Alteration of bone cell function by RANKL and OPG in different in vitro models.

BACKGROUND: Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) are well-documented potent regulators of osteoclast development. However, their effects in mature bone cells and in organ cultures have not been well studied. It is uncertain whether their activities in different experimental models are comparable. MATERIALS AND METHODS: RANKL and OPG were evaluated for their activities in mouse calvarial organ cultures, mouse bone marrow cultures, isolated rat mature osteoclast assays and rat primary osteoblast cultures. Results In murine calvarial organ culture, both muRANKL (> or = 10 ng mL(-1)) and rRANKL (> or = 100 ng mL(-1)) significantly stimulated (45)Ca release, while OPG (> or = 50 ng mL(-1)) was an inhibitor of bone resorption. Meanwhile, [(3)H]-thymidine incorporation in this assay was also modulated (indicating proliferation increases in the osteoblast lineage of cells) although these peptides had no direct effect on [(3)H]-thymidine incorporation in isolated osteoblast assays. In mouse bone marrow cultures, muRANKL (> or = 1 ng mL(-1)) and rRANKL (> or = 5 ng mL(-1)) significantly stimulated osteoclastogenesis. The number of nuclei per osteoclast was also significantly increased. OPG strongly inhibited this index, with over 90% suppression at 1 ng mL(-1). Both muRANKL (10 ng mL(-1)) and rRANKL (100 ng mL(-1)) stimulated, while OPG (10 ng mL(-1)) inhibited osteoclast activity in isolated mature osteoclast assays. CONCLUSION: The current study demonstrated that bone resorption modulated by RANKL and OPG, in murine calvarial organ culture, leads to changes in osteoblast proliferation, suggesting a feedback mechanism from osteoclasts to osteoblasts. In addition, it was found that RANKL and OPG have more potent effects on osteoclastogenesis than on the activity of mature osteoclasts.

Preptin, another peptide product of the pancreatic beta-cell, is osteogenic in vitro and in vivo.

Several hormones that regulate nutritional status also impact on bone metabolism. Preptin is a recently isolated 34-amino acid peptide hormone that is cosecreted with insulin and amylin from the pancreatic beta-cells. Preptin corresponds to Asp(69)-Leu(102) of pro-IGF-II. Increased circulating levels of a pro-IGF-II peptide complexed with IGF-binding protein-2 have been implicated in the high bone mass phenotype observed in patients with chronic hepatitis C infection. We have assessed preptin's activities on bone. Preptin dose-dependently stimulated the proliferation (cell number and DNA synthesis) of primary fetal rat osteoblasts and osteoblast-like cell lines at periphysiological concentrations (>10(-11) M). In addition, thymidine incorporation was stimulated in murine neonatal calvarial organ culture, likely reflecting the proliferation of cells from the osteoblast lineage. Preptin did not affect bone resorption in this model. Preptin induced phosphorylation of p42/p44 MAP kinases in osteoblastic cells in a dose-dependent manner (10(-8)-10(-10) M), and its proliferative effects on primary osteoblasts were blocked by MAP kinase kinase inhibitors. Preptin also reduced osteoblast apoptosis induced by serum deprivation, reducing the number of apoptotic cells by >20%. In vivo administration of preptin increased bone area and mineralizing surface in adult mice. These data demonstrate that preptin, which is cosecreted from the pancreatic beta-cell with amylin and insulin, is anabolic to bone and may contribute to the preservation of bone mass observed in hyperinsulinemic states such as obesity.

Lactoferrin and bone; structure-activity relationships.

