Am J Physiol Endocrinol Metab
279: E501–E507, 2000.
Effects of oral administration of a synthetic fragment
of human growth hormone on lipid metabolism
M. A. HEFFERNAN,1 W. J. JIANG,1 A. W. THORBURN,2 AND F. M. NG1
1
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3148;
and 2Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
Received 15 December 1999; accepted in final form 11 April 2000
Heffernan, M. A., W. J. Jiang, A. W. Thorburn, and
F. M. Ng. Effects of oral administration of a synthetic frag-
ment of human growth hormone on lipid metabolism. Am J
Physiol Endocrinol Metab 279: E501–E507, 2000.—A small
synthetic peptide sequence of human growth hormone (hGH),
AOD-9401, has lipolytic and antilipogenic activity similar to
that of the intact hormone. Here we report its effect on lipid
metabolism in rodent models of obesity and in human adi-
pose tissue to assess its potential as a pharmacological agent
for the treatment of human obesity. C57BL/6J (ob/ob) mice
were orally treated with either saline (n ⫽ 8) or AOD-9401
(n ⫽ 10) for 30 days. From day 16 onward, body weight gain
in AOD-9401-treated animals was significantly lower than
that of saline-treated controls. Food consumption did not
differ between the two groups. Analyses of adipose tissue ex
vivo revealed that AOD-9401 significantly reduced lipogenic
activity and increased lipolytic activity in this tissue. In-
creased catabolism was also reflected in an acute increase in
energy expenditure and glucose and fat oxidation in ob/ob
mice treated with AOD-9401. In addition, AOD-9401 in-
creased in vitro lipolytic activity and decreased lipogenic
activity in isolated adipose tissue from obese rodents and
humans. Together, these findings indicate that oral admin-
istration of AOD-9401 alters lipid metabolism in adipose
tissue, resulting in a reduction of weight gain in obese ani-
mals. The marked lipolytic and antilipogenic actions of AOD-
9401 in human adipose tissues suggest that this small syn-
thetic hGH peptide has potential in the treatment of human
obesity.
human growth hormone peptide; lipolysis; lipogenesis; en-
ergy expenditure
OBESITY is a major public health concern in most devel-
oped countries. For example, in Australia almost one in
five adults is obese, making them highly susceptible to
diabetes, coronary heart disease, and high blood pres-
sure, as well as reduced psychological health (1). Obe-
sity is normally treated by diet and exercise, but at-
tempts to sustain significant weight loss by dieting and
exercise nearly always meet with failure (6). There is a
great need to develop better pharmacotherapy for obe-
sity (18). Here, we begin an assessment of the potential
use of AOD-9401, a fragment of human growth hor-
mone (hGH), in the treatment of obesity.
Address for reprint requests and other correspondence: M. Hef-
fernan, Dept. of Biochemistry and Molecular Biology, Monash
Univ., Clayton, Melbourne, Victoria 3168, Australia (E-mail:
mark.heffernan@med.monash.edu.au).
http://www.ajpendo.org 0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society
The lipolytic/antilipogenic property of hGH is well
known (11, 19). For example, it is well documented that
hGH is a potent inhibitor of lipoprotein lipase and can
increase circulating free fatty acids, ultimately reduc-
ing fat cell mass (20). The association of circulating
hGH with fat mass is well characterized in adult GH-
deficient patients, where a strong correlation between
excess abdominal adiposity and reduced circulating
GH levels exists that can be normalized after GH
replacement therapy (10). However, clinical applica-
tions of hGH for long-term obesity treatment have not
been successful because of its diabetogenic and other
unwanted side effects (3). Advances in peptide synthe-
sis technology have made it possible to produce specific
and discrete functional domains of hGH (9), and there
is now considerable evidence supporting the concept of
discrete structural domains within hGH responsible
for the different metabolic functions of the intact hGH
(4). For example, Ng et al. (13) have reported that the
insulin-like actions of hGH may reside in the amino-
terminal region of the molecule [hGH-(6O13)]. This
was later confirmed to be within hGH-(1O43), another
well-known hypoglycemic fragment that exists in the
circulation of humans and results from posttransla-
tional cleavage in vivo (20, 29). On the other hand, the
carboxy terminus of the hGH molecule [hGH-(177O191),
or AOD-9401] has been identified as the lipid mobiliz-
ing domain of the intact hormone. This fragment in-
hibits the activity of acetyl-CoA carboxylase in adipo-
cytes and hepatocytes, and it acts to reduce glucose
incorporation into lipid in both isolated cells and tis-
sues (30). It has been suggested to be the lipolytic
domain of the hGH molecule. Previous studies in our
laboratory have shown that weight loss can be induced
by chronic intraperitoneal treatment of AOD-9401 (12).
