Neuropeptide Y knockout mice reveal a central role of NPY in the coordination of bone mass to body weight

Abstract

Changes in whole body energy levels are closely linked to alterations in body weight and bone mass. Here, we show that hypothalamic signals contribute to the regulation of bone mass in a manner consistent with the central perception of energy status. Mice lacking neuropeptide Y (NPY), a well-known orexigenic factor whose hypothalamic expression is increased in fasting, have significantly increased bone mass in association with enhanced osteoblast activity and elevated expression of bone osteogenic transcription factors, Runx2 and Osterix. In contrast, wild type and NPY knockout (NPY (-/-)) mice in which NPY is specifically over expressed in the hypothalamus (AAV-NPY+) show a significant reduction in bone mass despite developing an obese phenotype. The AAV-NPY+ induced loss of bone mass is consistent with models known to mimic the central effects of fasting, which also show increased hypothalamic NPY levels. Thus these data indicate that, in addition to well characterized responses to body mass, skeletal tissue also responds to the perception of nutritional status by the hypothalamus independently of body weight. In addition, the reduction in bone mass by AAV NPY+ administration does not completely correct the high bone mass phenotype of NPY (-/-) mice, indicating the possibility that peripheral NPY may also be an important regulator of bone mass. Indeed, we demonstrate the expression of NPY specifically in osteoblasts. In conclusion, these data identifies NPY as a critical integrator of bone homeostatic signals; increasing bone mass during times of obesity when hypothalamic NPY expression levels are low and reducing bone formation to conserve energy under 'starving' conditions, when hypothalamic NPY expression levels are high.

Figure 1. Generalized bone anabolic phenotype in 4 month old male and female NPY ^−/− mice.

(a) Body weight. Dual X-ray absorptiometry analysis of (b) fat mass, (c) lean mass indicate changed energy homeostasis in NPY ^−/− mice. Bone mass and density was also greater as seen in (d) whole body BMD, (e) whole body BMC, (f) lumbar BMD and (g) lumbar BMC. n = 8−14, data expressed as mean±SE.

Figure 2. Greater cortical bone in NPY ^−/− mice.

Peripheral quantitative computed tomography of mid-femur in male mice showing (a) total bone volume, (b) marrow volume, (c) cortical bone volume and (d) cortical thickness. (e) Mid femoral endocortical mineral apposition rate, (f) photomicrograph of endocortical bone formation. (g) 24 hour home cage activity. Scale bar represents 50 µm. # p<0.05 vs wild type. n = 8−14, data expressed as mean±SE.

Figure 3. Greater cancellous bone volume and formation in NPY ^−/− mice.

Histomorphometric analysis of distal femoral metaphysis in male mice showing (a) cancellous bone volume, (b) bone formation rate, (c) mineralizing surface, (d) mineral apposition rate, (e) osteoclast surface and (f) osteoclast number. (g) Representative micro-computed tomographs of the distal femoral metaphysis of 4 month old male wild type and NPY ^−/− mice. (h) 4th lumbar vertebral sections from male wild type and NPY ^−/− mice, displaying greater cancellous bone volume (BV/TV) in mutant mice. # p<0.05 vs wild type. n = 8−14, data expressed as mean±SE.

Figure 4. Hypothalamic NPY over-expression inhibits bone formation, despite increased body weight.

Photomicrograph of coronal brain section showing NPY over expression following unilateral rAAV-NPY+ injection in the arcuate nucleus (Arc). Changes in (b) body weight, (c) white adipose tissue, (d) tibial BMD, (e) tibial BMC, (f) mid femoral endocortical and (g) mid femoral periosteal mineral apposition rate in response to hypothalamic NPY over expression in wild type mice (WT NPY+) compared to control (WT empty). (h) Representative photomicrographs of femoral endocortical mineral apposition rate WT empty or WT NPY+ mice. Changes in (i) body weight, (j) fat mass, (k) distal femoral metaphyseal mineral apposition rate, (l) femoral BMD, and (m) distal femoral metaphyseal cancellous bone volume in germline NPY ^−/− mice after hypothalamic NPY over expression (NPY ^−/− NPY+) compared to controls (NPY ^−/− empty). # p<0.05 vs empty. Scale bar represents 20 µm. n = 7−12, data expressed as mean±SE.

Figure 5. NPY expression and action in bone tissue.

(a) Analysis of NPY expression by in situ hybridisation in wild type mice shows specific staining of osteoblasts employing a NPY antisense probe in cancellous and cortical bone (arrows) and megakaryocytes (arrowheads). (b) This staining is absent in NPY ^−/− mice. (c) Levels of ALP, Runx2 and Osterix are greater in NPY ^−/− mice compared to wild type as determined by quantitative real-time PCR on mRNA from long bones. (mean±SE. n = 5−6 mice per genotype.). (d) Bone marrow stromal cells isolated from NPY ^−/− mice produce more mineral in vitro than wild type mice. (mean±SE, n = 10−15 wells per time, 3 independent experiments). # p<0.05, ## p<0.01 vs wild type, scale bar represents 50 µm.

Figure 6. Schematic representation of the co-ordinate regulation of body weight and bone by NPY.

(a) Altered energy homeostatic demand regulates hypothalamic NPY production, which signals via arcuate Y2 receptors through efferent sympathetic relays to the osteoblast. At the osteoblast, tonic cell activity is modulated by exogenous neural inputs as well as local NPY production, acting through cell surface Y1 receptors. (b) NPY exhibits an inverse relationship with both energy homeostasis and bone mass. As body weight increases as a result of positive energy homeostasis, the decrease in NPY signaling stimulates the production of bone to match body weight to bone mass. Likewise, negative energy homeostasis, and the coincident reduction of body weight is accompanied by increased NPY, which inhibits the production of bone, thereby conserving energy and increasing the mobilization of nutrient stores form bone.