Jessica L. Costa

In Vitro and in Vivo Effects of Adiponectin on Bone

Fat mass impacts on both bone turnover and bone density and is a critical risk factor for osteoporotic fractures. Adipocyte-derived hormones may contribute to this relationship, and adiponectin is a principal circulating adipokine. However, its effects on bone remain unclear. We have, therefore, investigated the direct effects of adiponectin on primary cultures of osteoblastic and osteoclastic cells in vitro and determined its integrated effects in vivo by characterizing the bone phenotype of adiponectin-deficient mice. Adiponectin was dose-dependently mitogenic to primary rat and human osteoblasts ( approximately 50% increase at 10 microg/ml) and markedly inhibited osteoclastogenesis at concentrations of 1 microg/ml or greater. It had no effect on osteoclastogenesis in RAW-264.7 cells or on bone resorption in isolated mature osteoclasts. In adiponectin knockout (AdKO) male C57BL/6J mice, trabecular bone volume and trabecular number (assessed by microcomputed tomography) were increased at 14 wk of age by 30% (P = 0.02) and 38% (P = 0.0009), respectively. Similar, nonsignificant trends were observed at 8 and 22 wk of age. Biomechanical testing showed lower bone fragility and reduced cortical hardness at 14 wk. We conclude that adiponectin stimulates osteoblast growth but inhibits osteoclastogenesis, probably via an effect on stromal cells. However, the AdKO mouse has increased bone mass, suggesting that adiponectin also has indirect effects on bone, possibly through modulating growth factor action or insulin sensitivity. Because adiponectin does influence bone mass in vivo, it is likely to be a contributor to the fat-bone relationship.

Skeletal phenotype of the leptin receptor-deficient db/db mouse.

Leptin, a major hormonal product of the adipocyte, regulates appetite and reproductive function through its hypothalamic receptors. The leptin receptor is present in osteoblasts and chondrocytes, and previously we have shown leptin to be an anabolic bone factor in vitro, stimulating osteoblast proliferation and inhibiting osteoclastogenesis. Leptin increases bone mass and reduces bone fragility when administered peripherally but also can indirectly reduce bone mass when administered into the central nervous system. However, data from animal models deficient in either leptin (ob/ob) or its receptor (db/db) remain contradictory. We compared the bone phenotype of leptin receptor–deficient (db/db) and wild‐type mice using micro–computed tomographic (µCT) analysis of the proximal tibias and vertebrae. In the tibia, db/db mice had reduced percent trabecular bone volume (13.0 ± 1.62% in wild‐type versus 6.01 ± 0.601% in db/db mice, p = .002) and cortical bone volume (411 ± 21.5 µm3 versus 316 ± 3.53 µm3, p = .0014), trabecular thickness (48.4 ± 001.07 µm versus 45.1 ± 0.929 µm, p = .041) and trabecular number (2.68 ± 0.319 mm−1 versus 1.34 ± 0.148 mm−1, p = .0034). In the fifth lumbar vertebral body, the trabecular thickness and cortical thickness were decreased in the db/db versus wild‐type mice (0.053 ± 0.0011 mm versus 0.047 ± 0.0013 mm, p = .0002 and 0.062 ± 0.00054 mm versus 0.056 ± 0.0009 mm, p = .0001), respectively, whereas the trabecular and cortical percent bone volume and trabecular number did not reach significance. The total (endosteal and periosteal) cortical perimeter (12.2 ± 0.19 mm versus 13.2 ± 0.30 mm, p = .01) was increased. The serum osteocalcin levels were reduced in the db/db mice, suggesting that bone formation rates are decreased. The material properties of db/db femurs were determined by three‐point bending and nanoindentation, showing decreased bone strength (13.3 ± 0.280 N versus 7.99 ± 0.984 N, p = .0074) and material stiffness (28.5 ± 0.280 GPa versus 25.8 ± 0.281 GPa, p < .0001). These results demonstrate that bone mass and strength are reduced in the absence of leptin signaling, indicating that leptin acts in vivo as an anabolic bone factor. This concurs with results of in vitro studies and of peripheral leptin administration in vivo and suggests that leptins direct effects on bone cells are likely to override its actions via the central nervous system.