This study aims to extend these findings by examin-
ing whether oral administration of this fragment of
hGH can also reduce body weight and affect lipid
metabolism. This study had three parts. First, we inves-
tigated the effectiveness of oral administration of AOD-
9401 on body weight reduction, energy balance, lipol-
ysis, and lipogenesis in obese C57BL/6J (ob/ob) mice.
Next, we examined the in vitro antilipogenic, lipolytic,
The costs of publication of this article were defrayed in part by the
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solely to indicate this fact.
E501
E502 EFFECT OF HGH FRAGMENT ON LIPID METABOLISM
and fat oxidation activity of AOD-9401 in peripheral
adipose tissues from obese rodents. Finally, to assess the
feasibility of human treatment, the in vitro action of
AOD-9401 on lipolysis and lipogenesis in adipose tissue
from obese human adipose tissue was examined.
MATERIALS AND METHODS
Chemical synthesis of hGH functional domain (AOD-9401).
AOD-9401, a synthetic fragment of hGH consisting of the
amino acid residues 177–191, was prepared with solid-phase
synthesis procedure and purified with reverse-phase HPLC
methodology in our laboratories at Monash University (9).
The structure of the peptide analog was verified with mass
spectrometry and amino acid analysis.
In vitro tests for nonenzymic and enzymic degradation of
AOD-9401. AOD-9401 was tested for its in vitro stability
against potential gastrointestinal degradation, according to
the standard protocols of enzyme digestion for protein (26).
The procedure of De Laureto et al. (4) was used to evaluate
the rate of degradation by measuring the residual peptide
with RP-HPLC techniques as well as amino acid analysis.
Experimental animals and oral treatment. Eighteen male
C57BL/6J (ob/ob) mice aged 10–12 wk and weighing 46.4 ⫾
1.1 (SE) g were used in this study. The animals were divided
into saline (n ⫽ 8) or AOD-9401 treatment groups (n ⫽ 10)
and were matched for body weight and sex. Animals were
housed in a normal 12:12-h light-dark cycle at a constant
room temperature of 23°C in the Departmental Animal
House at Monash University. Animals were fed ad libitum a
standard laboratory nonpurified diet (Clark King, Mel-
bourne, Australia) and allowed free access to water at all
times. The mice were given daily oral doses of either AOD-
9401 (500 ␮g/kg body wt) dissolved in 0.3 ml saline or only
saline of equivalent volume for 30 days. Accurate dosing was
facilitated by a stainless steel gavage needle (7.5 ⫻ 0.1 cm
diameter). The dose was administered slowly to avoid reflux.
Measurements of body weight gain, food consumption, en-
ergy expenditure, and physical activity. The body weights of
the mice were measured before treatment and then every 2
days until the end of the study. Food consumption was
measured every 2 days after treatment started and averaged
to give a daily measurement. Grams of nonpurified diet
consumed were multiplied by 2.85 to give caloric intake in
kilocalories per day and then multiplied by 4.184 to convert
to kilojoules per day. Energy expenditure was measured at
the end of the treatment period with an indirect calorimeter
(Columbus Instruments, Columbus, Ohio).
Gravimetrically determined standard gas mixtures of
20.48% O2 and 0.5% CO2 were used to calibrate the machine
before use (BOC Gases Australia, Preston, Victoria, Austra-
lia). After gavage, mice were fasted for 2 h and then placed in
a 20 ⫻ 13 ⫻ 11-cm Perspex box through which fresh air was
drawn at a rate of 0.65 l/min. Mean rates of CO2 produced
and O2 consumed were calculated every 3 min over a 30-min
period. Rates of energy expenditure (kcal/min) were calcu-
lated using data from the final 15 min, after assuming a
urinary nitrogen excretion of 0.84 mg 䡠 min⫺1 䡠 kg body wt⫺1
(27). For the last 3 days of the treatment period, voluntary
physical activity levels were also measured in these mice by
use of 15-cm-diameter running wheels. Continuous 24-h
monitoring of the use of the running wheels was made using
a computerized meter (7).
