Growth hormone (GH) is of importance for normal bone remodelling. A recent clinical study demonstrated that MK-677, a member of a class of GH secretagogues (GHSs), increases serum concentrations of biochemical markers of bone formation and bone resorption. The aim of the present study was to investigate whether the GHSs, ipamorelin (IPA) and GH-releasing peptide-6 (GHRP-6), increase bone mineral content (BMC) in young adult female rats. Thirteen-week-old female Sprague-Dawley rats were given IPA (0.5 mg/kg per day; n=7), GHRP-6 (0.5 mg/kg per day; n=8), GH (3.5 mg/kg per day; n=7), or vehicle administered continuously s.c. via osmotic minipumps for 12 weeks. The animals were followed in vivo by dual X-ray absorptiometry (DXA) measurements every 4th week. After the animals were killed, femurs were analysed in vitro by mid-diaphyseal peripheral quantitative computed tomography (pQCT) scans. After this, excised femurs and vertebrae L6 were analysed by the use of Archimedes' principle and by determinations of ash weights. All treatments increased body weight and total tibial and vertebral BMC measured by DXA in vivo compared with vehicle-treated controls. However, total BMC corrected for the increase in body weight (total BMC:body weight ratio) was unaffected. Tibial area bone mineral density (BMD, BMC/area) was increased, but total and vertebral area BMDs were unchanged. The pQCT measurements in vitro revealed that the increase in the cortical BMC was due to an increased cross-sectional bone area, whereas the cortical volumetric BMD was unchanged. Femur and vertebra L6 volumes were increased but no effect was seen on the volumetric BMDs as measured by Archimedes' principle. Ash weight was increased by all treatments, but the mineral concentration was unchanged. We conclude that treatment of adult female rats with the GHSs ipamorelin and GHRP-6 increases BMC as measured by DXA in vivo. The results of in vitro measurements using pQCT and Archimedes' principle, in addition to ash weight determinations, show that the increases in cortical and total BMC were due to an increased growth of the bones with increased bone dimensions, whereas the volumetric BMD was unchanged.
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The GH secretagogues ipamorelin and GH-releasing peptide-6
increase bone mineral content in adult female rats
J Svensson
1
, S Lall
2
, S L Dickson
2
, B-Å Bengtsson
1
, J Rømer
3
,
I Ahnfelt-Rønne
3
, C Ohlsson
1
and J-O Jansson
1
1
Research Centre for Endocrinology and Metabolism, Sahlgrenska University Hospital, Göteborg, Sweden
2
Department of Physiology, University of Cambridge, Cambridge, UK
3
Health Care Discovery, Novo Nordisk A/S, Bagsvaerd, Denmark
(Requests for offprints should be addressed to J Svensson, Research Centre for Endocrinology and Metabolism, Gröna Stra ̊ ket 8, Sahlgrenska University
Hospital, SE-413 45 Göteborg, Sweden; Email: [email protected])
(J Svensson and S Lall have made an equal contribution to this paper)
Abstract
Growth hormone (GH) is of importance for normal bone
remodelling. A recent clinical study demonstrated that
MK-677, a member of a class of GH secretagogues
(GHSs), increases serum concentrations of biochemical
markers of bone formation and bone resorption. The aim
of the present study was to investigate whether the GHSs,
ipamorelin (IPA) and GH-releasing peptide-6 (GHRP-6),
increase bone mineral content (BMC) in young adult
female rats. Thirteen-week-old female Sprague–Dawley
rats were given IPA (0·5 mg/kg per day; n=7), GHRP-6
(0·5 mg/kg per day; n=8), GH (3·5 mg/kg per day; n=7),
or vehicle administered continuously s.c. via osmotic
minipumps for 12 weeks. The animals were followed in
vivo by dual X-ray absorptiometry (DXA) measurements
every 4th week. After the animals were killed, femurs
were analysed in vitro by mid-diaphyseal peripheral quan-
titative computed tomography (pQCT) scans. After this,
excised femurs and vertebrae L6 were analysed by the use
of Archimedes’ principle and by determinations of ash
weights. All treatments increased body weight and total
tibial and vertebral BMC measured by DXA in vivo
compared with vehicle-treated controls. However, total
BMC corrected for the increase in body weight (total
BMC:body weight ratio) was unaffected. Tibial area bone
mineral density (BMD, BMC/area) was increased, but
total and vertebral area BMDs were unchanged. The
pQCT measurements in vitro revealed that the increase in
the cortical BMC was due to an increased cross-sectional
bone area, whereas the cortical volumetric BMD was
unchanged. Femur and vertebra L6 volumes were in-
creased but no effect was seen on the volumetric BMDs as
measured by Archimedes’ principle. Ash weight was
increased by all treatments, but the mineral concentration
was unchanged. We conclude that treatment of adult
female rats with the GHSs ipamorelin and GHRP-6
increases BMC as measured by DXA in vivo. The results of
in vitro measurements using pQCT and Archimedes’
principle, in addition to ash weight determinations, show
that the increases in cortical and total BMC were due to an
increased growth of the bones with increased bone dimen-
sions, whereas the volumetric BMD was unchanged.
Journal of Endocrinology (2000) 165, 569–577
Introduction
Both clinical and animal studies have demonstrated that
growth hormone (GH) is important for normal bone
modelling and remodelling (Bouillon 1991, Eriksen et al.
1996, Ohlsson et al. 1998). In young adult rats, GH
treatment increases bone mass by increased longitudinal
growth at the growth plate and by increased subperiosteal
bone deposition (Ohlsson et al. 1998). In old male rats, 80
days of GH treatment increased cortical bone formation
mainly through increased subperiosteal bone formation
(Andreassen et al. 1995). In young and in old rats, GH
treatment increased cortical mechanical strength, mainly as
a result of an increase in bone dimensions ( Jørgensen et al.
1991, Andreassen et al. 1995). In primates, GH but not
insulin-like growth factor (IGF)-I given to female mon-
keys for 7 weeks increased bone formation as measured
with mineral apposition rate, and the bone formation
rate (Sass et al. 1997). In GH-deficient (GHD) humans,
long-term GH treatment increased total bone mineral
content (BMC) and area bone mineral density (BMD) in
several weight-bearing skeletal locations (Johannsson et al.
1996). Studies to date have not shown any impressive
effect of GH on bone mass in non-GHD subjects.
The mechanism for the stimulatory effect of GH on
the amount of bone is not fully understood. Normal
bone metabolism is regulated by a balance between bone
formation caused by osteoblasts and bone resorption caused
569
Journal of Endocrinology (2000) 165, 569–577
0022–0795/00/0165–569 2000 Society for Endocrinology Printed in Great Britain
Online version via http://www.endocrinology.org
by osteoclasts. Osteoblastic cells, in addition to being
directly responsible for bone formation, control bone
resorption by regulating the proliferation, differentiation
and recruitment of osteoclast progenitors (Takahashi et al.
1988). Human osteoblast-like cells express functional GH
receptors and GH has been shown to exert an anabolic
effect on the osteoblasts (Kassem et al. 1993, Nilsson et al.
