1 Departments of Medicine and 2 Molecular Medicine and Physiology, University of Auckland, Auckland 1001, New Zealand; and 3 Department of Clinical and Biomedical Sciences, Barwon Health, University of Melbourne, Melbourne 3220, Australia
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ABSTRACT |
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-Melanocyte-stimulating hormone
(
-MSH), a 13-amino acid peptide produced in the brain and
pituitary gland, is a regulator of appetite and body weight, and its
production is regulated by leptin, a factor that affects bone mass when
administered centrally.
-MSH acts via melanocortin receptors. Humans
deficient in melanocortin receptor 4 (MC4-R) have increased bone mass,
and MC4-R has been identified in an osteoblast-like cell line. Thus
-MSH may act directly on the skeleton, a question addressed by the
present studies. In primary cultures of osteoblasts and chondrocytes,
-MSH dose dependently (
10
9 M) stimulated cell
proliferation. In bone marrow cultures,
-MSH (>10
9 M)
stimulated osteoclastogenesis. Systemic administration of
-MSH to
mice (20 injections of 4.5 µg/day) decreased the trabecular bone
volume in the proximal tibiae from 19.5 ± 1.8 to 15.2 ± 1.4% (P = 0.03) and reduced trabecular number
(P = 0.001). Radiographic indexes of trabecular bone,
assessed by phase-contrast X-ray imaging, confirmed the bone loss. It
is concluded that
-MSH acts directly on bone, increasing bone
turnover, and, when administered systemically, it decreases bone
volume. The latter result may also be contributed to by
-MSH effects
elsewhere, such as the adipocyte, pancreatic
-cell, or central
nervous system.
osteoblast; osteoclast; chondrocyte; systemic administration
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INTRODUCTION |
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BODY WEIGHT IS AN
IMPORTANT DETERMINANT of bone mineral density (24, 41, 42,
53, 55) and is one of the most important risk factors for
osteoporotic fractures (1, 19, 21, 33, 36, 48, 62, 73).
The two major components of body weight, fat mass and lean mass,
probably each contribute to these relationships, but in a number of
studies fat mass has been shown to have a substantial, independent
effect on both bone density (35, 51, 53, 55, 56, 67) and
fracture rates (38, 62). The effects of fat mass on
skeletal load may contribute to this relationship, although they do not
explain it in non-weight-bearing sites (55). Similarly, estrogen production in the adipocyte may contribute to these
relationships in postmenopausal women, but it does not explain the
relationship between fat mass and bone density before menopause
(59). It is therefore of interest to assess the skeletal
impact of hormonal factors that either regulate fat mass or are
influenced by it. For these reasons, there has been a recent focus of
attention on the roles of insulin, amylin, leptin, and preptin on
skeletal metabolism (54). However, there are a number of
other hormones that could contribute to these relationships, one of
which is -melanocyte-stimulating hormone (
-MSH).
-MSH is a 13-amino acid peptide derived from proopiomelanocortin
(POMC) and produced in the brain and pituitary gland. It is a key
factor in the central regulation of appetite and body weight, and its
precursor, POMC mRNA, is regulated by leptin (18), a
factor recently shown to have substantial effects on bone mass when
administered into the central nervous system (16).
-MSH acts via the melanocortin receptors, and it has recently been reported
that humans deficient in melanocortin receptor 4 (MC4-R) have markedly
increased bone mass (22). Also, it has recently been noted
that MC4-R is present in an osteoblast-like cell line (UMR 106)
(17), raising the possibility that
-MSH may act
directly on the skeleton. The present studies address this question by assessing the effects of
-MSH on cells of the osteoblast and osteoclast lineages and assess the effect of its systemic
administration to intact adult mice.