The maintenance of the mechanical integrity of the skeleton depends on bone remodeling, the well-coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts. The coupled action of osteoblasts and osteoclasts is regulated by the action of many local and circulating hormones and factors as well as central regulation by a neurological mechanism. We have previously shown that lactoferrin can promote bone growth. At physiological concentrations, lactoferrin potently stimulates the proliferation and differentiation of primary osteoblasts and acts as a survival factor. Lactoferrin also affects osteoclasts, potently inhibiting their formation. In vivo, local injection of lactoferrin results in substantial increases in bone formation and bone area. In a critical bone-defect model in vivo, lactoferrin was also seen to promote bone growth. The mitogenic effect of lactoferrin in osteoblast-like cells is mediated mainly through low-density lipoprotein-receptor protein-1 (LRP1), a member of the low-density lipoprotein-receptor-related proteins that are primarily known as endocytic receptors; however, LRP1 is not necessary for the anti-apoptotic actions of lactoferrin. Lactoferrin also induces the activation of p42/44 mitogen-activated protein kinase (MAPK) signalling and the PI3-kinase-dependent phosphorylation of Akt in osteoblasts. In this study, we examined other properties of lactoferrin and the way they affect osteogenic activity. The degree of glycosylation, iron-binding, and the structure-activity relationships indicate that lactoferrin maintains osteogenic activity in deglycosylated, holo, and apo forms, and in with various small fragments of the molecule. These data suggest that lactoferrin signals through more than 1 membrane-bound receptor to produce its anabolic skeletal effects, and that it signals through diverse pathways. We conclude that lactoferrin might have a physiological role in bone growth and healing and a potential therapeutic role as an anabolic factor in osteoporosis.

Interleukin-18 is a novel mitogen of osteogenic and chondrogenic cells.

IL-18 was identified due to its ability to induce interferon-gamma (IFNgamma) production by T cells. It is a pleiotropic factor that shares structural features with IL-1 and functional activities with IL-12. IL-18 has a role in T cell development, where it has been demonstrated to act cooperatively with IL-12 to regulate IFNgamma. In bone, IL-18 is mainly produced by macrophages, but is also expressed by osteoblasts and inhibits osteoclast formation through granulocyte-macrophage colony-stimulating factor (GM-CSF) and not IFNgamma production by T cells. We have investigated the effects of IL-18 on mature osteoclast activity and for potential actions on osteoblasts or chondrocytes. The effects of IL-18 on mature osteoclast activity were determined using two assays: isolated mature osteoclast cell culture and neonatal murine calvarial organ culture. IL-18 did not affect bone resorption in either assay system. The actions of IL-18 on osteogenic cells (primary cell cultures of fetal rat and neonatal mouse osteoblasts, as well as neonatal mouse calvarial organ culture) and primary chondrocytes (canine) were assessed by proliferation assays (quantification of cell numbers and thymidine incorporation). In each assay system, IL-18 acted as a mitogen to the osteogenic and chondrogenic cells. Since IL-18 signal transduction may involve IFNgamma or GM-CSF, we assessed their involvement in the IL-18 response. IL-18 did not induce IFNgamma production by primary osteoblasts, but, of greater significance, IFNgamma had the opposing action to IL-18 in that it inhibited the primary osteoblast cell proliferation. Although IL-18 rapidly induced GM-CSF production by primary osteoblasts, IL-18 was still mitogenic in osteoblast preparations established from GM-CSF-deficient mice. Combined, these studies indicate that IL-18 may have an autocrine/paracrine mitogen role for both osteogenic and chondrogenic cells, independent of the production of IFNgamma or GM-CSF.

Leptin directly regulates bone cell function in vitro and reduces bone fragility in vivo.