1995). These findings indicate a direct stimulatory effect of
GH on bone formation.
Pituitary GH secretion is regulated by the stimulatory
peptide GH-releasing hormone (GHRH) and the in-
hibitory peptide somatostatin (Frohman & Jansson 1986).
The new class of synthetic GH secretagogues (GHSs) that
has been developed includes GH-releasing peptides (GH-
RPs) such as GHRP-2, GHRP-6 and hexarelin, and
non-peptidyl oral GHSs such as MK-677 (Smith et al.
1998). A G protein-coupled receptor has been found to be
a target for these substances (Howard et al. 1996). An
endogenous ligand specific for the GHS receptor was
recently described (Kojima et al. 1999). At the hypothal-
amic level, the GHSs stimulate GH secretion by acti-
vating arcuate nucleus GHRH neurones (Dickson &
Luckman 1997), resulting in increased release of GHRH
into portal blood (Guillaume et al. 1994). The GHSs also
stimulate GH secretion by a direct action at the pituitary
level (Bowers et al. 1984), where the GHSs may act as
functional antagonists of somatostatin action (Smith et al.
1998). The stimulatory effect on GH release seems to be
persistent, as the GHRPs hexarelin and GHRP-2 have
been reported to increase growth velocity in children with
short stature/GHD (Laron et al. 1995, Mericq et al. 1998).
In addition to GH release, most GHSs have been found to
elicit a transient cortisol response (Svensson et al. 1998a).
However, the novel GHS, ipamorelin, has not yet been
found to stimulate glucocorticoid release (Raun et al.
1998).
The GHSs enhance the amplitude of the physiological
GH pulses (Smith et al. 1998), whereas an s.c. injection of
GH induces a bell-shaped curve of serum GH concen-
trations that are continuously high for more than 12 h
(Oscarsson et al. 1997). Little is known about the effects on
bone of an increase in the amplitude of endogenous
pulsatile GH secretion. We have recently shown that
short-term treatment of obese men with the oral GHS,
MK-677, increases circulating biochemical markers of
bone formation and bone resorption (Svensson et al.
1998b). The aim of the present study was to determine the
effect of 12 weeks of treatment with the GHSs ipamorelin
and GHRP-6 on bone mass in young adult female
Sprague–Dawley rats.
Material and Methods
Animals
Female Sprague–Dawley rats (n=30; 13 weeks old, mean
body weight 228 g at onset; obtained from BK Universal,
Stockholm, Sweden) were housed under standardised
environmental conditions (24–26 C, 50–60% relative
humidity, artificial lighting on from 0500 to 1900 h). The
rats were given free access to water and to standard rat
chow containing 0·8% calcium and 0·6% phosphorus (BK
Universal). All experimental procedures were carried out
according to ethical guidelines for animal experimentation
at the University of Göteborg.
Study procedure
Over a 12-week period, rats were infused s.c. with human
GH (3·5 mg/kg per day; Norditropin; Novo Nordisk A/S,
Copenhagen, Denmark; n=7), GHRP-6 (0·5 mg/kg per
day, Bachem, Bubendorf, Switzerland; n=8), ipamorelin
(0·5 mg/kg per day; Novo Nordisk A/S; n=7) or vehicle
(0·05% acetic acid in sterile 0·5% saline; n=8) using
osmotic minipumps (Alzet 2004; Alza Corp., Palo Alto,
CA, USA). In vivo dual X-ray absorptiometry (DXA)
measurements were performed at baseline and after 4, 8
and 12 weeks of treatment. Before the DXA measure-
ments, the rats were anaesthetised with a combination of
ketamine (1·8 ml/kg; Ketalar, Park Davies, Morris Plains,
NJ, USA; 67 mg/kg) and xylazine (13 mg/kg; Rompun,
Bayer AG, Leverkusen, Germany) given i.p. During
anaesthesia, the osmotic minipumps were removed and
body weight and DXA measurements were performed,
followed by implantation of new osmotic minipumps.
Food intake was measured at baseline and after 4, 8 and 12
weeks of treatment by the use of metabolic cages. At the
end of the experiment, rats were killed by decapitation and
excised bones were stored in ethanol, then excised femurs
were analysed by using peripheral quantitative computed
tomography (pQCT). After this, femurs and vertebrae L6
were analysed by the use of Archimedes’ principle and by
determinations of ash weights.
Dual X-ray absorptiometry
BMC (in g) and area BMD (in g/cm
2
) were measured
in vivo by DXA (pDEXA Sabre; Norland Corp., Fort
Atkinson, WI, USA). The software Sabre Research
(version 3.6) was used. Total body BMC and area BMD
(BMC/area) were measured from the lower level of
thoracic vertebra 9 to the inferior level of the first tail
vertebra (the maximal scan area of the DXA). BMC and
area BMD of tibia were measured from the knee joint to
the joint between tibia and calcaneus (and thus these
measurements also include fibula). BMC and area BMD of
vertebra were measured from the superior level of lumbar
vertebra 3 to the inferior level of lumbar vertebra 6
(L3–L6). The interassay coefficients of variation (CVs) for
the DXA measurements were less than 4%.
Femur length and vertebral body L6 height
The length of femurs was determined in vitro with DXA.
The femurs were placed in an anatomical position (facing
J SVENSSON and others · GH secretagogues stimulate bone mineral content570
www.endocrinology.orgJournal of Endocrinology (2000) 165, 569–577
anteriorly) in a Petri dish. The length of each femur was
determined by lining up the cursors on the screen with the
trochanter at the top and the lateral condyle at the bottom
of the femur. The distance between the cursors was
measured by a ruler tool in the software. The CV was less
than 1%.
The body of vertebra L6 was cleaned of all soft tissue,
including the intravertebral discs. Then the height of
vertebral body L6 was determined at the middle of the
anterior surface using a micrometer. The CV was less
than 1%.
Peripheral quantitative computed tomography
Tomographic measurements were performed using the
Stratec pQCT XCT Research M (Norland Corp.) specifi-
cally modified for use on small bone specimens (software
version 5.4B; resolution 70 μm (Windahl et al. 1999)).
Cortical volumetric BMD (BMC/volume), the cortical
cross-sectional area and cortical BMC were determined by
mid-diaphyseal pQCT scans of excised femurs. The inter-
assay CVs for the pQCT measurements were less than 3%.
Archimedes’ principle and ash weights
The whole femurs and vertebrae were immersed in
distilled water and submitted to vacuum for at least 10 min
to remove trapped air bubbles. Then the wet weight and
the weight of the bones in distilled water were deter-
mined. After the bones had been defatted in acetone for 2
days followed by freeze-drying for 3 days, the dry defatted
weight was determined (Sandstedt et al. 1996). The (real)
volume was calculated as (dry defatted weightweight in
distilled water)/density of distilled water. The (real) volu-
metric BMD was calculated as dry defatted weight/(real)
volume of the bone. The ash weight (mineral weight) was
determined after incineration in a muffle oven at 105 C
for 2 h, followed by 580 C for 24 h. The mineral con-
centration was calculated by dividing mineral weight by
dry defatted weight.
