Novel Role of Y1 Receptors in the Coordinated Regulation of Bone and Energy Homeostasis*

The importance of neuropeptide Y (NPY) and Y2 receptors in the regulation of bone and energy homeostasis has recently been demonstrated. However, the contributions of the other Y receptors are less clear. Here we show that Y1 receptors are expressed on osteoblastic cells. Moreover, bone and adipose tissue mass are elevated in Y1-/- mice with a generalized increase in bone formation on cortical and cancellous surfaces. Importantly, the inhibitory effects of NPY on bone marrow stromal cells in vitro are absent in cells derived from Y1-/- mice, indicating a direct action of NPY on bone cells via this Y receptor. Interestingly, in contrast to Y2 receptor or germ line Y1 receptor deletion, conditional deletion of hypothalamic Y1 receptors in adult mice did not alter bone homeostasis, food intake, or adiposity. Furthermore, deletion of both Y1 and Y2 receptors did not produce additive effects in bone or adiposity. Thus Y1 receptor pathways act powerfully to inhibit bone production and adiposity by nonhypothalamic pathways, with potentially direct effects on bone tissue through a single pathway with Y2 receptors.

The importance of neuropeptide Y (NPY) and Y2 receptors in the regulation of bone and energy homeostasis has recently been demonstrated. However, the contributions of the other Y receptors are less clear. Here we show that Y1 receptors are expressed on osteoblastic cells. Moreover, bone and adipose tissue mass are elevated in Y1 ؊/؊ mice with a generalized increase in bone formation on cortical and cancellous surfaces. Importantly, the inhibitory effects of NPY on bone marrow stromal cells in vitro are absent in cells derived from Y1 ؊/؊ mice, indicating a direct action of NPY on bone cells via this Y receptor. Interestingly, in contrast to Y2 receptor or germ line Y1 receptor deletion, conditional deletion of hypothalamic Y1 receptors in adult mice did not alter bone homeostasis, food intake, or adiposity. Furthermore, deletion of both Y1 and Y2 receptors did not produce additive effects in bone or adiposity. Thus Y1 receptor pathways act powerfully to inhibit bone production and adiposity by nonhypothalamic pathways, with potentially direct effects on bone tissue through a single pathway with Y2 receptors.
Many physiological functions are regulated by signals processed within the brain. Y receptors, members of the G-protein-coupled receptor superfamily, play an important role in this regulatory axis, mediated by their endogenous ligands: neuropeptide Y (NPY), 6 peptide YY, and pancreatic polypeptide. The Y receptor system is complex, consisting of five Y receptors (Y1, Y2, Y4, Y5, and y6), each with varying distributions across peripheral and central tissues, including the hypothalamus. Among a number of responsive tissues, both bone and adipose tissue are known to be regulated, at least in part, via hypothalamic Y2 receptors (1)(2)(3)(4). Indeed, lack of central Y2 signaling, as in hypothalamus-specific Y2 receptor conditional knock-out mice, causes increased bone mass (1). Furthermore, deletion of Y2 receptors has recently been demonstrated to decrease Y1 receptor expression in stromal cells, associated with a greater population of progenitor cells, and accounting for the greater synthetic activity of these cells in vitro suggesting an important role of this Y receptor in bone formation as well (see the accompanying report (39).
Y1 receptors are widely expressed in the central nervous system, including the hypothalamus (5,6), as well as on peripheral tissues such as vascular smooth muscle cells (7) and pancreatic ␤ cells (8). Y1 receptors are expressed on bone marrow stromal cells and bone tissue (see accompanying report (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-naling and the previously identified Y2 receptor pathway was assessed in Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ double knock-out mice.

EXPERIMENTAL PROCEDURES
Animal Care-All research and animal care procedures were approved by the Garvan Institute/St. Vincent's Hospital Animal Experimentation Ethics Committee and were in agreement with the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose. All mice were fed a normal chow diet ad libitum (6% calories from fat, 21% calories from protein, 71% calories from carbohydrate, 2.6 kilocalories/g, Gordon's Specialty Stock Feeds, Yanderra, New South Wales, Australia), with ad libitum access to water.
Generation of Y1 Ϫ/Ϫ and Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ Double Mutant Mice -A targeting vector for the Y1 and Y2 receptor genes (Npy1r and Npy2r, respectively) has been used to produce both germ line (Y1 Ϫ/Ϫ or Y2 Ϫ/Ϫ ) and conditional (floxed, Y1 lox/lox or Y2 lox/lox ) knock-out mice, as previously published (2,14). Both of these knock-out strategies result in deletion of the entire coding region of the Y1 or Y2 receptor, including the neomycin selection cassette. Germ line Y1 Ϫ/Ϫ and Y2 Ϫ/Ϫ lines were bred to generate double heterozygote mice, which were then crossed to obtain the double knock-out mouse line. All mice were on a mixed C57BL/6 -129/SvJ background.