Fat mass is an important determinant of bone density, but the mechanism of this relationship is uncertain. Leptin, as a circulating peptide of adipocyte origin, is a potential contributor to this relationship. Recently it was shown that intracerebroventricular administration of leptin is associated with bone loss, suggesting that obesity should be associated with low bone mass, the opposite of what is actually found. Since leptin originates in the periphery, an examination of its direct effects on bone is necessary to address this major discrepancy. Leptin (>10(-11) m) increased proliferation of isolated fetal rat osteoblasts comparably with IGF-I, and these cells expressed the signalling form of the leptin receptor. In mouse bone marrow cultures, leptin (>or=10(-11) m) inhibited osteoclastogenesis, but it had no effect on bone resorption in two assays of mature osteoclasts. Systemic administration of leptin to adult male mice (20 injections of 43 micro g/day over 4 weeks) reduced bone fragility (increased work to fracture by 27% and displacement to fracture by 21%, P<0.001). Changes in tibial histomorphometry were not statistically significant apart from an increase in growth plate thickness in animals receiving leptin. Leptin stimulated proliferation of isolated chondrocytes, and these cells also expressed the signalling form of the leptin receptor. It is concluded that the direct bone effects of leptin tend to reduce bone fragility and could contribute to the high bone mass and low fracture rates of obesity. When administered systemically, the direct actions of leptin outweigh its centrally mediated effects on bone, the latter possibly being mediated by leptin's regulation of insulin sensitivity.

Effects of calcitonin, amylin, and calcitonin gene-related peptide on osteoclast development.

Amylin and calcitonin gene-related peptide (CGRP) are homologous 37 amino acid peptides that are found in the circulation. Both peptides belong to the calcitonin family. Similar to calcitonin, amylin and CGRP inhibit osteoclast activity, although they are much less potent than calcitonin. Calcitonin is known to act on the latter stages of osteoclast development, inhibiting the fusion of committed preosteoclasts to form mature multinucleated cells; however, whether or not calcitonin acts earlier in the formation of the precursor osteoclasts is controversial. The question of osteoclast development has never been examined with respect to amylin and CGRP. These issues are addressed in the present study. We studied the effects of calcitonin (salmon and rat), amylin (human and rat), and CGRP (human and rat) in mouse bone marrow cultures stimulated to generate osteoclasts using 1alpha,25-dihydroxyvitamin D3. Calcitonin dose-dependently decreased the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells as well as TRAP-positive mono-/binucleated cells at concentrations >10(-13) mol/L. Amylin and CGRP showed similar effects at concentrations >10(-9) mol/L. In addition, calcitonin substantially reduced the ratio of TRAP-positive multinucleated to mono-binucleated cells, indicating an effect on fusion of osteoclast precursors. The present data establish that this family of peptides not only acts on mature osteoclasts but also inhibits their development in bone marrow cultures. This activity is shared by amylin and CGRP. The much greater potency of calcitonin than amylin and CGRP is consistent with the action of these peptides being mediated by calcitonin receptors.

Systemic administration of adrenomedullin(27-52) increases bone volume and strength in male mice.

Adrenomedullin is a 52-amino acid peptide first described in a human phaeochromocytoma but since been found to be present in many tissues, including the vascular system and bone. Because of its structural similarity to amylin and calcitonin gene-related peptide, both of which have actions on bone cells, we have previously assessed the effects of adrenomedullin on the skeleton, and found that it increases osteoblast proliferation in vitro and bone formation following local injection in vivo. The present study carries this work forward by assessing the effects on bone of the systemic administration of a fragment of this peptide lacking the structural requirements for vasodilator activity. Two groups of 20 adult male mice received 20 injections of human adrenomedullin(27-52) 8.1 microg or vehicle over a 4-week period and bone histomorphometry and strength were assessed. In the tibia, adrenomedullin(27-52) produced increases in the indices of osteoblast activity, osteoid perimeter and osteoblast perimeter (P<0.05 for both using Student's t-test). Osteoclast perimeter was not affected. There was a 21% increase in cortical width and a 45% increase in trabecular bone volume in animals treated with adrenomedullin(27-52) (P<0.002 for both). Assessment of bone strength by three-point bending of the humerus showed both the maximal force and the displacement to the point of failure were increased in the animals treated with adrenomedullin(27-52) (P<0.03 for both). There was also a significant increase in the thickness of the epiphyseal growth plate. No adverse effects of the treatment were noted. It is concluded that adrenomedullin(27-52) acts as an anabolic agent on bone. These findings may be relevant to the normal regulation of bone mass and to the design of agents for the treatment of osteoporosis.

