alpha -Melanocyte-stimulating hormone is a novel regulator of bone

Jillian Cornish1, Karen E. Callon1, Kathleen G. Mountjoy2, Usha Bava1, Jian-Ming Lin1, Damian E. Myers3, Dorit Naot1, and Ian R. Reid1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

alpha -Melanocyte-stimulating hormone (alpha -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. alpha -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 alpha -MSH may act directly on the skeleton, a question addressed by the present studies. In primary cultures of osteoblasts and chondrocytes, alpha -MSH dose dependently (>= 10-9 M) stimulated cell proliferation. In bone marrow cultures, alpha -MSH (>10-9 M) stimulated osteoclastogenesis. Systemic administration of alpha -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 alpha -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 alpha -MSH effects elsewhere, such as the adipocyte, pancreatic beta -cell, or central nervous system.

osteoblast; osteoclast; chondrocyte; systemic administration


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -melanocyte-stimulating hormone (alpha -MSH).

alpha -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). alpha -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 alpha -MSH may act directly on the skeleton. The present studies address this question by assessing the effects of alpha -MSH on cells of the osteoblast and osteoclast lineages and assess the effect of its systemic administration to intact adult mice.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -MEM plus 15% FCS, 50 µg/ml L-ascorbic acid 2-phosphate and 10 mM beta -glycerophosphate, and one-half of the flasks were supplemented with 10-8 M alpha -MSH. The medium was changed to fresh medium with or without alpha -MSH on days 4 and 7. On days 4, 7, and 10, one flask treated with alpha -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 alpha 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 with alpha -MEM using a syringe with a 23-gauge needle. The marrow cells were collected in a 50-ml centrifuge tube, spun at 1,200 rpm for 2 min, and washed with 10% fetal bovine serum (FBS)-alpha -MEM (both from GIBCO). Marrow cells were then cultured for 2 h in 90-mm petri dishes. After 2 h, nonadherent cells were collected, spun at 1,200 rpm for 2 min, washed with 15% FBS-alpha -MEM, and seeded at 106 cells/ml in 48-well plates (0.5 ml/well). 1,25(OH)2-vitamin D3 (1,25(OH)2D3; 10-8 M) was added (day 0) to all wells except negative controls, which remained in the absence of 1,25(OH)2D3 throughout the experiment. Forty-eight hours later (day 2), cultures were fed 0.5 ml of fresh medium with 1,25(OH)2D3 to make a total of 1 ml/well. After a further 48 h (day 4), cultures were fed by replacing 0.5 ml of old medium with fresh medium with 1,25(OH)2D3. Test substances were added to test groups, and vehicle was added to control groups at 0, 2, and 4 days, depending on the particular protocol. All cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. After culture for 7 days, cells adherent to the well surface were fixed with citrate-acetone-formaldehyde (25:65:8, vol/vol/vol) for 30 s. Culture plates were then stained for tartrate-resistant acid phosphatase (TRAP) by use of SIGMA Kit 387-A (Sigma Diagnostics, St Louis, MO). TRAP-positive, multinucleated cells (containing >= 3 nuclei) were counted in all wells. Each experiment had three wells in which cells were grown on bone slices and checked for resorptive pits, indicating that the TRAP-positive, multinucleated cells in these cultures were capable of resorbing bone. There were at least eight wells for each group, and each experiment was repeated three or four times.

