Publications by authors named "Paul A Baldock"

76 Publications

Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption.

Cell 2021 Mar 25;184(5):1330-1347.e13. Epub 2021 Feb 25.

Healthy Ageing Theme, Garvan Institute of Medical Research, Sydney, NSW, Australia.

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.
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http://dx.doi.org/10.1016/j.cell.2021.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938889PMC
March 2021

The mTORC1 complex in pre-osteoblasts regulates whole-body energy metabolism independently of osteocalcin.

Bone Res 2021 Feb 8;9(1):10. Epub 2021 Feb 8.

Adelaide Medical School, Faculty of Health and Medical Science, University of Adelaide, Adelaide, 5005, SA, Australia.

Overnutrition causes hyperactivation of mTORC1-dependent negative feedback loops leading to the downregulation of insulin signaling and development of insulin resistance. In osteoblasts (OBs), insulin signaling plays a crucial role in the control of systemic glucose homeostasis. We utilized mice with conditional deletion of Rptor to investigate how the loss of mTORC1 function in OB affects glucose metabolism under normal and overnutrition dietary states. Compared to the controls, chow-fed Rptor mice had substantially less fat mass and exhibited adipocyte hyperplasia. Remarkably, upon feeding with high-fat diet, mice with pre- and post-natal deletion of Rptor in OBs were protected from diet-induced obesity and exhibited improved glucose metabolism with lower fasting glucose and insulin levels, increased glucose tolerance and insulin sensitivity. This leanness and resistance to weight gain was not attributable to changes in food intake, physical activity or lipid absorption but instead was due to increased energy expenditure and greater whole-body substrate flexibility. RNA-seq revealed an increase in glycolysis and skeletal insulin signaling pathways, which correlated with the potentiation of insulin signaling and increased insulin-dependent glucose uptake in Rptor-knockout osteoblasts. Collectively, these findings point to a critical role for the mTORC1 complex in the skeletal regulation of whole-body glucose metabolism and the skeletal development of insulin resistance.
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http://dx.doi.org/10.1038/s41413-020-00123-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868369PMC
February 2021

Roux-en-Y gastric bypass and gastric sleeve surgery result in long term bone loss.

Int J Obes (Lond) 2021 Jan 26;45(1):235-246. Epub 2020 Aug 26.

Osteoporosis and Bone Biology Division, Garvan institute of Medical Research, Sydney, NSW, Australia.

Objectives: Little is known about the long-term skeletal impact of bariatric procedures, particularly the increasingly commonly performed gastric sleeve surgery (GS). We examined bone density (BMD) change following three types of bariatric surgery Roux-en-Y gastric bypass (RYGB), GS and laparoscopic adjustable gastric banding (LAGB), compared with diet, over 36 months.

Methods: Non-randomized, prospective study of participants with severe obesity (n = 52), undergoing weight-loss interventions: RYGB (n = 7), GS (n = 21), LAGB (n = 11) and diet (n = 13). Measurements of calciotropic indices, gut hormones (fasting and post prandial) peptide YY (PYY), glucagon-like peptide 1 (GLP1) and adiponectin together with dual-X-ray absorptiometry and quantitative computed tomography scans were performed thorough the study.

Results: All groups lost weight during the first 12 months. Despite weight stability from 12 to 36 months and supplementation of calcium and vitamin D, there was progressive bone loss at the total hip (TH) over 36 months in RYGB -14% (95% CI: -12, -17) and GS -9% (95% CI: -7, -10). In RYGB forearm BMD also declined over 36 months -9% (95% CI: -6, -12) and LS BMD declined over the first 12 months -7% (95% CI: -3, -12). RYGB and GS groups experienced significantly greater bone loss until 36 months than LAGB and diet groups, which experienced no significant BMD loss. These bone losses remained significant after adjustment for weight loss and age. RYGB and GS procedures resulted in elevated postprandial PYY, adiponectin and bone turnover markers up to 36 months without such changes among LAGB and diet participants.

Conclusions: RYGB and GS but not LAGB resulted in ongoing TH bone loss for three postoperative years. For RYGB, bone loss was also observed at LS and non-weight-bearing forearms. These BMD changes were independent of weight and age differences. We, therefore, recommend close monitoring of bone health following RYGB and GS surgeries.
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http://dx.doi.org/10.1038/s41366-020-00660-xDOI Listing
January 2021

Neuropeptide Y Regulation of Energy Partitioning and Bone Mass During Cold Exposure.

Calcif Tissue Int 2020 11 17;107(5):510-523. Epub 2020 Aug 17.

Bone Biology Division, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW, 2010, Australia.

The maintenance of whole body energy homeostasis is critical to survival and mechanisms exist whereby an organism can adapt to its environment and the stresses placed upon it. Environmental temperature and thermogenesis are key components known to affect energy balance. However, little is known about how these processes are balanced against the overall energy balance. We show that even mild cold exposure has a significant effect on energy expenditure and UCP-1 levels which increase by 43% and 400%, respectively, when wild-type (WT) mice at thermoneutral (29 °C) were compared to mice at room temperature (22 °C) conditions. Interestingly, bone mass was lower in cold-stressed WT mice with significant reductions in femoral bone mineral content (- 19%) and bone volume (- 13%). Importantly, these cold-induced skeletal changes were absent in mice lacking NPY, one of the main controllers of energy homeostasis, highlighting the critical role of NPY in this process. However, energy expenditure was significantly greater in cold-exposed NPY null mice, indicating that suppression of non-thermogenic tissues, like bone, contributes to the adaptive responses to cold exposure. Altogether, this work identifies NPY as being crucial in coordinating energy and bone homeostasis where it suppresses energy expenditure, UCP-1 levels and lowers bone mass under conditions of cold exposure.
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http://dx.doi.org/10.1007/s00223-020-00745-9DOI Listing
November 2020

Direct evidence for transport of RNA from the mouse brain to the germline and offspring.