A potential role for adrenomedullin as a local regulator of bone growth.

Bone remodeling is a complex process of coordinated resorption and formation of bone, which is regulated by systemic hormones and by local factors. We have previously shown that the peptide hormone adrenomedullin is mitogenic to osteoblastic cells in vitro and that it promotes bone growth in vivo. The aim of the present study was to characterize the expression of molecules that may mediate adrenomedullin signaling in osteoblasts and to investigate the expression of adrenomedullin itself in these cells. The first adrenomedullin receptor that was cloned is the seven-transmembrane G protein-coupled receptor, L1. Two additional receptors for adrenomedullin, which arise from interactions between calcitonin receptor-like receptor and receptor activity modifying proteins 2 or 3, have now been described. In the current study, we used RT-PCR and Northern blot analysis to demonstrate that messenger RNA for the three adrenomedullin receptors, as well as for adrenomedullin itself, is expressed in primary rat osteoblasts. Treating primary osteoblasts with transforming growth factor-beta and insulin-like growth factor-I moderately reduced adrenomedullin RNA levels, whereas PTH had no effect. We have shown by immunocytochemistry that adrenomedullin peptide is present in osteoblasts, and by competitive binding assays that (125)I-adrenomedullin binds with high affinity to intact osteoblasts and to osteoblast cell membranes. Coexpression of adrenomedullin and adrenomedullin receptors in osteoblasts, taken together with our previous finding that adrenomedullin is mitogenic to these cells, raises the possibility that this peptide is a local regulator of bone growth.

Systemic administration of a novel octapeptide, amylin-(1---8), increases bone volume in male mice.

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.

Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes.

Peptides purified by HPLC are often in the form of a trifluoroacetate (TFA) salt, because trifluoroacetic acid is used as a solvent in reversed-phase HPLC separation. However, the potential effects of this contaminant in culture systems have not been addressed previously. TFA (10(-8) to 10(-7) M) reduced cell numbers and thymidine incorporation into fetal rat osteoblast cultures after 24 h. Similar effects were found in cultures of articular chondrocytes and neonatal mouse calvariae, indicating that the effect is not specific to one cell type or to one species of origin. When the activities of the TFA and hydrochloride salts of amylin, amylin-(1-8), and calcitonin were compared in osteoblasts, cell proliferation was consistently less with the TFA salts of these peptides, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding is likely to be relevant to all studies of purified peptides in concentrations above 10(-9) M in whatever cell or tissue type. Such peptides should be converted to a hydrochloride or biologically equivalent salt before assessment of their biological effects is undertaken.

Comparison of the effects of calcitonin gene-related peptide and amylin on osteoblasts.

Calcitonin gene-related peptide (CGRP) and amylin are homologous 37-amino-acid peptides which have been demonstrated to have anabolic effects on bone. It is not clear whether these effects are mediated by a common receptor, nor is it known which ligand is the more potent. These questions are addressed in the present study using cultures of fetal rat osteoblasts. CGRP increased cell number when present in a concentration >/=10-9 M, but 10-8 M CGRP was required to stimulate thymidine and phenylalanine incorporation. Amylin was effective on these indices at 100-fold lower concentrations, and its maximal effects were about twice as great as those of CGRP. ED50's for the effects of amylin and CGRP on cell number were 10-12 M and 10-10 M, respectively. There was no additivity between maximal doses of the peptides on these indices. The effects of specific receptor blockers on the maximal stimulation of cell number by these peptides were also studied. The CGRP receptor-blocker, CGRP-(8-37), completely blocked the effect of CGRP at blocker concentrations >/=10-9 M. In contrast, the amylin receptor blocker, amylin-(8-37), completely blocked the effects of CGRP when the blocker was present in concentrations as low as 10-11 M. The KI of CGRP-(8-37) was 2 x 10-10 M and that of amylin-(8-37) was 7 x 10-12 M. In converse experiments studying the blockade of maximal doses of amylin, amylin-(8-37) 10-10 M was effective (KI 1 x 10-10 M), whereas a 100-fold greater concentration of CGRP-(8-37) was necessary to achieve the same effect (KI 6 x 10-9 M). It is concluded that amylin and CGRP probably act through a common receptor to stimulate osteoblast growth, and that this receptor has a higher affinity for amylin than for CGRP.

