Systemic administration of a novel
octapeptide, amylin-(1---8), increases bone volume in male
mice
Jillian
Cornish1,
Karen
E.
Callon1,
Juerg A.
Gasser2,
Usha
Bava1,
Edith M.
Gardiner3,
David H.
Coy4,
Garth J. S.
Cooper1,5, and
Ian R.
Reid1
1 Department of Medicine, 5 School of Biological
Sciences, University of Auckland, Auckland 1001, New Zealand;
2 Bone Metabolism Unit, Novartis Pharma, Basel CH-4002,
Switzerland; 3 Garvan Institute, Sydney NSW 2010, Australia; and
4 Peptide Research Laboratories, Tulane University Medical
Center, New Orleans, Louisiana 70112
 |
ABSTRACT |
Amylin increases bone mass
when administered systemically to mice. However, because of its size,
the full peptide is not an ideal candidate for the therapy of
osteoporosis. The fragment, amylin-(1---8), stimulates osteoblast
proliferation in vitro, although it is without effect on carbohydrate
metabolism. The present study assessed the effects of daily
administration of this peptide on sexually mature male mice for 4 wk.
Amylin-(1---8) almost doubled histomorphometric indices of osteoblast
activity but did not change measures of bone resorption. Trabecular
bone volume increased by 36% as a result of increases in both
trabecular number and trabecular thickness, and tibial cortical width
increased by 8%. On three-point bending tests of bone strength,
displacement to fracture was increased by amylin-(1---8), from
0.302 ± 0.013 to 0.351 ± 0.017 mm (P = 0.02). In a separate experiment using dynamic histomorphometry with
bone-seeking fluorochrome labels, amylin-(1---8) was administered by
local injection over the calvariae of female mice. Amylin-(1---8) (40 nM) increased the double-labeled surface threefold. The effect was dose
dependent from 0.4 to 40 nM and was greater than that of an equimolar
dose of human parathyroid hormone-(1---34) [hPTH-(1---34)]. Mineral
apposition rate was increased by 40 nM amylin-(1---8) but not by
hPTH-(1---34). Amylin-(1---8) thus has significant anabolic effects in
vivo, suggesting that this peptide or analogs of it should be further
evaluated as potential therapies for osteoporosis.
amylin; osteoporosis; peptide hormones; bone growth; osteoblast
 |
INTRODUCTION |
THE PANCREATIC
PEPTIDE amylin has previously been shown to stimulate
proliferation of osteoblasts and to inhibit osteoclastic bone
resorption, both in vitro and in vivo (4, 8, 13). These
effects result in substantial increases in bone mass when the peptide
is administered either locally or systemically to sexually mature mice
(4, 6). The anabolic effects of amylin on osteoblasts are
shared by the related peptide adrenomedullin, although adrenomedullin
does not modulate osteoclast activity (5). These findings
suggest that this class of peptides might have a role in the therapy of
osteoporosis, although because of its size the intact peptide is not an
ideal therapeutic agent. One way of circumventing this problem is
suggested by our recent observation that an octapeptide fragment of
this hormone, amylin-(1---8), which is inactive on fuel metabolism
(3), is still anabolic to osteoblasts (8).
This small ring peptide is a more attractive candidate for
pharmaceutical development and might be used as a model for the
creation of orally active, nonpeptide analogs. However, this fragment
has a lower potency than the intact molecule and lacks any inhibitory
effects on osteoclastic bone resorption, so it is uncertain how
substantial its effects on bone in vivo might be. We have now addressed
this question by assessing the effects of 1 mo of daily systemic
treatment with amylin-(1---8) on the histomorphometry and mechanical
properties of the tibiae in sexually mature mice. In addition, we have
studied its effects on dynamic bone histomorphometry after local
injection over the calvariae of mice and compared these effects with
those of human parathyroid hormone-(1---34) [hPTH-(1---34)].
 |
METHODS |
Systemic Study
Experimental design.
Two groups of 20 sexually mature male Swiss mice aged between 40 and 50 days and weighing 25-32 g were given daily subcutaneous injections
[2.2 µg rat amylin-(1---8) in 50 µl of water or water alone] in
the loose skin at the nape of the neck for 5 days/wk over four
consecutive weeks. This dose was chosen because the same molar dose (93 nmol/kg) of the intact peptide in this model produces substantial
effects on bone turnover and bone area (6). Animals were
housed in a room maintained at 20°C on 12:12-h light-dark cycles.