Molecular mechanisms involved in the mitogenic effect of lactoferrin in osteoblasts

Lactoferrin, an iron-binding glycoprotein present in milk and other exocrine secretions in mammals, is anabolic to bone at physiological concentrations. Lactoferrin stimulates the proliferation, differentiation and survival of osteoblasts, as well as potently inhibiting osteoclastogenesis in bone marrow cultures. In the current study we further investigated the mechanism of action of lactoferrin in osteoblasts. We used low-density arrays to measure the level of expression of 45 genes in MC3T3-E1 osteoblast-like cells treated with lactoferrin, and identified transient, dose-dependent increases in the transcription levels of interleukin-6, of the pro-inflammatory factor prostaglandin-endoperoxide synthase 2 (Ptgs2), and of the transcription factor nuclear factor of activated T cells (Nfatc1). We demonstrated similar changes in primary osteoblast cultures from human and rat. Levels of prostaglandin E2 were increased in conditioned media collected from osteoblasts treated with lactoferrin, indicating that the activity of the enzyme cyclooxygenase 2 (COX2), which is encoded by Ptgs2, was also up-regulated. Using a luciferase reporter construct we showed that lactoferrin induced transcription from the NFAT consensus sequence. We found that inhibiting either COX2 or NFATc1 activity blocked the mitogenic effect of lactoferrin in osteoblasts and that inhibition of NFATc1 activity partially blocked the transcriptional activation of Ptgs2. Our study has provided the first evidence that COX2 and NFATc1 activities are increased by lactoferrin, and demonstrated a role for each of these molecules as mediators of the mitogenic effects of lactoferrin in osteoblasts.

Exposure to inflammatory cytokines IL-1β and TNFα induces compromise and death of astrocytes; implications for chronic neuroinflammation.

Background Astrocytes have critical roles in the human CNS in health and disease. They provide trophic support to neurons and are innate-immune cells with keys roles during states-of-inflammation. In addition, they have integral functions associated with maintaining the integrity of the blood-brain barrier. Methods We have used cytometric bead arrays and xCELLigence technology to monitor the to monitor the inflammatory response profiles and astrocyte compromise in real-time under various inflammatory conditions. Responses were compared to a variety of inflammatory cytokines known to be released in the CNS during neuroinflammation. Astrocyte compromise measured by xCELLigence was confirmed using ATP measurements, cleaved caspase 3 expression, assessment of nuclear morphology and cell death. Results Inflammatory activation (IL-1β or TNFα) of astrocytes results in the transient production of key inflammatory mediators including IL-6, cell surface adhesion molecules, and various leukocyte chemoattractants. Following this phase, the NT2-astrocytes progressively become compromised, which is indicated by a loss of adhesion, appearance of apoptotic nuclei and reduction in ATP levels, followed by DEATH. The earliest signs of astrocyte compromise were observed between 24-48h post cytokine treatment. However, significant cell loss was not observed until at least 72h, where there was also an increase in the expression of cleaved-caspase 3. By 96 hours approximately 50% of the astrocytes were dead, with many of the remaining showing signs of compromise too. Numerous other inflammatory factors were tested, however these effects were only observed with IL-1β or TNFα treatment. Conclusions Here we reveal direct sensitivity to mediators of the inflammatory milieu. We highlight the power of xCELLigence technology for revealing the early progressive compromise of the astrocytes, which occurs 24-48 hours prior to substantive cell loss. Death induced by IL-1β or TNFα is relevant clinically as these two cytokines are produced by various peripheral tissues and by resident brain cells.

Ghrelin is an Osteoblast Mitogen and Increases Osteoclastic Bone Resorption In Vitro.

Ghrelin is released in response to fasting, such that circulating levels are highest immediately prior to meals. Bone turnover is acutely responsive to the fed state, with increased bone resorption during fasting and suppression during feeding. The current study investigated the hypothesis that ghrelin regulates the activity of bone cells. Ghrelin increased the bone-resorbing activity of rat osteoclasts, but did not alter osteoclast differentiation in a murine bone marrow assay nor bone resorption in ex vivo calvarial cultures. Ghrelin showed mitogenic activity in osteoblasts, with a strong effect in human cells and a weaker effect in rat osteoblasts. The expression of the human ghrelin receptor, GHSR, varied among individuals and was detectable in 25–30% of bone marrow and osteoblast samples. However, the rodent Ghsr expression was undetectable in bone cells and cell lines from rat and mouse. These data suggest that elevated levels of ghrelin may contribute to the higher levels of bone turnover that occurs in the fasted state.

