0021-972X/00/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2000 by The Endocrine Society
Vol. 85, No. 2
Printed in U.S.A.
Exogenous 20K Growth Hormone (GH) Suppresses
Endogenous 22K GH Secretion in Normal Men
YOSHIHIDE HASHIMOTO, TAKESHI KAMIOKA, MASAHARU HOSAKA,
KAZUNORI MABUCHI, AKIRA MIZUCHI, YUKIO SHIMAZAKI, MICHIO TSUNOO,
AND TOSHIAKI TANAKA
Institute of Biological Science (Y.H., M.H., K.M.), Mitsui Pharmaceuticals, Inc., Chiba 297-0017;
Clinical Development Department (T.K., A.M., Y.S.), Mitsui Pharmaceuticals, Inc., Tokyo 103-0027;
Komagome-higashi Clinic (M.T.), Kiyaku-kai Medical Corporation Hohsen Clinic, Tokyo 170-0003; and
Department of Endocrinology and Metabolism (T.T.), National Children’s Medical Research Center,
Tokyo 154-8509, Japan
ABSTRACT
The physiological and pharmacological functions of the 20-kDa
human GH (20K-hGH) isoform are unknown. We conducted a pharmacokinetic study of recombinant 20K-hGH in human subjects
(Phase I clinical trial). Placebo or 20K-hGH was administered sc to
normal men (20 –31 yr of age, n ⫽ 6 – 8 per group) at 2100 h. Serum
20K- and 22K-hGH levels were monitored every 30 min for 24 h by
specific enzyme-linked immunosorbent assays. Serum free fatty acid,
insulin-like growth factor I, insulin, and glucose levels were measured
for 24 h. In the placebo group, the secretion profiles of endogenous
20K- and 22K-hGH were pulsatile and similar to each other. The
proportion of 20K- to 22K-hGH was fairly constant. In the 20K-hGHtreated groups, serum 20K-hGH levels increased in a dose-dependent
manner over the dose range of 0.01– 0.1 mg/kg. Maximum serum
20K-hGH levels were reached at 3– 4 h and decreased with half-lives
of 2–3 h. Marked suppression of endogenous 22K-hGH secretion was
observed in a time-dependent manner. Serum free fatty acid and
insulin-like growth factor I levels were significantly elevated (P ⬍
0.01) at 4, 8, and 12 h and at 8, 12, and 24 h after 20K-hGH administration, respectively. Serum insulin and glucose levels did not
change significantly within 24 h. These results suggested that: 1)
regulation of 20K-hGH secretion is physiologically the same as that
of 22K-hGH; 2) the pharmacokinetics after sc injection of 20K-hGH
are comparable with those of 22K-hGH; 3) 20K-hGH regulates hGH
secretion through “GH-induced negative feedback mechanisms”; and
4) administration of 20K-hGH is expected to exert GH actions
(growth-promoting activity and lipolytic activity). Monitoring of serum 20K- and 22K-hGH levels may be useful in evaluating the effects
of administered GH isoforms on their own release from the pituitary.
(J Clin Endocrinol Metab 85: 601– 606, 2000)
T
HE 20-kDa HUMAN GH (20K-hGH) is a naturally occurring isoform lacking residues 32– 46 of 22K-hGH
(1–3). This deleted region is involved in the interface with
both the hGH receptor (4) and the PRL receptor (5) in 22KhGH. The 20K-hGH comprises approximately 10% of pituitary hGH, but its physiological and pharmacological functions remain to be elucidated (6, 7). The 20K-hGH stimulates
linear growth in hypophysectomized rats (8), exerts lipolytic
activity in vitro (9), and binds to hGH receptors (10) similarly
to 22K-hGH, but differs in some metabolic effects, such as
acute insulin-like activity (11) and binding to lactogenic receptors (12, 13). Recently, recombinant 20K-hGH has been
produced in high purity and in large amounts (9). We have
also constructed an enzyme-linked immunosorbent assay
(ELISA) system, which specifically reacts with 20K-hGH but
not 22K-hGH (14, 15). The ELISA system has been applied to
the determination of serum 20K-hGH levels in both normal
subjects and patients with endocrine or metabolic disorders
(15, 16). The level of circulating 20K-hGH was highly correlated to that of 22K-hGH in both normal subjects and
patients, and the proportion of 20K-hGH in each individual
subject was fairly constant even after pharmacological and
physiological stimuli. Here, we conducted a pharmacokinetic study of recombinant 20K-hGH in human subjects
(Phase I clinical trial). The aim of the present study was to
investigate: 1) physiological secretion profiles of serum 20Kand 22K-hGH; 2) the pharmacokinetics after sc injection of
20K-hGH; and 3) the GH actions of 20K-hGH [i.e. its effects
on peripheral 22K-hGH, insulin-like growth factor I (IGF-I),
free fatty acid (FFA), insulin, and glucose levels].