Generation of Adult-onset Hypothalamus-specific Y1 Receptor Knock-out Mice (Y1 Hyp )-9-to 10-week-old Y1 lox/lox mice were anesthetized with 100/20 mg/kg ketamine/xylazine (Parke Davis-Pfizer, Sydney, Australia and Bayer AG, Leverkusen, Germany). With the head in the flat skull position using a stereotaxic table (David Kopf, Tujunga, CA), brain injection coordinates relative to Bregma were posterior 0.8 mm, lateral Ϯ 0.5 mm, ventral 4.7 mm, corresponding to the paraventricular nucleus of the hypothalamus (15). 0.5 l of virus (1 ϫ 10 9 plaque-forming units/l) was injected bilaterally over 10 min using a 26-gauge guide cannula and a 33-gauge injector (PlasticsOne, Roanoke, VA) connected to a Hamilton syringe and a syringe infusion pump (World Precision Instruments, Sarasota, FL). Injection with adeno-associated viral vector (rAAV) expressing Cre-recombinase produced hypothalamus-specific Y1 receptor knock-out mice (Y1 Hyp ). Control Y1 lox/lox mice were injected with virus carrying an empty adeno-associated viral vector and are referred to as Y1 lox/lox . Mice were housed individually for the ensuing 9 weeks, and body weight was measured three to five times per week at the same time of day.
Temperature Measurements-At 5 weeks after rAAV vector injection, body temperature was measured at ϳ9.00 h with a rectal thermometer (Physitemp Instruments Inc., Clifton, NJ). Temperature readings were taken within 10 s of removing the mouse from its cage. Repeat readings were taken from each mouse on 3 consecutive days, and the average of the three readings was used for statistical analysis.
Feeding and Behavioral Studies-At 6 weeks after rAAV vector injection, mice were transferred from housing on soft bedding to cages with only a single paper towel on the bottom of the cage and allowed to acclimatize for 3 nights. 24-h food and water intake were determined as the average of triplicate readings taken over 3 consecutive days. Actual food intake was cal-culated as the weight of pellets taken from the food hopper minus the weight of food spilled in the cage. Fecal weight was also determined in triplicate during these analyses. Wild-type mice housed on paper toweling typically used this to build a nest. This "nesting behavior" was quantified by weighing the amount of paper towel that had been shredded. 7 weeks post injection, the effect of 24 h fasting on body weight was determined. Food and water consumption, food spillage, and fecal output were determined as described above after 1 and 2 days of re-feeding, and body weight was also tracked during the first 3 days of re-feeding. Mice were then returned to soft bedding.
Glucose Tolerance Tests-At 8 weeks after rAAV vector injection, mice were fasted for 24 h before intraperitoneal injection of a 10% D-glucose solution (1.0 g/kg) with tail blood sampling (ϳ20 -50 l) at 0, 15, 30, 60, and 120 min after injection. Serum was stored at Ϫ20°C for subsequent analysis.
Tissue Collection-Mice were injected with the fluorescent compound calcein (15 mg/kg, Sigma) 10 and 3 days prior to tissue collection to enable subsequent calculation of bone formation rate. Y1 Hyp mice and Y1 lox/lox controls were culled at 18 -19 weeks of age, 9 weeks after rAAV vector injection. Germ line Y1 Ϫ/Ϫ , Y2 Ϫ/Ϫ , and Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ mice and wild-type controls were culled at 15-17 weeks of age. Mice were culled in the freely fed state between 12:00 noon to 3:00 p.m. by cervical dislocation followed by decapitation for collection of trunk blood. Serum was collected and stored at Ϫ20°C until subsequent analysis as described below. The interscapular brown adipose tissue (BAT) as well as white adipose tissue (WAT) depots (right inguinal, right epididymal or periovarian (gonadal), right retroperitoneal, and mesenteric) were removed and weighed. The weight of these WAT depots were summed together and expressed as total WAT weight, normalized as a percentage of body weight. Both femurs and the caudal vertebrae were excised and fixed in 4% paraformaldehyde for 16 h at 4°C.
Bone Histomorphometry-The right femur was bisected transversely at the midpoint of the long axis, the distal half was embedded undecalcified in methacrylate resin (Medim-Medizinische Diagnostik, Giessen, Germany), and 5-m sagittal sections were analyzed, as previously described (16). The 4th caudal vertebra was sectioned in the sagittal plane, and mid vertebral sections were analyzed as previously described (17). Briefly, sections were stained for mineralized bone, and trabecular bone volume, thickness, and trabecular numbers were calculated. Bone formation (mineralizing surface), mineral apposition rate, and bone formation rate were calculated, as previously described (16) using fluorescence microscopy (Leica, Heerbrugg, Switzerland). Osteoclast surface and osteoclast number were estimated using tartrate-resistant acid phosphatase-stained sections, with only multinucleated, tartrate-resistant acid phosphatase-positive cells associated with the bone surface being included in the analysis. Cortical mineral apposition rate was measured on the anterior periosteal surface in a region extending 1000 m distal from the midpoint and in an endosteal region extending 1000 m proximal from the posterior aspect of the growth plate, as previously described (18).
