Ninjin'yoeito modulates feeding and activity under negative energy balance conditions via the NPY system.

The central and peripheral neuropeptide Y (NPY) system is critically involved in feeding and energy homeostasis control. Disease conditions as well as aging can lead to reduced functionality of the NPY system and boosting it represents a promising option to improve health outcomes in these situations. Here we show that Ninjin-yoeito (NYT), a Japanese kampo medicine comprising twelve herbs, and known to be effective to treat anorexia and frailty, mediates part of its action via NPY/peptide YY (PYY) related pathways. Especially under negative energy homeostasis conditions NYT is able to promote feeding and reduces activity to conserve energy. These effects are in part mediated via signalling through the NPY system since lack of Y4 receptors or PYY leading to modification in these responses highlighting the possibility for combination treatment to improve aging related conditions on energy homeostasis control.

Ninjin'yoeito, a herbal medicine, enhances glucose tolerance in mice.

The prevalence of Type 2 diabetes increases under conditions of obesity but also due to aging. While a variety of treatment options are being explored there are still many unanswered questions about the underlying mechanisms for the aetiology and progression of this illness. Here we show that pre-treatment with Ninjin'yoeito (NYT), a herbal medicine composed of 12 different ingrediencies, before a glucose challenge results in significantly improved glucose tolerance. This occurs in the absence of significant alterations in insulin excursion compared to vehicle treatment, indicating NYT improves insulin responsiveness and/or insulin-independent glucose disposal. Furthermore, we identify Ginseng - one of the 12 ingredients of NYT - as one key component contributing to NYT's effect on glucose clearance. Importantly, lack of Y4 receptor signalling abolishes the positive effects of NYT on glucose tolerance suggesting Y4 receptor-controlled pathways are crucial in mediating this action of NYT. Using c-fos as neuronal activation marker, we show NYT activates the area postrema - a circumventricular organ in the brainstem that expresses high level of Y4 receptors, supporting an involvement of brainstem Y4 signalling in NYT-activated central networks. Together, these data suggest that NYT is a positive influencer of glucose metabolism in insulin-sensitive tissues and the mechanistic actions of NYT include brainstem Y4 circuitries.

Energy partitioning between fat and bone mass is controlled via a hypothalamic leptin/NPY relay.

BACKGROUND/OBJECTIVES: Maintaining energy balance is important to ensure a healthy organism. However, energy partitioning, coordinating the distribution of sufficient energy to different organs and tissues is equally important, but the control of this process is largely unknown. In obesity, an increase in fat mass necessitates the production of additional bone mass to cope with the increase in bodyweight and processes need to be in place to communicate this new weight bearing demand. Here, we investigate the interaction between leptin and NPY, two factors critically involved in the regulation of both energy metabolism and bone mass, in this process. METHODS: We assessed the co-localization of leptin receptors on NPY neurons using RNAScope followed by a systematic examination of body composition and energy metabolism profiling in male and female mice lacking leptin receptors specifically in NPY neurons (Leprlox/lox;NPYCre/+). The effect of short-term switching between chow and high-fat diet was also examined in these mice. RESULTS: We uncovered that leptin receptor expression is greater on a subpopulation of NPY neurons in the arcuate that do not express AgRP. We further show that Leprlox/lox;NPYCre/+ mice exhibit significantly increased adiposity while bone mass is diminished. These body composition changes occur in the absence of alterations in food intake or energy expenditure, demonstrating a prominent role for leptin signaling in NPY neurons in the control of energy partitioning. Importantly however, when fed a high-fat diet, these mice display a switch in energy partitioning whereby they exhibit a significantly enhanced ability to increase their bone mass to match the increased bodyweight caused by higher caloric intake concurrent with attenuated adiposity. CONCLUSIONS: Taken together, these results demonstrate that leptin signaling in NPY neurons is critical for coordinating energy partitioning between fat and bone mass especially during situations of changes in energy balance.

Lack of NPY in neurotensin neurons leads to a lean phenotype.