BMC Biol 2020 04 30;18(1):45. Epub 2020 Apr 30.

QIMR Berghofer Medical Research Institute, Herston, QLD, 4006, Australia.

Background: The traditional concept that heritability occurs exclusively from the transfer of germline-restricted genetics is being challenged by the increasing accumulation of evidence confirming the existence of experience-dependent transgenerational inheritance. However, questions remain unanswered as to how heritable information can be passed from somatic cells. Previous studies have implicated the critical involvement of RNA in heritable transgenerational effects, and the high degree of mobility and genomic impact of RNAs in all organisms is an attractive model for the efficient transfer of genetic information.

Results: We hypothesized that RNA may be transported from a somatic tissue, in this case the brain, of an adult male mouse to the germline, and subsequently to embryos. To investigate this, we injected one hemisphere of the male mouse striatum with an AAV1/9 virus expressing human pre-MIR941 (MIR941). After 2, 8 and 16 weeks following injection, we used an LNA-based qPCR system to detect the presence of virus and human MIR941 in brain, peripheral tissues and embryos, from injected male mice mated with uninjected females. Virus was never detected outside of the brain. Verification of single bands of the correct size for MIR941 was performed using Sanger sequencing while quantitation demonstrated that a small percentage (~ 1-8%) of MIR941 is transported to the germline and to embryos in about a third of the cases.

Conclusions: We show that somatic RNA can be transported to the germline and passed on to embryos, thereby providing additional evidence of a role for RNA in somatic cell-derived intergenerational effects.
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http://dx.doi.org/10.1186/s12915-020-00780-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191717PMC
April 2020

Energy partitioning between fat and bone mass is controlled via a hypothalamic leptin/NPY relay.

Int J Obes (Lond) 2020 10 9;44(10):2149-2164. Epub 2020 Mar 9.

Neuroscience Division, Garvan Institute of Medical Research, St Vincent's Hospital Sydney, Sydney, NSW, 2010, Australia.

Background/objectives: Maintaining energy balance is important to ensure a healthy organism. However, energy partitioning, coordinating the distribution of sufficient energy to different organs and tissues is equally important, but the control of this process is largely unknown. In obesity, an increase in fat mass necessitates the production of additional bone mass to cope with the increase in bodyweight and processes need to be in place to communicate this new weight bearing demand. Here, we investigate the interaction between leptin and NPY, two factors critically involved in the regulation of both energy metabolism and bone mass, in this process.

Methods: We assessed the co-localization of leptin receptors on NPY neurons using RNAScope followed by a systematic examination of body composition and energy metabolism profiling in male and female mice lacking leptin receptors specifically in NPY neurons (Lepr;NPY). The effect of short-term switching between chow and high-fat diet was also examined in these mice.

Results: We uncovered that leptin receptor expression is greater on a subpopulation of NPY neurons in the arcuate that do not express AgRP. We further show that Lepr;NPY mice exhibit significantly increased adiposity while bone mass is diminished. These body composition changes occur in the absence of alterations in food intake or energy expenditure, demonstrating a prominent role for leptin signaling in NPY neurons in the control of energy partitioning. Importantly however, when fed a high-fat diet, these mice display a switch in energy partitioning whereby they exhibit a significantly enhanced ability to increase their bone mass to match the increased bodyweight caused by higher caloric intake concurrent with attenuated adiposity.

Conclusions: Taken together, these results demonstrate that leptin signaling in NPY neurons is critical for coordinating energy partitioning between fat and bone mass especially during situations of changes in energy balance.
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http://dx.doi.org/10.1038/s41366-020-0550-6DOI Listing
October 2020

Treatment of type 2 diabetes with the designer cytokine IC7Fc.

Nature 2019 10 25;574(7776):63-68. Epub 2019 Sep 25.

Baker Heart & Diabetes Institute, Melbourne, Victoria, Australia.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.
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http://dx.doi.org/10.1038/s41586-019-1601-9DOI Listing
October 2019

Using mouse genetics to understand human skeletal disease.

Bone 2019 09 15;126:27-36. Epub 2019 Feb 15.

Division of Bone Biology, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia; St Vincent's Clinical School, Faculty of Medicine, UNSW Australia, Sydney, NSW, 2010, Australia; University of Notre Dame Australia, Sydney, NSW, 2010, Australia.