Mature 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 acidified alpha -MEM (72 µl concentrated HCl/80 ml medium) with 10% FBS (GIBCO-BRL-Life Technologies, Auckland, NZ) and antibiotics. Bones were homogenized gently, and the cell suspension was collected in a 15-ml conical tube. The remaining bone tissue was placed in a small petri dish with 1 ml of medium and chopped quickly with a scalpel blade. The resulting cell suspension was collected and added to the same conical tube. The bone tissue was homogenized again in 1 ml of medium, and the suspension was once again collected. This osteoclast-rich suspension was placed onto bovine bone slices, ~9 mm2, in 96-well plates and incubated at 37°C in a humidified atmosphere of 5% CO2 in air for 35 min to allow the mature osteoclasts to settle. The bone slices were washed several times in PBS to remove contaminating nonosteoclastic cells, followed by a rinse in medium. The bone slices were then placed in 6-well plates (4 slices/well) containing 6 ml of medium and incubated with test substances or vehicle for 20 h. After incubation, the bone slices were fixed with 2.5% glutaraldehyde-PBS and stained for TRAP using SIGMA Kit 387-A. The number of TRAP-positive multinucleated cells (i.e., containing >= 3 nuclei) on each bone slice was quantified, and the cells were removed by gentle scrubbing and then stained for 30 s with toluidine blue. After numerous washes in water, the bone slices were dried and assessed for the "pits" excavated by the osteoclasts. This was achieved using reflected-light microscopy with metallurgic lenses. The results were expressed as a ratio of the number of pits to the number of osteoclasts per bone slice. There were 6-12 bone slices in each group, and each experiment was repeated two or three times.

Bone 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 alpha -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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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Primary Osteoblasts and Chondrocytes

alpha -MSH dose dependently stimulated thymidine incorporation into primary cultures of fetal rat osteoblasts, indicating a stimulation of DNA synthesis. This was accompanied by an increase in cell number after 24 h of culture. Both of these effects were seen with alpha -MSH concentrations of 10-9 M and greater (Fig. 1).


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Fig. 1.   Effect of alpha -melanocyte-stimulating hormone (alpha -MSH) on cell number and thymidine incorporation in primary cultures of fetal rat osteoblasts at 24 h. * Significantly different from control, P <=  0.04; ** significantly different from control, P <=  0.01.

The effect of alpha -MSH on osteoblast differentiation was assessed by comparing the levels of expression of alkaline phosphatase, collagen I, and osteocalcin in osteoblasts treated with alpha -MSH with the expression in control cultures. Primary osteoblasts were treated with 10-8 M alpha -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.

alpha -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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Fig. 2.   Effect of alpha -MSH on cell number and thymidine incorporation in primary cultures of canine chondrocytes at 24 h. * Significantly different from control, P <=  0.05.

Osteoclast Effects

The effect of alpha -MSH on the development of osteoclasts was studied in cultures of mouse bone marrow (Fig. 3A). The number of osteoclasts present after 7 days of culture was doubled by alpha -MSH. In contrast, alpha -MSH did not affect the activity of mature osteoclasts. This was assessed from the number of resorption pits formed on bovine bone exposed for 20 h to osteoclasts prepared from neonatal rats (Fig. 3B). These results were confirmed in studies of a second model of mature osteoclast function, the neonatal mouse calvaria (Fig. 3C). Because there is virtually no bone marrow in this tissue, these explants reflect mature osteoclast function and again showed no effect of alpha -MSH treatment.


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Fig. 3.   A: effect of alpha -MSH on osteoclast development in mouse bone marrow cultures. The number of multinucleated (MNC) cells staining positively (+ve) for tartrate-resistant acid phosphatase (TRAP) was significantly increased by alpha -MSH (P < 0.03). Salmon calcitonin (sCT) was included as a positive control and significantly reduced osteoclast numbers (P < 0.01). B: effect of alpha -MSH on pit formation on bovine bone by isolated mature rat osteoclasts. No significant effects were detected. C: effect of alpha -MSH on calcium release from cultured neonatal mouse calvariae. There were no significant effects.