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METHODS |
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Osteoblast-Like Cell Culture
Osteoblast proliferation assay ([3H]thymidine incorporation). Osteoblasts were isolated from 20-day fetal rat calvariae as previously described (13). Briefly, calvariae were excised, and the frontal and parietal bones, free of suture and periosteal tissue, were collected. The calvariae were sequentially digested using collagenase, and the cells from digests 3 and 4 were collected, pooled, and washed. Cells were grown to confluence and then subcultured into 24-well plates. Cells were growth arrested in minimum essential medium (MEM; GIBCO, Grand Island, NY)-bovine serum albumin (ICP, Auckland, New Zealand) for 24 h. Fresh medium and experimental compounds were added for a further 24 h. Cells were pulsed with [3H]thymidine 2 h before the end of the experimental incubation. The experiment was terminated, and cell counts and thymidine incorporation were assessed. There were six wells in each group, and each experiment was repeated three or four times.
Assessment of expression of osteoblast differentiation markers.
Primary rat osteoblasts were plated in MEM plus 10% FCS at 1 × 106 cells/T75 flask. The next day
(day 1), the medium was changed to -MEM plus 15% FCS, 50 µg/ml L-ascorbic acid 2-phosphate and 10 mM
-glycerophosphate, and one-half of the flasks were supplemented with
10
8 M
-MSH. The medium was changed to fresh medium
with or without
-MSH on days 4 and 7. On
days 4, 7, and 10, one flask treated with
-MSH
and one control culture were harvested, and RNA was extracted using
RNeasy Mini Kit and RNase-Free DNase Set (Qiagen, Valencia, CA).
Semiquantitative RT-PCR was performed as previously described
(9). The primers used to amplify rat alkaline phosphatase (accession no. Y00714) were 5'-CCCAAAGGCTTCTTCTTG for forward primer
and 5'-CCTGGTAGTTGTTGTGAGCA for reverse, and PCR products were analyzed
after 28, 30, and 35 cycles of amplification. Rat osteocalcin
(accession no. NM_013414) was amplified using 5'-CTCTCTGCTCACTCTGCTGG forward primer and 5'-AAGCCGATGTGGTCAGC reverse primer, and PCR products were analyzed after 35 cycles. Rat collagen
1, type I
(accession no. Z78279) was amplified using 5'-GTGGTCAGGCTGGTGTGATG forward primer and 5'-GACCACGGACGCCATCTT reverse primer, and PCR products were analyzed after 20, 25, and 30 cycles. For rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
5'-CATCATCTCCGCCCCTTCCG was used for forward primer and
5'-CCTGCTTCACCACCTTCTTG for reverse primer, and products were analyzed
after 20 and 25 cycles. The PCR experiments were repeated three times
with similar results.
Bone Marrow Culture
Bone marrow was obtained from long bones of normal Swiss male mice aged 4-6 wk. Mice were killed by cervical dislocation while under halothane anesthesia. Femurs and tibias were aseptically removed and dissected free of adhering tissues. The epiphyses were cut off with a scalpel blade, and the marrow cavity was flushed withMature Isolated Osteoclast Culture
Rat osteoclasts were isolated from 1-day-old neonatal rats. The rats were killed by decapitation and the long bones aseptically removed. The bones were dissected free of adherent soft tissues. Epiphyses were removed and the remaining diaphyses split longitudinally and then placed in a tissue homogenizer containing 2 ml of acidifiedBone Organ Culture
Bone resorption studies were carried out in neonatal mouse calvariae as described previously (58). Mice were injected subcutaneously with 5 µCi of 45Ca at 2 days of age, and hemicalvariae were dissected out 4 days later. Hemicalvariae were preincubated for 24 h in medium 199 with 0.1% bovine serum albumin and then changed to fresh medium containing test substances or vehicle. Incubation was continued for a further 48 h. To assess DNA synthesis, [3H]thymidine (0.6 µCi/ml) was added in the last 4 h of the incubation, as described previously (40). The experiment was terminated, and both calcium release and thymidine incorporation were assessed. There were five to seven hemicalvariae in each group, and each experiment was repeated three or four times.Chondrocyte Cell Culture