Technological advances have enabled the study of the human genome in incredible detail with relative ease. However, our ability to interpret the functional significance of the millions of genetic variants present within each individual is limited. As a result, the confident assignment of disease-causing variant calls remains a significant challenge. Here we explore how mouse genetics can help address this deficit in functional genomic understanding. Underpinned by marked genetic correspondence, skeletal biology shows inter-species similarities which provide important opportunities to use data from mouse models to direct research into the genetic basis of skeletal pathophysiology. In this article we outline critical resources that may be used to establish genotype/phenotype relationships in skeletal tissue, identify genes with established skeletal effects and define the transcriptome of critical skeletal cell types. Finally, we outline how these mouse resources might be utilized to progress from a list of human sequence variants toward plausible gene candidates that contribute to skeletal disease.
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http://dx.doi.org/10.1016/j.bone.2019.02.015DOI Listing
September 2019

Slc20a2, Encoding the Phosphate Transporter PiT2, Is an Important Genetic Determinant of Bone Quality and Strength.

J Bone Miner Res 2019 06 19;34(6):1101-1114. Epub 2019 Mar 19.

Molecular Endocrinology Laboratory, Department of Medicine, Imperial College London, London, UK.

Osteoporosis is characterized by low bone mineral density (BMD) and fragility fracture and affects over 200 million people worldwide. Bone quality describes the material properties that contribute to strength independently of BMD, and its quantitative analysis is a major priority in osteoporosis research. Tissue mineralization is a fundamental process requiring calcium and phosphate transporters. Here we identify impaired bone quality and strength in Slc20a2 mice lacking the phosphate transporter SLC20A2. Juveniles had abnormal endochondral and intramembranous ossification, decreased mineral accrual, and short stature. Adults exhibited only small reductions in bone mass and mineralization but a profound impairment of bone strength. Bone quality was severely impaired in Slc20a2 mice: yield load (-2.3 SD), maximum load (-1.7 SD), and stiffness (-2.7 SD) were all below values predicted from their bone mineral content as determined in a cohort of 320 wild-type controls. These studies identify Slc20a2 as a physiological regulator of tissue mineralization and highlight its critical role in the determination of bone quality and strength. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
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http://dx.doi.org/10.1002/jbmr.3691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618161PMC
June 2019

RANK deletion in neuropeptide Y neurones attenuates oestrogen deficiency-related bone loss.

J Neuroendocrinol 2019 02 1;31(2):e12687. Epub 2019 Feb 1.

Neuroscience Division, Garvan Institute, St Vincent's Hospital, Darlinghurst, New South Wales, Australia.

The RANKL pathway is known to be an important aspect of the pathogenesis of oestrogen deficiency-induced bone loss. RANK deletion specifically in neuropeptide Y (NPY) neurones has been shown to enhance the ability of the skeleton to match increases in body weight caused by high-fat diet feeding, likely via the modulation of NPY levels. In the present study, we used ovariectomy in female mice to show that RANK deletion in NPY neurones attenuates bone loss caused by long-term oestrogen deficiency, particularly in the vertebral compartment. Ovariectomy led to a reduction in NPY expression levels in the arcuate nucleus of NPY ;RANK mice compared to NPY ;RANK controls. Because NPY deficient mice also displayed a similar protection against ovariectomy-induced bone loss, modulation of hypothalamic NPY signalling is the likely mechanism behind the protection from bone loss in the NPY ;RANK mice.
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http://dx.doi.org/10.1111/jne.12687DOI Listing
February 2019

Diet-induced adaptive thermogenesis requires neuropeptide FF receptor-2 signalling.

Nat Commun 2018 11 9;9(1):4722. Epub 2018 Nov 9.

Neuroscience Division, Garvan Institute of Medical Research, St. Vincent's Hospital, Darlinghurst, NSW, Australia.

Excess caloric intake results in increased fat accumulation and an increase in energy expenditure via diet-induced adaptive thermogenesis; however, the underlying mechanisms controlling these processes are unclear. Here we identify the neuropeptide FF receptor-2 (NPFFR2) as a critical regulator of diet-induced thermogenesis and bone homoeostasis. Npffr2 mice exhibit a stronger bone phenotype and when fed a HFD display exacerbated obesity associated with a failure in activating brown adipose tissue (BAT) thermogenic response to energy excess, whereas the activation of cold-induced BAT thermogenesis is unaffected. NPFFR2 signalling is required to maintain basal arcuate nucleus NPY mRNA expression. Lack of NPFFR2 signalling leads to a decrease in BAT thermogenesis under HFD conditions with significantly lower UCP-1 and PGC-1α levels in the BAT. Together, these data demonstrate that NPFFR2 signalling promotes diet-induced thermogenesis via a novel hypothalamic NPY-dependent circuitry thereby coupling energy homoeostasis with energy partitioning to adipose and bone tissue.
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http://dx.doi.org/10.1038/s41467-018-06462-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226433PMC
November 2018

Effects of Leptin on the Skeleton.

Endocr Rev 2018 12;39(6):938-959

University of Auckland, Auckland, New Zealand.