Metabolic cages
The rats were housed for 2 days in metabolic cages at
baseline and after 4, 8 and 12 weeks of treatment (before
the DXA measurements at each time point). Water and
powdered food were freely available and food intake was
measured daily.
Statistics
All the descriptive statistical results that are presented are
given as the arithmetic mean and standard error of the
mean (...). Bartlett’s test was performed to evaluate if
data were normally distributed. For skewly distributed data
(cortical BMD, cross-sectional cortical bone area and
cortical BMC obtained from the pQCT measurements of
femurs in vitro), a logarithmic transformation was per-
formed before the analysis of between-group differences.
For body weight and for data obtained from DXA
measurements in vivo, a two-way analysis of variance
(ANOVA) of the percent change from baseline were
performed with time and treatment as the independent
variables, followed by Student–Newman–Keuls post hoc
test. The data obtained from in vitro measurements
(pQCT, Archimedes’ principle, ash weight) were analysed
by a one-way ANOVA with treatment as the independent
variable, followed by Student–Newman–Keuls post hoc
test. A two-tailed P< 0·05 was considered significant.
Results
Body weight, food intake and dimensions of bones
Baseline body weight was similar in all study groups.
However, the increase in body weight was larger after all
Table 1 Body weight and bone dimensions after 12 weeks of treatment of female Sprague–Dawley rats with IPA, GHRP-6, GH or vehicle.
All values are presented as the mean (S.E.M.)
Vehicle
(n=8)
IPA
(n=7)
GHRP-6
(n=8)
GH
(n=7)
Variable
Body weight at baseline (g) 234 (8) 223 (9) 230 (8) 223 (8)
Body weight after 12 weeks of treatment (g) 320 (8) 340 (9)** 354 (5)** 338 (5)**
Body weight increase (%) 37·6(3·5) 52·9(5·9)** 55·1(4·9)** 53·3(5·8)**
Femur length (mm) 37·2(0·4) 37·9(0·2) 38·4(0·2)* 37·8(0·2)
Vertebral body L6 height (mm) 7·67 (0·07) 7·82 (0·10) 7·85 (0·05) 7·74 (0·08)
For body weight after 12 weeks of treatment, P-values (compared with vehicle) were calculated by two-way ANOVA of the percent change from baseline,
followed by Student–Newman–Keuls post hoc test. P values for differences in femur length and vertebral body L6 height were calculated by one-way
ANOVA followed by Student–Newman–Keuls post hoc test.
*P<0·05, **P<0·01, compared with vehicle.
GH secretagogues stimulate bone mineral content · J SVENSSON and others 571
www.endocrinology.org Journal of Endocrinology (2000) 165, 569–577
treatments than after vehicle (Table 1). Food intake was
unchanged (data not shown). Femur length was increased
in the GHRP-6-treated rats compared with vehicle-
treated rats, but that in the GH- and IPA-treated rats was
not statistically changed (Table 1). The height of vertebral
body L6 was unchanged (Table 1).
DXA measurements of BMC in vivo
There were no differences at baseline in any of the bone
parameters measured by DXA (data not shown). As
measured by DXA in vivo, both of the GHSs (IPA and
GHRP-6) and GH increased total, tibial and vertebral
BMCs compared with vehicle (Fig. 1a–c). However,
Figure 1 Percentage change in (a) total body BMC, (b) tibia BMC, (c) vertebral BMC (L3–L6), and (d) total
BMC/body weight ratio during 12 weeks of treatment of female Sprague–Dawley rats with IPA, GHRP-6,
GH or vehicle. The BMC values were measured by DXA in vivo. The P values shown (compared with
vehicle) were calculated by two-way ANOVA of the percent change from baseline, followed by
Student–Newman–Keuls post hoc test.
J SVENSSON and others · GH secretagogues stimulate bone mineral content572
www.endocrinology.orgJournal of Endocrinology (2000) 165, 569–577
when the increase in total BMC was corrected for the
increase in body weight (total BMC/body weight ratio),
the increase in total BMC lost statistical significance (Fig.
1d). Spine and tibia BMC also were unchanged when
corrected for body weight (data not shown).
All substances increased the tibial area BMD, as
measured by DXA, but no significant effect was seen on
total and vertebral area BMDs (Fig. 2a–c).
Effects on cortical bone as measured using pQCT
Mid-diaphyseal pQCT scans of excised right femurs
demonstrated that the cortical BMC was increased in the
IPA and GH groups (IPA P< 0·05 compared with vehicle;
GH P< 0·05 compared with vehicle; GHRP-6 P=0·06
compared with vehicle; Table 2). The increase in mid-
diaphyseal cortical BMC was due to an increase in
Figure 2 Percent change in (a) total body BMD, (b) tibia BMD, and (c) vertebral BMD (L3–L6) as measured
by DXA in vivo during 12 weeks of treament of female Sprague–Dawley rats with IPA, GHRP-6, GH or
vehicle. No effect was found on total or vertebral BMD, but all treatments induced a significant increase in
tibia BMD compared with the vehicle-treated controls. The P values shown (compared with vehicle) were
calculated by two-way ANOVA of the percent change from baseline, followed by Student–Newman–Keuls
post hoc test.
GH secretagogues stimulate bone mineral content · J SVENSSON and others 573
www.endocrinology.org Journal of Endocrinology (2000) 165, 569–577
cross-sectional cortical bone area, whereas the cortical
volumetric BMD was unaffected (Table 2).
Archimedes’ principle and ash weight
The dry defatted weight of femur and vertebra L6 was
increased by all treatments compared with the vehicle-
treated controls (Table 3). Measurements using Archimedes’
principle showed that the volumes of femur and vertebra
L6 were increased, but the volumetric BMD was
unchanged (Table 3). Ash weight was increased for all
treatments, but the mineral concentration, determined
as ash weight per dry defatted weight, was unchanged
(Table 3).
Discussion
The present study demonstrates that treatment with the
GHSs, ipamorelin and GHRP-6, increases BMC in
normal adult female rats. The results of several different
measurements clearly indicate that the increases in cortical
and total BMC were caused by increased bone volume,
whereas the volumetric BMD was unchanged. The
pQCT measurements of femurs in vitro showed an increase
in cortical BMC that was due to an increase in the cross
sectional bone area. Femur and vertebra L6 volumes were
increased as measured by Archimedes’ principle, whereas
no effect was seen on the volumetric BMDs. Finally,
ash weight (i.e. mineral content) was increased in all
treatment groups, whereas the mineral concentration was
unchanged.
In the present study, treatment with the GHSs and with
GH itself increased body weight compared with vehicle,
suggesting that the doses used and the route of ad-
ministration of treatments were effective. The sustained
increase in body weight in the GH-treated rats made it
possible to rule out the possibility of species-dependent
development of active antibodies against the human GH.