Bone Densitometry-Whole femoral bone mineral content and bone mineral density were measured using a dedicated mouse dual x-ray absorptiometer (Lunar Piximus II, GE Medical Systems, Madison, WI) in excised left hind limbs. Femurs were scanned with tibiae attached and the knee joint in flexion to 90°, to ensure consistent placement and scanning of the sagittal profile.
Quantitative Computed Tomography-Quantitative computed tomography was used to isolate cortical bone for analysis in male mice, using a Stratec XCT Research SA (Stratec Medizintechnik, Pforzheim, Germany). Scans were conducted using a voxel size of 70 m, scan speed of 5 mm/s, and slice width of 0.2 mm every 0.5 mm on excised left femurs, as previously described (16). Bones were scanned in two consecutive slices, 7 and 7.5 mm from the distal margin of the femur, representing a mid femoral aspect. Bone strength index, an indicator of bending strength, was calculated (18).
Serum Analyses-Hormone levels in serum samples collected at cull were determined with commercial radioimmunoassay kits: insulin (Linco Research, St. Louis, MO), corticosterone, free T4 (ICN Biomedicals, Costa Mesa, CA), and insulin-like growth factor-1 (Bioclone Australia, Marrickville, New South Wales, Australia). Basal and glucose-induced serum glucose and insulin levels were determined with a glucose oxidase kit (Trace Scientific, Melbourne, Australia) and insulin with an enzyme-linked immunosorbent assay kit from Linco Research, respectively.
Gene Expression in Mouse Calvarial Osteoblast Cultures-Bone cells were isolated from calvariae of 2-to 3-day-old CsA mice using a modified time sequential enzyme-digestion technique (19). Cells from populations 6 to 10 were used. These cells showed an osteoblastic phenotype as assessed by their cAMP responsiveness to parathyroid hormone, expression of alkaline phosphatase, osteocalcin and bone sialoprotein, and the capacity to form mineralized bone noduli (data not shown). The cells were seeded in culture flasks containing ␣-minimal essential medium supplemented with 10% fetal calf serum, L-glutamine, and antibiotics at 37°C in humidified air containing 5% CO 2 .
After 4 days in flasks, the cells were seeded in culture dishes. Osteoblasts were plated at a density of 10 4 cells/cm 2 in culture dishes containing ␣-minimal essential medium/10% fetal bovine serum. After attachment overnight, medium was changed to ␣-minimal essential medium/10% fetal bovine serum. After 7 days of culture, RNA was extracted and used for quantitative real-time reverse transcription-PCR analyses.
RNA Extraction and cDNA Synthesis-Total RNA was extracted from mouse calvarial osteoblasts using the RNAqueous-4PCR kit following the manufacturer's protocol (Ambion Inc., Austin, TX). The RNA was quantified spectrophotometrically, and the integrity of the RNA preparations was examined by agarose gel electrophoresis. Extracted total RNA was treated with deoxyribonuclease I to eliminate genomic DNA according to manufacturer instructions. One microgram of total RNA, following DNase treatment, was reverse transcribed into single-stranded cDNA with a 1st Strand cDNA Synthesis Kit using oligo-p(dT)15 primers. After incubation at 25°C for 10 min and at 42°C for 60 min, the avian myeloblastosis virus reverse transcriptase was denatured at 99°C for 5 min, followed by cooling to ϩ4°C for 5 min. The cDNA was kept at Ϫ20°C until used for PCR.
Quantitative Real-time PCR-Expression of Y1 and Y2 receptor mRNA were analyzed by quantitative real-time PCR using the TaqMan Universal PCR master mix (Applied Biosystems, Foster City, CA) and a sequence detection system (ABI Prism 7900 HT Sequence Detection System and Software, Applied Biosystems) with fluorescence-labeled probes (reporter fluorescent dye VIC at the 5Ј-end and quencher fluorescent dye tetramethylrhodamine at the 3Ј-end). Primers and probes for Y1 and Y2 receptors were analyzed using a kit from Applied Biosystems. To control for variability in amplification due to differences in starting mRNA concentrations, ␤-actin was used as an internal standard. The specific primers and probes used are as follows for ␤-actin (sense: 5Ј-GGACCT-GACGGACTACCTCATG-3Ј, antisense: 5Ј-TCTTTGATGT-CACGCACGATTT-3Ј, probe: 5Ј-CCTGACCGAGCGTGGC-TACAGCT TC-3Ј). The relative expression of target mRNA was computed from the target Ct values and the ␤-actin C t value using the standard curve method (User Bulletin 2, Applied Biosystems).