Leptin originates in adipocytes, including those in bone marrow, and circulates in concentrations 20 to 90 times higher than those in the cerebrospinal fluid. It has direct anabolic effects on osteoblasts and chondrocytes, but it also influences bone indirectly, via the hypothalamus and sympathetic nervous system, via changes in body weight, and via effects on the production of other hormones (e.g., pituitary). Leptin's role in bone physiology is determined by the balance of these conflicting effects. Reflecting this inconsistency, the leptin-deficient mouse has reduced length and bone mineral content of long bones but increased vertebral trabecular bone. A consistent bone phenotype in human leptin deficiency has not been established. Systemic leptin administration in animals and humans usually exerts a positive effect on bone mass, and leptin administration into the cerebral ventricles usually normalizes the bone phenotype in leptin-deficient mice. Reflecting the role of the sympathetic nervous system in mediating the central catabolic effects of leptin on the skeleton, β-adrenergic agonists and antagonists have major effects on bone in mice, but this is not consistently seen in humans. The balance of the central and peripheral effects of leptin on bone remains an area of substantial controversy and might vary between species and according to other factors such as body weight, baseline circulating leptin levels, and the presence of specific pathologies. In humans, leptin is likely to contribute to the positive relationship observed between adiposity and bone density, which allows the skeleton to respond appropriately to changes in soft tissue mass.
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http://dx.doi.org/10.1210/er.2017-00226DOI Listing
December 2018

Diet-induced obesity suppresses cortical bone accrual by a neuropeptide Y-dependent mechanism.

Int J Obes (Lond) 2018 11 9;42(11):1925-1938. Epub 2018 Mar 9.

Osteoporosis and Bone Biology Division, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW, 2010, Australia.

Objective: To determine whether age and neuropeptide Y (NPY) were involved in the skeletal response to extended periods of diet-induced obesity.

Methods: Male wild-type (WT) and NPY null (NPYKO) mice were fed a mild (23% fat) high-fat diet for 10 weeks from 6 or 16 weeks of age. Metabolism and bone density were assessed during feeding. Skeletal changes were assessed by microCT and histomorphometry.

Results: High-fat feeding in 6-week-old WT mice led to significantly increased body weight, adiposity and serum leptin levels, accompanied with markedly suppressed cortical bone accrual. NPYKO mice were less susceptible to fat accrual but, importantly, displayed a complete lack of suppression of bone accrual or cortical bone loss. In contrast, when skeletally mature (16 week old) mice underwent 10 weeks of fat feeding, the metabolic response to HFD was similar to younger mice, however bone mass was not affected in either WT or NPYKO. Thus, growing mice are particularly susceptible to the detrimental effects of HFD on bone mass, through suppression of bone accrual involving NPY signalling.

Conclusion: This study provides new insights into the relationship between the opposing processes of a positive weight/bone relationship and the negative 'metabolic' effect of obesity on bone mass. This negative effect is particularly active in growing skeletons, which have heightened sensitivity to changes in obesity. In addition, NPY is identified as a fundamental driver of this negative 'metabolic' pathway to bone.
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http://dx.doi.org/10.1038/s41366-018-0028-yDOI Listing
November 2018

High dietary fat and sucrose results in an extensive and time-dependent deterioration in health of multiple physiological systems in mice.

J Biol Chem 2018 04 13;293(15):5731-5745. Epub 2018 Feb 13.

From the Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Camperdown, New South Wales 2006, Australia.

Obesity is associated with metabolic dysfunction, including insulin resistance and hyperinsulinemia, and with disorders such as cardiovascular disease, osteoporosis, and neurodegeneration. Typically, these pathologies are examined in discrete model systems and with limited temporal resolution, and whether these disorders co-occur is therefore unclear. To address this question, here we examined multiple physiological systems in male C57BL/6J mice following prolonged exposure to a high-fat/high-sucrose diet (HFHSD). HFHSD-fed mice rapidly exhibited metabolic alterations, including obesity, hyperleptinemia, physical inactivity, glucose intolerance, peripheral insulin resistance, fasting hyperglycemia, ectopic lipid deposition, and bone deterioration. Prolonged exposure to HFHSD resulted in morbid obesity, ectopic triglyceride deposition in liver and muscle, extensive bone loss, sarcopenia, hyperinsulinemia, and impaired short-term memory. Although many of these defects are typically associated with aging, HFHSD did not alter telomere length in white blood cells, indicating that this diet did not generally promote all aspects of aging. Strikingly, glucose homeostasis was highly dynamic. Glucose intolerance was evident in HFHSD-fed mice after 1 week and was maintained for 24 weeks. Beyond 24 weeks, however, glucose tolerance improved in HFHSD-fed mice, and by 60 weeks, it was indistinguishable from that of chow-fed mice. This improvement coincided with adaptive β-cell hyperplasia and hyperinsulinemia, without changes in insulin sensitivity in muscle or adipose tissue. Assessment of insulin secretion in isolated islets revealed that leptin, which inhibited insulin secretion in the chow-fed mice, potentiated glucose-stimulated insulin secretion in the HFHSD-fed mice after 60 weeks. Overall, the excessive calorie intake was accompanied by deteriorating function of numerous physiological systems.
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http://dx.doi.org/10.1074/jbc.RA117.000808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900752PMC
April 2018

Homozygous Dkk1 Knockout Mice Exhibit High Bone Mass Phenotype Due to Increased Bone Formation.

Calcif Tissue Int 2018 01 6;102(1):105-116. Epub 2017 Nov 6.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Research Building, Locked Bag 4001, Westmead, NSW, 2145, Australia.