Subjects and Methods
Subjects and protocol
Thirty-two healthy male subjects, aged 20 –31-yr-old, were studied
after giving their informed consent. The protocol for the study was
approved by the Kiyaku-kai Medical Corporation Hohsen Clinic (Tokyo,
Japan) Institutional Review Board for human investigation. The study
was performed according to a double-masked, randomized and noncrossover protocol. Four groups of eight individuals (six on active, two
on placebo) received a single sc dose of 20K-hGH: 0.01, 0.025, 0.05, and
0.1 mg/kg. Treatment was sequential; beginning with the lowest dose
of 20K-hGH, the tolerability of each level was established before the next
higher dose was administered. Placebo or 20K-hGH was administered
sc into the thigh at 2100 h. Blood samples were collected every 30 min
for 24 h, except at 16.5 and 20.5 h for meals, centrifuged, and the resulting
serum samples were frozen and kept at ⫺80 C until assay.
Received August 3, 1999. Revision received October 12, 1999. Accepted October 20, 1999.
Address correspondence and requests for reprints to: Yoshihide
Hashimoto, Ph.D., Senior Scientist, Institute of Biological Science, Mitsui
Pharmaceuticals, Inc., 1900 –1 Togo, Mobara, Chiba 297-0017, Japan.
Adverse events
Healthy male volunteers tolerated exposure to single doses of 0.01–
0.1 mg/kg 20K-hGH well. Few adverse effects were observed. Those that
were seen were predominantly mild, with no apparent relationship to
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the dose, and were similar between the 20K-GH-treated and placebo
groups. For example, transient increases in temperature were observed
in four subjects: one who received placebo, two who received 20K-hGH
0.01 mg/kg, and one who received 20K-hGH 0.1 mg/kg.
half-life (T1/2) of 2–3 h. There was a linear relationship between dose and Cmax or area under the serum level-time
curve (AUC), indicating linear pharmacokinetics.
Materials
Effects of 20K-hGH administration on endogenous 22KhGH secretion
Recombinant 20K-hGH (Lot DB9805) (9) was prepared for clinical use
at a concentration of 2 mg/mL in sodium phosphate solution containing
creatinine, polysorbate 80, L-arginine and D-mannitol.
Assays
Serum 20K- and 22K-hGH were measured by specific ELISAs, as
described previously (15). Briefly, in 20K-hGH ELISA, 0.1 mL assay
buffer and 0.025 mL standard or serum samples were added to monoclonal anti-20K-hGH antibody (D05; Mitsui Pharmaceuticals, Inc., Tokyo, Japan)-precoated microtiter plates, followed by incubation for 2 h
at room temperature. After washing, 0.1 mL peroxidase-labeled anti20K-hGH monoclonal antibody (POD-D14; Mitsui Pharmaceuticals,
Inc., 0.5 mg/L) was added and incubated for 2 h at room temperature.
After washing, 0.1 mL TMB/H2O2 substrate was added, followed by
incubation for 30 min at room temperature. The absorbances were read
with a microtiter plate reader at 450 nm (reference, 620 nm) after stopping the enzyme reaction. In 22K-hGH ELISA, the microtiter plates were
coated with monoclonal anti-hGH antibody (A36020047P; BiosPacific,
Inc., Emeryville, CA). Other procedures were the same as described
above, except that the concentration of POD-D14 was 0.05 mg/L. The
cutoff values were 10 pg/mL for 20K-hGH and 100 pg/mL for 22K-hGH.
Serum IGF-I and insulin were determined with an immunoradiometric
assay kit (Somatomedin C; Chiba-Corning, Inc. Tokyo, Japan) and a RIA
kit (Phadisef Insulin; Pharmacia-Upjohn, Tokyo, Japan), respectively.
Serum nonesterified FFA and glucose were measured with commercial
kits using an auto-analyzer (TBA-80FR; Toshiba, Tokyo, Japan).