They were fed a diet of 86 rodent pellets (New Zealand Stockfeed) ad
libitum throughout the experiment. Each animal's weight was recorded
at the beginning and end of the experiment. The study had the approval
of the local institutional review board.
Histomorphometry.
Indices of bone formation and resorption and bone volume were assessed
in the proximal tibiae where the predominance of trabecular bone with
its high surface-to-volume ratio results in larger changes in these
measures than are seen elsewhere. One tibia from each animal was used
for histomorphometric analyses. The tibiae were dissected free of
adherent tissue, and bone lengths were recorded by measuring the
distance between the proximal epiphysis and the distal tibio-fibular
junction by use of an electronic micrometer (Digimatic Calipers,
Mitutoyo, Japan). Bones were placed in 10% phosphate-buffered Formalin
for 24 h, dehydrated in a graded series of ethanol solutions, and
embedded, undecalcified, in methyl methacrylate resin (Acros Organics,
Geel, Belgium). Tibiae were sectioned longitudinally through the
frontal plane. Sections (4 µm thick) were cut using a Leitz rotary
microtome (Leica Instruments, Nussloch, Germany) and a
tungsten-carbide knife (Microknife Sharpening) and then were mounted on
gelatin-coated slides and air-dried. They were stained with Goldner's
trichrome and examined using an Olympus BX 50 microscope (Olympus
Optical, Tokyo, Japan) that was attached to an Osteomeasure Image
Analyzer (Osteometrics, Atlanta, GA).
Tibial histomorphometric analyses were made from three adjacent
sections one-third of the way through the anterior/posterior depth of
the proximal tibiae. All trabecular bone tissue in the secondary
spongiosa was quantified for bone volume in each section using a ×10
objective, and parameters were derived using the formulas of Parfitt et
al. (10). Parameters of bone formation and resorption were
measured using a ×20 objective in all trabecular bone tissue in the
secondary spongiosa in the middle of the three adjacent sections. The
surfaces measured were those immediately adjacent to unmineralized
matrix, those adjacent to osteoblasts, and those adjacent to
osteoclasts (osteoid, osteoblast, and osteoclast perimeters, respectively). The osteoclast index is the number of osteoclasts per
millimeter trabecular perimeter. Perimeters and cell numbers were
expressed per section. Cortical width was measured on both sides of the
tibial shaft, 2.5 mm below the epiphysial growth plate. Epiphyseal
growth plate thickness was measured at three sites evenly spaced along
its length. All measurements were made by one operator (J. Cornish) who
was blinded to the treatment group of each bone. The precisions of
these histomorphometric measurements in our laboratory (expressed as
coefficients of variation of paired measurements) were as follows:
mineralized bone area 1.3%, osteoid perimeter 6.9%, osteoblast
perimeter 6.8%, osteoblast number 1.7%, osteoclast perimeter 7.9%,
osteoclast number <1.0%, width measurements 1.7%.
Mechanical strength of the tibia.
The remaining tibia from each animal was fixed in 70% ethanol and was
used for mechanical strength estimations. The mechanical strength of
the tibiae was determined by three-point bending tests. Each tibia was
dissected free of soft tissue and tested on a MTS 858 Bionix Testing
Machine (MTS Systems, Minneapolis, MN). Samples were tested at room
temperature with a support span of 10 mm. Load was applied at a
constant deformation rate of 2 mm/min with a force application at the
upper anterior midpoint of the tibia. Load-deformation curves were
recorded, and displacement values (a measurement of how much the bone
bends from the time that the force is applied until its final failure
point) were obtained directly from the curve and expressed in millimeters.
Fat mass estimations.
Fat mass estimations were made from measurements of the animals' body
densities calculated from water displacement. Immediately after death
the mice were submerged head first to the base of the tail in a 250-ml
measuring cylinder containing 150 ml of water, and the displacement
volume was recorded. The fraction of body weight that was fat mass was
calculated using a modification of the Siri equation for use in rodents
(9). The coefficient of variation for repeated measures of
fat mass was 7%.