Wnt antagonist Dkk1 is a negative regulator of bone formation and Dkk1 heterozygous mice display a high bone mass phenotype. Complete loss of Dkk1 function disrupts embryonic head development. Homozygous Dkk1 mice that were heterozygous for Wnt3 loss of function mutation (termed Dkk1 KO) are viable and allowed studying the effects of homozygous inactivation of Dkk1 on bone formation. Dkk1 KO mice showed a high bone mass phenotype exceeding that of heterozygous mice as well as a high incidence of polydactyly and kinky tails. Whole body bone density was increased in the Dkk1 KO mice as shown by longitudinal dual-energy X-ray absorptiometry. MicroCT analysis of the distal femur revealed up to 3-fold increases in trabecular bone volume and up to 2-fold increases in the vertebrae, compared to wild type controls. Cortical bone was increased in both the tibiae and vertebrae, which correlated with increased strength in tibial 4-point bending and vertebral compression tests. Dynamic histomorphometry identified increased bone formation as the mechanism underlying the high bone mass phenotype in Dkk1 KO mice, with no changes in bone resorption. Mice featuring only Wnt3 heterozygosity showed no evident bone phenotype. Our findings highlight a critical role for Dkk1 in the regulation of bone formation and a gene dose-dependent response to loss of DKK1 function. Targeting Dkk1 to enhance bone formation offers therapeutic potential for osteoporosis.
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http://dx.doi.org/10.1007/s00223-017-0338-4DOI Listing
January 2018

A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts.

Cell Rep 2017 Oct;21(1):274-288

Neuromuscular and Regenerative Medicine Unit, University of New South Wales, Sydney, NSW 2010, Australia.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.
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http://dx.doi.org/10.1016/j.celrep.2017.09.022DOI Listing
October 2017

Osteocalcin-dependent regulation of glucose metabolism and fertility: Skeletal implications for the development of insulin resistance.

J Cell Physiol 2018 05 18;233(5):3769-3783. Epub 2017 Sep 18.

Faculty of Health and Medical Science, Myeloma Research Laboratory, Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia.

The skeleton has recently emerged as a critical insulin target tissue that regulates whole body glucose metabolism and male reproductive function. While our understanding of these new regulatory axes remains in its infancy, the bone-specific protein, osteocalcin, has been shown to be centrally involved. Undercarboxylated osteocalcin acts as a secretagogue in a feed-forward loop to stimulate pancreatic β-cell proliferation and insulin secretion, improve insulin sensitivity, and promote testosterone production. Importantly, dysregulation of insulin signaling in bone causes a reduction in serum osteocalcin levels that is associated with elevated blood glucose and reduced serum insulin levels, suggesting that the skeleton may play a significant role in the development of diet-induced insulin resistance. Insulin signaling is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1) which becomes hyper-activated in response to nutrient overload. Loss- and gain-of function models suggest that mTORC1 function in bone is essential for normal skeletal development; however, the role of this complex in the regulation of glucose metabolism remains to be determined. This review highlights our current understanding of the role played by osteocalcin in the skeletal regulation of glucose metabolism and fertility. In particular, it examines data emerging from transgenic mouse models which have revealed a pancreas-bone-testis regulatory axis and discusses recent human studies which seek to corroborate findings from mouse models with clinical observations. Moreover, we review recent studies which suggest dysregulation of insulin signaling in bone leads to the development of insulin resistance and discuss the potential role of mTORC1 signaling in this process.
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http://dx.doi.org/10.1002/jcp.26163DOI Listing
May 2018

Inhibiting the osteocyte-specific protein sclerostin increases bone mass and fracture resistance in multiple myeloma.

Blood 2017 06 17;129(26):3452-3464. Epub 2017 May 17.

The Garvan Institute of Medical Research, Sydney, NSW, Australia.

Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM.
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http://dx.doi.org/10.1182/blood-2017-03-773341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492093PMC
June 2017

Osteoblastic Actions of the Neuropeptide Y System to Regulate Bone and Energy Homeostasis.

Curr Osteoporos Rep 2016 Feb;14(1):26-31

Osteoporosis and Bone Biology Division, Garvan Institute of Medical Research, St Vincent's Hospital, 390 Victoria St, Darlinghurst, Sydney, NSW, 2010, Australia.

Neural pathways are now a well-appreciated factor in the regulatory milieu controlling the maintenance of bone mass. A number of neural pathways from the brain to bone have been identified. These pathways often involve elements of the energy homeostatic apparatus, indicating links between the regulation of bone metabolism and energy balance. Neuropeptide Y is one such factor that co-regulates these two processes. Initial studies outlined the skeletal actions of NPY from within the brain and the interactions with energy homeostatic processes. However, in recent years, an appreciation for the actions of NPY within bone cells has expanded. Cells of the osteoblastic lineage express both NPY ligand and a cognate receptor NPY, Y1R. Murine studies have demonstrated that both ligand and receptor actively control bone mass and osteoblast activity and interact with mechanical signals to integrate with the local loading environment. Local NPY signalling regulates osteoprogenitor production and differentiation, to cover the entire osteoblastic lineage. In addition, several recent studies have demonstrated extra-skeletal actions of osteoblastic NPY signalling, to regulate energy expenditure and with it adiposity, and in a separate study, to control release of a factor-controlling beta cell mass and insulin production/release and with it glucose tolerance. Thus, osteoblastic neuropeptide production and signalling illustrates the rapidly widening sphere of influence of skeletal tissue, and suggests a far more complex and interconnected physiology then is currently appreciated.
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http://dx.doi.org/10.1007/s11914-016-0300-9DOI Listing
February 2016

Prader-Willi Critical Region, a Non-Translated, Imprinted Central Regulator of Bone Mass: Possible Role in Skeletal Abnormalities in Prader-Willi Syndrome.