Isolation and Culture of Bone Marrow Stromal Cells-Bone marrow stromal cells were isolated from 5-to 9-week-old male wild-type and germ line Y1 Ϫ/Ϫ mice as previously described (see accompanying report (39)). Briefly, marrow was flushed from femurs and tibias with control media, and cells were plated at a density of 1.9 ϫ 10 6 cells/cm 2 in 50-cm 2 plastic tissue culture plates. The non-adherent cell population was removed by medium changes 3 and 5 days later. Cells were trypsinized after 7 days in culture with 0.25% trypsin containing 0.53 mM EDTA and re-plated at 3 ϫ 10 4 cells/cm 2 in 24-well plates in either control media or control media containing 100 nM human NPY (Auspep, Parkville, Victoria, Australia). After an additional 5 or 20 days in culture, cells were trypsinized, and viable cell numbers were determined by trypan blue staining. Statistical Analyses-All data are expressed as mean Ϯ S.E. Differences between two groups were assessed by two-tailed Student's t test. Differences among multiple groups of mice were assessed by analysis of variance or repeated measures analysis of variance, followed by Fisher's or Contrast post-hoc comparisons if appropriate (StatView version 4.51 or Super-ANOVA, Abacus Concepts Inc., Berkeley, CA). Statistical significance was defined as p Ͻ 0.05.

Greater Bone Formation in Y1 Receptor Null Mice
To examine the mechanism behind the elevation in bone mass evident in Y1 Ϫ/Ϫ mice (see accompanying report (39)), we examined distal femurs and caudal vertebrae from skeletally mature male and female mice.
Cancellous Bone-Germ line Y1 Ϫ/Ϫ mice displayed significantly greater cancellous bone volume in the distal femoral metaphysis, with greater trabecular number and thickness ( Figs. 1 and 2). Interestingly, this was associated with increased activity of both osteoblastic and osteoclastic lineages. Similar to Y2 Ϫ/Ϫ mice, bone formation rate was greater in Y1 Ϫ/Ϫ mice compared with wild-type mice with enhanced mineral apposition rate (MAR) in both sexes, but no change in mineralizing surface (Fig. 2). However, in contrast to Y2 Ϫ/Ϫ mice, bone resorption was also altered in Y1 Ϫ/Ϫ mice, with significantly greater osteoclast surface in both sexes (Fig. 2).
These changes in cancellous bone homeostasis were also evident in the caudal vertebrae. At this site, cancellous bone volume was significantly increased in knock-out mice compared with wild-type values (Y1 Ϫ/Ϫ , 30.9 Ϯ 1.2% versus wild type, 25.5 Ϯ 2.4%, n ϭ 5-9 male mice, p Ͻ 0.05). As in the femurs, this change was also associated with greater trabecular thick-ness (Y1 Ϫ/Ϫ , 65.4 Ϯ 2.0 m versus wild type, 44.0 Ϯ 2.9 m, p Ͻ 0.0001). These cancellous changes are consistent with the previously noted elevation in bone mineral density and content in Y1 Ϫ/Ϫ long bones; however, because cortical bone rather than  cancellous bone is the major contributor to changes in bone density and content, the cortical content of bones from Y1 Ϫ/Ϫ mice was further examined.
Cortical Bone-Cortical changes in Y1 Ϫ/Ϫ long bones were examined by quantitative computed tomography. At the mid-femur, Y1 Ϫ/Ϫ mice had greater cortical mineral content and density, consistent with greater cortical area and thickness (Fig. 3). Polar moment of inertia and strength index were also significantly greater in the femoral crosssections of Y1 Ϫ/Ϫ compared with wild-type mice (Fig. 3). The observed increase in cortical mineral content in Y1 Ϫ/Ϫ mice combined with the 25% increase in strength index indicate a response sufficient to produce functional relevant changes in bone strength in vivo and highlights the therapeutic potential of such pathways.
To further investigate the cellular basis for these differences, both endocortical and periosteal surfaces were examined. Cortical osteoblast activity was increased in Y1 Ϫ/Ϫ mice, with MAR elevated on the femoral endosteal surface an average of 70% in both genders and on the periosteum of male Y1 Ϫ/Ϫ mice with an increase of nearly 7-fold (Fig. 4). Taken together, these data reveal that loss of Y1 receptor signaling results in a generalized elevation in parameters of osteoblast activity, at both axial and appendicular sites leading to greater cancellous and cortical bone accrual.
Sustained Elevation of Adiposity in Y1 Receptor Null Mice-Body weight and adiposity of young and aged Y1 Ϫ/Ϫ mice were compared with age-matched wild-type controls to determine progression of the Y1 Ϫ/Ϫ energy homeostasis phenotype. Both male and female Y1 Ϫ/Ϫ mice developed an obese phenotype with advancing age (Fig. 5), indicated by significantly greater body weight, WAT and BAT depots, and with a more pronounced phenotype in female Y1 Ϫ/Ϫ mice. These changes in body weight were not due to changes in stature, because femur  length was not significantly different between Y1 Ϫ/Ϫ mice and wild type (data not shown). Thus, whereas wild-type mice showed no changes in body weight and adiposity with age beyond 12 months, Y1 Ϫ/Ϫ mice showed marked and significant increases, indicating a sustained and continuing effect on energy homeostasis and adipocyte function in these mice.