Stimulation of osteoblast proliferation by C-terminal fragments of parathyroid hormone-related protein.

Parathyroid hormone (PTH)-related protein (107-139) (PTHrP(107-139)) and PTHrP(107-111) have been reported to be potent inhibitors of isolated osteoclast activity, and inhibition of bone resorption by PTHrP(107-139) occurs in vivo. However, the actions of C-terminal PTHrP on osteoblast activity has not been studied much. The present study addresses this issue by examining the effect of PTHrP(107-139), PTHrP(107-119), PTHrP(120-139), and PTHrP(107-111) on the proliferation of fetal rat osteoblasts. Treatment with PTHrP(107-139) for 24 h caused a dose-dependent increase in cell number, [3H]thymidine and [3H]phenylalanine incorporation in cultured osteoblasts. The effect was apparent at concentrations of 10-10 M and greater and was sustained over time. PTHrP(107-119) and PTHrP(107-111) had effects on cell number, DNA, and protein synthesis which were comparable to those of PTHrP(107-139), whereas PTHrP(120-139) was without effect. Retroverted PTHrP(107-111) also stimulated all three activities but was only one tenth as potent as PTHrP(107-139). PTHrP(107-139) had no effect on osteoblast apoptosis. It is concluded that PTHrP(107-139) is not only an inhibitor of osteoclastic bone resorption but that it also stimulates osteoblast growth. This activity resides within the pentapeptide fragment PTHrP(107-111). These findings support a possible role for C-terminal fragments of PTHrP in the normal regulation of bone cell function and, possibly, bone mass.

Systemic administration of amylin increases bone mass, linear growth, and adiposity in adult male mice.

Amylin is a peptide hormone cosecreted with insulin from the pancreatic beta-cells that can act as an osteoblast mitogen and as an inhibitor of bone resorption. The effects on bone of its systemic administration are uncertain. The present study addresses this question in adult male mice that were given daily subcutaneous injections of amylin (10.5 microgram) or vehicle (n = 20 in each group) for 4 wk. Histomorphometric indices of bone formation increased 30-100% in the amylin-treated group, whereas resorption indices were reduced by approximately 70% (P < 0.005 for all indices). Total bone volume in the proximal tibia was 13.5 +/- 1.4% in control animals and 23.0 +/- 2.0% in those receiving amylin (P = 0.0005). Cortical width, tibial growth plate width, tibial length, body weight, and fat mass were all increased in the amylin-treated group. It is concluded that systemic administration of amylin increases skeletal mass and linear bone growth. This peptide has potential as a therapy for osteoporosis if its bone effects can be dissociated from those on soft tissue mass.

Dissociation of the effects of amylin on osteoblast proliferation and bone resorption.