PLoS One 2016 29;11(1):e0148155. Epub 2016 Jan 29.

Bone and Mineral Research Division, Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, Australia.

Prader-Willi Syndrome (PWS), a maternally imprinted disorder and leading cause of obesity, is characterised by insatiable appetite, poor muscle development, cognitive impairment, endocrine disturbance, short stature and osteoporosis. A number of causative loci have been located within the imprinted Prader-Willi Critical Region (PWCR), including a set of small non-translated nucleolar RNA's (snoRNA). Recently, micro-deletions in humans identified the snoRNA Snord116 as a critical contributor to the development of PWS exhibiting many of the classical symptoms of PWS. Here we show that loss of the PWCR which includes Snord116 in mice leads to a reduced bone mass phenotype, similar to that observed in humans. Consistent with reduced stature in PWS, PWCR KO mice showed delayed skeletal development, with shorter femurs and vertebrae, reduced bone size and mass in both sexes. The reduction in bone mass in PWCR KO mice was associated with deficiencies in cortical bone volume and cortical mineral apposition rate, with no change in cancellous bone. Importantly, while the length difference was corrected in aged mice, consistent with continued growth in rodents, reduced cortical bone formation was still evident, indicating continued osteoblastic suppression by loss of PWCR expression in skeletally mature mice. Interestingly, deletion of this region included deletion of the exclusively brain expressed Snord116 cluster and resulted in an upregulation in expression of both NPY and POMC mRNA in the arcuate nucleus. Importantly, the selective deletion of the PWCR only in NPY expressing neurons replicated the bone phenotype of PWCR KO mice. Taken together, PWCR deletion in mice, and specifically in NPY neurons, recapitulates the short stature and low BMD and aspects of the hormonal imbalance of PWS individuals. Moreover, it demonstrates for the first time, that a region encoding non-translated RNAs, expressed solely within the brain, can regulate bone mass in health and disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148155PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732947PMC
July 2016

Snord116 is critical in the regulation of food intake and body weight.

Sci Rep 2016 Jan 4;6:18614. Epub 2016 Jan 4.

Neuroscience Division, Garvan Institute of Medical Research, St Vincent's Hospital, Darlinghurst, NSW, Australia.

Prader-Willi syndrome (PWS) is the predominant genetic cause of obesity in humans. Recent clinical reports have suggested that micro-deletion of the Snord116 gene cluster can lead to PWS, however, the extent of the contributions of the encoded snoRNAs is unknown. Here we show that mice lacking Snord116 globally have low birth weight, increased body weight gain, energy expenditure and hyperphagia. Consistent with this, microarray analysis of hypothalamic gene expression revealed a significant alteration in feeding related pathways that was also confirmed by in situ hybridisation. Importantly, selective deletion of Snord116 only from NPY expressing neurons mimics almost exactly the global deletion phenotype including the persistent low birth weight, increased body weight gain in early adulthood, increased energy expenditure and hyperphagia. Mechanistically, the lack of Snord116 in NPY neurons leads to the upregulation of NPY mRNA consistent with the hyperphagic phenotype and suggests a critical role of Snord116 in the control of NPY neuronal functions that might be dysregulated in PWS.
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http://dx.doi.org/10.1038/srep18614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698587PMC
January 2016

The y6 receptor suppresses bone resorption and stimulates bone formation in mice via a suprachiasmatic nucleus relay.

Bone 2016 Mar 22;84:139-147. Epub 2015 Dec 22.

Osteoporosis and Bone Biology Division, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia; Faculty of Medicine, University of NSW, Kensington, Sydney, NSW 2052, Australia. Electronic address:

The neuropeptide Y system is known to play an important role in the regulation of bone homeostasis and while the functions of its major receptors, Y1R and Y2R, in this process have become clearer, the contributions of other Y-receptors, like the y6 receptor (y6R), are unknown. Y6R expression is restricted to the suprachiasmatic nucleus (SCN) of the hypothalamus, an area known to regulate circadian rhythms, and the testis. Here we show that lack of y6R signalling, results in significant reduction in bone mass, but no changes in bone length. Male and female y6R knockout (KO) mice display reduced cortical and cancellous bone volume in axial and appendicular bones. Mechanistically, the reduction in cancellous bone is the result of an uncoupling of bone remodelling, leading to an increase in osteoclast surface and number, and a reduction in osteoblast number, osteoid surface, mineralizing surface and bone formation rate. y6R KO mice displayed increased numbers of osteoclast precursors and produced greater numbers of osteoclasts in RANKL-treated cultures. They also produced fewer CFU-ALP osteoblast precursors in the marrow and showed reduced mineralization in primary osteoblastic cultures, as well as reduced expression for the osteoblast lineage marker, alkaline phosphatase, in bone isolates. The almost exclusive location of y6Rs in the hypothalamus suggests a critical role of central neuronal pathways controlling this uncoupling of bone remodelling which is in line with known actions or other Y-receptors in the brain. In conclusion, y6R signalling is required for maintenance of bone mass, with loss of y6R uncoupling bone remodelling and resulting in a negative bone balance. This study expands the scope of hypothalamic regulation of bone, highlighting the importance for neural/endocrine coordination and their marked effect upon skeletal homeostasis.
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http://dx.doi.org/10.1016/j.bone.2015.12.011DOI Listing
March 2016

Osteoclasts control reactivation of dormant myeloma cells by remodelling the endosteal niche.