Neuropeptide Y (NPY) producing neurons in the arcuate nucleus (Arc) of the hypothalamus are essential to the regulation of food intake and energy homeostasis. Whilst they have classically been thought to co-express agouti-related peptide (AgRP), it is now clear that there is a sub-population of NPY neurons in the Arc that do not. Here, we show that a subset of AgRP-negative, NPY-positive neurons in the Arc also express neurotensin (NTS) and we use an NTS-Cre line to investigate the function of this sub-population of NPY neurons. The lack of NPY in NTS-positive neurons led to a marked reduction in fat mass and bodyweight as well as a significant reduction in food intake in male NPYlox/lox; NTScre/+ mice compared to controls. Despite the reduction in food intake, overall energy expenditure was similar between genotypes due to concomitant reduction in activity in NPYlox/lox; NTScre/+ mice. Furthermore, cortical bone mass was significantly reduced in NPYlox/lox;NTScre/+ mice with no evident alterations in the cancellous bone compartment, likely due to reduced leptin levels as a result of their reduced adiposity. Taken together, these data suggest that the sub-population of Arc NPY neurons expressing NTS are critical for regulating food intake, activity and fat mass but are not directly involved in the control of bone mass.

Amygdala NPY Circuits Promote the Development of Accelerated Obesity under Chronic Stress Conditions.

Neuropeptide Y (NPY) exerts a powerful orexigenic effect in the hypothalamus. However, extra-hypothalamic nuclei also produce NPY, but its influence on energy homeostasis is unclear. Here we uncover a previously unknown feeding stimulatory pathway that is activated under conditions of stress in combination with calorie-dense food; NPY neurons in the central amygdala are responsible for an exacerbated response to a combined stress and high-fat-diet intervention. Central amygdala NPY neuron-specific Npy overexpression mimics the obese phenotype seen in a combined stress and high-fat-diet model, which is prevented by the selective ablation of Npy. Using food intake and energy expenditure as readouts, we demonstrate that selective activation of central amygdala NPY neurons results in increased food intake and decreased energy expenditure. Mechanistically, it is the diminished insulin signaling capacity on central amygdala NPY neurons under combined stress and high-fat-diet conditions that leads to the exaggerated development of obesity.

GPR88 is a critical regulator of feeding and body composition in mice.

GPR88 is an orphan G-protein-coupled receptor with predominant expression in reward-related areas in the brain. While the lack of GPR88 has been demonstrated to induce behavioral deficits, the potential function of the receptor in the control of food intake and energy balance remains unexplored. In this work, the role of GPR88 in energy homeostasis was investigated in Gpr88 -/- mice fed either standard chow or high fat diet (HFD). Gpr88 -/- mice showed significantly reduced adiposity accompanied with suppressed spontaneous food intake, particularly pronounced under HFD treatment. While energy expenditure was likewise lower in Gpr88 -/- mice, body weight gain remained unchanged. Furthermore, deregulation in glucose tolerance and insulin responsiveness in response to HFD was attenuated in Gpr88 -/- mice. On the molecular level, distinct changes in the hypothalamic mRNA levels of cocaine-and amphetamine-regulated transcript (Cartpt), a neuropeptide involved in the control of feeding and reward, were observed in Gpr88 -/- mice. In addition, GPR88 deficiency was associated with altered expressions of the anorectic Pomc and the orexigenic Npy in the arcuate nucleus, especially under HFD condition. Together, our results indicate that GPR88 signalling is not only important for reward processes, but also plays a role in the central regulatory circuits for energy homeostasis.

Sucralose Promotes Food Intake through NPY and a Neuronal Fasting Response.

Non-nutritive sweeteners like sucralose are consumed by billions of people. While animal and human studies have demonstrated a link between synthetic sweetener consumption and metabolic dysregulation, the mechanisms responsible remain unknown. Here we use a diet supplemented with sucralose to investigate the long-term effects of sweet/energy imbalance. In flies, chronic sweet/energy imbalance promoted hyperactivity, insomnia, glucose intolerance, enhanced sweet taste perception, and a sustained increase in food and calories consumed, effects that are reversed upon sucralose removal. Mechanistically, this response was mapped to the ancient insulin, catecholamine, and NPF/NPY systems and the energy sensor AMPK, which together comprise a novel neuronal starvation response pathway. Interestingly, chronic sweet/energy imbalance promoted increased food intake in mammals as well, and this also occurs through an NPY-dependent mechanism. Together, our data show that chronic consumption of a sweet/energy imbalanced diet triggers a conserved neuronal fasting response and increases the motivation to eat.

Temperature dependence of the control of energy homeostasis requires CART signaling.