This study assesses the structure-activity relationships of the actions of amylin on bone. In fetal rat osteoblasts, only intact amylin and amylin-(1-8) stimulated cell proliferation (half-maximal concentrations 2.0 x 10(-11) and 2.4 x 10(-10) M, respectively). Amylin-(8-37), COOH terminally deamidated amylin, reduced amylin, and reduced amylin-(1-8) (reduction results in cleavage of the disulfide bond) were without agonist effect but acted as antagonists to the effects of both amylin and amylin-(1-8). Calcitonin gene-related peptide-(8-37) also antagonized the effects of amylin and amylin-(1-8) on osteoblasts but was substantially less potent in this regard than amylin-(8-37). In contrast, inhibition of bone resorption in neonatal mouse calvariae only occurred with the intact amylin molecule and was not antagonized by any of these peptides. The rate of catabolism of the peptides in calvarial cultures was not accelerated in comparison with that of intact amylin. This dissociation of the actions of amylin suggests that it acts through two separate receptors, one on the osteoclast (possibly the calcitonin receptor) and a second on the osteoblast.

Leukemia inhibitory factor is mitogenic to osteoblasts.

Leukemia inhibitory factor (LIF) regulates cell growth and is produced by a variety of tissues, including bone. Previously we have shown that recombinant human LIF induced an increase in osteoclast number, bone formation, and DNA synthesis. In the present study, we have defined the cells in intact bone at which the proliferative effects of LIF occur, using simultaneous enzyme histochemistry and autoradiographic techniques. The area of alkaline phosphatase-positive staining was increased twofold (p = 0.0008) and the number of [3H]thymidine-positive cells was increased twofold (p = 0.0024) in LIF-treated bones. The radiolabeled cells either colocalized with alkaline phosphatase or were in the osteoprogenitor region. They were not found in the acid phosphatase-positive staining osteoclasts. These results indicate that cells which have a mitogenic response to LIF are bone-forming rather than bone-resorbing cells.

Parathyroid hormone-related protein-(107-139) inhibits bone resorption in vivo.

PTH-related protein-(107-139) [PTHrP-(107-139)] has been reported previously to be a potent inhibitor of osteoclast activity. However, this finding has not been reproduced in other in vitro models. We have now examined the effects of this peptide in an in vivo model, employing intact adult mice. Four groups of eight male mice were given injections of either vehicle or one of three doses of PTHrP-(107-139) (4 x 10(-13), 4 x 10(-11), or 4 x 10(-9) mol) over the periosteum of the right hemicalvaria for 5 consecutive days. The animals were killed 1 week after the last injection. There were significant decreases in bone resorption indexes after all doses of PTHrP-(107-139), with a 70% decrease in osteoclast number (P < 0.001), a 70% decrease in osteoclast perimeter (P = 0.004) and a 50% decrease in eroded perimeter (P = 0.001). In addition, some indexes of bone formation were significantly decreased, with 40% decreases in both osteoblast number (P = 0.05) and osteoblast perimeter (P = 0.02), but no significant change in osteoid area. There was a dose-related upward trend in mineralized bone area, reaching 12% at the highest dose, but this was not statistically significant. It is concluded that PTHrP-(107-139) is a potent inhibitor of bone resorption in vivo. As this model is very dissimilar to that of isolated osteoclasts, in which this peptide is also active, the present findings suggest that osteoclast inhibition is an authentic action of the C-terminus of PTHrP, which may, therefore, play a role in the regulation of bone turnover in vivo.

Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo.

Adrenomedullin is a 52-amino acid vasodilator peptide produced in many tissues, including bone. It has 20% sequence identity with amylin, a regulator of osteoblast growth, and circulates in picomolar concentrations. The present study assesses whether adrenomedullin also acts on osteoblasts. At concentrations of 10(-12) M and greater, adrenomedullin produced a dose-dependent increase in cell number and [3H]thymidine incorporation in cultures of fetal rat osteoblasts. This effect was also seen with adrenomedullin-(15-52), -(22-52), and -(27-52), but adrenomedullin-(40-52) was inactive. These effects were lost in the presence of amylin blockers, suggesting they were mediated by the amylin receptor. Adrenomedullin also increased [3H]thymidine incorporation into cultured neonatal mouse calvaria but, unlike amylin, did not reduce bone resorption in this model. Adrenomedullin stimulated phenylalanine incorporation into both isolated osteoblasts and calvaria. When injected daily for 5 days over the calvariae of adult mice, it increased indexes of bone formation two- to threefold (P < 0.0001) and increased mineralized bone area by 14% (P = 0.004). It is concluded that adrenomedullin regulates osteoblast function and that it increases bone mass in vivo. The potential of this family of peptides in the therapy of osteoporosis should be further evaluated.