Nat Commun 2015 Dec 3;6:8983. Epub 2015 Dec 3.

Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia.

Multiple myeloma is largely incurable, despite development of therapies that target myeloma cell-intrinsic pathways. Disease relapse is thought to originate from dormant myeloma cells, localized in specialized niches, which resist therapy and repopulate the tumour. However, little is known about the niche, and how it exerts cell-extrinsic control over myeloma cell dormancy and reactivation. In this study, we track individual myeloma cells by intravital imaging as they colonize the endosteal niche, enter a dormant state and subsequently become activated to form colonies. We demonstrate that dormancy is a reversible state that is switched 'on' by engagement with bone-lining cells or osteoblasts, and switched 'off' by osteoclasts remodelling the endosteal niche. Dormant myeloma cells are resistant to chemotherapy that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling myeloma cell dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse.
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http://dx.doi.org/10.1038/ncomms9983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686867PMC
December 2015

The brain in bone and fuel metabolism.

Bone 2016 Jan 9;82:56-63. Epub 2015 Nov 9.

Osteoporosis and Bone Biology Division, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, NSW, Australia; Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia; School of Medicine, The University of Notre Dame Australia, Sydney, NSW, Australia. Electronic address:

Obesity and osteoporosis have become major public health challenges worldwide. The brain is well established as a pivotal regulator of energy homeostasis, appetite and fuel metabolism. However, there is now clear evidence for regulation between the brain and bone. Similarly, evidence also indicates that the involvement of the brain in bone and adipose regulation is both related and interdependent. The hypothalamus, with its semi-permeable blood brain barrier, is one of the most powerful regulatory regions within the body, integrating and relaying signals not only from peripheral tissues but also from within the brain itself. Two main neuronal populations within the arcuate nucleus of the hypothalamus regulate energy homeostasis: The orexigenic, appetite-stimulating neurons that co-express neuropeptide Y and agouti-related peptide and the anorexigenic, appetite-suppressing neurons that co-express proopiomelanocortin and cocaine- and amphetamine related transcript. From within the arcuate, these four neuropeptides encompass some of the most powerful control of energy homeostasis in the entire body. Moreover, they also regulate skeletal homeostasis, identifying a co-ordination network linking the processes of bone and energy homeostasis. Excitingly, the number of central neuropeptides and neural factors known to regulate bone and energy homeostasis continues to grow, with cannabinoid receptors and semaphorins also involved in bone homeostasis. These neuronal pathways represent a growing area of research that is identifying novel regulatory axes between the brain and the bone, and links with other homeostatic networks; thereby revealing a far more complex, and interdependent bone biology than previously envisioned. This review examines the current understanding of the central regulation of bone and energy metabolism.
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http://dx.doi.org/10.1016/j.bone.2015.10.020DOI Listing
January 2016

Neuronal control of bone and muscle.

Bone 2015 Nov;80:95-100

Osteoporosis and Bone Biology Division, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, NSW, Australia; Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia; School of Medicine, The University of Notre Dame Australia, Sydney, NSW, Australia. Electronic address:

The functional interplay between bone and muscle that enables locomotion is a fundamental aspect of daily life. However, other interactions between bone and muscle continue to attract attention as our understanding of the breath and importance of this inter-relationship continues to expand. Of particular interest is the regulatory connection between bone and muscle, which adds a new insight to the coordination of the bone/muscle unit. We have appreciated the importance of neuronal signaling to the control of bone turnover and muscle contraction, but recent data indicate that neuronal inputs control a far wider range of bone and muscle physiology than previously appreciated. This review outlines the role of the sympathetic nervous system and neuronal/neuropeptide inputs upon the regulation of bone and muscle tissue, and the potential for co-regulatory actions, particularly involving the sympathetic nervous system. This article is part of a Special Issue entitled "Muscle Bone Interactions".
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http://dx.doi.org/10.1016/j.bone.2015.05.006DOI Listing
November 2015

Uncoupling protein-1 is protective of bone mass under mild cold stress conditions.

Bone 2018 Jan 6;106:167-178. Epub 2015 Jun 6.