Acute effect of AOD-9401 on fat oxidation in vivo. The acute
effect of AOD-9401 on the rates of fat and glucose oxidation
was assessed at the conclusion of the 30-day oral treatment
period in the obese ob/ob mice after food intake, physical
activity, and resting energy expenditure studies had been
completed. On the morning of the last day of the study, a
group of three saline-treated mice and four AOD-9401-
treated mice were food-deprived for 1 h; then basal fat oxi-
dation, glucose oxidation, and energy expenditure were mea-
sured for 10 min with the indirect calorimetry procedure
described previously. The mice were then given an intraperi-
toneal injection of saline (in the saline-treated group) or
AOD-9401 (250 ␮g/kg in the AOD-treated group), and rates
of fat oxidation, glucose oxidation, and energy expenditure
were measured for a further 18 min.
Isolation of adipose tissues. Groups of mice were killed
with a lethal dose of pentobarbitone (0.2 ml) 24 h after the
last treatment with oral AOD-9401 on day 30. Energy expen-
diture measurements after the intraperitoneal AOD-9401
dosing were not conducted on these mice. Epididymal fat
pads from male mice were isolated as in our previous studies
(14). The tissues were washed in room-temperature saline
before being weighed into ⬃200-mg pieces for ex vivo assays.
For in vitro assays, male C57BL/6J (ob/ob) mice were used.
Male Zucker rats (200–300 g, 12 wk of age) were used to
assess adipose tissue fat oxidation rates in vitro in response
to AOD-9401. Human subcutaneous abdominal adipose tis-
sue was obtained with consent from an overweight female
patient (age: 42 yr; body mass index: 28.4) who had no other
known medical complications and who had undergone fat-
reduction surgery for cosmetic reasons.
Assay for lipogenic activity in adipose tissue. The rate of
incorporation of exogenous [14C]glucose into total lipid in
adipose tissue was used as an index of lipogenic activity.
Tissues were placed in 2 ml of Krebs-Ringer bicarbonate
(KRB) buffer (pH 7.4) containing 2% defatted BSA and 0.1
mg/ml glucose and then were gassed with 95% O2-5% CO2 in
a shaking water bath, with temperature controlled at 37°C.
After 30 min of preincubation, the tissues were transferred to
another 2 ml of fresh medium containing [14C]glucose (final
specific activity of 0.05 ␮Ci/␮mol) for a further 90 min (same
conditions as above). Tissues were then removed and rinsed
thoroughly in saline, and lipid was extracted with a chloro-
form-methanol (2:1) mixture. 14C radioactivity was counted
on a Wallac 1410 liquid scintillation counter (Turku, Fin-
land). The rates of total lipid synthesis were expressed as
picomoles of glucose incorporated into fat per milligram of
tissue per hour.
Assay for lipolytic activity in adipose tissue. The rate of
lipolytic activity was measured by the release of glycerol into
the incubation medium. Tissue pieces were placed in 2 ml
KRB buffer with 2% BSA and 0.1 mg/ml glucose and incu-
bated for 60 min (same conditions as above). The tissues were
then removed and discarded, and the amount of glycerol
present in the incubation medium was enzymatically as-
sayed using glycerol phosphate oxidase reactions (Sigma
Diagnostics, catalog no. GPO-337; St Louis, MO). Glycerol
was determined with a spectrophotometer and converted to
micromoles of glycerol released per gram of tissue per hour.
Plasma measurements. Blood samples were collected from
the tail vein of anesthetized animals in capillary tubes after
chronic treatment. Plasma was stored at ⫺20°C until used.
Glucose was measured using a 2300 STAT glucose analyzer.
Free fatty acids (FFAs) were determined by the method
developed from Noma (15). Triglycerides (TGs) were mea-
sured with a kit according to the recommendations of the
manufacturer (Sigma).
In vitro FFA oxidation assay. FFA oxidation was determined
by measuring the converted [14C]O2 from [1-14C]palmitic acid
(23). [14C]O2, a final product of ␤-oxidation of FFA, was trapped
by hyamine hydroxide and measured by a liquid scintillation
 EFFECT OF HGH FRAGMENT ON LIPID METABOLISM
counter. Adipose tissues removed from laboratory animals were
sliced into segments of ⬃200 mg each. The tissues were placed
in 25-ml vials containing 2 ml of Krebs-Ringer phosphate buffer
and 4% defatted BSA and then were preincubated at 37°C for
30 min under an atmosphere of carbogen (95% O2-5% CO2).