Statistical analysis
The results are expressed as means ⫾ sd unless otherwise noted.
Differences between groups were evaluated by ANOVA using the computer software StatLight (Yukms Corp., Tokyo, Japan), with P ⬍ 0.05
taken to indicate significance.
Results
Physiological 24-h secretion profiles of serum 20K- and
22K-hGH
Fig. 1 shows the 24-h profiles of serum 20K- and 22K-GH
levels in a representative subject for each of the groups. In the
placebo group (Fig. 1A), a large degree of variability was
noted between subjects, and the secretion profiles of endogenous 20K- and 22K-hGH were typical pulsatile (17) and
similar to each other. The proportion of 20K-hGH to 22KhGH was fairly constant (ca. 5%). In the 20K-hGH-treated
groups (Fig. 1, B-E), serum 20K-hGH levels increased within
30 min after injection, reached a peak between 2 and 6 h, and
decreased by the end of the sampling period. In contrast, the
spontaneous 22K-hGH surges were suppressed after a delay
of a few hours after injection, especially at higher doses.
Pharmacokinetics following sc injection of 20K-hGH
Serum 20K-hGH levels after a single sc injection of 20KhGH are shown in Fig. 2A. In the placebo group, mean 24-h
serum 20K-hGH levels were 0.13 ⫾ 0.12 ng/mL. Serum 20KhGH level increased in a dose-dependent manner. The pharmacokinetic parameters of 20K-hGH are summarized in Table 1. Maximum serum 20K-hGH levels (Cmax) after injection
were reached at 3– 4 h, declining thereafter with a mean
Fig. 2B illustrates serum 22K-hGH levels after 20K-hGH
administration. In the placebo group, mean 24-h serum 22KhGH levels were 2.5 ⫾ 1.7 ng/mL, and the typical nyctohemeral variations in hGH secretion (17) were observed. The
AUC0 –12 h of serum 22K-hGH was almost 3-fold higher than
the AUC12–24 h. In the 20K-hGH-treated groups (0.01, 0.025,
0.05, and 0.1 mg/kg), mean 24-h serum 22K-hGH levels were
1.1 ⫾ 0.4, 0.6 ⫾ 0.6, 0.9 ⫾ 0.9, and 0.9 ⫾ 0.7 ng/mL, respectively. Serum 22K-hGH levels decreased in a time-dependent
manner. Although the mean serum 22K-GH levels after injection were not different even at higher doses compared to
the placebo group during the first 4 h, the 22K-hGH levels
were reduced even at lower doses from approximately 4 – 6
h up to 12 h. During the subsequent 24-h observation period,
the 22K-hGH levels gradually returned to the placebo level.
Fig. 3 summarizes the AUC of serum 22K-hGH over 6 h,
which was used as an index of total 22K-hGH secretion.
There were no significant changes in the AUC0 – 6 h between
the 20K-hGH-treated and placebo groups. Marked suppression of the AUC6 –12 h (P ⬍ 0.01) was observed at all doses,
and almost 10-fold reductions were seen in comparison to the
placebo group. Both the AUC12–18 h and AUC18 –24 h tended
to be suppressed in the 20K-hGH-treated groups, although
differences were not significant compared with the placebo
group.
Effects of 20K-hGH administration on serum FFA and IGFI levels
The 20K-hGH-treated groups showed significant elevations in serum FFA and IGF-I levels with different time
courses after 20K-hGH administration (Fig. 4). Serum FFA
levels increased more rapidly than that of IGF-I, with maximum levels at 4 – 8 h. The FFA levels were significantly
higher than those of the placebo group at 4, 8, and 12 h (P ⬍
0.01), and returned to the control levels by 24 h (Fig. 4A). On
the other hand, serum IGF-I levels were not increased significantly at 4 h, but were increased at 8, 12, 24 (Fig. 4B), and
36 h (data not shown) (P ⬍ 0.01). Serum insulin and glucose
levels were not changed significantly during the 24-h observation period (data not shown).