This finding is supported by several previous studies
Table 2 Cortical BMD, cross-sectional cortical bone area and cortical BMC, as measured by mid-diaphyseal pQCT scans of femurs in vitro,
after 12 weeks of treatment of female Sprague–Dawley rats with IPA, GHRP-6, GH or vehicle. All values are presented as the mean (S.E.M.)
Vehicle
(n=8)
IPA
(n=7)
GHRP-6
(n=8)
GH
(n=7)
Variable
Cortical BMD (mg/mm
3
)1·355 (0·004) 1·355 (0·004) 1·348 (0·004) 1·357 (0·004)
Cross-sectional cortical bone area (mm
2
)5·50 (0·15) 5·92 (0·09)* 5·80 (0·11)* 5·92 (0·07)*
Cortical BMC (mg/mm) 7·46 (0·22) 8·02 (0·12)* 7·82 (0·16)** 8·04 (0·09)*
Between-group differences were calculated by one-way ANOVA followed by Student–Newman–Keuls post hoc test.
*P<0·05, **P=0·06, compared with vehicle.
Table 3 Results of in vitro measurements using Archimedes’ principle and ash weight determinations after 12 weeks of treatment of female
Sprague–Dawley rats with IPA, GHRP-6, GH or vehicle. All values are presented as the mean (
S.E.M.)
Vehicle
(n=8)
IPA
(n=7)
GHRP-6
(n=8)
GH
(n=7)
Variable
Dry defatted weight (mg)
Femur 580·2(13·8) 624·0(9·9)* 613·8(7·7)* 605·2(7·9)*
Vertebra L6 191·8(5·4) 216·4(3·8)** 207·2(3·0)* 203·4(3·0)*
Bone volume (mm
3
)
Femur 255·8(5·6) 274·8(4·4)* 268·4(3·7)* 270·4(3·8)*
Vertebra L6 86·1(2·5) 99·1(1·8)** 94·8(1·6)** 94·4(1·8)**
Volumetric BMD (g/cm
3
)
Femur 2·27 (0·01) 2·27 (0·01) 2·29 (0·01) 2·24 (0·02)
Vertebra L6 2·23 (0·02) 2·18 (0·02) 2·19 (0·02) 2·16 (0·03)
Ash weight (mg)
Femur 407·7(8·4) 437·1(4·5)** 430·6(4·7)* 427·0(4·3)*
Vertebra L6 131·2(3·3) 148·3(2·5)** 143·9(2·7)** 141·6(2·3)**
Mineral concentration†
Femur 70·3(0·3) 70·1(0·8) 70·2(0·2) 70·6(0·4)
Vertebra L6 68·5(0·5) 68·6(0·7) 69·4(0·8) 69·6(0·6)
Between-group differences were calculated by one-way ANOVA followed by Student–Newman–Keuls post hoc test.
†Ash weight/dry defatted weight.
*P<0·05, **P<0·01, compared with vehicle.
J SVENSSON
and others · GH secretagogues stimulate bone mineral content574
www.endocrinology.orgJournal of Endocrinology (2000) 165, 569–577
demonstrating a sustained effect of human GH on BMC in
rats (Andreassen et al. 1995, 1996). A desensitisation of the
initial GH response has previously been observed after
repeated GHS administration (Smith et al. 1998). In
contrast, in the present study, a sustained increase in body
weight and BMC was seen for IPA and GHRP-6,
indicating that GHSs can also exert stimulatory effects in
treatment prolonged over several months.
Total food intake was unaffected by both GHS and GH
treatment in the present study. An acute increase in food
intake has been observed after intracerebroventricular
administration of GHSs (Locke et al. 1995, Okada et al.
1996), but in this study, as in a previous clinical study
(Svensson et al. 1998a), no effect has been observed after
prolonged systemic or oral administration of GHSs. GH
administration is known to increase food efficiency in rats
(weight gain/food weight (Verrat-Durebex et al. 1999)).
Therefore, increased food efficiency due to increased
circulating GH is a likely explanation for the increases in
body weight induced by all treatments in this study.
GH treatment and GHS treatment increased tibia area
BMD as measured by DXA in vivo in the present study.
However, the pQCT measurements of femurs in vitro
revealed an unaffected cortical volumetric BMD and the
measurements using Archimedes’ principle showed an
unchanged total femur and vertebra L6 volumetric BMD.
Other studies have also shown that long-term GH treat-
ment increases area BMD as measured by DXA (Yeh et al.
1994, Ohlsson et al. 1998), but the volumetric BMD
(BMC/volume), as measured by Archimedes’ principle or
pQCT ( Jørgensen et al. 1991, Andreassen et al. 1995,
Rosen et al. 1995), is unchanged. These findings could be
explained by an overestimation of the increase in BMD by
DXA. Area BMD (BMC/area) does not account for the
bone thickness perpendicular to the two-dimensional
DXA image. Therefore, when cortical bone mass is
increased because of enhanced subperiosteal bone depo-
sition, this itself may result in enhanced area BMD as
measured by DXA.
The increases in BMCs were of a magnitude similar to
that in body weight. In almost all studies in rats, GH
administration has resulted in increased body weight
(Ohlsson et al. 1998). It can not be excluded that the
increase in BMC is due to increased mechanical load
exerted on the skeleton by the increased body weight.
However, administration of GH or IGF-I also increased
subperiosteal bone formation under weightless conditions
during space flight, and the amount of added bone was
similar to that obtained by GH or IGF-I administration
on the ground (Turner 1995, Bateman et al. 1998).
Therefore, the present increase in bone dimensions is
likely to be due to a direct effect of GH on total bone
growth. This may be caused by a radial bone growth
resulting from increased subperiosteal bone formation. GH
has been shown to increase subperiosteal bone deposition
in both young and old adult rats (Andreassen et al. 1995,
Ohlsson et al. 1998), and an increased cross-sectional
cortical bone area was also seen in this study.
The present increase in bone volume may also have
been due to linear bone growth derived from the growth
plate. In rats, the growth plates first begin to close when
the rats are very old (Ohlsson et al. 1998). For this reason,
most studies have also found a small stimulatory effect on
longitudinal growth in adult rats (Ohlsson et al. 1998).
Therefore, it is likely that linear bone growth was also
stimulated in our present study. The measurements of
femur length and vertebral body L6 height only showed a
significant increase for femur height in the GHRP-6-
treated rats. However, small stimulatory effects on linear
bone growth may not have been detected statistically in
the GH- and ipamorelin-treated groups, as a result of the
study design (seven or eight rats in each group).
GH administered by osmotic minipumps induces a
continuous increase in circulating GH concentrations,
whereas the GHSs, also when administered continuously,
increase the amplitude of the endogenous GH pulses
(Huhn et al. 1993, Jaffe et al. 1993). Furthermore, it has
been suggested that different subtypes of the recently
cloned GHS receptor may exist and that different GHSs
may differ in their binding affinity for the different
subtypes (Ong et al. 1998). Despite these differences, the
effects on body growth and BMC were similar for all
treatments with the doses and the route of administration
used in the present study. There are also non-peptidyl
GHSs that produce a potent GH response after oral
administration (Ankersen et al. 1998, Hansen et al. 1998,
Smith et al. 1998). Further studies are needed to investi-
gate whether orally administered non-peptidyl GHSs also
increase BMC.