Tissues were then transferred to Konte flasks containing fresh
incubation medium, with 0.15 mM sodium palmitate and 0.20
␮Ci/␮mol of 14C specific activity and different concentrations of
hGH-(177–191) peptide. A filter paper roll was placed in a well
inside the flask and then was sealed with a rubber septum
stopper. Flasks were incubated at 37°C for 1 h, and the reaction
was terminated by injecting 250 ␮l of 4.5 M H2SO4 with a
needle through the rubber septum into the medium of a flask.
Hyamine hydroxide (250 ␮l) was then injected into the filter
paper roll in the center well. Incubation proceeded for another
60 min to ensure the complete absorption of released [14C]O2 by
the paper roll. The filter paper rolls were then carefully re-
moved and transferred to scintillation vials, and the 14C radio-
activity was measured by a liquid scintillation counter. The rate
of [14C]palmitic acid oxidation to [14C]O2 was calculated and
expressed as micromoles per gram of tissue per hour.
Statistical analysis. The Student’s t-test was used to ana-
lyze the results. All data are expressed as means ⫾ SE. P
values of ⬍0.05 were accepted as statistically significant.
RESULTS
Molecular stability of AOD-9401. Synthetic AOD-
9401 was stable in the aqueous KRB buffer at pH 7.4.
Less than 5% of the analog was found degraded after
16-h incubation at 37°C (data not shown). The half-life
(t1/2) of AOD-9401 was ⬃50 and 170 min in enzymic
digestion with pepsin and trypsin, respectively (Fig. 1).
These t1/2 values indicate that the structure of the
peptide was reasonably stable in the presence of tryp-
sin and pepsin, the two major gastrointestinal enzymes
in the digestive tract of the body. These findings are
supported by our observation that AOD-9401 is de-
Fig. 1. Results from the in vitro digestion of AOD-9401 in the
presence of trypsin and pepsin. Conditions for digestion are as
described by Stone and Williams (26).
E503
Fig. 2. Effect of AOD-9401 on cumulative body weight gain (means ⫾
SE) of obese (ob/ob) mice during the 30-day treatment period. Ani-
mals were gavaged daily with either 0.3 ml of saline (●) (n ⫽ 8) or
AOD-9401 (500 ␮g 䡠 kg body wt⫺1 䡠 day⫺1, E) (n ⫽ 10) via a gavage
needle (* P ⬍ 0.05).
tected in rat plasma 30 min after oral administration
to animals (unpublished data).
In vivo effect of AOD-9401 on energy balance in obese
(ob/ob) mice. Figure 2 shows the change in body weight
after mice were orally treated with AOD-9401 or saline
for 30 days. The rate of weight gain was 58% lower in
mice treated with AOD-9401 from day 16 of treatment
onward (0.33 to 0.14 g/day, P ⬍ 0.05). This is the first
time that oral administration of an hGH peptide frag-
ment was shown to reduce body weight gain. Body
weights for control animals started at 44.5 ⫾ 2.3 g on day
0 and finished at 54.7 ⫾ 1.8 g after 30 days of saline
treatment (⫹10.2 g). In contrast, AOD-9401-treated
animals weighed 47.8 ⫾ 0.9 g on day 0 and at the
conclusion of treatment weighed 52.5 ⫾ 0.6 g (⫹4.7 g).
The reduction in body weight gain in the AOD-9401-
treated obese mice was not due to a decrease in food
intake. Figure 3 shows that the average daily caloric
intake from day 2 to day 30 was the same in the saline-
and AOD-9401-treated groups. Nor did the reduction
in body weight gain correlate with increased resting
energy expenditure measured 2 h after gavage treat-
ment (0.00525 ⫾ 0.00028 vs. 0.00518 ⫾ 0.00042 kcal/
min in AOD-9401- and saline-treated mice, respec-
tively). Resting energy expenditure was the same in
AOD-9401- and saline-treated mice even when normal-
ized for body weight (0.103 ⫾ 0.004 vs. 0.101 ⫾ 0.009
kcal 䡠 min⫺1 䡠 kg⫺1 in AOD-9401- and saline-treated
mice, respectively). AOD-9401 did not increase run-
ning wheel activity in obese ob/ob mice (data not
shown). The mouse strain is very inactive compared
with other strains of mice and remains so even after
AOD-9401 treatment.