To gain insight into possible mechanisms of obesity associated with Y1 deficiency, we measured metabolic parameters and energy expenditure in Y1 Ϫ/Ϫ mice. There was no significant effect of genotype on serum concentrations of corticosterone, glucose, insulin-like growth factor 1, and free T4 and temperature (data not shown); however, serum insulin levels were significantly higher in Y1 Ϫ/Ϫ mice (female Y1 Ϫ/Ϫ , 177 Ϯ 27 pM versus wild type, 56 Ϯ 7 pM, n ϭ 17, p Ͻ 0.001; male Y1 Ϫ/Ϫ , 220 Ϯ 52 pM versus wild type, 120 Ϯ 18 pM, n ϭ 18, p Ͻ 0.05). Because insulin is lipogenic (20 -22), it is possible that these higher serum insulin levels may be causally linked to the greater adiposity observed in young and aged germ line Y1 Ϫ/Ϫ mice.

Hypothalamic Y1 Receptors Do Not Regulate Bone Mass-
Previously we showed that hypothalamus-specific Y2 receptor deletion leads to pronounced anabolic effects on bone (1). We therefore hypothesized that the phenotype observed in the bones of germ line Y1 Ϫ/Ϫ mice might also involve signals mediated by the hypothalamus. To test this, we investigated bone homeostasis in mice with adult-onset, hypothalamus-specific deletion of Y1 receptors. Localized Y1 receptor deletion in the hypothalamus was verified by PCR on genomic DNA extracted from the hypothalamus, with forebrain and liver of rAAV vector-injected mice used as negative controls. PCR primers were designed to produce a detectable product only when the Y1 receptor had been deleted. This was the case only in amplified DNA extracted from the hypothalamus of virus-injected Y1 lox/lox mice but not in DNA from control samples (data not shown), confirming the successful ablation of Y1 receptor genes in this area.
Cancellous Bone-Deletion of hypothalamic Y1 receptors from adult mice (Y1 Hyp ) did not alter cancellous bone volume compared with age-matched Y1 lox/lox controls of either sex ( Fig.  1 and Table 1). Importantly, and consistent with the lack of change in cancellous bone volume, hypothalamus-specific deletion of Y1 receptors did not alter bone cell activity. Mineral apposition rate and osteoclast surface and number were not different between Y1 Hyp and Y1 lox/lox controls of either gender (Table 1).

Y1 Receptor Mediates Direct Effects of NPY on Bone Marrow Stromal Cell Number-To investigate
the possibility that Y1 receptors influence bone tissue via direct effects, Y1 receptor expression was investigated in mouse calvarial osteoblasts expressing the osteoblastic marker genes alkaline phosphatase, osteocalcin, and bone sialoprotein, and demonstrating the capability of forming mineralized bone noduli (data not shown). Quantitative real-time PCR revealed that Y1 receptor gene transcripts were present in osteoblasts at day 7 of culture ( Fig.  6A). Expression of Y2 receptors was

TABLE 1 Cancellous bone parameters in the femur following adult-onset hypothamus-specific deletion of Y1 receptors (Y1 Hyp ) compared to control mice with Y1 receptors intact (Y1 lox/lox )
Data are means Ϯ S.E. of three to five male and seven to ten female mice per group. At p Ͻ 0.05, no significant differences were detected in within-gender comparisons. not detected. These data are consistent with a direct, Y1-mediated effect in these cells. To further investigate this possibility, the effect of NPY treatment on cultured bone marrow stromal cells from wild-type and Y1 Ϫ/Ϫ mice was examined. Administration of NPY to cultures from wild-type tissue markedly reduced cell numbers (Fig. 6B). Cell numbers in Y1 Ϫ/Ϫ cultures were comparable to those of wild-type control cultures and were not altered by NPY treatment, providing the first evidence of direct, Y1-mediated regulation of this system. Hypothalamic Y1 Receptors Do Not Alter Energy Homeostasis-Hypothalamic Y1 receptors are hypothesized to mediate anabolic effects, namely hyperphagia, under conditions of elevated hypothalamic NPY levels such as fasting or genetic obesity (10,11). We investigated energy homeostasis in Y1 Hyp mice but did not see significant differences from control Y1 lox/lox mice with respect to body weight in the first 6 weeks after rAAV vector injection, at time of cull 9 weeks after vector injection, or in response to 24-h fasting and 72-h re-feeding ( Fig. 7 and Table  2). The actual food intake of Y1 Hyp mice was not significantly different from that of control mice (Fig. 7), either in the non-fasted state or in the first 2 days of re-feeding after a 24-h fast. In keeping with a lack of effect of hypothalamic Y1 receptor deletion on food intake, water intake and fecal output were not significantly different between Y1 Hyp and Y1 lox/lox mice ( Table 2).

