(39)), by contrast, Y2 receptors have not been detected on bone. In addition to effects in bone, Y1 receptors have been considered as important regulators of energy homeostasis, consistent with pharmacological evidence from Y receptor agonists and antagonists to stimulate or inhibit feeding (9). Fasting-induced re-feeding is reduced in germ line Y1 receptor knock-out mice (10), and deletion of Y1 receptors in genetically obese ob/ob mice, in which hypothalamic NPY-ergic activity is chronically increased, significantly reduces food intake and body weight (11). Paradoxically, germ line Y1 receptor knock-out mice develop late-onset obesity in the absence of hyperphagia (10,12,13). One hypothesis to reconcile this apparent discrepancy is that hypothalamic and non-hypothalamic Y1 receptors have different effects on energy homeostasis.Given the clear involvement of Y1 receptors in the regulation of energy homeostasis as well as new evidence of a putative role for Y1 receptors on osteoblast-like cells, we investigated the effect of germ line and conditional (adult-onset, hypothalamus-specific) deletion of Y1 receptors in mice. In addition, the potential interaction between Y1 receptor sig-

Neuropeptide Y (NPY) is a downstream modulator of leptin action, possibly at the level of the arcuate nucleus where NPY neurons are known to express both leptin receptors and Y2 receptors. In addition to the well-described role of NPY and leptin in energy balance and obesity, intracerebroventricular administration of NPY or leptin also causes bone loss. Here we show that Y2 receptor-deficient mice have a twofold increase in trabecular bone volume as well as greater trabecular number and thickness compared with control mice. We also demonstrate that central Y2 receptors are crucial for this process, since selective deletion of hypothalamic Y2 receptors in mature conditional Y2 knockout mice results in an identical increase in trabecular bone volume within 5 weeks. This hypothalamus-specific Y2 receptor deletion stimulates osteoblast activity and increases the rate of bone mineralization and formation, with no effect on osteoblast or osteoclast surface measurements. The lack of any changes in plasma total calcium, leptinemia, or hypothalamo-pituitary-corticotropic, -thyrotropic, -somatotropic, or -gonadotropic output suggests that Y2 receptors do not modulate bone formation by humoral mechanisms, and that alteration of autonomic function through hypothalamic Y2 receptors may play a key role in a major central regulatory circuit of bone formation.

Vertebrate T cells express either an alpha beta or gamma delta T cell receptor (TCR). The developmental relatedness of the two cell types is unresolved. alpha beta + T cells respond to specific pathogens by collaborating with immunoglobulin-producing B cells in distinct lymphoid organs such as the spleen and Peyer's patches. The precise influence of alpha beta + T cells on B cell development is poorly understood. To investigate the developmental effects of alpha beta + T cells on B cells and gamma delta + T cells, mice homozygous for a disrupted TCR alpha gene were generated. The homozygotes showed elimination of alpha beta + T cells and the loss of thymic medullae. Despite this, gamma delta + T cells developed in normal numbers, and there was an increase in splenic B cells.

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Leptin and Y2 receptors on hypothalamic NPY neurons mediate leptin effects on energy homeostasis; however, their interaction in modulating osteoblast activity is not established. Here, direct testing of this possibility indicates distinct mechanisms of action for leptin anti-osteogenic and Y2 −/− anabolic pathways in modulating bone formation.Introduction: Central enhancement of bone formation by hypothalamic neurons is observed in leptin-deficient ob/ob and Y2 receptor null mice. Similar elevation in central neuropeptide Y (NPY) expression and effects on osteoblast activity in these two models suggest a shared pathway between leptin and Y2 receptors in the central control of bone physiology. The aim of this study was to test whether the leptin and Y2 receptor pathways regulate bone by the same or distinct mechanisms. Materials and Methods:The interaction of concomitant leptin and Y2 receptor deficiency in controlling bone was examined in Y2 −/− ob/ob double mutant mice, to determine whether leptin and Y2 receptor deficiency have additive effects. Interaction between leptin excess and Y2 receptor deletion was examined using recombinant adeno-associated viral vector overproduction of NPY (AAV-NPY) to produce weight gain and thus leptin excess in adult Y2 −/− mice. Cancellous bone volume and bone cell function were assessed. Results: Osteoblast activity was comparably elevated in ob/ob, Y2