Local Injection Study
A further experiment was carried out using the local injection
of peptide over the calvariae of female OF-1 mice to assess the effects
of amylin-(1---8) on dynamic histomorphometry, to assess dose-response
effects, and to allow comparison with an established anabolic agent,
parathyroid hormone. Five groups of sexually mature mice
(n = 5 in each group) were allocated to receive 50-µl
injections two times a day over the central calvaria for 5 days. In the
respective groups, the injections consisted of PBS, amylin-(1---8) in
concentrations of 0.4, 4, or 40 nM, or 40 nM hPTH-(1---34). The mice
were killed 14 days after the first injection, and the frontal and
parietal bones were removed for histology. To permit dynamic
histomorphometry, animals were labeled with alizarine (20 mg/kg sc
injected at the lower back) at baseline. Further fluorochrome labels
were administered at 10 days before necropsy (30 mg/kg sc calceine) and
at 3 days before necropsy (20 mg/kg alizarine). Bones were fixed in
Karnovski's fixative and embedded in HistoDur (Leica Instruments). The
distribution of fluorescent labels was assessed using a Bioquant (R&M
Biometrics, Nashville TN).
Materials.
Rat amylin-(1---8) was synthesized as a COOH-terminal amide on
methylbenzhydrylamine resin by standard solid-phase techniques, as
described previously (8). It was cyclized in a dilute
solution of 90% acetic acid by treatment with methanol solutions of
iodine and was purified to >96% homogeneity by reverse-phase HPLC. To avoid losses when handling the peptide, an anti-static device (Zerostat
3; Discwasher, Reconton, Lake Mary, FL) was used to remove static
electric charge from the peptide itself and from any containers in
which it was placed. The hydrochloride salt of the peptide was produced
by dissolving it in 3 mM hydrochloric acid (10 µmol peptide in 50 µl) and leaving it for 1 h at room temperature before
freeze-drying (model SVC 100H; Savant Instruments). Before use, it was
redissolved in pure water with sonication (Soniprep 150, West Sussex,
UK), cooled on ice for 15 s, and then stored at 4°C until
required for injection. Amylin-(1---8) was dissolved for a minimum of
48 h before injection, because we have found that this increases
the concentration of peptide in solution, as measured by HPLC. The
molecule is very adherent to glass, so only plastic containers were
used in handling it. hPTH-(1---34) was made using solid-phase synthesis (Novartis).
Statistical analysis.
Data are presented as means ± SE. Where parameters were measured
more than one time in each animal (e.g., cortical thickness), these
values were averaged to produce a single value for each animal before
further analysis. The significance of treatment effects was evaluated
using Student's t-tests for unpaired data. All tests were
two tailed.
 |
RESULTS |
Systemic Study
Amylin-(1---8) produced substantial increases in all three indices
of osteoblast activity assessed (osteoid perimeter from 3.5 ± 0.3 to 6.0 ± 0.3 mm, osteoblast perimeter from 1.4 ± 0.2 to 2.6 ± 0.2 mm, and number of osteoblasts from 157 ± 15 to
254 ± 17, P < 0.0001 for each; Fig.
1). In contrast, there was no effect on
bone resorption (Fig. 2). These changes
resulted in an 8.1% increase in cortical width [from 0.160 ± 0.005 mm in control animals to 0.173 ± 0.004 mm in those
receiving amylin-(1---8)] and a 36% increase in trabecular bone
volume [from 13.7 ± 0.8% in the control animals to 18.7 ± 0.8% in those treated with amylin-(1---8); Fig. 3]. The increase in trabecular volume
was contributed to by increases in trabecular thickness and trabecular
number and from a decline in trabecular separation (Fig.
4). These effects can be directly appreciated by comparing the sections of bone from control animals with
those treated with amylin-(1---8), as shown in Fig.
5. When the contralateral tibiae were
subjected to three-point bending to assess bone strength, the
displacement to the point of failure was increased from 0.302 ± 0.013 mm in control animals to 0.351 ± 0.017 mm in those treated
with amylin-(1---8) (P = 0.02), suggesting that the
bones of the treated animals were stronger (Fig.
6).

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Fig. 1.