Genotype
Some factors that regulate energy homeostasis do so by altering fat mass or glucose metabolism even in the absence of effects on body weight or food intake (10,21,23,24). We therefore investigated whether hypothalamic Y1 receptor knock-out induced alterations in fat mass or glucose homeostasis. Y1 Hyp mice showed no significant difference from Y1 lox/lox mice with respect to WAT and BAT depots or rectal temperature ( Table  2), suggesting no change in thermogenesis. In addition, there was no significant effect on fasting serum glucose or insulin levels or the change in serum glucose or insulin levels in response to intraperitoneal glucose injection, albeit the area under the insulin curve after glucose injection tended to be higher in knock-out than in wild-type mice ( Table 2).
The conditional deletion of Y1 receptors in adult mice has, for the first time, enabled examination of the role of these signals in FIGURE 7. Effect of hypothalamus-specific Y1 receptor deletion on non-fasted and fasting-induced body weight, feeding, and nesting behavior. Region-specific Y1 receptor knock-out, in the hypothalamus (Y1 Hyp ) on day 0, had no significant effect on body weight (A and B) compared with control mice (Y1 lox/lox ). Shown are the percent body weight values lost during 24-h fasting and 72-h re-feeding (C and D) and feeding behavior incorporating the amount of food taken from the food hopper but spilled on the cage floor as well as the amount of food actually eaten, either in the non-fasted state (before fasting) or in the first 48 h of re-feeding (E and F). G, nesting behavior, quantified as the weight of paper toweling shredded per day. Data in panels C-G were collected at 7 weeks after adenoviral vector injection. Data are means Ϯ S.E. of four or five male and seven to ten female mice per group. *, p Ͻ 0.05 versus the amount of food spilled or the amount of paper toweling shredded by control (Y1 lox/lox ) mice of the same gender and of the same nutritional status (non-fasted or re-fed).

TABLE 2 Effect of hypothalamus-specific deletion of Y1 receptors on parameters of energy balance in adult mice
Data are means Ϯ S.E. of three to five male and seven to ten female mice. At p Ͻ 0.05, no significant differences were detected in within-gender comparisons. energy homeostasis. In a similar manner to skeletal effects, the regulation of energy homeostasis and food intake by Y1 is not controlled by those hypothalamic receptors that were deleted in the current study, indicating a consistent pattern of non-hypothalamic Y1 action in the regulation of these processes. However, hypothalamic Y1 receptor knock-out did alter specific aspects of behavior in relation to feeding. Food grinding was markedly increased in these mice, with the amount of ground food spilled on the cage floor significantly elevated in both male and female Y1 Hyp mice compared with controls (Fig. 7).

Genotype
Hypothalamic Y1 Receptors Modify Behavior-Interestingly, although hypothalamus-specific Y1 receptor deletion had no impact on parameters of energy homeostasis, it significantly influenced a related maternal behavior, showing a loss of nest-building abilities. Whereas control Y1 lox/lox mice consistently used the paper towel provided as bedding material for constructing a nest, this behavior was completely absent in Y1 Hyp mice. Indeed, the measured weight of shredded paper, as an indicator for nesting behavior, was dramatically reduced in both male and female Y1 Hyp versus Y1 lox/lox control mice (Fig. 7).

Coincident Deletion of Y1 and Y2 Receptors Does Not Induce Additive Changes in Bone
Cancellous Bone-Our previous study revealed a Y2-dependent inhibition of Y1 expression in osteoblastic and adipocytic lineage cells (see accompanying report (39)), thereby suggesting a putative mechanism whereby central Y2 signaling modulates peripheral tissue homeostasis. To gain further insights into whether Y1 and Y2 receptors are linked in the regulation of bone physiology, we generated Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ receptor double knock-out mice and investigated whether additive effects were apparent. Importantly, although Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ receptor double knock-out mice of both genders had significantly greater cancellous bone volume compared with wild-type controls (Fig. 8), there were no significant differences from Y1 Ϫ/Ϫ and Y2 Ϫ/Ϫ mice. Mineral apposition rate was significantly increased in all three Y receptor-deficient models compared with wild-type mice. There were however, some minor differences, with MAR in male Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ mice significantly reduced compared with Y2 Ϫ/Ϫ mice. Unlike Y1 Ϫ/Ϫ mice, Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ mice showed no increase in osteoclast surface (Fig. 8). Thus, whereas double deletion of Y1 and Y2 receptors induces significant effects on cancellous bone, there were no obvious additive effects over those of Y1 or Y2 receptor deletion in isolation.
Cortical Bone-Similarly, quantitative computed tomography analysis revealed that Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ femurs had significantly greater cortical bone area and thickness than wild-type mice, although there was no significant effect on cortical mineral content or density (Fig. 9). As in the Y1 Ϫ/Ϫ and Y2 Ϫ/Ϫ single knockout models, these architectural changes were coincident with greater cortical bone formation in Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ mice, with endosteal MAR elevated compared with wild type (Fig. 9). The strength index of femurs from Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ double knock-out mice was comparable to those in single Y1 Ϫ/Ϫ or Y2 Ϫ/Ϫ animals (Fig. 9). Although a common feedback control for independent pathways cannot be ruled out, the lack of additive responses in Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ mice is consistent with a common pathway from the hypothalamus to bone involving both Y2 and Y1 signaling.

