Neuropeptide Y attenuates stress-induced bone loss through suppression of noradrenaline circuits

Chronic stress and depression have adverse consequences on many organ systems, including the skeleton, but the mechanisms underlying stress-induced bone loss remain unclear. Here we demonstrate that neuropeptide Y (NPY), centrally and peripherally, plays a critical role in protecting against stress-induced bone loss. Mice lacking the anxiolytic factor NPY exhibit more anxious behavior and elevated corticosterone levels. Additionally, following a 6-week restraint, or cold-stress protocol, Npy-null mice exhibit three-fold greater bone loss compared to wild-type mice, owing to suppression of osteoblast activity. This stress-protective NPY pathway acts specifically through Y2 receptors. Centrally, Y2 receptors suppress corticotropin-releasing factor expression and inhibit activation of noradrenergic neurons in the paraventricular nucleus. In the periphery, they act to control noradrenaline release from sympathetic neurons. Specific deletion of arcuate Y2 receptors recapitulates the Npy-null stress response, coincident with elevated serum noradrenaline. Importantly, specific reintroduction of NPY solely in noradrenergic neurons of otherwise Npy-null mice blocks the increase in circulating noradrenaline and the stress-induced bone loss. Thus, NPY protects against excessive stress-induced bone loss, through Y2 receptor-mediated modulation of central and peripheral noradrenergic neurons

Peptide YY Regulates Bone Remodeling in Mice: A Link between Gut and Skeletal Biology

<div><h3>Background & Aims</h3><p>Gastrointestinal peptides are increasingly being linked to processes controlling the maintenance of bone mass. Peptide YY (PYY), a gut-derived satiety peptide of the neuropeptide Y family, is upregulated in some states that also display low bone mass. Importantly, PYY has high affinity for Y-receptors, particularly Y1R and Y2R, which are known to regulate bone mass. Anorexic conditions and bariatric surgery for obesity influence circulating levels of PYY and have a negative impact on bone mass, but the precise mechanism behind this is unclear. We thus examined whether alterations in PYY expression affect bone mass.</p> <h3>Methods</h3><p>Bone microstructure and cellular activity were analyzed in germline PYY knockout and conditional adult-onset PYY over-expressing mice at lumbar and femoral sites using histomorphometry and micro-computed tomography.</p> <h3>Results</h3><p>PYY displayed a negative relationship with osteoblast activity. Male and female PYY knockout mice showed enhanced osteoblast activity, with greater cancellous bone mass. Conversely, PYY over-expression lowered osteoblast activity <em>in vivo</em>, via a direct Y1 receptor mediated mechanism involving MAPK stimulation evident <em>in vitro</em>. In contrast to PYY knockout mice, PYY over expression also altered bone resorption, as indicated by greater osteoclast surface, despite the lack of Y-receptor expression in osteoclastic cells. While evident in both sexes, cellular changes were generally more pronounced in females.</p> <h3>Conclusions</h3><p>These data demonstrate that the gut peptide PYY is critical for the control of bone remodeling. This regulatory axis from the intestine to bone has the potential to contribute to the marked bone loss observed in situations of extreme weight loss and higher circulating PYY levels, such as anorexia and bariatric obesity surgery, and may be important in the maintenance of bone mass in the general population.</p> </div

Bone cell activities in the femora of PYY^−/− mice.

<p>In the distal femoral metaphysis, lack of PYY signaling results in greater bone formation rate (A,C) due to greater mineral apposition rate (MAR) with no change in mineralizing surface (C,I) in both genders. Endocortical MAR at the mid femur was also greater in PYY^−/− compared to wild-type. Parameters of cancellous bone resorption as indicated by osteoclast surface and number (B,D) are similar between PYY^−/− and wild-type controls. Representative images of endocortical MAR are shown in E. Scale bar represents 10 µm. Mean ± SEM of 5–18 mice per group are shown. *p<0.05, **p<0.005, ***p<0.001 versus wild-type (WT).</p

Greater bone mass in PYY^−/− mice.