Extended treatment with selective phosphatidylinositol 3-kinase and mTOR inhibitors has effects on metabolism, growth, behaviour and bone strength.

The class I phosphatidylinositol 3‐kinases (PtdIns3Ks) mediate the effects of many hormones and growth factors on a wide range of cellular processes, and activating mutations or gene amplifications of class I PtdIns3K isoforms are known to contribute to oncogenic processes in a range of tumours. Consequently, a number of small‐molecule PtdIns3K inhibitors are under development and in clinical trial. The central signalling role of PtdIns3K in many cellular processes suggests there will be on‐target side effects associated with the use of these agents. To gain insights into what these might be we investigated the effect of extended daily dosing of eight small‐molecule inhibitors of class Ia PtdIns3Ks. Animals were characterized in metabolic cages to analyse food intake, oxygen consumption and movement. Insulin tolerance and body composition were analysed at the end of the experiment, the latter using EchoMRI. Bone volume and strength was assessed by micro‐CT and three‐point bending, respectively. Surprisingly, after sustained dosing with pan‐PtdIns3K inhibitors and selective inhibitors of the p110α isoform there was a resolution of the impairments in insulin tolerance observed in drug‐naïve animals treated with the same drugs. However, pan‐PtdIns3K inhibitors and selective inhibitors of the p110α have deleterious effects on animal growth, animal behaviour and bone volume and strength. Together, these findings identify a range of on target effects of PtdIns3K inhibitors and suggest use of these drugs in humans may have important adverse effects on metabolism, body composition, behaviour and skeletal health.

The atypical anti-psychotic clozapine decreases bone mass in rats in vivo

BACKGROUND Fracture risk is increased in patients with schizophrenia, who often receive long-term therapy with anti-psychotic drugs. The mechanisms by which skeletal fragility is increased in patients with psychosis include increased risk of falling, but direct skeletal toxicity of anti-psychotic drugs is a possibility that has not been investigated. METHODS We examined the skeletal effects, in vivo and in vitro, of a typical anti-psychotic drug, haloperidol, which primarily inhibits dopaminergic signaling, and an atypical anti-psychotic drug, clozapine, which predominantly inhibits serotonergic signaling. RESULTS In growing rats, 42 days of clozapine treatment reduced whole body bone mineral density by 15% (P<0.01 vs vehicle), and trabecular and cortical bone volume, as assessed by microcomputed tomography, by 29% and 15%, respectively (P<0.05 vs vehicle for each). Treatment with haloperidol did not affect bone density. Clozapine, but not haloperidol, transiently increased levels of serum corticosterone, and decreased levels of serum testosterone. In vitro, clozapine dose-dependently decreased osteoblast mitogenesis, osteoblast differentiation and osteoclastogenesis, while haloperidol did not affect any of these parameters. CONCLUSIONS These data demonstrate that clozapine, but not haloperidol, exerts adverse skeletal effects in rodents, and that this effect may be attributable to direct actions to reduce osteoblast growth and function. Long-term administration of clozapine may therefore negatively affect bone health, and clinical studies to investigate this possibility are warranted.