In a previous clinical study we found that a 2-month
treatment with the GHS, MK-677, increases circulating
markers of bone formation and resorption (Svensson et al.
1998b). We have now shown that treatment of female rats
with the GHSs ipamorelin and GHRP-6 for 3 months
increased BMC as measured by DXA in vivo. The results
of in vitro measurements using pQCT and Archimedes’
principle, in addition to the ash weight determinations,
show that the increases in cortical and total BMC were due
to an increased growth of the bones with increased bone
dimensions, the volumetric BMD remaining unchanged.
Acknowledgements
This study was supported by Novo Nordisk A/S, the
Swedish Medical Research Council (9894 and 11621),
the British Medical Council (G 9521630), the Lundberg
Foundation, the Bergvall Foundation, the Swedish Medical
Society, the Göteborg Medical Society, and the Swedish
Association Against Rheumatic Diseases.
Presented in part at the 1998 Growth Hormone
Research Society (GRS) Conference, San Francisco,
USA, September 1998.
GH secretagogues stimulate bone mineral content · J SVENSSON and others 575
www.endocrinology.org Journal of Endocrinology (2000) 165, 569–577
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... Despite the ability of GHSs to generate impressive pulsatile GH secretion, attempts to stimulate growth in animals by GHS exposure have shown only modest body weight gain, mostly by once daily administration (Bowers e ta l, 1984;Hansen et al, 1999;McDowell et a l, 1995;Svensson et al, 2000). Attempts to stimulate additional growth in animals must overcome the problem that the GH responses to GHSs do sometimes desensitise with time (Jacks et a l, 1994). ...
... Despite the ability of GHSs to generate impressive pulsatile GH secretion, attempts to stimulate additional growth in animals and humans have not been as impressive. Still, several studies in rats have shown modest body weight gain in response to GHS treatment, mostly by once daily oral administration (Bowers et a l, 1984;Hansen et a l, 1999;Svensson et a l, 2000;McDowell et a l, 1995). Appropriate animal models for studying the growth promoting action of GHSs in the context of dwarfism have not been available. ...
... infusion (Wells et a l, 2000; Wells et a l, 1997) and continuous s.c. infusion (Svensson et a l, 2000). ...
Nina Balthasar
The synthetic growth hormone secretagogues (GHS) exert their growth hormone (GH) releasing effects by activating their receptor (GHS-R) in hypothalamic neurones and pituitary GH cells. The aim of this study was to investigate hypothalamic and pituitary GHS physiology by in vitro studies and transgenesis, over-expressing the GHS-R in hypothalamic GH releasing hormone (GHRH) neurones or pituitary GH cells. Initially, to analyse transgene expression driven by two targeting cosmids - a rat GHRH or a human GH cosmid - transgenic mice expressing enhanced green fluorescent protein (eGFP) were generated. In rGHRH-eGFP transgenic mice eGFP protein was visible in brightly fluorescing GHRH neurone cell bodies and median eminence nerve terminals. hGH-eGFP transgenic mice showed high levels of eGFP fluorescence limited to GH expressing pituitary cells. These mice provide useful new tools to study GHRH and GH cell physiology. In vitro studies with a stably GHS-R transfected GH cell line showed that processing of the GHS-R cDNA resulted in a functionally active receptor. In transgenic mice overexpression of the GHS-R in GHRH neurones caused alteration of the GH axis with increased pituitary GH content and body weight. GH responses to a GHS injection seemed enhanced. However, prolonged GHS exposure did not cause enhanced body weight gain compared to WT littermates. These studies suggested that enhancement of GHS-R abundance in GHRH neurones caused alteration of the GH and GHS axis, implicating GHRH neurones as an important GHS target in GH release. In contrast, transgenic mice with 50-fold GHS-R over-expression in GH cells showed reduced pituitary GH content, body weight and GH responses to GHRH. These data suggest that somatotroph function might be compromised by high levels of GHS-R over-expression. Crossing the hGHS-R over-expressing mice with their eGFP counterpart mice will facilitate the further investigation of their physiological phenotype by enabling in vivo analysis of GHS effects directly in identified cells.
... De-fattening is an important procedure carried out so as to prevent bones from looking dirty, attracting dust and also to make bleaching easier. Historically many chemicals have been used as a form of de-fattening agent but the most widely accepted is acetone which is currently used till date [1]. Acetone is a colorless liquid with a density of 0.79g/cm [2], a melting point of -94.9 0 C and a boiling point of 56.53 0 C. It is a compound with the formula OC (CH 3 ) 2 and is miscible in water with a viscosity of 0.32cp (20 0 C). ...
... Owing to the fact that acetone is miscible with water, it serves as an important solvent in its own right, typically as the solvent of choice for cleaning purposes in the laboratory. Some prefer to de-fatten using the mixture of acetone and dehydrated ether [3] while some prefer to use it alone, when using it alone the bone sample is immersed in 100% acetone for one hour after maceration so as to remove potentially superposed adipose tissue before bleaching [1]. ...
Huthman Oluwaseun Ibrahim Ghandi Abdur Razak Nagwari Umar Huthman Adebukola Selimot
Sunlight, which in broad sense is the total frequency spectrum of electromagnetic radiation given off by the sun. The experiment was carried out in order to appraise the de-fattening effect of sunlight on various animal bones as compared to conventional de-fatteners (acetone and kerosene). The bones were divided into four groups: Group one-to be de-fattened with acetone after maceration, Group two-to be de-fattened with kerosene after maceration, Group three-to be exposed to sunlight after maceration in order to evaluate the de-fattening effects and Group four-to be neither be de-fattened nor exposed to sunlight after maceration. All the animals were then killed in a humane way by carbon dioxide asphyxiation, after which they were de-fleshed with a scalpel and macerated in boiling detergent for an hour. The bones in group one were de-fattened with acetone for 4 hours, the bones in group two were de-fattened with kerosene for 4 hours, the bones in group three were exposed to sunlight for 6 hours while the bones from group four were not de-fattened with any de-fattening agent and were also kept away from sunlight as a control. Afterwards the de-fattening effects were then compared by a using a filter paper and by visual observation. Our preliminary observation showed that sunlight had a strong de-fattening effect on the bones, and this effect competed favorably with the bones de-fattened with acetone and kerosene. By these preliminary observations, sunlight may economically and functionally replace acetone and kerosene in biomedical analysis and osteological preparation.
... injection of the synthetic peptide GHRP2 (23). In adult rats, GHRP6 also increased body weight (24) as well as other GHSR agonists, like SM-130686 administered orally (25) and BIM-28131 administered by s.c. injection (26). ...
