Neuropeptide Y is implicated in energy homeostasis, and contributes to obesity when hypothalamic levels remain chronically elevated. To investigate the specific role of hypothalamic Y2 receptors in this process, we used a conditional Y2 knockout model, using the Cre-lox system and adenoviral delivery of Cre-recombinase. Hypothalamus-specific Y2-deleted mice showed a significant decrease in body weight and a significant increase in food intake that was associated with increased mRNA levels for the orexigenic NPY and AgRP, as well as the anorexic proopiomelanocortin (POMC) and cocaine-and amphetamine-regulated transcript (CART) in the arcuate nucleus. These hypothalamic changes persisted until at least 34 days after Y2 deletion, yet the effect on body weight and food intake subsided within this time. Plasma concentrations of pancreatic polypeptide and corticosterone were 3-to 5-fold increased in hypothalamus-specific Y2 knockout mice. Germ-line Y2 receptor knockout also produced a significant increase in plasma levels of pancreatic polypeptide. However, these mice differed from conditional knockout mice in that they showed a sustained reduction in body weight and adiposity associated with increased NPY and AgRP but decreased POMC and CART mRNA levels in the arcuate nucleus. The transience of the observed effects on food intake and body weight in the hypothalamus-specific Y2 knockout mice, and the difference of this model from germ-line Y2 knockout mice, underline the importance of conditional models of gene deletion, because developmental, secondary, or extrahypothalamic mechanisms may mask such effects in germ-line knockouts.neuropeptide Y ͉ pancreatic polypeptide ͉ cre-lox ͉ arcuate nucleus N europeptide Y (NPY) in the hypothalamus is known to be a strong stimulus for food intake (1, 2), and induces many neuroendocrine and metabolic changes that favor energy storage. Such changes include decreased thermogenesis in brown adipose tissue, hyperinsulinemia, hypercorticosteronemia, and insulin hyperresponsiveness in white adipose tissue (3, 4). All of these neuroendocrine and metabolic effects of central NPY administration persist even when NPY-induced hyperphagia is prevented by pair-feeding (3, 4), demonstrating that hyperphagia is not the only mechanism by which central NPY increases adiposity.Although numerous other hormones and peptides act within the hypothalamus to regulate energy homeostasis, many exert an important component of their effects via actions on the hypothalamic NPY-ergic system (5-7), demonstrating the pivotal role of NPY in coordinating energy homeostasis. However, it is not clear which of the 5 cloned Y-receptors (Y1, Y2, Y4, Y5, and y6) are responsible for these effects (8).There is increasing evidence that Y2 receptors are involved in energy homeostasis. Over 80% of NPY-containing neurons in the arcuate nucleus (Arc) coexpress Y2 receptor mRNA (9). This presynaptic Y2 receptor has therefore been proposed to regulate the release of NPY (10, 11) and other colocalized neurotransmitters involved ...

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.

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Aims/hypothesis: Obese people exhibit reduced circulating peptide YY (PYY) levels, but it is unclear whether this is a consequence or cause of obesity. We therefore investigated the effect of Pyy ablation on energy homeostasis. Methods: Body composition, i.p. glucose tolerance, food intake and hypothalamic neuropeptide expression were determined in Pyy knock-out and wildtype mice on a normal or high-fat diet. Results: Pyy knock-out significantly increased bodyweight and increased fat mass by 50% in aged females on a normal diet. Male chow-fed Pyy −/− mice were resistant to obesity but became significantly fatter and glucose-intolerant compared with wild-types when fed a high-fat diet. Pyy knock-out animals exhibited significantly elevated fasting or glucose-stimulated serum insulin concentrations vs wild-types, with no increase in basal or fasting-induced food intake. Pyy knock-out decreased or had no effect on neuropeptide Y expression in the arcuate nucleus of the hypothalamus, and significantly increased proopiomelanocortin expression in this region. Male but not female knock-outs exhibited significantly increased growth hormone-releasing hormone expression in the ventromedial hypothalamus and significantly elevated serum IGF-I and testosterone levels. This sex difference in activation of the hypothalamo-pituitary somatotrophic axis by Pyy ablation may contribute to the resistance of chow-fed male knockouts to late-onset obesity. Conclusions/interpretation: PYY signalling is important in the regulation of energy balance and glucose homeostasis, possibly via regulation of insulin release. Therefore reduced PYY levels may predispose to the development of obesity, particularly with ageing or under conditions of high-fat feeding.

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...

Hypothalamic neuropeptide Y (NPY) is implicated in the regulation of a variety of physiological functions, notably energy homeostasis and reproduction. Chronically elevated NPY levels in the hypothalamus, as in genetically obese ob/ob mice, are associated with obesity, a syndrome of type 2 diabetes, and infertility. However, it is not known which of the five cloned Y receptors mediate these effects. Here, we show that crossing the Y2 receptor knockout mouse (Y2 ؊/؊ ) onto the ob/ob background attenuates the increased adiposity, hyperinsulinemia, hyperglycemia, and increased hypothalamo-pituitary-adrenal (HPA) axis activity of ob/ob mice. Compared with lean controls, ob/ob mice had elevated expression of NPY and agouti-related protein (AgRP) mRNA in the arcuate nucleus and decreased expression of proopiomelanocortin (POMC) and cocaine-and amphetamine-regulated transcript (CART) mRNA. Y2 deletion in ob/ob mice significantly increased the hypothalamic POMC mRNA expression, with no effect on NPY, AgRP, or CART expression. [Y2 ؊/؊ ob/ob] mice were no different from ob/ob littermates with respect to food intake and body weight, and Y2 receptor deficiency had no beneficial effect on the infertility or the reduced hypothalamo-pituitary-gonadotropic function of ob/ob mice. These data demonstrate that Y2 receptors mediate the obese type 2 diabetes phenotype of ob/ob mice, possibly via alterations in melanocortin tonus in the arcuate nucleus and/or effects on the HPA axis. Diabetes 51:3420 -3427, 2002 N europeptide Y (NPY) in the hypothalamus is known to be a strong stimulus for food intake (1,2) and induces many neuroendocrine and metabolic changes that favor energy storage. Such changes include decreased thermogenesis in brown adipose tissue (BAT) (3), hyperinsulinemia, insulin resistance in muscles, insulin hyperresponsiveness in white adipose tissue (WAT) (4), activation of the hypothalamopituitary-adrenal (HPA) axis (4,5), and decreased activity of the hypothalamo-pituitary-thyrotropic (6), -somatotropic, and -gonadotropic axes (7). All of these adipogenic neuroendocrine and metabolic effects of central NPY administration persist, even when NPY-induced hyperphagia is prevented by pair-feeding (3,4,6,8), demonstrating that hyperphagia is not the only mechanism by which central NPY increases adiposity. However, it is not clear which of the five cloned Y receptors (Y1, Y2, Y4, Y5, and y6) are responsible for these effects.The Y2 receptor is expressed in the central and peripheral nervous system. High concentrations of Y2 receptors can be found on NPY-ergic neurons in the hypothalamic arcuate nucleus (9,10), where it is thought to act as an inhibitory autoreceptor that can regulate the expression and secretion of NPY and other neurotransmitters (11-13). These arcuate neurons are also known to express the leptin receptor (14) and are located in an area accessible to peripheral hormones (15), enabling modulation of hypothalamic circuits important in the maintenance of energy homeostasis.Previously, we have shown that germli...