DISCUSSION
In this study we show that Y1 receptors exert powerful control over bone production and have significant effects on adiposity. These findings indicate that these effects are most likely mediated by non-hypothalamic Y1 receptors. Germ line disruption of Y1 receptor signaling revealed a generalized increase in osteoblast activity on both cancellous and cortical surfaces, with consistent changes in femoral, tibial, and vertebral bones. Expression of Y1 receptors on osteoblastic and bone marrow stromal cells suggested a peripheral, possibly direct mechanism of action. In keeping with this, deletion of hypothalamic Y1 receptors did not alter bone homeostasis and direct action of Y1 signaling was confirmed ex vivo in bone marrow stromal cell cultures, where the NPY-mediated inhibition of cell number was absent in Y1 Ϫ/Ϫ cells. These findings are consistent with our previous study, with reduced expression of Y1 receptors in stromal cells associated with a greater number of mesenchymal progenitor cells in Y2 Ϫ/Ϫ mice (see accompanying report (39)). Moreover, a common pathway controlling bone formation by Y1 and Y2 receptor subtypes was suggested by a lack of additive effects on bone in Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ mice.
In addition, adiposity was significantly elevated in young and aged Y1 Ϫ/Ϫ mice, in association with significant decreases in fasting-induced hyperphagia and increases in serum insulin levels. Because insulin is lipogenic, promoting partitioning of fuels toward WAT and away from muscle (20,22), the hyperinsulinemia observed in germ line Y1 knock-out mice could contribute to the increased adiposity observed in these animals despite the lack of hyperphagia. As with effects on bone, these changes in food intake and adiposity were not evident in mice following adult-onset deletion of hypothalamic Y1 receptors, indicating mediation of these effects by Y1 receptors other than those hypothalamic receptors that were deleted in the current study. Although hypothalamic Y1 receptors are not likely involved in regulation of bone or adipose tissue nor of nonfasted or fasting-induced food intake or body weight, they profoundly influenced feeding and nesting behaviors, with hypothalamus-specific Y1 receptor knock-out mice showing marked increases in food grinding behavior and a pronounced lack of nest-building behavior. Overall, these findings indicate a generalized and powerful peripheral action of Y1 signaling in the regulation of bone and adipose tissue.
Current understanding of the role of the NPY system in the regulation of bone tissue and energy balance is rapidly expanding. Y2 receptors have been established as significant regulators of both bone and adipose tissue, with Y2 receptor knock-out enhancing bone formation and reducing adiposity (1,2,25,26). Coincident deletion of Y2 and Y4 receptors enhanced effects on bone and adipose tissue, with Y2 Ϫ/Ϫ Y4 Ϫ/Ϫ knock-out mice showing even more pronounced increases in bone mass and synergistic decreases in adiposity (3). We now demonstrate that loss of Y1 receptor signaling also altered bone tissue and energy homeostasis; however, the changes show important differences from Y2 Ϫ/Ϫ or Y2Y4 Ϫ/Ϫ mice, revealing unique actions of individual Y receptors. Loss of Y1 signaling led to an increase in adipose deposition, the opposite to that evident in lean Y2 Ϫ/Ϫ and Y2Y4 Ϫ/Ϫ mice. The regulation of fat and bone are to a certain extent related by the actions of leptin, acting in the hypothalamus to decrease adiposity and cancellous bone formation (27)(28)(29). This relationship is evident in Y2Y4 Ϫ/Ϫ mice, whose lean phenotype and consequently reduced serum leptin is a likely mechanism for the increased bone volume compared with Y2 Ϫ/Ϫ mice (3). Similarly, the obesity evident in Y1 Ϫ/Ϫ mice might be expected to affect bone volume through action of increased circulating leptin concentrations. However, as previously shown, despite greater fat mass, serum leptin levels are not increased in our Y1 Ϫ/Ϫ mice and indeed are not altered in Y2 Ϫ/Ϫ or Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ mice (13), suggesting that the leptin and NPY-mediated pathways act separately in this model, as in previous studies (3,16,25).
Given the central nature of Y2-mediated effects on energy homeostasis (2), the Y1-deficient phenotype was also assessed by conditional deletion of hypothalamic Y1 receptors. The paraventricular nucleus was chosen due to its strategic location in the energy homeostasis circuit and because of the presence of Y1 receptor expression in this area as well as known NPY projections to this region from the arcuate nucleus (6). Interestingly, the effects on adipose tissue evident in germ line Y1 Ϫ/Ϫ mice were absent in hypothalamus-specific Y1 receptor knock-out mice, demonstrating that the regulation of energy homeostasis is not directly mediated through those hypothalamic Y1 receptors deleted in this study but rather via other sites. Additionally, Y1 receptors are expressed in peripheral tissues, including pancreatic ␤ cells (8), and are likely to mediate direct effects such as inhibition of insulin secretion (23,30). Thus the hyperinsulinemia of Y1 Ϫ/Ϫ mice may be a direct response to lack of Y1 receptors in pancreatic islet tissue and, given that insulin is lipogenic (20,22), may contribute to the greater adiposity of these mice (13). Consistent with this action, the greatest change in adiposity was evident in Y1 Ϫ/Ϫ females, which also had the greatest increase in serum insulin.