Rebeca Martínez
Katerina Gonzalez Alain Gonzalez
Mario Pablo Estrada
Growth hormone (GH) has pleiotropic functions in all vertebrates. In addition to its essential role in the regulation of body growth and development, it can also influence reproduction, immunity, osmoregulation, and behavior Immune and neuroendocrine systems have bidirectional communications and it is well document the enhancing action of GH on teleost immune system .It is desirable controlled GH administration to allow growth and stimulation of the innate immune system of fish. In this study, we have characterized a chemical peptide compound, A228, designed by molecular modeling, which is able to perform the function of a GH peptide secretagogue. In pituitary cell culture, the peptide A228 induces GH secretion. It is able to increase superoxide production in tilapia peripheral blood leukocytes cultures and in a macrophage cell line J774 from mice, therefore using this molecule, innate immune system stimulation is obtained in vitro both in fish and in mammals cell cultures. In this paper is also shown the biological action in vivo of the molecule, to assess growth stimulation in tilapia larvae. RESUMEN La hormona del crecimiento (GH) tiene funciones pleiotrópicas en todos los vertebrados. Además de su papel esencial en la regu-lación del crecimiento y el desarrollo del cuerpo, sino que también puede influir en la reproducción, la inmunidad, la osmorregu-lación, y el comportamiento inmunológico y los sistemas neuroendocrinos tener comunicaciones bidireccionales y es documento bien la acción de mejorar la GH sobre el sistema inmune teleósteo .Es es la administración de GH controlada deseable permitir el crecimiento y la estimulación del sistema inmune innato de los peces. En este estudio, hemos caracterizado un compuesto peptídico química, A228, diseñado por modelado molecular, que es capaz de realizar la función de un péptido secretagogo de GH. En el cultivo de células pituitaria, el A228 péptido induce la secreción de GH. Es capaz de aumentar la producción de superóxido en tilapia periféricos culturas leucocitos de la sangre y en una línea celular de macrófagos J774 de ratones, por lo tanto, el uso de esta molécula, la estimulación del sistema inmune innato se obtiene in vitro tanto en los peces y en cultivos de células de mamíferos. En este documento también se muestra la acción biológica in vivo de la molécula, para evaluar la estimulación del crecimiento en larvas de tilapia. Palabras clave: actividad biológica, scretagogo, hormona del crecimiento, línea celular murina.
... 119 A study in rats showed that daily s.c. ipamorelin (0.5 mg/kg) and GHreleasing peptide-6 (0.5 mg/kg), a synthetic analogue of hexarelin where 2-methyl-D-Trp is substituted for D-Trp, increased cortical and total bone mass to a similar extent as caused by GH. 120 However, unlike GHRP-6, which increases plasma levels of cortisol, ipamorelin showed selectivity for GH release. 121 ...
Zaid Amso
Jillian Cornish
Margaret A Brimble
Loss of bone occurs in the age-related skeletal disorder, osteoporosis, leading to bone fragility and increased incidence of fractures, which are associated with enormous costs and substantial morbidity and mortality. Recent data indicate that osteoporotic fractures are more common than other diseases, which usually attract public attention (e.g., heart attack and breast cancer). The prevention and treatment of this skeletal disorder are therefore of paramount importance. Majority of osteoporosis medications restore skeletal balance by reducing osteoclastic activity, thereby reducing bone resorption. These agents, however, do not regenerate damaged bone tissue, leaving limited options for patients once bone loss has occurred. Recently, attention has turned to bone-anabolic agents. Such agents have the ability to increase bone mass and strength, potentially reversing structural damage. To date, only one bone-anabolic drug is available in the market. The discovery of more novel, cost-effective bone anabolic agents is therefore a priority to treat those suffering from this disabling condition. Short peptides offer an important alternative for the development of novel bone-anabolic agents given their high target binding specificity, which translates into potent activity with limited side effects. This review summarizes attempts in the identification of bone-anabolic peptides, and their development for promoting bone growth.
... In vivo studies showed that intraperitoneal [5] as well as central administration [12] of ghrelin increases bone mineral density and mass through a mechanism independent of GH-IGF1 axis and appetite regulation, respectively. Consistently, synthetic GH secretagogues increase bone mineral density [13] and prevent gonadectomy-induced bone loss in the rat [14,15]. Data from clinical studies are limited but indicate a positive association between circulating ghrelin levels and bone mineral density in women [16,17] and elderly men [18]. ...
Emanuela Mrak
Lavinia Casati
Francesca Pagani
Valeria Sibilia
Ghrelin, by binding growth hormone secretagogue receptor (GHS-R), promotes osteoblast proliferation but the signaling mechanism of GHS-R on these cells remains unclear. Since canonical Wnt/β-catenin pathway is critically associated with bone homeostasis, we investigated its involvement in mediating ghrelin effects in osteoblasts and in osteoblast-osteoclast cross talk. Ghrelin (10(-10)M) significantly increased β-catenin levels in rat osteoblasts (rOB). This stimulatory action on β-catenin involves a specific interaction with GHS-R1a, as it is prevented by the selective GHS-R1a antagonist, D-Lys(3)-GHRP-6 (10(-7)M). The effect of ghrelin on β-catenin involves the phosphorylation and inactivation of GSK-3β via protein kinase A (PKA). Inhibition of PKA activity reduces the facilitatory action of ghrelin on β-catenin stabilization. Ghrelin treatment of rOB significantly increases the expression of osteoprotegerin (OPG), which plays an important role in the regulation of osteoclastogenesis, and this effect is blocked by D-Lys(3)-GHRP-6. Furthermore, ghrelin reduced RANKL/OPG ratio thus contrasting osteoclastogenesis. Accordingly, conditioned media from rOB treated with ghrelin decreased the number of multinucleated TRAcP+ cells as compared with the conditioned media from untreated-control rOB. Our data suggest new roles for ghrelin in modulating bone homeostasis via a specific interaction with GHSR-1a in osteoblasts with subsequent enhancement of both β-catenin levels and OPG expression.
... Long-term GHS and ghrelin treatments cause modest increases in body weight in a variety of rodent models (19, [55][56][57]. Although GHS effects on body weight were initially attributed to their GH-releasing effects, it is now clear that a significant proportion of the weight gain is due to increased body fat, and reflects GH-independent effects (2,19). ...
... Among the disorders described in AN, osteopenia/osteoporosis is also one major problem that cause long term outcomes with in particular a strong increase of the bone fracture incidence (Lucas et al., 1999). Ghrelin has and ghrelin mimetics have been shown to increase bone mineral density (Svensson et al., 2000;Fukushima et al., 2005;Delhanty et al., 2006) by a mechanism that appears to include the promotion of both proliferation and differentiation of osteoblasts (cells involved in bone formation), involving GHS-R1a and GHS-R1b receptors (Fukushima et al., 2005;Delhanty et al., 2006). The etiopathogenesis of bone disease in AN is complex and multifaceted. ...