Chondrocytes were isolated by removing cartilage (full-depth slices) from the tibial and femoral surfaces of adult dogs under aseptic conditions. Slices were placed in Dulbecco's modified Eagle's medium (DMEM, GIBCO) containing 5% FBS and antibiotics (in µg/l: 50 penicillin, 50 streptomycin, and 100 neomycin) and chopped finely with a scalpel blade. Tissue was removed and incubated at 37°C with pronase (0.8% wt/vol for 90 min) followed by collagenase (0.1% wt/vol for 18 h) to complete the digestion. The cells were isolated from the digest by centrifugation (10 min at 1,300 rpm), resuspended in DMEM-5% FBS, passed through a nylon mesh screen of 90-µm pore size to remove any undigested fragments, and recentrifuged. The cells were washed and resuspended twice in the same medium and seeded into a 75-cm2 flask containing DMEM-10% FBS and 50 µg/ml ascorbic acid. The cells were incubated under 5% CO2-95% air at 37°C. Confluence was reached by 7 days, at which time the cells were subcultured. After trypsinization using trypsin-EDTA (0.05%/0.53 mM), the cells were rinsed in DMEM-5% FBS, resuspended in fresh medium, and then seeded into 24-well plates (5 × 104 cells/ml, 0.5 ml/well). Measurement of cell numbers and thymidine incorporation were performed in growth-arrested cell populations as for the osteoblast-like cell cultures.Systemic Study
Experimental design.
Two groups of 20 sexually mature male Swiss mice, aged between 40 and
50 days and weighing 25-36 g, were given daily subcutaneous injections (4.5 µg of -MSH in 50 µl of water or water alone) in
the loose skin at the nape of the neck for 5 days/wk over 4 consecutive
weeks. This dose was chosen because the same molar doses of amylin and
adrenomedullin in this model produce substantial effects on bone
turnover and bone area (7, 12), and these peptides have
similar effects on osteoblast proliferation. Animals were
housed in a room maintained at 20°C on a 12:12-h light-dark cycle.
They were fed Diet 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. Histomorphometric analyses were carried out in the proximal tibia. The tibias were dissected free of adherent tissue, and bone lengths were recorded by measuring the distance between the proximal and the distal epiphyses with an electronic micrometer (Digimatic Calipers, Mitutoyo, Japan). Tibias were then processed as previously described (11). Briefly, bones were fixed in 10% phosphate-buffered formalin for 24 h and then dehydrated in a graded series of ethanol solutions and embedded undecalcified in methyl methacrylate resin. Tibias were sectioned longitudinally through the frontal plane. Sections (4 µm thick) were cut using a Leitz rotary microtome and a tungsten-carbide knife and then mounted on gelatin-coated slides and air-dried. They were stained with Goldner's trichrome and examined using an Olympus BX 50 microscope, which was attached to an Osteomeasure Image Analyzer.
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. (49). Parameters of bone formation and resorption were measured using a 20× objective in all trabecular bone tissue in the secondary spongiosa in the second of the three adjacent sections. Cell numbers were expressed per unit of bone area. Cortical width was measured on both sides of the tibial shaft 2.5 mm below the epiphysial growth plate. Epiphysial 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.Phase-contrast X-ray imaging. X-ray microimaging was performed at CSIRO Manufacturing Science and Technology (Clayton, Victoria, Australia). This method has been described previously (72). Excised tibias from treated animals were fixed and stored in 70% alcohol. The tibias were prepared for imaging by orienting the bones in a brass clamp to enable mounting in the X-ray beam. These images were obtained using a microfocus X-ray source operated at 30 kV and with a 4-µm source size (approximate, as source is actually slightly elliptical). Samples were mounted at distance R1 = 10 cm from the X-ray source. An imaging plate was placed at distance R2 = 190 cm from the sample, resulting in an experimental magnification of ×20. Images were collected with Fuji imaging plates scanned using a BAS5000 Phosphorimager (Fuji, Japan). As described previously, imaging plates are particularly suited to the quantitative in-line phase-contrast X-ray technique because of their large linear dynamic range (29). The features of the images were quantified using MCID version 6 Elite software (Berthold, Melbourne, Australia). The trabecular region was outlined, and the circumference and length of the trabecular area were determined. A "form factor," indicative of the shape of the trabecular area, was also calculated.