Cocaine- and amphetamine-regulated transcript (CART) is a key neuropeptide with predominant expression in the hypothalamus central to the regulation of diverse biological processes, including food intake and energy expenditure. While there is considerable information on CART's role in the control of feeding, little is known about its thermoregulatory potential. Here we show the consequences of lack of CART signaling on major parameters of energy homeostasis in CART-/- mice under standard ambient housing (RT, 22°C), which is considered a mild cold exposure for mice, and thermoneutral conditions (TN, 30°C). WT mice kept at RT showed an increase in food intake, energy expenditure, BAT UCP-1 expression, and physical activity compared with TN condition, reflecting the augmented energy demand for thermogenesis at RT. On the molecular level, RT housing led to upregulated mRNA expression of TH, CRH, and TRH at the PVN, while NPY, AgRP and CART mRNA levels in the Arc were downregulated. CART-/- mice displayed elevated adiposity and diminished lean mass across both RT and TN. At RT, CART-/- mice showed unchanged food consumption yet greater body weight gain. In addition, an increase in energy expenditure and heightened BAT thermogenesis marked by UCP-1 protein expression was observed in the CART-/- mice. In contrast, TN-housed CART-/- mice exhibited lower weight gain than WT mice accompanied with pronounced reduction in basal feeding. These findings were correlated with reduced BAT temperature, but unchanged energy expenditure and UCP-1 levels. Interestingly, the respiratory exchange ratio for CART-/- mice, which shifted from lower at RT to higher at TN with respect to WT controls, indicates a transition of relative fuel source preference from fat to carbohydrate in the absence of CART signaling. Taken together, these results demonstrate that CART is a critical regulator of energy expenditure, energy partitioning and utilization dependent on the thermal environment.

Snord116 is critical in the regulation of food intake and body weight.

Prader-Willi syndrome (PWS) is the predominant genetic cause of obesity in humans. Recent clinical reports have suggested that micro-deletion of the Snord116 gene cluster can lead to PWS, however, the extent of the contributions of the encoded snoRNAs is unknown. Here we show that mice lacking Snord116 globally have low birth weight, increased body weight gain, energy expenditure and hyperphagia. Consistent with this, microarray analysis of hypothalamic gene expression revealed a significant alteration in feeding related pathways that was also confirmed by in situ hybridisation. Importantly, selective deletion of Snord116 only from NPY expressing neurons mimics almost exactly the global deletion phenotype including the persistent low birth weight, increased body weight gain in early adulthood, increased energy expenditure and hyperphagia. Mechanistically, the lack of Snord116 in NPY neurons leads to the upregulation of NPY mRNA consistent with the hyperphagic phenotype and suggests a critical role of Snord116 in the control of NPY neuronal functions that might be dysregulated in PWS.

Intermittent Moderate Energy Restriction Improves Weight Loss Efficiency in Diet-Induced Obese Mice.

BACKGROUND: Intermittent severe energy restriction is popular for weight management. To investigate whether intermittent moderate energy restriction may improve this approach by enhancing weight loss efficiency, we conducted a study in mice, where energy intake can be controlled. METHODS: Male C57/Bl6 mice that had been rendered obese by an ad libitum diet high in fat and sugar for 22 weeks were then fed one of two energy-restricted normal chow diets for a 12-week weight loss phase. The continuous diet (CD) provided 82% of the energy intake of age-matched ad libitum chow-fed controls. The intermittent diet (ID) provided cycles of 82% of control intake for 5-6 consecutive days, and ad libitum intake for 1-3 days. Weight loss efficiency during this phase was calculated as (total weight change) ÷ [(total energy intake of mice on CD or ID)-(total average energy intake of controls)]. Subsets of mice then underwent a 3-week weight regain phase involving ad libitum re-feeding. RESULTS: Mice on the ID showed transient hyperphagia relative to controls during each 1-3-day ad libitum feeding period, and overall ate significantly more than CD mice (91.1±1.0 versus 82.2±0.5% of control intake respectively, n = 10, P<0.05). There were no significant differences between CD and ID groups at the end of the weight loss or weight regain phases with respect to body weight, fat mass, circulating glucose or insulin concentrations, or the insulin resistance index. Weight loss efficiency was significantly greater with ID than with CD (0.042±0.007 versus 0.018±0.001 g/kJ, n = 10, P<0.01). Mice on the CD exhibited significantly greater hypothalamic mRNA expression of proopiomelanocortin (POMC) relative to ID and control mice, with no differences in neuropeptide Y or agouti-related peptide mRNA expression between energy-restricted groups. CONCLUSION: Intermittent moderate energy restriction may offer an advantage over continuous moderate energy restriction, because it induces significantly greater weight loss relative to energy deficit in mice.