Systemic Administration of alpha -MSH

Normal adult male mice were treated systemically with alpha -MSH or vehicle over a 4-wk period. Histomorphometry of the proximal tibias indicated that bone turnover was increased in the alpha -MSH-treated animals [osteoblast index: control 20.1 ± 1.5, alpha -MSH 28.6 ± 1.7, P < 0.0007; osteoclast index: control 0.26 ± 0.02, alpha -MSH 0.29 ± 0.03, not significant (NS)]. However, trabecular bone volume was decreased by almost one-quarter in the animals receiving alpha -MSH (Fig. 4). This resulted from a decrease in trabecular number and an increase in trabecular separation rather than from any change in trabecular thickness (control 24.5 ± 1.3, alpha -MSH 25.1 ± 1.1 µm, P = 0.74). There was no difference in cortical width between the groups (control 0.19 ± 0.01, alpha -MSH 0.19 ± 0.01 mm), and growth plate thickness was also comparable in the two groups (control 0.087 ± 0.004, alpha -MSH 0.087 ± 0.004 mm).


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Fig. 4.   Effect of daily administration of alpha -MSH (20 times over 4 wk) on trabecular bone volume, trabecular number, and trabecular separation in the proximal tibias of normal adult male mice. * Significantly different from control, P = 0.03; ** significantly different from control, P < 0.01.

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 alpha -MSH-treated group. Simple inspection indicates that the extent of trabecular bone is diminished following alpha -MSH treatment. In the alpha -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 alpha -MSH caused a reduction in trabecular bone mass.


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Fig. 5.   A: phase-contrast X-ray images of the trabecular region of tibias from control (left) and alpha -MSH-treated mice (right). B: perimeter and length of the trabecular regions were quantified as described in METHODS and were significantly reduced in the animals treated with alpha -MSH, *P < 0.0005, **P < 0.002.

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 alpha -MSH. However, the percent fat mass of the animals at the end of the experiment was lower in the alpha -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, alpha -MSH 18.7 ± 0.1 mm).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study is the first to address the direct effects of alpha -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, alpha -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 alpha -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 alpha -MSH on adipocytes [where it promotes lipolysis (4)] and the pancreatic beta -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 alpha -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 beta -cells (64) and are consistent with the EC50 values for cAMP production stimulated by alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -MSH on bone cells are modulated by its effects on other tissues such as adipose tissue, the pancreatic beta -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 alpha -MSH-treated animals is likely to contribute to the negative effect of alpha -MSH on bone mass. This may have been contributed to, in part, by the lower circulating leptin levels following treatment with alpha -MSH (9, 23). The effect of alpha -MSH on fat mass is well established: from studies of alpha -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 alpha -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 alpha -MSH are the product of appetite suppression and increased metabolism resulting from alpha -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.

alpha -MSH has been shown to directly inhibit secretion of insulin from the pancreatic beta -cell (64), and decreased circulating insulin concentrations have also been reported in humans treated systemically with alpha -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 beta -cell and will be reduced by alpha -MSH treatment. Blockade of this pathway may contribute to the bone loss seen in the in vivo study.

Systemically administered alpha -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 alpha -MSH is an important mediator of the central effects of leptin. Leptin action in the hypothalamus stimulates alpha -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 alpha -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 alpha -MSH in this model may act in concert with its direct effects on the pancreatic beta -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 alpha -MSH. They reported that the intracerebroventricular administration of an alpha -MSH analog to ob/ob mice does not affect bone mass; that the yellow agouti mouse, which is resistant to alpha -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 alpha -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 alpha -MSH does have an important effect on bone mass.

There is a striking parallel in the present data between the effects of alpha -MSH on chondrocytes and osteoblasts. alpha -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 alpha -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 alpha -MSH have also increased growth plate width and tibial length in vivo (7, 12). It is likely that the catabolic effects of systemically administered alpha -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 alpha -MSH administration on bone. Stenstrom et al. (65) showed that alpha -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 alpha -MSH on bone may be modified in vivo.

In conclusion, alpha -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 alpha -MSH to decrease fat mass and to diminish the secretion of anabolic hormones from the pancreatic beta -cell. It may contribute to the bone loss that follows from the intracerebroventricular administration of leptin. If alpha -MSH analogs were to be developed for the treatment of obesity, then monitoring of their effects on bone would be an important safety consideration.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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