Fat mass estimations. Fat mass estimations were made from measurements of the animals' body densities calculated from water displacement. Immediately after they were killed, the mice were submerged head-first to the base of the tail into 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 (46). The coefficient of variation for repeated measures of fat mass is 7%.
Statistical Analysis
Data are presented as means ± SE. When parameters were measured more than once 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 and a 5% significance level. ![]() |
RESULTS |
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Primary Osteoblasts and Chondrocytes
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The effect of -MSH on osteoblast differentiation was assessed by
comparing the levels of expression of alkaline phosphatase, collagen I,
and osteocalcin in osteoblasts treated with
-MSH with the expression
in control cultures. Primary osteoblasts were treated with
10
8 M
-MSH, RNA was extracted after 4, 7, and 10 days
of treatment, and semiquantitative RT-PCR was used to compare the
levels of expression of the osteoblast differentiation markers. With
this semiquantitative system, no significant differences were seen between the treatment and the control groups. Alkaline phosphatase and
collagen I were expressed at high levels at all the time points studied, whereas osteocalcin was visible only on day 10 in
both the treatment and the control cultures.
-MSH had a similar effect on the proliferation of primary cultures
of canine chondrocytes. Both thymidine incorporation and cell number
were increased (Fig. 2).
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Osteoclast Effects
The effect of
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Systemic Administration of -MSH
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The results of the histomorphometric analysis of trabecular structure
were confirmed by phase-contrast X-ray images of the tibias. Figure
5A shows images of three
tibias from the control group and three from the -MSH-treated
group. Simple inspection indicates that the extent of
trabecular bone is diminished following
-MSH treatment. In the
-MSH group, the perimeter and length of the trabecular area were
decreased by 34 (P < 0.0005) and 32% (P < 0.002), respectively (Fig. 5B). The
form factor of the trabecular zone was unchanged. These results
indicate that
-MSH caused a reduction in trabecular bone mass.
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Weight gains during the study were not significantly different in the
two groups; weight increased in the course of the study from 27.5 ± 0.8 to 33.6 ± 0.8 g in animals treated with vehicle and
from 27.4 ± 0.8 to 33.2 ± 0.5 g in those receiving
-MSH. However, the percent fat mass of the animals at the end of the experiment was lower in the
-MSH-treated group (8.7 ± 0.6%)
compared with the control group (10.2 ± 0.5%, P = 0.05). Tibial lengths were not different between the groups (control
18.6 ± 0.1,
-MSH 18.7 ± 0.1 mm).
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DISCUSSION |
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The present study is the first to address the direct effects of
-MSH on skeletal cells. It demonstrates that this peptide increases
the proliferation of osteoblasts without affecting their differentiation, increases chondrocyte proliferation, and stimulates the development of osteoclasts from their precursors in bone marrow. However,
-MSH does not act directly on the mature osteoclast either
when studied in isolated cell culture or when assessed in an organ
culture model lacking osteoclast precursors. Although
-MSH is a
neuropeptide, it does have direct actions on cells outside the central
nervous system (3). As its name implies, it regulates
pigment production in melanocytes (44, 60), and there is
also evidence of direct actions of
-MSH on adipocytes [where it
promotes lipolysis (4)] and the pancreatic
-cell [where it decreases insulin secretion (64)]. Its
adipocyte effects are particularly relevant to the present study, since
osteoblasts and adipocytes share a common precursor in the bone marrow
stromal cell. The concentrations of
-MSH found to be effective in
skeletal cells in the present studies are comparable to those that are active in in vitro studies of adipocytes (4) and
pancreatic
-cells (64) and are consistent with the
EC50 values for cAMP production stimulated by
-MSH via
MC1-R, MC3-R, or MC4-R (~10
9 M) in transfected cell
lines (26, 27, 43, 45, 61). The lowest active
concentrations of
-MSH in osteoblasts and chondrocytes are
comparable to the circulating concentrations in humans (34, 47) and rats (69), suggesting that these direct
actions in bone may be physiologically relevant.