Mathieu Méquinion
Fanny Langlet Sara Zgheib
Odile Viltart
Increasing clinical and therapeutic interest in the neurobiology of eating disorders reflects their dramatic impact on health. Chronic food restriction resulting in severe weight loss is a major symptom described in restrictive anorexia nervosa (AN) patients, and they also suffer from metabolic disturbances, infertility, osteopenia, and osteoporosis. Restrictive AN, mostly observed in young women, is the third largest cause of chronic illness in teenagers of industrialized countries. From a neurobiological perspective, AN-linked behaviors can be considered an adaptation that permits the endurance of reduced energy supply, involving central and/or peripheral reprograming. The severe weight loss observed in AN patients is accompanied by significant changes in hormones involved in energy balance, feeding behavior, and bone formation, all of which can be replicated in animals models. Increasing evidence suggests that AN could be an addictive behavior disorder, potentially linking defects in the reward mechanism with suppressed food intake, heightened physical activity, and mood disorder. Surprisingly, the plasma levels of ghrelin, an orexigenic hormone that drives food-motivated behavior, are increased. This increase in plasma ghrelin levels seems paradoxical in light of the restrained eating adopted by AN patients, and may rather result from an adaptation to the disease. The aim of this review is to describe the role played by ghrelin in AN focusing on its central vs. peripheral actions. In AN patients and in rodent AN models, chronic food restriction induces profound alterations in the « ghrelin » signaling that leads to the development of inappropriate behaviors like hyperactivity or addiction to food starvation and therefore a greater depletion in energy reserves. The question of a transient insensitivity to ghrelin and/or a potential metabolic reprograming is discussed in regard of new clinical treatments currently investigated.
Elisa Dieci
Lavinia Casati
Francesca Pagani
Valeria Sibilia
Increasing evidence suggests a role for oxidative stress in age-related decrease in osteoblast number and function leading to the development of osteoporosis. This study was undertaken to investigate whether ghrelin, previously reported to stimulate osteoblast proliferation, counteracts tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in MC3T3-E1 osteoblastic cells as well as to characterize the ghrelin receptor (GHS-R) involved in such activity. Pretreatment with ghrelin (10(-7)-10(-11 )M) significantly increased viability and reduced apoptosis of MC3T3-E1 cells cultured with t-BHP (250 μM) for three hours at the low concentration of 10(-9 )M as shown by MTT assay and Hoechst-33258 staining. Furthermore, ghrelin prevented t-BHP-induced osteoblastic dysfunction and changes in the cytoskeleton organization evidenced by the staining of the actin fibers with Phalloidin-FITC by reducing reactive oxygen species generation. The GHS-R type 1a agonist, EP1572 (10(-7)-10(-11 )M), had no effect against t-BHP-induced cytotoxicity and pretreatment with the selective GHS-R1a antagonist, D-Lys(3)-GHRP-6 (10(-7 )M), failed to remove ghrelin (10(-9) M)-protective effects against oxidative injury, indicating that GHS-R1a is not involved in such ghrelin activity. Accordingly, unacylated ghrelin (DAG), not binding GHS-R1a, displays the same protective actions of ghrelin against t-BHP-induced cytotoxicity. Preliminary observations indicate that ghrelin increased the trimethylation of lys4 on histones H3, a known epigenetic mark activator, which may regulate the expression of some genes limiting oxidative damage. In conclusion, our data demonstrate that ghrelin and DAG promote survival of MC3T3-E1 cell exposed to t-BHP-induced oxidative damage. Such effect is independent of GHS-R1a and is likely mediated by a common ghrelin/DAG binding site.
Johan Svensson
Several antiresorptive agents have been developed for the treatment of osteoporosis and bone disorders. Few agents have been introduced that stimulate bone formation. Treatment with parathyroid hormone induces, however, a potent increase in hip and lumbar bone mineral density (BMD) and is now available in some countries. The present review focuses on growth hormone (GH) / GH secretagogues (GHSs). GH/GHSs stimulates bone turnover, thereby increasing bone mass and density.
Thomas Kruse
Michael Ankersen Birgit Sehested Hansen
Peter Hongaard Andersen
A novel class of growth hormone-releasing compounds with a molecular weight in the range from 500 to 650 has been discovered. The aim of this study was to obtain growth hormone secretagogues with oral bioavailability. By a rational approach we were able to reduce the size of the lead compound ipamorelin (4) and simultaneously to reduce hydrogen-bonding potential by incorporation of backbone isosters while retaining in vivo potency in swine. A rat pituitary assay was used for screening of all compounds and to evaluate which compounds should be tested further for in vivo potency in swine and oral bioavailability, fpo, in dogs. Most of the tested compounds had fpo in the range of 10−55%. In vivo potency in swine after iv dosing is reported, and ED50 was found to be 30 nmol/kg of body weight for the most potent compound.
Michael Ankersen Nils Langeland Johansen Kjeld Madsen
Peter Hongaard Andersen
A new series of GH secretagogues derived from ipamorelin is described. In an attempt to obtain oral bioavailability, by reducing the size and the number of potential hydrogen-bonding sites of the compounds, a strategy using the peptidomimetic fragment 3-(aminomethyl)benzoic acid and sequential backbone N-methylations was applied. Several compounds from this series release GH with high in vitro potency and efficacy in a rat pituitary cell assay and high in vivo potency and efficacy in anesthetized rats. The tetrapeptide NNC 26-0235 (3-(aminomethyl)benzoyl-d-2Nal-N-Me-d-Phe-Lys-NH2) shows, following iv administration, comparable in vivo potency to ipamorelin, GHRP-2, and GHRP-6 with an ED50 in swine at 2 nmol/kg. NNC 26-0235 demonstrated a 10% oral bioavailability in dogs, and NNC 26-0235 and ipamorelin were able to increase basal GH level by more than 10-fold after oral administration of a dose of 1.8 and 2.7 mg/kg, respectively. The tripeptide NNC 26-0323 (3-(aminomethyl)benzoic acid-N-Me-d-2Nal-N-Me-d-Phe-ol) which showed moderate in vitro potency but lacked in vivo potency demonstrated a 20% oral bioavailability in rats.
Zvi Laron Jenny Frenkel R. Deghenghl A. Siibergeld
OBJECTIVE Hexarelin is a recently synthesized small growth hormone releasing peptide (GHRP) (His-D-methyl-Trp-Ala-Trp-D-Phe-Lys-NH2). It Is actlve by intra-venous, oral and intranasal administration In animals and man. The aim of this study was to find out whether longterm administration of this peptlde would promote growth In short chlldren. DESIGN AND PATIENTS intranasal hexareiin was administered in a dose of 60 pglkg thrice dally to 8 prepubertal short children aged 4-11-6 years for perlods of up to months. RESULTS Hexarelln treatment stimulated Insulin-like growth factor-i (IGF-I) secretion ralsing the level from 10.4 ± 3.9 (SD) to 141 f 46 nmoill (P < 0.004). The rise IGF-l led to a significant Increase In the mean (±SD) linear growth velocity from 53 ± 08 to 8 3 ± 1.7 cm/year (P < 0.0001). There was also a slgniflcant decrease skinfold thickness despite Increase in body weight and an Increase in head circumference. Additional flndlngs were a rlse in serum phosphate from 1–5 ± 01 to 1–8 ± 01 mmoi/l (P < 0004) and of aikallne phosphatase from 219 ± 74 to 261 ± 75 U/l (P < 005). CONCLUSIONS The long-term GHIIGF-I stimulating, anabolic and growth promoting effects achieved by intranasai administration of this hexapeptide, seemingly without undeslrable slde-effects, suggests clinical potential for this new class of drugs.