Effects of daily systemic administration of
amylin-(1---8) for 4 wk on histomorphometric indices of bone formation
in the tibiae of normal sexually mature male mice (n = 20 in each group). Data are means ± SE. * Significantly
different from control, P < 0.0001.
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Fig. 2.
Effects of daily systemic administration of
amylin-(1---8) for 4 wk on histomorphometric indices of bone resorption
in the tibiae of normal sexually mature male mice (n = 20 in each group). Data are means ± SE. There were no significant
effects.
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Fig. 3.
Effects of daily systemic administration of
amylin-(1---8) for 4 wk on histomorphometric indices of bone volume in
the tibiae of normal sexually mature male mice (n = 20 in each group). Data are means ± SE. * Significantly different
from control, P = 0.04. ** P < 0.0001.
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Fig. 4.
Effects of daily systemic administration of
amylin-(1---8) for 4 wk on histomorphometric indices of trabecular bone
in the tibiae of normal sexually mature male mice (n = 20 in each group). Data are means ± SE. * Significantly
different from control, P < 0.008.
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Fig. 5.
Photomicrographs of the proximal tibiae of mice treated
with vehicle (A) or amylin-(1---8) (B)
demonstrating the increase in trabecular bone volume and cortical width
associated with amylin-(1---8) treatment (magnification ×80). Sections
have been stained with Goldner's trichrome, with mineralized bone
appearing green.
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Fig. 6.
Effects of daily systemic administration of
amylin-(1---8) for 4 wk on mechanical strength of the tibiae, estimated
using displacement values from load-deformation curves obtained from
3-point bending tests (n = 20 in each group). Data are
means ± SE. * Significantly different from control,
P = 0.02.
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The effects of amylin-(1---8) were not confined to bone; there was also
a near doubling of the thickness of the epiphysial growth plate (Fig.
7). However, there was no significant
difference in tibial lengths between the groups [control 11.05 ± 0.33 mm, amylin-(1---8) 11.15 ± 0.25 mm].

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Fig. 7.
Effects of daily systemic administration of
amylin-(1---8) for 4 wk on the thickness of the epiphysial growth plate
in the tibiae of normal sexually mature male mice (n = 20 in each group). Data are means ± SE. * Significantly
different from control, P < 0.0001.
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Body weight increased from 27.8 ± 0.4 to 32.8 ± 0.5 g
in the control animals and from 27.8 ± 0.4 to 33.1 ± 0.6 g in those treated with amylin-(1---8). Fat masses at the end
of the study were 2.42 ± 0.13 and 2.27 ± 0.19 g in the
control and amylin-(1---8)-treated animals, respectively. These results
were not significantly different between the groups.
Local Injection Study
Amylin-(1---8) injections increased the extent of the
double-labeled surface (assessed using the second and third labels and expressed as a percentage of total bone surface) in a dose-dependent fashion (Fig. 8A). The effect
was significant with 4 and 40 nM amylin-(1---8) and with 40 nM
hPTH-(1---34). The increase in double-labeled surface observed with
amylin-(1---8) was greater than with the same concentration of
hPTH-(1---34). All of the labels showed sharp delineation, and no woven
bone was observed with either peptide. Mineral apposition rate was also
increased in mice receiving 40 nM amylin-(1---8) but not with
hPTH-(1---34) (Fig. 8B).

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Fig. 8.
Effect of amylin-(1---8) and human parathyroid hormone
(PTH)-(1---34) on the extent of the fluorochrome double-labeled surface
(A) and mineral apposition rate (B) in mice
calvariae. Mice were treated with local subcutaneous injections over
the central calvaria for 5 consecutive days and were killed 10 days
later. [Peptide], peptide concentration. * Significantly different
from control, P < 0.04. ** Significantly different
from control, P < 0.001. The groups receiving 40 nM
amylin and 40 nM PTH were also significantly different from each other,
P < 0.02.