Discussion
In this study, single sc administration of recombinant 20KhGH in normal men induced significant elevations in spontaneous serum FFA and IGF-I levels associated with a
marked reduction in the serum 22K-hGH level in a different
time-dependent manner. hGH secretion is mainly controlled
by hypothalamic hormones, GHRH, and somatostatin (18),
and also controlled negatively by hGH itself (19 –21) or hGHdependent substances: IGF-I (21), FFA (23, 24), glucose (18),
and so on. The increases in the main hGH-dependent substances (FFA and IGF-I) after 20K-hGH administration suggested that 20K-hGH has direct GH actions on adipose tissue
20K-hGH SUPPRESSES 22K-hGH SECRETION
603
FIG. 1. Individual representative 24-h serum 20K-hGH (F) and 22K-hGH (E) profiles in normal men administered the placebo (A) in comparison
with those in normal men administered 20K-hGH at the indicated doses (B-E). Placebo and 20K-hGH (0.01– 0.1 mg/kg) were administered at
2100 h. In the placebo group, the typical pulsatile pattern of hGH secretion was observed.
or the liver through hGH receptors similarly to 22K-hGH.
Therefore, 20K-hGH is expected to exert GH actions (growthpromoting activity and lipolytic activity) in humans. Fur-
thermore, the suppression of endogenous 22K-hGH secretion could be a result of so-called “GH-induced negative
feedback mechanisms.”
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FIG. 2. Mean serum 20K-hGH (A) and
22K-hGH (B) levels in normal men after
single sc administration of placebo and
20K-hGH. Sera were analyzed by 20KhGH and 22K-hGH ELISA, respectively. The values are means ⫾ SE (n ⫽
6 – 8). Placebo and 20K-hGH (0.01– 0.1
mg/kg) were administered at 2100 h. In
the placebo group, the typical nyctohemeral variations in hGH secretion were
observed.
TABLE 1. Pharmacokinetic parameters of serum 20K-hGH after a single sc administration of 20K-hGH to normal men
Dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC0-⬁ (ng 䡠 h/mL)
0.01
0.025
0.05
0.1
8.1 ⫾ 4.1
18.6 ⫾ 5.3
46.2 ⫾ 11.2
88.3 ⫾ 21.7
3.7 ⫾ 1.2
3.5 ⫾ 1.4
3.7 ⫾ 0.5
3.7 ⫾ 0.5
1.9 ⫾ 0.7
1.9 ⫾ 0.3
1.9 ⫾ 0.4
2.9 ⫾ 1.9
42 ⫾ 14
104 ⫾ 24
295 ⫾ 40
614 ⫾ 83
Mean ⫾ SD (n ⫽ 6).
Cmax, Maximum serum level; Tmax, time of observed Cmax; T1/2, half-life; AUC0-⬁, area under the serum level-time curve from 0 to ⬁.
We found that the 24-h profile of 20K-hGH secretion in the
placebo group was similar to that of 22K-hGH and that the
proportion of 20K- to 22K-hGH was fairly constant. These
observations suggested that regulation of 20K-hGH secretion
is physiologically the same as that of 22K-hGH. Baumann
and Stolar (25) suggested that 20K- and 22K-hGH may be stored
together in secretory granules in the somatotroph and, hence,
released together in response to various stimuli. Our observations support this hypothesis. Furthermore, these results suggested that the endogenous kinetics of 20K-hGH may be com-
parable with those of 22K-hGH. Interestingly, the pharmacokinetics after sc injection of recombinant 20K-hGH were nearly
comparable with those of recombinant 22K-hGH (26, 27). In
20K-hGH-treated groups, the serum 20K-hGH levels contained
both exogenously administered and endogenously secreted
20K-hGH, but the endogenous 20K-hGH levels were ignored in
this study because the mean secreted 20K-hGH levels in the
placebo group were fairly low (0.13 ⫾ 0.12 ng/mL). It has been
reported that 20K-hGH is cleared more slowly than 22K-hGH
in rats (28, 29), but this observation has not been confirmed in
20K-hGH SUPPRESSES 22K-hGH SECRETION
605
FIG. 3. Endogenous 22K-hGH secretion (AUC; ng Eh/mL per 6 h)
in normal men after placebo and 20K-hGH administration. Each
bar represents the mean ⫾ SE (n ⫽ 6 – 8). **, P ⬍ 0.01 (vs. placebo
by the Kruskal-Wallis and Steel test). Administration of 20K-hGH
(0.01– 0.1 mg/kg) resulted in marked reduction in AUC6 –12 h of
22K-hGH, but no significant differences were observed between
AUC0 – 6 h, AUC12–18 h and AUC18 –24 h of 22K-hGH.
guinea pigs (30). These discrepancies may be related to the
differences in the species studied (rat, guinea pig, human)
and/or assay methods used.
