Brain-derived neurotrophic factor (BDNF) plays important roles in neuronal differentiation/survival, in the regulation of food intake, and in the pathobiology of obesity and type 2 diabetes mellitus. BDNF and its receptor are expressed in osteoblasts and chondrocyte. BDNF in vitro has a positive effect on bone; whether central BDNF affects bone mass in vivo is not known. We therefore examined bone mass and energy utilization in brain-targeted BDNF conditional knock-out mice (Bdnf2lox/2lox/93). The deletion of BDNF in the brain led to a metabolic phenotype characterized by hyperphagia, obesity and increased abdominal white adipose tissue (WAT). Central BDNF deletion produces a marked skeletal phenotype characterized by increased femur length, elevated whole bone mineral density and bone mineral content. The skeletal changes are developmentally regulated and appear concurrently with the metabolic phenotype, suggesting that the metabolic and skeletal actions of BDNF are linked. The increased bone development is evident in both the cortical and trabecular regions. Compared to control, Bdnf2lox/2lox/93 mice show greater trabecular bone volume (BV/TV +50% for distal femur, p<0.001; +35% for vertebral body, p<0.001) and mid-femoral cortical thickness (+11 to 17%, p<0.05), measured at 3 and 6 months of age. The skeletal and metabolic phenotypes were gender-dependent with female being more affected than male mice. However, uncoupling Protein-1 expression in brown fat, a marker of sympathetic tone, was not different between genotypes. We show that deletion of central BDNF expression in mice results in increased bone mass and WAT, with no significant changes in sympathetic signaling or peripheral serotonin, associated with hyperphagia, obesity and leptin resistance.

Brown adipose tissue (BAT) is controlled by the sympathetic nervous system (SNS) and has the ability to dissipate energy through uncoupling protein-1 (UCP-1), influencing energy expenditure. Besides BAT, the SNS influences bone, and recent studies have demonstrated a positive correlation between BAT activity and bone. Nerve Growth Factor (NGF) genes are expressed in brain, white adipose tissue (WAT), BAT and bone where it coordinates brain and body reactions to challenges. Similarly Osteocalcin besides its role in bone metabolism acts as an hormone regulating glucose metabolism and brain. We previously showed that NGF and its receptor p75NTR genes are highly expressed in BAT versus brain in mice, suggesting that NGF may act as a regulator of energy. To further investigate the role of NGF and osteocalcin in bone and energy regulation we analyzed NGF and its receptor p75NTR and Osteocalcin mRNA from 3 months old mice after cold exposure. UCP-1 was used as positive control. Mice were divided into three groups: room temperature (RT), cold stress for 6h and 5 days (n = 5 for each group). Mice as control group were all placed at RT (23 °C) for 5 days, while the cold groups were placed at 4 °C for the abovementioned times. The mice were subsequently sacrificed and the interscapular BAT, bone and brain were analyzed for mRNA extraction. The exposure of 6 h to cold stress enhanced mRNA levels of UCP-1 and NGF genes by 3 and 2.5 fold vs controls, respectively, reducing mRNA of p75NTR by 19 fold. The UCP-1 gene was still up-regulated after 5 days of cold stress, the NGF gene was not affected and mRNA of the p75NTR gene was reduced by 7 fold vs controls. The mRNA NGF/NGFR genes were not affected in bone or brain following cold stress. The mRNA levels of osteocalcin in bone were upregulated following 6h and 5 days cold exposure vs controls. Osteocalcin gene in brain was downregulated following cold stress. Our study shows that NGF mRNA expression significantly increases after 6 hours of cold stress however with minor extent with respect to UCP-1. The enhanced mRNA level of NGF is observed in parallel with the decrease of NGF receptor gene expression. We found no change in NGF and p75NTR expression in brain or bone, but osteocalcin genes were upregulated in bone following cold stress. These results suggest that during cold stress when BAT-dependent thermogenesis is required, NGF activity is required, and osteocalcin may exert a local protective effect on bone mass.