Neuroscience Division, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia; Faculty of Medicine, University of NSW, Kensington, Sydney, NSW 2052, Australia. Electronic address:

Brown adipose tissue (BAT), largely controlled by the sympathetic nervous system (SNS), has the ability to dissipate energy in the form of heat through the actions of uncoupling protein-1 (UCP-1), thereby critically influencing energy expenditure. Besides BAT, the SNS also strongly influences bone, and recent studies have demonstrated a positive correlation between BAT activity and bone mass, albeit the interactions between BAT and bone remain unclear. Here we show that UCP-1 is critical for protecting bone mass in mice under conditions of permanent mild cold stress for this species (22°C). UCP-1 mice housed at 22°C showed significantly lower cancellous bone mass, with lower trabecular number and thickness, a lower bone formation rate and mineralising surface, but unaltered osteoclast number, compared to wild type mice housed at the same temperature. UCP-1 mice also displayed shorter femurs than wild types, with smaller cortical periosteal and endocortical perimeters. Importantly, these altered bone phenotypes were not observed when UCP-1 and wild type mice were housed in thermo-neutral conditions (29°C), indicating a UCP-1 dependent support of bone mass and bone formation at the lower temperature. Furthermore, at 22°C UCP-1 mice showed elevated hypothalamic expression of neuropeptide Y (NPY) relative to wild type, which is consistent with the lower bone formation and mass of UCP-1 mice at 22°C caused by the catabolic effects of hypothalamic NPY-induced SNS modulation. The results from this study suggest that during mild cold stress, when BAT-dependent thermogenesis is required, UCP-1 activity exerts a protective effect on bone mass possibly through alterations in central NPY pathways known to regulate SNS activity.
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http://dx.doi.org/10.1016/j.bone.2015.05.037DOI Listing
January 2018

Vitamin D, muscle and bone: Integrating effects in development, aging and injury.

Mol Cell Endocrinol 2015 Jul 30;410:3-10. Epub 2015 Mar 30.

Salk Institute of Biological Studies, La Jolla, CA, USA.

Beyond the established effects of muscle loading on bone, a complex network of hormones and growth factors integrates these adjacent tissues. One such hormone, vitamin D, exerts broad-ranging effects in muscle and bone calcium handling, differentiation and development. Vitamin D also modulates muscle and bone-derived hormones, potentially facilitating cross-talk between these tissues. In the clinical setting, vitamin D deficiency or mutations of the vitamin D receptor result in generalized atrophy of muscle and bone, suggesting coordinated effects of vitamin D at these sites. In this review, we discuss emerging evidence that vitamin D exerts specific effects throughout the life of the musculoskeletal system - in development, aging and injury. From this holistic viewpoint, we offer new insights into an old debate: whether vitamin D's effects in the musculoskeletal system are direct via local VDR signals or indirect via its systemic effects in calcium and phosphate homeostasis.
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http://dx.doi.org/10.1016/j.mce.2015.03.020DOI Listing
July 2015

Bone muscle interactions and vitamin D.

Bone 2015 Nov 6;80:89-94. Epub 2015 Mar 6.

Department of Internal Medicine/Endocrinology, VU University Medical Centre, Amsterdam, The Netherlands.

Beyond the established roles of vitamin D in bone and mineral homeostasis, we are becoming increasingly aware of its diverse effects in skeletal muscle. Subjects with severe vitamin D deficiency or mutations of the vitamin D receptor develop generalized atrophy of muscle and bone, suggesting coordinated effects of vitamin D in musculoskeletal physiology. At a mechanistic level, vitamin D exerts wide-ranging effects in muscle and bone calcium handling, differentiation and development. Vitamin D also modulates muscle and bone-derived hormones, facilitating cross-talk between these tissues. In this review, we discuss emerging evidence that vitamin D regulates bone and muscle in a direct, integrated fashion, positioning the vitamin D pathway as a potential therapeutic target for musculoskeletal diseases. This article is part of a Special Issue entitled "Muscle Bone Interactions".
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http://dx.doi.org/10.1016/j.bone.2015.02.029DOI Listing
November 2015

NPY signalling in early osteoblasts controls glucose homeostasis.

Mol Metab 2015 Mar 16;4(3):164-74. Epub 2015 Jan 16.

Neuroscience Division, Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales, Australia ; Faculty of Medicine, University of NSW, Sydney, New South Wales, Australia.

Objective: The skeleton has recently emerged as an additional player in the control of whole-body glucose metabolism; however, the mechanism behind this is not clear.

Methods: Here we employ mice lacking neuropeptide Y, Y1 receptors solely in cells of the early osteoblastic lineage (Y1f3.6Cre), to examine the role of osteoblastic Y1 signalling in glycaemic control.

Results: Y1f3.6Cre mice not only have a high bone mass phenotype, but importantly also display altered glucose homeostasis; significantly decreased pancreas weight, islet number and pancreatic insulin content leading to elevated glucose levels and reduced glucose tolerance, but with no effect on insulin induced glucose clearance. The reduced glucose tolerance and elevated bone mass was corrected in Y1f3.6Cre mice by bone marrow transplant from wildtype animals, reinforcing the osteoblastic nature of this pathway. Importantly, when fed a high fat diet, Y1f3.6Cre mice, while equally gaining body weight and fat mass compared to controls, showed significantly improved glucose and insulin tolerance. Conditioned media from Y1f3.6Cre osteoblastic cultures was unable to stimulate insulin expression in MIN6 cells compared to conditioned media from wildtype osteoblast, indicating a direct signalling pathway. Importantly, osteocalcin a secreted osteoblastic factor previously identified as a modulator of insulin secretion was not altered in the Y1f3.6Cre model.

Conclusion: This study identifies the existence of other osteoblast-derived regulators of pancreas function and insulin secretion and illustrates a mechanism by which NPY signalling in bone tissue is capable of regulating pancreatic function and glucose homeostasis.
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http://dx.doi.org/10.1016/j.molmet.2014.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338316PMC
March 2015