The bone-fat mass relationship: Laboratory studies

Over the past 20 years, a large number of clinical studies have shown a positive correlation between bone density and soft tissue mass. Furthermore, studies of fracture epidemiology have shown that low body weight is a risk factor for fractures. The greater mechanical load exerted on the skeleton by additional weight can only provide a partial explanation for the correlation between adipose tissue and the skeleton and a number of other physiological mechanisms operate in maintaining this correlation. Adipose tissue itself secretes factors known as “adipokines” that circulate and affect other target tissues. Levels of circulating adipokines vary with changes in food intake and weight, and there is evidence that numerous adipokines can regulate bone metabolism through direct and indirect mechanisms of action. Another source of hormones that regulate energy metabolism are β-pancreatic cells, which in obese states hypersecrete hormones with direct and indirect effects on bone. Hormones secreted from the gut are also likely to act as mediators of the bone-fat mass relationship, and in addition there is evidence for direct effects of ingested nutrients such as glucose and fatty acids on bone turnover. This review focuses mainly on results from laboratory studies investigating possible mechanisms involved in the positive relationship between bone mass and fat mass. IBMS BoneKEy. 2009 September;6(9):311-322. 2009 International Bone & Mineral Society Body weight influences both bone turnover and bone density, such that low body weight is a risk factor for osteoporotic fracture (1). The effects of body weight on skeletal health may be attributable to independent effects of fat mass and lean mass, but in postmenopausal women fat mass appears to be more important. Total body fat mass is positively related to bone density and inversely related to fracture risk, and changes in fat mass are positively related to changes in bone density (1). These clinical findings were the impetus for our group and others to investigate the possible mechanisms involved in the correlation of fat mass to bone mass. Many studies describe the bone effects of hormones, secreted by different tissues and cell types, which are responsive to changes in food intake and body mass, and are therefore likely to contribute to the relationship between adipose tissue and bone. This review will summarize some of the findings primarily from laboratory studies in the field. Adipokines Whereas in the past the adipocyte was not considered to play a significant regulatory role, it is now well-accepted that it is an important source of circulating factors, or “adipokines”, which act as regulators of metabolism. Several adipokines have been studied extensively and their effects in bone and other tissues are well-described, while a list of additional adipokines is steadily growing and their roles in bone biology remain to be elucidated.

Skeletal Actions of Fasting-Induced Adipose Factor (FIAF)

Several adipokines are known to influence skeletal metabolism. Fasting-induced adipose factor (FIAF) is an adipokine that gives rise to 2 further peptides in vivo, the N-terminal coiled-coil domain (FIAF(CCD)) and C-terminal fibrinogen-like domain (FIAF(FLD)). The skeletal action of these peptides is still uncertain. Our results show that FIAF(CCD) is a potent inhibitor of osteoclastogenesis and function, as seen in mouse bone marrow and RAW264.7 cell cultures, and in a resorption assay using isolated primary mature osteoclasts. The inhibitory effects at 500 ng/mL were approximately 90%, 50% and 90%, respectively, in these assays. FIAF(CCD) also stimulated osteoblast mitogenesis by approximately 30% at this concentration. In comparison, FIAF(FLD) was only active in decreasing osteoblast mitogenesis, and intact FIAF had no effect in any of these assays. In murine bone marrow cultures, FIAF(CCD) reduced the expression of macrophage colony-stimulating factor (M-CSF), nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP), and to lesser extent suppressed the expression of connective tissue growth factor (CTGF). FIAF(CCD) also decreased expression of M-CSF and CTGF in stromal/osteoblastic ST2 cells. Its effect on receptor activator of nuclear factor κB (RANKL) and osteoprotegerin expression in bone marrow was not consistent with its inhibitory action on osteoclastogenesis, but it decreased RANKL expression in ST2 cells. In RAW264.7 cell cultures, FIAF(CCD) significantly reduced the expression of NFATc1 and DC-STAMP. In conclusion, FIAF(CCD) inhibits osteoclast differentiation and function in vitro and decreases expression of genes encoding key osteoclastogenic factors such as M-CSF, CTGF, NFATc1, and DC-STAMP. FIAF(CCD)s action on osteoclasts may be independent of the RANKL/osteoprotegerin pathway. These results suggest a novel mechanism by which adipose tissue may regulate bone resorption and skeletal health.

Genetic modifications of mouse proopiomelanocortin peptide processing.

Pro-opiomelanocortin (POMC) is a prohormone which undergoes extensive tissue and cell specific post-translational processing producing a number of active peptides with diverse biological roles ranging from control of adrenal function to pigmentation to the regulation of feeding. One approach to unraveling the complexities of the POMC system is to engineer mouse mutants which lack specific POMC peptides. We describe here the design, generation, validation, and preliminary analysis of one such partial POMC mutant specifically lacking α-MSH. In contrast to POMC null mutant mice, mice lacking α-MSH in the presence of all other POMC peptides maintain adrenal structures and produce corticosterone comparable to wildtype littermates; however, they still have decreased levels of aldosterone, as found in POMC null mutant mice. Our findings demonstrate that α-MSH is not needed for maintenance of adrenal structure or for corticosterone production, but is needed for aldosterone production. These data demonstrate that mouse strains generated with precise genetic modifications of POMC peptide processing can answer questions about POMC peptide function. Further analysis of this and additional strains of mice with modified POMC peptide processing patterns will open up a novel avenue for studying the roles of individual POMC peptides.