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DISCUSSION |
The present study demonstrates that the positive effects of intact
amylin on both trabecular and cortical bone can be reproduced by the
NH2-terminal octapeptide of the molecule. The effect is less than what we found with equimolar doses of amylin itself (36%
increase in trabecular bone volume compared with 70% with the intact
peptide; see Ref. 6), consistent with the lower anabolic potency of the
fragment in vitro and with its lack of effect on osteoclasts
(8). The fact that positive effects on bone volume remain
despite the absence of anti-osteoclast effects suggests that the
increase in bone volume previously found with intact amylin is
substantially attributable to its action on osteoblasts. The positive
effects on double-labeled surfaces and mineral apposition rates in the
local injection studies shown here are consistent with this. Although
the systemic dose response of this effect requires further study, the
effect on trabecular bone volume found here is substantial. It will be
of interest to determine whether this activity can be increased in
analogs of this small cyclic peptide and whether nonpeptide analogs
that might be orally active can also be developed. However, the
significance of the present data in the therapy of osteoporosis will
ultimately be determined by the assessment of amylin-(1---8) or its
analogs in human studies.
This study provides further evidence for the anabolic action on
osteoblasts of the family of related peptides consisting of amylin,
adrenomedullin, and calcitonin gene-related peptide (CGRP), all of
which have been shown to exert this action both in vitro and in vivo
(2, 5, 11). Valentijn et al. (12) have
recently shown partial prevention of postovariectomy bone loss in rats after daily injections of CGRP-
for 4 wk. The same group has shown
that transgenic mice overexpressing the CGRP gene in bone have a 5%
increase in distal femoral bone density at the age of 12 wk
(1). Calcitonin does not stimulate bone formation in these
models (4, 11). Amylin and adrenomedullin are
approximately equipotent in their actions on osteoblasts, but CGRP is
significantly less active (2, 4). Furthermore, CGRP
activity on osteoblasts is completely blocked by amylin receptor
blockers, whereas the converse is not true (7). These
findings are consistent with the fact that the changes found in the
present studies of amylin were achieved using lower doses of peptide
than those of CGRP used by Valentijn et al. (12) in their
study of ovariectomized rats. Thus the osteogenic effects demonstrated
in the present study are likely to be mediated by a receptor with a
higher affinity for amylin than for CGRP.
The effects of amylin-(1---8) are different from those of amylin itself
with respect to the effects on fat mass in this model. The intact
molecule increases fat mass in these mice when administered according
to the schedule used in the present study (6). In contrast, the octapeptide has no effect on either body weight or fat
mass in the present study. This is consistent with other evidence that
any shortening of the parent molecule results in a loss of its activity
on intermediary metabolism (3). Similarly, the formation
of amyloid from amylin is dependent on residues in the COOH-terminal
region of the molecule and would not be expected with this octapeptide
(3).
A further effect of the systemic administration of intact amylin
is an increase in the thickness of the tibial growth plate and an
acceleration of linear growth of the tibia (6). This suggests that this peptide also affects chondrocyte activity, which is
something we have now confirmed in cultures of isolated canine
articular chondrocytes (unpublished observation). In the present study,
a thickening of the tibial growth plate is again observed, although no
significant effect on bone length was found. This is consistent with
the findings of our in vitro studies that show that 10-fold higher
concentrations of amylin-(1---8) than those of amylin itself are
necessary to achieve comparable stimulation of chondrocyte
proliferation. The chondrocyte actions of amylin-(1---8) may also have
contributed to the increase in bone volume in the present study, but
this is unlikely to be the major mechanism, because anabolic effects
have been demonstrated in models that are free of chondrocytes (e.g.,
isolated osteoblast cultures and with local injection over calvariae in
vivo; see Ref. 4). It will be of interest to determine whether the
local or systemic administration of these peptides has a significant
effect on articular cartilage in vivo, because this might have
relevance to conditions such as osteoarthritis.
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ACKNOWLEDGEMENTS |
We thank Martin Svehla and Dr. Bill Walsh, University of Sydney,
and Ronaldo Enriquez, Garvan Institute, Sydney, Australia, for
expertise and assistance in measuring the bone strengths.
 |
FOOTNOTES |
This work was supported by the Health Research Council of New Zealand
and the Auckland Medical Research Foundation.
Address for reprint requests and other correspondence: J. Cornish, Dept. of Medicine, Univ. of Auckland, Private Bag 92019, Auckland, New Zealand (E-mail: j.cornish{at}auckland.ac.nz).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 October 1999; accepted in final form 2 May 2000.
 |
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