E504 EFFECT OF HGH FRAGMENT ON LIPID METABOLISM

Fig. 3. Average daily food consumption (kJ/day) of obese (ob/ob) mice

during the 30-day treatment period after the administration of saline
(●) or AOD-9401 (E). Results are expressed as means ⫾ SE. No
statistical significance was observed throughout the treatment pe-
riod.

Ex vivo effect of AOD-9401 on lipogenesis and lipol-

ysis. The data in Fig. 4A show that AOD-9401 increased
the lipolytic rate from 0.63 ⫾ 0.20 ␮mol 䡠 g tissue⫺1 䡠 h⫺1
in control animals to 1.02 ⫾ 0.25 ␮mol 䡠 g tissue⫺1 䡠 h⫺1
(P ⬍ 0.001) as measured by the rate of glycerol release.
The lipogenic activity of the tissue (Fig. 4B) was con-
versely inhibited by 22% (4.23 ⫾ 0.08 vs. 3.31 ⫾ 0.2
pmol 䡠 mg tissue⫺1 䡠 h⫺1, P ⬍ 0.0025) after 30 days of
oral administration of the compound as measured by
the incorporation of [14C]glucose into lipid. These al-
terations in lipid metabolism are consistent with the
observed decrease in cumulative body weight gain of
the treated animals. Fig. 4. Ex vivo lipogenic activity (A) and lipolytic activity (B) in
In addition to these observations, effects on plasma adipose tissues of the obese (ob/ob) mice after 30-day oral adminis-
metabolites were also noted (Table 1). Results indicate tration of AOD-9401. Data are expressed as means ⫾ SE of control
(n ⫽ 10) and treated (n ⫽ 10) tissues. * P ⬍ 0.0025.
that, after chronic administration of AOD-9401, a sig-
nificant increase in plasma FFAs can be recorded.
Levels of glucose and TGs are slightly lower in treated shows a clear dose-dependent effect of AOD-9401 on
animals but are not significantly different from con- lipogenesis (Fig. 6A) and lipolysis (Fig. 6B). Peripheral
trols. epididymal adipose tissue from male C57BL/6J (ob/ob)
Acute effect of AOD-9401 on in vivo fat oxidation. An mice was incubated in the presence of AOD-9401 for
acute dose of AOD-9401 can markedly increase fat 1 h.
oxidation in vivo in obese ob/ob mice. Figure 5 shows The effect of AOD-9401 on fat oxidation was also
the effect of a single intraperitoneal injection of AOD- measured, this time in obese male Zucker rats. AOD-
9401 or saline on the rates of energy expenditure, fat, 9401 acted dose dependently to increase the rate of fat
and glucose oxidation (Fig. 5, A, B, and C, respectively). oxidation (Fig. 7), with a significant 25% increase in fat
After 18 min, AOD-9401 increased energy expenditure
45% above basal (P ⬍ 0.02) and increased fat oxidation Table 1. Effects of AOD-9401 treatment on plasma
83% above basal values (P ⬍ 0.02). It also increased glucose and lipids
glucose oxidation 2.4-fold after 9 min (P ⬍ 0.05); how-
ever, this effect disappeared 15 min after the AOD- Glucose, mg/dl FFA, mmol/l TG, mmol/l
9401 injection. In contrast, an intraperitoneal dose of
Control (saline) 381.6 ⫾ 3.2 1.0 ⫾ 0.04 343.2 ⫾ 5.5
saline had no significant effect on either energy expen- AOD-9401,
diture or fat or glucose oxidation in the obese ob/ob 500 ␮g 䡠 kg⫺1 䡠 day⫺1 366.85 ⫾ 5.96 1.38 ⫾ 0.04* 326.85 ⫾ 3.65
mice.
Values of plasma glucose and lipids are shown here as means ⫾ SE
In vitro effect of AOD-9401 on lipogenesis, lipolysis, of control (n ⫽ 5) and AOD-9401 treated (n ⫽ 7) ob/ob mice after
and fat oxidation. The effect of AOD-9401 on lipogen- chronic oral administration (* P ⬍ 0.005). FFA, free fatty acid; TG,
esis and lipolysis is also evident in vitro. Figure 6 triglyceride.
 EFFECT OF HGH FRAGMENT ON LIPID METABOLISM E505

a threefold increase in glycerol release, and decreased

lipogenic activity by 50%, similar to our in vitro find-
ings in ob/ob mouse tissue.