Insulin increases histomorphometric indices of bone formation In vivo.

Recent clinical studies have established that bone density is related to both fat mass and circulating insulin levels. A direct action of insulin on the osteoblast may contribute to these relationships. Osteoblast-like cells have insulin receptors, and insulin has been shown to stimulate proliferation of these cells in vitro. However, it has not been possible to study the effects of insulin administration on bone in vivo because of the metabolic effects of insulin, particularly hypoglycemia. A model involving the local injection of insulin over one hemicalvaria of an adult mouse overcomes these difficulties and permits the histomorphometric study of insulin's action on bone. Insulin or vehicle was injected daily for 5 days over the right hemicalvariae of adult mice, and the animals were sacrificed 1 week later. All indices of bone formation were significantly increased in insulin-treated hemicalvariae compared with the noninjected hemicalvariae. There was a 2.73 +/- 0. 50-fold increase in osteoid area (P = 0.005), a 2.20 +/- 0.37-fold increase in osteoblast surface (P = 0.021) and a 2.04 +/- 0.29-fold increase in osteoblast number (P = 0.012). Indices of bone resorption tended to decline and mineralized bone area tended to increase in insulin-treated animals. The direct action of insulin on bone may contribute to the increased bone density seen in obesity and to the osteopenia of type I diabetes, conditions associated with insulin excess and deficiency, respectively.

An in vivo model for the rapid assessment of the local effects of parathyroid hormone on bone histomorphometry.

The process of bone remodelling is likely to be controlled to a large extent by factors acting locally in a paracrine or autocrine manner, along with some systemic control. In our laboratory we routinely use an in vivo model in which the local effects of factors on bone histomorphometry can be determined. The factor under investigation is injected just above the periosteum of the right hemicalvaria in the adult male mouse. These subcutaneous injections are given daily over a 1-week period and the animals sacrificed at intervals after the last injection. With appropriate staining techniques it is possible to determine the effects of a particular agent on osteoblast and osteoclast numbers; the area of total bone, mineralized bone, osteoid and periosteum; osteoblast, osteoclast and eroded surfaces, and thus to infer the rate of bone formation and bone resorption in the right hemicalvaria compared to the uninjected left hemicalvaria and to vehicle-injected control animals. All these parameters are measured using a bone-dedicated image analyzer. Utilizing this in vivo model, we and others have studied a number of bone-active factors. We report the effects of parathyroid hormone, as well as reviewing results of our studies of leukemia inhibitory factor, amylin, calcitonin and calcitonin gene-related peptide in the model. The results obtained are similar to those found in most other animal models and in man. In conclusion, we describe an in vivo model whereby bone-active factors, injected locally, can be rapidly assessed.

Amylin stimulates osteoblast proliferation and increases mineralized bone volume in adult mice.

Amylin, a 37-amino-acid peptide co-secreted with insulin from the beta-cells of the pancreatic islets, has previously been demonstrated to inhibit bone resorption in vitro. However, its effects on bone formation and bone mass have not been assessed. We report that periphysiological concentrations of amylin stimulate proliferation of fetal rat osteoblasts in vitro. When amylin is injected daily for 5 days over the calvariae of adult mice in vivo, there are substantial increases in histomorphometric indices of bone formation, a reduction in bone resorption, and a significant increase in mineralized bone area. Equimolar doses of calcitonin in this in vivo model produced an inhibition of bone resorption but no significant effect on bone area. These findings support a role for amylin as a physiological regulator of bone and suggest that it should also be evaluated as a potential treatment for osteoporosis.