Regulation of energy homeostasis by the NPY system.

Obesity develops when energy intake exceeds energy expenditure over time. Numerous neurotransmitters, hormones, and factors have been implicated to coordinately control energy homeostasis, centrally and peripherally. However, the neuropeptide Y (NPY) system has emerged as the one with the most critical functions in this process. While NPY centrally promotes feeding and reduces energy expenditure, peptide YY (PYY) and pancreatic polypeptide (PP), the other family members, mediate satiety. Importantly, recent research has uncovered additional functions for these peptides that go beyond the simple feeding/satiety circuits and indicate a more extensive function in controlling energy homeostasis. In this review, we will discuss the actions of the NPY system in the regulation of energy balance, with a particular focus on energy expenditure.

Actions of NPY, and its Y1 and Y2 receptors on pulsatile growth hormone secretion during the fed and fasted state.

The hypothalamic NPY system plays an important role in regulating food intake and energy expenditure. Different biological actions of NPY are assigned to NPY receptor subtypes. Recent studies demonstrated a close relationship between food intake and growth hormone (GH) secretion; however, the mechanism through which endogenous NPY modulates GH release remains unknown. Moreover, conclusive evidence demonstrating a role for NPY and Y-receptors in regulating the endogenous pulsatile release of GH does not exist. We used genetically modified mice (germline Npy, Y1, and Y2 receptor knock-out mice) to assess pulsatile GH secretion under both fed and fasting conditions. Deletion of NPY did not impact fed GH release; however, it reversed the fasting-induced suppression of pulsatile GH secretion. The recovery of GH secretion was associated with a reduction in hypothalamic somatotropin release inhibiting factor (Srif; somatostatin) mRNA expression. Moreover, observations revealed a differential role for Y1 and Y2 receptors, wherein the postsynaptic Y1 receptor suppresses GH secretion in fasting. In contrast, the presynaptic Y2 receptor maintains normal GH output under long-term ad libitum-fed conditions. These data demonstrate an integrated neural circuit that modulates GH release relative to food intake, and provide essential information to address the differential roles of Y1 and Y2 receptors in regulating the release of GH under fed and fasting states.

The endogenous opioid dynorphin is required for normal bone homeostasis in mice.

Chronic opiate usage, whether prescribed or illicit, has been associated with changes in bone mass and is a recognized risk factor for the development of osteoporosis; however, the mechanism behind this effect is unknown. Here we show that lack of dynorphin, an endogenous opioid, in mice (Dyn-/-), resulted in a significantly elevated cancellous bone volume associated with greater mineral apposition rate and increased resorption indices. A similar anabolic phenotype was evident in bone of mice lacking dynorphin's cognate receptor, the kappa opioid receptor. Lack of opioid receptor expression in primary osteoblastic cultures and no change in bone cell function after dynorphin agonist treatment in vitro indicates an indirect mode of action. Consistent with a hypothalamic action, central dynorphin signaling induces extracellular signal-regulated kinase (ERK) phosphorylation and c-fos activation of neurons in the arcuate nucleus of the hypothalamus (Arc). Importantly, this signaling also leads to an increase in Arc NPY mRNA expression, a change known to decrease bone formation. Further implicating NPY in the skeletal effects of dynorphin, Dyn-/-/NPY-/- double mutant mice showed comparable increases in bone formation to single mutant mice, suggesting that dynorphin acts upstream of NPY signaling to control bone formation. Thus the dynorphin system, acting via NPY, may represent a pathway by which higher processes including stress, reward/addiction and depression influence skeletal metabolism. Moreover, understanding of these unique interactions may enable modulation of the adverse effects of exogenous opioid treatment without directly affecting analgesic responses.

Y1 and Y5 receptors are both required for the regulation of food intake and energy homeostasis in mice.