DISCUSSION

Administration of exogenous GH to GH-deficient hu-

man subjects and experimental animals affects body
composition by stimulating lipid mobilization and en-
ergy expenditure (16, 22). However, hGH is not a
potentially useful drug for treating obesity, because
prolonged use results in several adverse side effects,
namely glucose intolerance, insulin resistance, edema,
and hypertension (2, 3). There is strong evidence im-
plicating the effects of hGH to specific fragments of its
sequence (22). There is therefore the potential of de-
veloping a peptide analog of hGH that modifies body
composition without the adverse effects of the intact
molecule. We have synthesized a carboxy-terminal
fragment of the hGH molecule [hGH-(177–191)] that
appears to regulate lipid metabolism (12) but lacks the

Fig. 5. Measurements of basal energy expenditure (A), fat oxidation

(B), and glucose oxidation (C) before and after ip injection of AOD-
9401 (250 ␮g/kg, n ⫽ 4) or saline (n ⫽ 3) in obese (ob/ob) mice.
Results are expressed as means ⫾ SE (* P ⬍ 0.05).

oxidation observed at concentrations of 1.0 ␮M AOD-

9401 (P ⬍ 0.05). Maximal fat oxidation rates appeared
to be achieved at 3.0 ␮M.
In vitro effect of AOD-9401 on human adipose tissue.
AOD-9401 and synthetic analogs have the potential to
be developed for therapeutic applications, including
management of human obesity. Thus, we also evalu-
Fig. 6. In vitro lipogenesis (A) and lipolysis (B) in epididymal adipose
ated the effect of AOD-9401 on human subcutaneous tissue from obese (ob/ob) mice in the presence of saline (control) or
adipose tissue. Table 2 indicates that AOD-9401 en- various concentrations of AOD-9401. Each bar represents means ⫾
hanced lipolysis in human adipose tissue, resulting in SE of 6 determinations (* P ⬍ 0.05).
E506 EFFECT OF HGH FRAGMENT ON LIPID METABOLISM
Fig. 7. In vitro rates of fat oxidation in male Zucker rat epididymal
adipose tissue incubated in the presence of various concentrations of
AOD-9401. Bars express means ⫾ SE of 6 determinations. (* P ⬍
0.05).
hGH fragment hGH-(1–43) that is associated with
diabetes (13). Glucose clamp experiments from our
laboratory have also indicated that AOD-9401 does not
induce glucose intolerance, as shown by euglycemic
clamp experiments (M. A. Heffernan, M. J. Waters and
F. M. Ng, unpublished observations). Our previous
studies have shown that intraperitoneal AOD-9401
treatment is capable of inducing weight loss in obese
mice (30). The present study extends this work by
demonstrating that the peptide is as effective when
given orally, reducing the rate of weight gain in obese
ob/ob mice by 58%. This has significant implications for
its potential use as a therapeutic agent. Enzyme diges-
tion data presented in this study suggest that AOD-
9401 is relatively resistant to enzymatic degradation in
the gastrointestinal (GI) tract and is available for ab-
sorption across the GI tract and into the bloodstream.
Interestingly, the lower weight gain after 30 days of
oral treatment with AOD-9401 was not due to reduced
energy intake. Nor did there appear to be an increase
in resting energy expenditure or voluntary physical
activity that was associated with this lower weight
gain. There are several possible reasons for this. First,
even a small alteration in the metabolic efficiency of an
organism may result in weight loss or gain (27), and it
is possible that the method we used could not detect
these small changes. Studies in our laboratory, for
example, indicate that, for a single mouse, the day-to-
day coefficient of variation in food intake is substantial
(23%). In addition, measuring energy expenditure in
mice that have been deprived of food for 2 h after
gavage with AOD-9401 may not have been the optimal
time to look for changes in energy expenditure. AOD-
9401 may increase diet-induced thermogenesis or noc-
turnal energy expenditure, which we did not measure
in this study. We could, however, detect increases in
energy expenditure, fat oxidation, and glucose oxida-
tion in the ob/ob mice after acute intraperitoneal ad-
ministration of AOD-9401, which means that this pep-
tide is capable of altering metabolic efficiency. The
profound effect of AOD-9401 on fat oxidation was also
illustrated in vitro in epididymal fat tissue isolated
from obese Zucker rats.