The results of systemic administration of -MSH to adult mice are
generally consistent with the in vitro findings in that increased bone
turnover was observed in both models. In vivo, this was significant
with respect to the histomorphometric assessment of osteoblast numbers
but not those of osteoclasts, possibly reflecting the inaccuracy of the
assessment of bone resorption indexes from static histomorphometry.
However, the balance of these effects was toward bone loss, resulting
in a 22% decrease in trabecular bone volume in the animals receiving
-MSH and similar changes in the radiographic indexes of trabecular
mass. It would be of interest to extend these studies to a wider range
of doses of
-MSH and its analogs to determine whether the balance of
the opposing effects on osteoblasts and osteoclasts is affected by either of these variables. The present findings suggest that, in vivo,
the stimulatory effects of
-MSH on osteoclastogenesis predominate
over its actions on osteoblast development, although the
histomorphometry does not provide clear evidence of this. A second
explanation is that the direct effects of
-MSH on bone cells are
modulated by its effects on other tissues such as adipose tissue, the
pancreatic
-cell, or the central nervous system. These possibilities
will now be discussed.
As reviewed above, fat mass and bone mass have frequently been found to
be positively related to one another (35, 51, 53, 55, 59),
so the lower fat mass of the -MSH-treated animals is likely to
contribute to the negative effect of
-MSH on bone mass. This may
have been contributed to, in part, by the lower circulating leptin
levels following treatment with
-MSH (9, 23). The
effect of
-MSH on fat mass is well established: from studies of
-MSH administration in humans (23), from studies in
mice (32) and humans (22, 28, 31, 70) lacking
a functioning MC4-R, from findings in mice and humans deficient in the
-MSH-precursor peptide POMC (37, 74), and in the agouti
mouse, which overexpresses an antagonist of MC4-R (32).
The weight-regulatory actions of centrally administered
-MSH are the
product of appetite suppression and increased metabolism resulting from
-MSH effects in the hypothalamus (6), although there
may also be some contribution from its direct lipolytic effects on
adipocytes (4). When the peptide is administered
peripherally, as in the present studies, effects on fat mass presumably
result from the same two mechanisms of action, although the central
effect must be considerably less.
-MSH has been shown to directly inhibit secretion of insulin from
the pancreatic
-cell (64), and decreased circulating insulin concentrations have also been reported in humans treated systemically with
-MSH analogs (23). In vivo, insulin
concentrations correlate closely with bone density (57,
66), and this relationship may have a number of mechanisms.
Insulin interacts directly with osteoblasts, stimulating cell
proliferation in vitro and in vivo (14, 30, 52). Also,
insulin inhibits sex hormone-binding globulin production in the liver,
thereby increasing free concentrations of sex hormones (25, 39,
50). Furthermore, insulin is cosecreted with other bone-active
factors such as amylin [a direct stimulator of osteoblast growth and
an inhibitor of osteoclasts (10)], and preptin, a
fragment of pro-IGF-II, (5) is also a potent osteoblast
growth factor (8). Thus a cascade of
bone-anabolic factors comes from the pancreatic
-cell and will be
reduced by
-MSH treatment. Blockade of this pathway may contribute
to the bone loss seen in the in vivo study.
Systemically administered -MSH is most likely acting primarily
through peripheral melanocortin receptors, although there is evidence
that it may cross the blood-brain barrier to a small extent
(15), so a central mechanism of action cannot be
completely ruled out. The concept of a centrally acting agent impacting
on bone was first introduced by Ducy et al. (16), who
demonstrated that the intracerebroventricular injection of the
adipocyte hormone leptin resulted in trabecular bone loss similar to
that observed in the present in vivo study. The present findings may
contribute to an explanation of the work of Ducy et al., since
-MSH
is an important mediator of the central effects of leptin. Leptin
action in the hypothalamus stimulates
-MSH release from neurons of
the arcuate nucleus (63), resulting in appetite
suppression and weight loss and leading to effects mediated by the
autonomic nervous system. These include the regulation of insulin
secretion, which is decreased by as much as 80% by central
-MSH
administration (20). This effect is abrogated by
sympathetic blockade with phentolamine (20), confirming
that it is mediated by the autonomic nervous system. Thus any central
effects of
-MSH in this model may act in concert with its direct
effects on the pancreatic
-cell, described above, to decrease bone mass.