Neuropeptide Y (NPY) acting in the hypothalamus is one of the most powerful orexigenic agents known. Of the five known Y receptors, hypothalamic Y1 and Y5 have been most strongly implicated in mediating hyperphagic effects. However, knockout of individual Y1 or Y5 receptors induces late-onset obesity--and Y5 receptor knockout also induces hyperphagia, possibly due to redundancy in functions of these genes. Here we show that food intake in mice requires the combined actions of both Y1 and Y5 receptors. Germline Y1Y5 ablation in Y1Y5(-/-) mice results in hypophagia, an effect that is at least partially mediated by the hypothalamus, since mice with adult-onset Y1Y5 receptor dual ablation targeted to the paraventricular nucleus (PVN) of the hypothalamus (Y1Y5(Hyp/Hyp)) also exhibit reduced spontaneous or fasting-induced food intake when fed a high fat diet. Interestingly, despite hypophagia, mice with germline or hypothalamus-specific Y1Y5 deficiency exhibited increased body weight and/or increased adiposity, possibly due to compensatory responses to gene deletion, such as the decreased energy expenditure observed in male Y1Y5(-/-) animals relative to wildtype values. While Y1 and Y5 receptors expressed in other hypothalamic areas besides the PVN--such as the dorsomedial nucleus and the ventromedial hypothalamus--cannot be excluded from having a role in the regulation of food intake, these studies demonstrate the pivotal, combined role of both Y1 and Y5 receptors in the mediation of food intake.

Y2 and Y4 receptor signalling attenuates the skeletal response of central NPY.

Both the neuropeptide Y (NPY) and the leptin systems have been shown to be important central mediators of bone metabolism. However, the interaction between these two systems is complex and not fully understood. Here, we show that a unique interaction exists between Y2 and Y4 receptors in the regulation of bone homeostasis that is not evident when combined with lack of Y1 signalling. Despite the hypoleptinaemia shown in male Y2/Y4 double knockout (Y2⁻/⁻ Y4⁻/⁻) mice, when on the leptin-deficient ob/ob background, these mice display reduced cancellous bone mass. However, combined Y2/Y4 deletion enhances the effect of leptin deficiency on the cortical bone compartment. By replicating the enhanced central NPY expression evident in ob/ob mice using virally mediated overexpression of NPY in the hypothalamus of Y receptor knockout mice, we demonstrate that Y2⁻/⁻ Y4⁻/⁻ mice have an exaggerated response to the anti-osteogenic effects of elevated hypothalamic NPY in both cancellous and cortical bone and that this effect appears to be dependent on Y1 receptor signalling. This study highlights the complex interaction between Y receptors in the control of bone mass. Moreover, it suggests that the reduction in cortical bone observed in the absence of leptin is due to the anti-osteogenic effect of elevated hypothalamic NPY levels.

Neuropeptide Y and sex hormone interactions in humoral and neuronal regulation of bone and fat.

The hypothalamus regulates the skeleton and adipose tissue via endocrine mechanisms. Changes in sex steroid levels in menopause and aging are central to the associated changes in bone mass and adiposity. Whereas many of these effects occur via direct actions on osteoblasts or adipocytes, sex hormones can also mediate effects on bone and adipose tissue via interaction with neuronal pathways. A key hypothalamic regulator of bone and adipose tissue is neuropeptide Y (NPY), which coordinately influences these tissues via effects on neuroendocrine and sympathetic nervous output. Better understanding of the interaction between NPY and sex steroids in regulating skeletal and energy homeostasis could lead to more effective treatments for osteoporosis and obesity.

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

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.

Prodynorphin-derived peptides are critical modulators of anxiety and regulate neurochemistry and corticosterone.

Stress and anxiety are mainly regulated by amygdala and hypothalamic circuitries involving several neurotransmitter systems and providing physiological responses to peripheral organs via the hypothalamic-pituitary-adrenal axis and other pathways. The role of endogenous opioid peptides in this process is largely unknown. Here we show for the first time that anxiolytic parameters of explorative behavior in mice lacking prodynorphin were increased 2-4-fold in the open field, the elevated plus maze and the light-dark test. Consistent with this, treatment of wild-type mice with selective kappa-opioid receptor antagonists GNTI or norbinaltorphimine showed the same effects. Furthermore, treatment of prodynorphin knockout animals with U-50488H, a selective kappa-opioid receptor agonist, fully reversed their anxiolytic phenotype. These behavioral data are supported by an approximal 30% reduction in corticotropin-releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus and central amygdala and an accompanying 30-40% decrease in corticosterone serum levels in prodynorphin knockout mice. Although stress-induced increases in corticosterone levels were attenuated in prodynorphin knockout mice, they were associated with minor increases in depression-like behavior in the tail suspension and forced swim tests. Taken together, our data suggest a pronounced impact of endogenous prodynorphin-derived peptides on anxiety, but not stress coping ability and that these effects are mediated via kappa-opioid receptors. The delay in the behavioral response to kappa-opioid receptor agonists and antagonist treatment suggests an indirect control level for the action of dynorphin, probably by modulating the expression of CRH or neuropeptide Y, and subsequently influencing behavior.