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Bone mass, metabolism and reproduction are regulated coordinately. The bone-derived osteocalcin(Ost) favors insulin sensitivity, male fertility and neurogenesis. The neurotrophins BDNF and NGF are involved in energy and bone metabolism. NGF regulates fertility elevating LH in the female, Ost-/- mice show obesity and high LH in spite of decreased testosterone. To investigate the NGF-osteocalcin interaction we analyzed by RT-PCR the mRNA levels of NGF, BDNF, Ost. and their receptors p75NTR/NTRK1, TRKb and Gprc6a respectively in adipose WAT/BAT, reproductive organs, brain and bone(positive controls) of 3 months old female and male mice. Here, the mRNA levels of NGF and p75NTR are 50% higher in BAT than the brain. NGF and its receptors are down-regulated in WAT and bone in both genders. Osteocalcin and Gprc6a are up-regulated in bone and brain, downregulated in BAT/WAT. BDNF and TRKb expression in bone is higher than brain, but lower in BAT/WAT; TRKb is down-regulated in bone and up-regulated in adipose tissue. NGF is up-regulated in the ovaries/uterus, but down-regulated in the testes. The mRNA levels of p75NTR is respectively 300%, 100% and 50% higher in testis, ovaries and uterus than the brain. NTRK1 is down-regulated in all tissues. The Gprc6a is expressed in the testes, not in the ovaries and uterus. BDNF and TRKb are down-regulated in the sexual organs. Therefore, the up-regulation of NGF and related-receptors in fat is consistent with NGF as an energy regulator. The inverse correlation of NGF and BDNF in fat and bone, shows these exerting opposite effects on leptin with BDNF regulating bone. The up-regulation of p75NTR in the testes match the Gprc6a expression, and is responsible for higher LH in the Ost-/- mice. The animal care was performed in accordance with the DIRECTIVE 2010/63/EU. The protocol was approved by the Ethics Committee of the University of Bari, Italy.

Bone mass, metabolism and reproduction are regulated coordinately. The bone-derived osteocalcin (Ost) favors insulin sensitivity, male fertility and neurogenesis. The neurotrophins BDNF/NGF and oxytocin (Oxt) are involved in energy and bone metabolism. NGF regulates fertility elevating LH in female, Ost-/- mice show obesity and high LH in spite of decreased testosterone. To investigate the NGF/BDNF-Oxt-Ost interactions we analyzed by RT-PCR the mRNA levels of NGF, BDNF, Oxt, Ost and their receptors p75NTR/NTRK1, TRKb, Oxtr and Gprc6a in brain, bone, WAT/BAT and reproductive organs, of 3 months old female and male mice. Brain and bone were used as positive controls respectively. NGF and p75NTR expression is 50% higher in BAT than brain. NGF and its receptors are downregulated in WAT and bone in both genders. Ost and Gprc6a are upregulated in bone and brain, down-regulated in BAT/WAT. BDNF and TRKb expression in bone is higher than brain, but lower in BAT/WAT; TRKb is downregulated in bone and up-regulated in adipose tissue. NGF is up-regulated in ovaries/uterus, but down-regulated in the testes. p75NTR is respectively 300%, 100% and 50% higher in testis, ovaries and uterus than brain. NTRK1 is downregulated in all tissues. The Gprc6a is expressed in testes, not in ovaries and uterus. BDNF and TRKb are downregulated in sexual organs. Oxt is markedly expressed in brain and with minor extend in bone in either genders, while Oxtr in ovaries although a significant expression level is observed in fat and bone. The up-regulation of NGF and related-receptors in fat is consistent with NGF as an energy regulator. The inverse correlation of NGF and BDNF in fat and bone, shows these exerting opposite effects on leptin with BDNF regulating bone. The up-regulation of p75NTR in testes matches the Gprc6a expression in the same organ. Gene correlation analysis shows the existence of an interaction between NGF and osteocalcin. Therefore, we speculate that NGF can be a physiologic mediator of osteocalcin both peripherally regulating steroid production in Leydig cell in the testosterone deficient OST-/-, and centrally thought the sprouting of new synapses in this cognitively impaired mice. The pattern of expression of these molecules and their receptors show a similar trend with Ost, NGF, Oxt and BDNF genes highly expressed in brain of both genders, while their receptors were expressed in the reproductive organs showing a gender expression profile. These data add evidences that the signalling of bone metabolism and reproduction are released from CNS to act on peripheral tissues.