Takeda et al. (68) have recently provided significant new
information regarding the relationship between the bone effects of
leptin and -MSH. They reported that the intracerebroventricular administration of an
-MSH analog to ob/ob mice does not
affect bone mass; that the yellow agouti mouse, which is resistant to
-MSH, has a normal bone mass and shows the same bone response to
leptin as wild-type mice; and that mice deficient in MC4-R have normal
bone mass. However, the latter finding directly contradicts the report
of Farooqi et al. (22) in humans with this deficiency, in
whom bone mass is markedly increased. This discrepancy could be related
to differences between species in the role of
-MSH, differences in
the gene mutation, effects of the mutations on skeletal size, or
differences in techniques for assessment of bone mass (histomorphometry
of trabecular bone in the vertebral body in the mouse studies vs. whole
body dual-energy X-ray absorptiometry in the humans). Confirmation of
the results of Takeda et al. will be important to clarify understanding
in this area. In the meantime, the present data tend to support
the suggestion from the study of Farooqi et al. that
-MSH does have
an important effect on bone mass.
There is a striking parallel in the present data between the effects of
-MSH on chondrocytes and osteoblasts.
-MSH is a potent mitogen
for both cell types in vitro, but there is no evidence of this effect
in vivo. In the case of chondrocytes, this is reflected in the lack of
effect of
-MSH on the width of the growth plate or on tibial growth.
This contrasts with our previous work in this mouse model, in which
factors stimulating chondrocyte proliferation to a degree comparable to
that seen with
-MSH have also increased growth plate width and
tibial length in vivo (7, 12). It is likely that the
catabolic effects of systemically administered
-MSH mediated by the
hormonal and neuronal mechanisms discussed in the three preceding
paragraphs also account for this dissociation of its in vitro and in
vivo effects on cartilage.
This is not the first study of the effects of systemic -MSH
administration on bone. Stenstrom et al. (65) showed that
-MSH administration for 20 days increased cortical width in
hypophysectomized rats. In contrast, Aspenberg et al. (2)
found no effect in similar studies in normal rats, implying that their
result was specific to hormone-deficient animals. Thus both of these
results agree with those of the present study, which also found no
effect on cortical bone. Neither of these studies assessed trabecular bone. An ACTH analog has been shown to either stimulate or depress osteoblast proliferation in vivo, depending on dose and time of day
(71). The variability of these effects probably reflects an interaction with endogenous ACTH release, which itself shows a
marked diurnal rhythm. Interference with secretion of endogenous peptides is another mechanism by which the effects of
-MSH on bone
may be modified in vivo.
In conclusion, -MSH acts directly on skeletal cells to stimulate
their proliferation, but in vivo it leads to a net loss of trabecular
bone. This may be mediated, to some extent, by the effects of
-MSH
to decrease fat mass and to diminish the secretion of anabolic hormones
from the pancreatic
-cell. It may contribute to the bone loss that
follows from the intracerebroventricular administration of leptin. If
-MSH analogs were to be developed for the treatment of obesity, then
monitoring of their effects on bone would be an important safety consideration.
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. Andrew Stevenson and the support of CSIRO Manufacturing Science and Technology, Clayton, Victoria, Australia, in the performance of the phase-contrast X-ray imaging. We also acknowledge the generous assistance of X-Ray Technologies Pty Ltd. (XRT), Fuji, Japan, and Berthold Australia Pty Ltd., Melbourne, Australia. Doreen Presnall's assistance with the preparation of the manuscript is also gratefully acknowledged.
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FOOTNOTES |
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This work was funded 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.
First published March 4, 2003;10.1152/ajpendo.00412.2002
Received 18 September 2002; accepted in final form 17 February 2003.
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