IntroductionGenetically obese rodents that lack leptin (ob/ob mice) or its receptor (db/db mice) show increased activity of neuropeptide Y (NPY) circuits in the hypothalamus due to lack of leptin-induced inhibition of NPY expression and secretion (1). Such increased hypothalamic NPY signaling contributes to the massive obesity, hypercorticism, stunted growth, and reproductive defects of these mice (2). Since NPY ablation in ob/ob mice attenuates all of these defects (3), these findings demonstrate that NPY is a downstream mediator of leptin's central effects.Recently it was shown that leptin can inhibit bone synthesis by action within the hypothalamus, and that mice with no or reduced leptin signaling (ob/ob, db/db, and transgenic A-ZIP/F-1 fat-deficient mice) have high bone density associated with increased bone formation (4). This effect of leptin deficiency occurred independently of increased body fat or body weight, and despite the hypercorticism, decreased somatotropic activity, and hypogonadism in ob/ob and db/db mice. Intriguingly, central infusion of NPY for 28 days in wild-type mice had the same inhibitory effect on bone function as leptin had, suggesting that the increased hypothalamic NPY expression of leptin-deficient mice does not mediate the associated increase in bone density (4). However, it is unclear from this study whether the inhibitory effects on bone of central NPY infusion are a direct consequence of hypothalamic NPY action or a secondary effect of the resulting increase in expression (5) and circulating concentrations (6) of leptin. Furthermore, NPY mediates its effects through the activation of at least five different receptors: Y1, Y2, Y4, Y5, and in the mouse also y6, all of which are expressed in the hypothalamus (7-9). The potential for simultaneous activation of all of these receptors by central NPY infusion makes it difficult to distinguish the true role of the different types of hypothalamic Y receptors in bone physiology.NPY synthesis is particularly high in neurons of the arcuate nucleus, with many of these neurons also expressing leptin receptors (10). A high percentage of these arcuate NPY-expressing neurons coexpress the Y2 receptor (11), which is thought to act as an autoreceptor that can modulate the expression and secretion of NPY and other neurotransmitters (12). Since leptin receptors and Y2 receptors are present on NPY-expressing neurons of the arcuate nucleus and are likely to share some common signaling pathways (1, 10-12), we hypothesized that the Y2 receptor might be involved in the central regulation of bone metabolism. To test this hypothesis, we generated both germline Y2 receptor knockout mice (Y2 -/- Neuropeptide Y (NPY) is a downstream modulator of leptin action, possibly at the level of the arcuate nucleus where NPY neurons are known to express both leptin receptors and Y2 receptors. In addition to the well-described role of NPY and leptin in energy balance and obesity, intracerebroventricular administration of NPY or leptin also causes bone loss. Here we sho...

Neuropeptide Y regulates numerous physiological processes via at least five different Y receptors, but the specific roles of each receptor are still unclear. We previously demonstrated that Y2 receptor knockout results in a lean phenotype, increased cancellous bone volume, and an increase in plasma pancreatic polypeptide (PP), a ligand for Y4 receptors. PP-overexpressing mice are also known to have a lean phenotype. Deletion of the Y4 receptor also produced a lean phenotype and increased plasma PP levels. We therefore hypothesized that part of the Y2 phenotype results from increased PP action on Y4 receptors and tested this in PP transgenic Y4 and Y2؊/؊ Y4 ؊/؊ double knockout mice. Bone mass was not altered in Y4 knockout mice. Surprisingly, despite significant hyperphagia, Y2؊/؊ Y4 ؊/؊ mice retained a markedly lean phenotype, with reduced body weight, white adipose tissue mass, leptinemia, and insulinemia. Furthermore, bone volume was also increased threefold in Y2 ؊/؊ Y4 ؊/؊ mice, and this was associated with enhanced osteoblastic activity. These changes were more pronounced than those observed in Y2 ؊/؊ mice, suggesting synergy between Y2 and Y4 receptor pathways. The lack of bone changes in PP transgenic mice suggests that PP alone is not responsible for the bone mass increases but might play a major role in the lean phenotype. However, a synergistic interaction between Y2 and Y4 pathways seems to regulate bone volume and adiposity and could have important implications for possible interventions in obesity and for anabolic treatment of osteoporotic bone loss.Neuropeptide Y (NPY) is an important regulator of numerous physiological processes, such as food intake (9, 43), energy homeostasis (22, 42), and growth and reproduction (2, 10). These processes are mediated at least partially within the hypothalamus, via five different Y receptors (Y1, Y2, Y4, Y5, and y6). All of these receptors are expressed in the hypothalamus, the brain stem, and peripheral tissues (12). Despite having highly divergent primary structures, different Y receptors have very similar pharmacological profiles, with few agonists and antagonists that are selective for specific Y receptors and active in vivo being presently available. Consequently, the specific roles of individual receptors and the mechanisms that mediate the diverse physiological effects of NPY are not yet clearly understood.Recent studies with germ line and conditional Y receptor knockout mouse models have begun to shed some light on the functions of different Y receptors. We have shown that both hypothalamus-specific and germ line Y2 receptor knockout resulted in a lean phenotype in male mice (reduced body weight and white adipose tissue [WAT] mass) (35) and a twofold increase in cancellous bone volume associated with accelerated bone formation (3). Consistent with the general inhibitory functions of NPY, it appears from these data that Y2 receptors directly or indirectly mediate the tonic stimulation of adipocyte activity while also mediating tonic inhibition of osteoblast...

The microarchitecture of bone is regulated by complex interactions between the bone-forming and resorbing cells, and several compounds regulate both actions. For example, vitamin D, which is required for bone mineralization, also stimulates bone resorption. Transgenic mice overexpressing the vitamin D receptor solely in mature cells of the osteoblastic bone-forming lineage were generated to test the potential therapeutic value of shifting the balance of vitamin D activity in favor of bone formation. Cortical bone was 5% wider and 15% stronger in these mice due to a doubling of periosteal mineral apposition rate without altered body weight or calcium homeostatic hormone levels. A 20% increase in trabecular bone volume in transgenic vertebrae was also observed, unexpectedly associated with a 30% reduction in resorption surface rather than greater bone formation. These findings indicate anabolic vitamin D activity in bone and identify a previously unknown pathway from mature osteoblastic cells to inhibit osteoclastic bone resorption, counterbalancing the known stimulatory action through immature osteoblastic cells. A therapeutic approach that both stimulates cortical anabolic and inhibits trabecular resorptive pathways would be ideal for treatment of osteoporosis and other osteopenic disorders.