<p>Male (A) and female (B) PYY^−/− mice display no difference in body weight, but greater whole body BMD, whole body BMC in female mice. Regional analysis revealed greater lumbar BMD and BMC in PYY^−/− of both genders. Representative images of distal femur histological sections and 3D surface rendered models of µCT scanned lumbar vertebra of male (C) and female (D) mice showing greater cancellous bone volume in the distal femoral metaphysis of PYY^−/− mice in both genders and in the lumbar vertebral body of female PYY^−/− mice (D). Mean ± SEM of 7–18 mice per group are shown. *p<0.05, **88p<0.005 versus wild-type (WT).</p

Lower bone mass and reduced bone size in female PYYtg mice.

<p>Although male PYYtg mice are similar to wild-type littermates (A-B), female PYYtg mice have lower femoral BMD, femur length, vertebral height and cancellous bone volume of distal femoral metaphysis (C-D); representative images from male (E) and female (F) mice of distal femoral histological sections and 3D model images of µCT scanned lumbar vertebra. Mean ± SEM of 4–12 mice per group are shown. *p<0.05, **p<0.05 versus wild-type (WT).</p

PYY signalling in osteoblasts via Y1 receptors.

<p>Y receptor gene expression was evident in brain tissue, but was absent in bone marrow macrophages (BMM) or cultured osteoclasts (Oc) from wild type mice(A). Y1R was not detected at any time point throughout RANKL-induced osteoclast differentiation (A). Calcitonin Receptor (CTR) and Cathepsin K gene expression were used as osteoclast markers. NTC, no template control. PYY induced signaling in primary calvarial osteoblasts through strong phosphorylation of ERK, a response which was blunted in the presence of Y1 receptor specific antagonist (1229U91). (B) p-ERK, phosphorylated ERK; t-ERK, total ERK from western blots of calvarial cultures. (C) Western blots were quantified by densitometry of p-ERK and t-ERK bands. Proliferation of osteoblastic cultures from wild type and Y1 receptor null mice were not altered by PYY (D). Mean ± SEM of 4 replicates per group are shown.</p

Evaluation of PYY overproduction.

<p>As shown in the schematic diagram of the PYY transgene construct (A), liver genomic DNA was analyzed for presence of PYY transgene construct (B) and deletion of stop cassette (hence activation of PYY transgene) by southern analysis. Hepatic PYY mRNA expression was detected only in PYYtg mice and not littermate controls as shown by RT?PCR (C) and qPCR (D). PYY staining (brown) is more intense in the islets of Langerhans in the pancreas of PYY transgenic than wild-type littermates (E). PYY mRNA levels are greater in PYYtg hypothalamus and the rest of the brain, compared to wild-type littermates, but are lower compared to NPY mRNA levels of the same genotype (F). Number of PCR cycles is indicated at right, followed by size of PCR product (B-C). Values shown are normalised to ribosomal protein L13 (RPL-13) levels and relative to wild-type expression (D, F). n.d., not detected (E). Scale bar represents 50 µm (E). Pictures are representative of staining observed in tissues obtained from 16-week-old mice (2?3 mice per group) (E). *p<0.05, ***p<0.001 versus wild-type (WT).</p

Bone cell activities in the femora of PYYtg mice.

<p>Although cancellous bone volume is lower in female but not male PYYtg, alterations in bone cell activities are observed in both genders. Mineral apposition rate (MAR) was reduced in both genders (A,C), with bone formation rate reduced only in females, with no change in mineralizing surface. Endocortical MAR at the mid femur was reduced in female PYYtg compared to wild-type. Osteoclast surface was greater in both genders of PYYtg and osteoclast number only greater in females. Representative images of endocortical MAR are shown in E. Scale bar represents 10 µm. Mean ± SEM of 4–12 mice per group are shown. *p<0.05, **p<0.005, ***p<0.001 versus wild-type (WT).</p