Peter Holmberg Jørgensen Bue Bak T T Andreassen
The influence of biosynthetic human growth hormone (b-hGH) on female rat cortical femur and tibia was studied after administration of hormone doses of 0.16, 1.10, or 8.33 mg/kg body weight/day for 90 days. The mechanical properties, dimensions, real density, ash weight, and the mineral and collagen concentrations of the bones were measured. In both femur and tibia a positive linear relation was found between the dose of hormone and ultimate load, ultimate stiffness, energy absorption at ultimate load, load at failure, energy absorption at failure, and deflection at failure. In the femur a positive correlation between dose and deflection at ultimate load was also found. After normalizing the mechanical data for the dimensions of the bones, no differences were found in the hormone treated groups compared to placebo, except for the elastic modulus (Young's modulus), which was decreased in the femur in the group given 8.33 mg b-hGH. The mineral and collagen concentration were unaffected in both femur and tibia, whereas the real density was decreased in the femur. The growth-hormone-induced changes in the mechanical properties seem to be caused mainly by increased dimensions of the bones.
Naoyuki Takahashi Takuhiko Akatsu Nobuyuki Udagawa
Tatsuo Suda
We developed a co-culture system with mouse spleen cells and osteoblastic cells to examine the role of osteoblasts in osteoclast formation. When mouse spleen cells and osteoblastic cells isolated from fetal mouse calvariae were co-cultured in the presence of 10 nM 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], numerous tartrate-resistant acid phosphate (TRACP)-positive mononuclear and multinucleated cells were formed within 8 days. Neither the same co-cultures without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced TRACP-positive cells. Salmon calcitonin (CT) markedly increased cAMP production in the co-cultures treated with 1 alpha,25(OH)2D3. Autoradiographic studies clearly demonstrated that [125I]-CT specifically bound to the TRACP-positive cells formed in the co-cultures with the vitamin. When spleen cells and osteoblastic cells were co-cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, numerous resorption lacunae were formed on the slices. Neither co-cultures of alveolar macrophages and osteoblastic cells nor those of spleen cells and mouse skin-derived fibroblasts induced TRACP-positive cells even in the presence of 1 alpha,25(OH)2D3. When spleen cells and osteoblastic cells were cultured separately from each other by a membrane filter (0.45 micron), no TRACP-positive cells were formed. These results indicate that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts.
Lawrence Frohman
John-Olov Jansson
The identification of GRH has been followed by an extraordinarily rapid rate of knowledge accumulation. Within a period of slightly more than 3 yr since the structure of the GRH was determined, nearly 500 papers have been published pertaining to the hormone. Extensive knowledge of its anatomy, chemistry, molecular biology, physiology, and pathology has been gathered and, in particular, studies in humans have proceeded faster than with any other of the hypophysiotropic hormones. New insights have been gained with respect to the pathogenesis of both GH deficiency and GH excess states, and the use of GRH and its analogs as diagnostic and therapeutic agents already represents a reality.
Cyril Bowers
Frank A Momany
Gemma Reynolds A Hong
His-DTrp-Ala-Trp-DPhe-LysNH2, [His1,Lys6] GHRP, is a new synthetic hexapeptide which specifically elicits a dosage-related release of GH in vitro and in vivo without a concomitant release of LH, FSH, TSH, or PRL and, in limited in vivo studies, insulin or glucagon. Our results indicate that this small peptide has the attributes of a hypophysiotropic hormone. In vitro the minimum and maximum active dosages ranged from 1-10 ng/ml in the pituitary incubate assay. It was active in rats, monkeys, lambs, calves, and under special experimental conditions chicks, indicating its lack of species dependency. It was active when administered iv, sc, or ip to rats. After iv injection, GH levels rose within 2 min, peaked at +10-20 min, and by 2 h usually had returned to normal. It was not possible to directly compare the potencies of [His1,Lys6]GHRP, and the GH-releasing factors GHRF-44 and GHRF-40 after a single sc injection in rats because the time course of the GH response of these peptides was different. The GH response of [His1,Lys6]GHRP was longer in duration than either of these larger peptides. Both SRIF-14 and SRIF-28 inhibited the GH response of the hexapeptide; however, SRIF-28 was about four times more active than SRIF-14 in vitro and 7.5 times more active in vivo. When this small peptide was administered sc once or twice daily to immature rats for 9 or 25 days, the BW gain increased above the control. At the end of the weight gain studies the pituitary remained fully responsive to the peptide. Thus, [His1,Lys6] GHRP may be a valuable peptide for investigating the function of the pituitary somatotrophs and, in addition, it has the potential for increasing BW gain of a variety of normal animals by inducing GH release via a direct pituitary site of action.
T T Andreassen Peter Holmberg Jørgensen
Allan Flyvbjerg H Oxlund
The influence of growth hormone on bone formation, mechanical strength, and composition has been investigated in femur middiaphyseal cortical bone from 2-year-old male rats. The rats were given biosynthetic human growth hormone (bhGH) at 2.7 mg/kg/day in two daily injections for 20, 40, or 80 days, and all animals were killed 80 days after the start of bhGH administration. Control animals were given saline. All animals were labeled with tetracycline on days 41 and 69. Only in the bhGH-80-day group was subperiosteal tetracycline double labeling seen all around the femur diaphysis, and this pattern was found in all animals of the group. Double labeling subperiosteally at the posteromedial aspect was found in all animals of the experiment, but compared with the control group, a 400% and an 800% increase in mineral apposition rate was seen in the bhGH-40-day and bhGH-80-day groups, respectively. Light microscopy and polarization microscopy showed that this newly deposited bone was organized in the same concentric lammellae and had the same direction of the collagen fibers when compared with the surrounding bone formed before the start of bhGH injections. The cortical bone cross-sectional area was increased in the bhGH-40-day and bhGH-80-day groups. At the endosteum, scattered labeling was found in animals from all groups, and no differences in medullary cross-sectional areas were seen. The mechanical analysis revealed an increased mechanical strength of the whole diaphyseal bone after bhGH administration. When the data were corrected for dimensions of the diaphyseal bone, no differences in intrinsic mechanical properties of the bone tissue were found. No differences in apparent density of dry defatted bone, ash, and collagen were seen, whereas apparent density of dry defatted bone minus ash was decreased in all groups given bhGH. Correspondingly, a slight increase in ash concentrations of the bhGH-injected animals was seen. bhGH administration also increased the body weight, muscle mass, and total serum IGF-I and thyroxine concentrations.