The fact that AOD-9401 is able to increase fat oxi-
dation in vivo and in vitro, and is able to increase
lipolysis and reduce lipogenesis ex vivo as well as in
vitro, makes it likely that AOD-9401 acts directly on
adipose tissue. It must also be noted that an increase in
circulating FFAs is difficult to explain; however, many
acute factors may induce such transient metabolic
changes. Plasma levels of glucose and TGs are slightly
lower in treated animals compared with controls, but
not significantly different. This may indicate that
AOD-9401 can somehow improve glucose tolerance in
hyperglycemic mice. A slight reduction in the TG level
may be ascribed to the normalization of hyperphagia
and reduction in activity of lipogenic enzymes involved
in lipid reesterification.
Whether central administration of AOD-9401 is also
capable of influencing peripheral tissue lipid metabo-
lism has not yet been investigated. The mechanism of
action of AOD-9401 is not known but is currently being
examined in our laboratory. hGH is thought to increase
lipolysis by increasing hormone-sensitive lipase ex-
pression and phosphorylation (4a), increasing ␤3-ad-
renergic receptor level (27), inhibiting Gi protein (5),
and/or preventing insulin-induced antilipolysis (20).
Previous work in our laboratory has shown that the
increase in lipolysis induced by AOD-9401 is also as-
sociated with an increase in activity of the lipolytic
enzyme hormone-sensitive lipase in adipose tissue (9).
In addition, we have shown that the reduction in lipo-
genesis is associated with a reduction in activity of the
lipogenic enzyme acetyl-CoA carboxylase (12). Identi-
fying the receptor that AOD-9401 binds to would help
to decipher the mode of action of AOD-9401. It is pos-
sible that the hGH receptor is not involved, because
other peptide fragments isolated from hGH [hGH-(95–
133) and hGH-(108–129)] appear to work in a receptor-
mediated fashion distinct from the hGH receptor to
induce mitogenic activity in 3T3-F442A fibroblasts (8,
25). Whatever the site and mode of action, the fact that
AOD-9401 influences lipid metabolism when orally ad-
Table 2. In vitro lipolysis and antilipogenesis in
human subcutaneous adipose tissue by AOD-9401
Lipolysis, Lipogenesis,
␮mol glycerol 䡠 g pmol 䡠 mg
tissue⫺1 䡠 h⫺1 tissue⫺1 䡠 h⫺1
Control 0.52 ⫾ 0.03 4.98 ⫾ 0.34
AOD-9401 (0.1 ␮M) 1.39 ⫾ 0.34 (⫹167%)* 2.74 ⫾ 0.32 (⫺45%)*
AOD-9401 (1.0 ␮M) 1.22 ⫾ 0.19 (⫹134%)* 3.01 ⫾ 0.17 (⫺40%)*
In vitro lipolysis and antilipogenesis induced with two concentra-
tions of AOD-9401 in obese human subcutaneous adipose tissue.
Tissue was obtained from abdmoninoplasty surgery and was incu-
bated in the presence of AOD-9401 at various concentrations for 1 h.
Lipolysis and lipogenesis were determined. Results represent
means ⫾ SE of 4 samples in each group (with % change), isolated
from a single obese human (* P ⬍ 0.05).
 EFFECT OF HGH FRAGMENT ON LIPID METABOLISM
ministered and is capable of increasing lipolysis and
decreasing lipogenesis in human adipose tissue means
that it is worthwhile pursuing this peptide as a possi-
ble treatment for human obesity.
It is possible that AOD-9401 is a naturally occurring
peptide that exists in the circulation. Wroblewski et al.
(29) have shown that proteolytically cleaved two-chain
forms of hGH do exist in vivo, which together may
account for much of the activity of hGH. Such protein
fragments can persist longer in the circulation than
hGH and have led to the hypothesis that hGH may
behave as a prohormone, which requires proteolytic
cleavage to induce the diversity of effects observed. The
existence of such peptides, which may or may not share
the biological outcomes of hGH, helps to explain the
diversity of action of hGH.
Because conventional dietary modification and phys-
ical exercise programs are usually unable to maintain
weight loss in the long term, there is an urgent need to
develop safe, effective pharmacotherapy to treat hu-
man obesity. Although further studies are required,
this study suggests that AOD-9401 given orally may be
useful in the treatment of obesity because of its ability
to increase fat oxidation and lipolysis and decrease
lipogenesis in adipose tissue.
This study was supported by Special Research Grants to F. M. Ng
from Monash University, and by Metabolic Pharmaceuticals Lim-
ited, Australia. M. A. Heffernan is a recipient of an Australian
Postgraduate Research Scholarship.
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