Although there is a lack of involvement of hypothalamic Y1 receptors in the regulation of energy homeostasis, these receptors play a significant role in the regulation of feeding and other feeding-related behavior. The exact reasons for food grinding in mice are not clear (31), although it has been shown that electrical and chemical stimulation of the hypothalamus influences the activity of masticatory trigmental neurons in the brain stem important for jaw movement (32). It is therefore possible that ablation of Y1 receptors in the hypothalamus leads to altered responses to these neurons in the brainstem, leading to increased grinding of food. Interestingly, electrical lesioning of the paraventricular nucleus has also been found to disrupt the initiation of maternal behavior, e.g. nest building in the rat (33). Considering the high level of Y1 receptors in the paraventricular nucleus, lack of Y1 receptors in this area might cause a similar alteration in mice and could explain at least part of the altered nest-building behavior seen in these conditional knockout mice.
Consistent with the adipose phenotype, and in contrast to the centrally mediated effects on bone in Y2 Ϫ/Ϫ mice, the Y1dependent changes in bone did not involve those receptors expressed in the hypothalamus (1). The importance of this peripheral action of Y1 is heightened by the presence of Y1 receptors in osteoblastic cells, indicating, as in adipose tissue and pancreatic ␤ cells, the potential for direct effects. Moreover, the lack of NPY-mediated inhibition in Y1 Ϫ/Ϫ bone marrow stromal cell cultures shows, for the first time, direct regulation of osteoblastic cells by NPY-mediated stimulation of Y1 receptors and is consistent with previous NPY effects in bone cells (34,35). The putative regulatory role of these osteoblastic Y1 receptors is consistent with a growing understanding of the direct effects of neural signaling molecules on bone cell activity. Adrenergic (36), glutamatergic (37), and cannabinoid (38) receptors are among those neural signals recently described as directly mediating changes in bone homeostasis. Although similar to Y2 Ϫ/Ϫ mice with respect to osteoblastic effects, the bone phenotype of Y1 Ϫ/Ϫ mice was different from that of Y2 Ϫ/Ϫ mice, in that Y1 receptor knockouts displayed involvement of the osteoclastic lineage. The greater osteoclast surface in Y1 Ϫ/Ϫ mice, not evident in Y2 Ϫ/Ϫ , suggests that, although there is some evidence these receptors may share a common pathway to control osteoblastic activity, they differ in their ability to control cells of the osteoclast lineage. Importantly, this elevation in osteoclast indices was not sufficient to counteract the anabolic changes.
The similarity of osteoblast phenotypes between Y1 Ϫ/Ϫ and Y2 Ϫ/Ϫ mice suggested a common signaling pathway to regulate bone formation. In keeping with this, deletion of both the Y1 and Y2 receptors did not result in additive effects on bone mass. There were, however, some microarchitectural changes evident in these mice, with fewer, but thicker, trabeculae compared with single knockouts, which may relate to subtle shifts in the balance of resorption and formation. Consistent with this, the osteoclast surface was not elevated in Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ mice, suggesting a Y2 Ϫ/Ϫ -like phenotype. The dominance of the Y2 Ϫ/Ϫ phenotype was not complete, however, with Y1 Ϫ/Ϫ Y2 Ϫ/Ϫ mice showing a more Y1 Ϫ/Ϫ like cortical bone phenotype. We previously showed that the effects of Y1 deletion to increase circulating insulin levels and enhance adiposity in mice were no longer evident when Y2 receptors were also missing (13), consistent with a dominant Y2 Ϫ/Ϫ -like effect on adipose homeostasis. Although Y1 and Y2 appear to share common pathways in the regulation of bone and adipose tissue, discreet actions of individual Y receptors are still apparent in these tissues.
In conclusion, this work provides clear evidence of a role for Y1 receptors in the regulation of skeletal homeostasis and indicates a direct role for these receptors to inhibit osteoblast activity. A peripheral site of action is supported by the lack of skeletal changes after hypothalamus-specific Y1 receptor deletion as well as the presence of Y1 receptors on osteoblasts and bone marrow stromal cells and the abolition of the effect of NPY on bone marrow stromal cells from Y1 receptor knock-out mice. Although these data also demonstrate that the hypothalamic Y1 receptors deleted in this study do not mediate the increased adiposity observed in germ line Y1 receptor knock-out mice, they may play a role in modulating other feeding-related behaviors such as grinding and nesting, revealing an altered regulatory axis for the homeostatic and behavioral aspects of energy balance. The magnitude of changes evoked in bone and fat tissue by germ line loss of Y1 signaling suggests that targeting such pathways may represent effective therapeutic strategies for both skeletal fragility and obesity.