially well-known to stimulate secretion of hGH in man. However, there has been no report that glycine, which is the simplest amino acid, might modulate hypothalamohypophyseal functions. From both neurochemical and neurophysiologic studies, 4s it has been suggested that glycine Table 5. Plasma levels of Glycine (fig/ml, Subjects With Gastrectomy
Time Sex(N)
Meun * SE) in Normal Subiects and
Before and After Glycine Administration After Administration
0
(min) 90
60
120
180
M(l)+F(2)
25.2 + 5.1
76.0 f
16.8
62.6 zt 24.3
61.9 + 31.4
38.1 + 13.1
Gastr. (p.0.)
M(3)+F(2)
24.4 f
66.4 f
19.1
79.6 f 22.5
46.9 f 4.1
47.4 zt 5.1
Normal (i.d.)t
M(l)+F(l)
27.1
67.4
41.4
30.2
Normal
(p.o.)*
2.7
50.9
No significant difference in glycine level among these groups. ‘Oral administration. tlntroduodenal administration.
GLYCINE-STIMULATED
hGH SECRETION
207
may be a neurotransmitter in mammalian CNS, in addition to the amines mentioned above and gamma-aminobutyric acid, etc. The present study was undertaken to determine whether or not glycine stimulates the pituitary gland to secrete hGH. At present this agent is commonly used in clinical provocative tests to release gastrin in the stomach.12 The results in the first study indicated that the drug was able to stimulate secretion of hGH from the gland as well as the other amino acids and that serum hGH response was greater in the subjects with gastrectomy than in the normal controls. Because plasma glycine levels were not significantly different between the normals and the subjects with gastrectomy (Table 5), it was suggested that unknown factors might facilitate the hGH release by glycine in the intestine. However, there is also the possibility that glycine might be absorbed rapidly and adequately within 60 min, as is alanine,13 and that it might facilitate hGH release more pronouncedly in the subjects with gastrectomy than in the normals (oral administration). Since glycine is a putative neurotransmitter in CNS, the hypothesis would be attractive that the mechanism of hGH release by glycine might be mediated through the growth hormone-releasing hormoneI or somatostatin” in the hypothalamus and/or stimulate the gland directly. However, understanding of the exact mechanism of glycine stimulation of hGH secretion from the gland must await further investigation. Although it is known that growth hormone may have a diabetogenic action 3‘6~17 the exact role of hGH in the development of diabetes mellitus and its vascular complications is still uncertain. Recently we reported that the effectiver8,” stimulation of secretion of hGH ness of L-dopa, arginine, and tolbutamide from the pituitary was less in diabetics than in normals; we thus hypothesized that the secretory ability of hGH in diabetics might be retarded. Therefore, the present study extended to diabetics to confirm our previous reports. In nonobese diabetics, glycine was administered intraduodenally, but no pronounced rise of hGH was found (Table 4 and Fig. 3). Although there were several problems in this new stimulation of hGH by glycine, such as absorption rate in the intestine, it may be concluded that glycine given intraduodenally to stimulate release of hGH is less effective in diabetics than in the normal controls.
REFERENCES 1. Knopf RF, Conn JW, Fajans SS, et al: Plasma growth hormone response to intravenous administration of amino acids. J Clin Endocrinol25: I 140, 1965 2. Imura H, Nakai Y, Yoshimi T: Effect of S-hydroxytryptophan (5HTP) on growth hormone and ACTH release in man. J Clin Endocrinol36:204, 1973 3. Boyd AE, Levovitz HE, Pfeiffer JB: Stimulation of human growth hormone by L-dopa. N Engl J Med 2381425, 1970 4. Cutler RWP, Hammerstad JP, Cornick LR, et al: Efflux of amino acid neurotransmitters from rat spinal cord slices. 1. Factors
influencing the spontaneous efflux of (14C) glytine and ‘H-GABA. Brain Res 35:337, 1971 5. Bruin WJ, Frantz BM, Sallach HJ: The occurrence of a glycine cleavage system in mammalian brain. J Neurochem 20:1649, 1973 6. Somogyi M: Notes on sugar determination. J Biol Chem 195:19, 1952 7. Arimura A. Davis TS, Nishi N, et al: Catecholamines and the pituitary, in Charro Salgado AL, et al (eds): Basic Applications and Clinical Uses of Hypothalmic Hormones. Amsterdam, Excerpta Medica, 1976, p 200 8. Mtiller EE, Gil-AD I, Panerai AE, et al: Involvement of brain monoamines in the con-
208
trol of growth hormone secretion, in Charro Salgado AL, et al (eds): Basic Applications and Clinical Uses of Hypothalmic Hormones. Amsterdam, Excerpta Medica, 1976, p 2 I 1 9. Imura H, Kato Y. lkeda M, et al: ElTect of adrenergic-blocking or -stimulating agents on plasma growth hormone, immunoreactive insulin, and blood free fatty acid levels in man. J Clin Invest 50:1069, 1971 10. Kleinberg DL, Noel GL, Frantz AG: Chrolpromazine stimulation and L-dopa suppression of plasma prolactin in man. J Clin Endocrinol 33:873, 1971 Il. Kato Y, Nakai Y, Imura H, et al: Effect of 5hydroxytryptophan (5HTP) on plasma prolactin levels in man. J Clin Endocrinol 38:695, 1974 12. McGuian JE, Trudeau WL: Studies with antibodies to gastrin: Radioimmunoassay in human serum and physiological studies. Gastroenterology 58: 139, 1970 13. Rossini AA, Aoki TT, Ganda OP, et al: Alanine-induced amino acid interrelationships. Metabolism 24: 1185, 1975 14. Schally AV. Baba Y, Nair PMG, et al:
KASAI.
KOBAYASHI,
AND SHIMODA
The amino acid sequence of a peptide with isolated growth hormoneereleasing activity from porcine hypothalamus. J Biol Chem 246: 6647, 1971 15. Brazeau P, Vale W, Burgus R, et al: Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:77, 1973 16. Houssay BA, Anderson E: Diabetogenic action of purified anterior pituitary hormones. Endocrinology 45:627, 1949 17. Luft R, Guillemin R: Growth hormone and diabetes in man: Old concepts-New implications. Diabetes 23:783, 1974 18. Shimoda S, Takemura Y, Ieiri T, et al: A low response of pituitary growth hormone secretion in the patient with diabetes mellitus after intravenous administration of L-dopa. Folia Endocrinol Jpn 51:9, 1975 (in Japanese) 19. Kasai K. Kikuchi T. Shimoda S: Growth hormone secretion from the pituitary after a single dose of tolbutamidr injection in normal subjects, hyperthyroidism, hypothyroidism, diabetes mellitus and nephrotic syndrome. Folia Endocrinol Jpn 50: 1353. 1974 (in Japanese)