We have demonstrated the time course of the suppressive
effect induced by exogenous 20K-hGH on endogenous 22KhGH secretion in humans. The reduction of serum 22K-hGH
level after 20K-hGH administration required a period of ca.
4 h, and the level tended to recover by 24 h. However, the
delay in suppression of endogenous 22K-hGH by exogenous
20K-hGH is difficult to define precisely because of the intermittent nature of hGH secretion. Additional studies are
required to clarify the time lag between 20K-hGH exposure
and suppression of endogenous 22K-hGH. In previous studies (31, 32), single intramuscularly or sc administration of
hGH (with monitoring of the resulting plasma profiles)
showed a delayed and prolonged suppressive effect on rat
GH secretion. The time course of endogenous GH suppression in rats was similar to but faster than that in humans
reported here. The fast time course in rats was probably due
to the rapid absorption of hGH in this species (14, 33). Willoughby et al. (31) suggested that suppression is achieved
through metabolic or other intermediary processes, rather
than acutely by a direct membrane effect of the hGH
molecule.
The marked suppression of endogenous 22K-hGH secretion occurred in parallel with the FFA elevation; serum FFA
levels increased with maximum levels at 4 – 8 h and recovered by 24 h after 20K-hGH administration. In contrast, serum IGF-I levels increased after 8 h and were prolonged up
to 24 h or more, and no increase in circulating glucose levels
was observed for 24 h. Our data are consistent with those of
Rosenthal et al. (34), who found that 6-h methionyl 22K-hGH
infusion raised plasma FFA levels but not IGF-I or glucose
levels and blunted GHRH-induced GH secretion in normal
FIG. 4. Mean serum FFA (A) and IGF-I (B) increases (⌬) over the
basal values after placebo and 20K-hGH administration in normal
men. Placebo and 20K-hGH (0.01– 0.1 mg/kg) were administered at
2100 h. The values are means ⫾ SE (n ⫽ 6 – 8).
men. Of the main hGH-dependent substances, elevation of
FFA rather than IGF-I levels may play a leading role at least
in the marked 22K-hGH suppression at AUC6 –12 h after a
single sc administration of 20K-hGH. Administration of FFA
markedly reduced the basal GH secretion and blocked GH
secretion induced by pharmacological and physiological
stimuli in humans (23, 35). Recently, Briard et al. (36) reported
that FFA acts both at the hypothalamic level, through increased somatostatin secretion, and at the pituitary level in
sheep.
The suppression of 22K-hGH secretion was observed even
at the lowest dose of 20K-hGH administered (0.01 mg/kg),
with a Cmax of 8.1 ⫾ 4.1 ng/mL. Rosenthal et al. (34) reported
that the GHRH-induced GH response in humans was significantly inhibited during 6-h methionyl 22K-hGH infusion,
whereas the plasma GH level remained constant (9 –13 ng/
mL). Therefore, the effect of 20K-hGH on negative feedback
may be as potent as that of 22K-hGH.
There are experimental limitations to differentiating between exogenous and endogenous hGH in humans. The time
course of GH-induced negative feedback in humans can only
be studied indirectly by using the peripheral GH response to
GH provocation (21, 34, 37, 38) or the amplitude of sleep-
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HASHIMOTO ET AL.
related GH secretion (20) as an indicator of suppression of
GH secretion. Our observations extended these studies and
indicated that an exogenously administered GH isoform
could suppress the other endogenously secreted GH isoform
in a time-dependent manner. The proportion of 20K- to 22KhGH is fairly constant under physiological conditions. Therefore, by measuring the serum 20K- and 22K-hGH levels and
using the other hGH isoform as an indicator of the endogenous hGH, it may be possible to monitor the internal behavior of exogenously administered hGH in clinical application of 20K-hGH and, especially, 22K-hGH. Measurement
of serum 20K- and 22K-hGH may be useful in evaluating the
effects of circulating GH isoforms on their own release from
the pituitary.
Acknowledgment
We thank Drs. Kohei Yazawa, Fumiaki Ikeda, and Masaru Honjo for
advice and encouragement during these studies. We also thank Ms.
Noriko Takayama, Ms. Keiko Kawano, and Ms. Hiromi Takeda for
technical assistance.
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