A rare human variant that disrupts GPR10 signalling causes weight gain in mice.

Disruption of brain-expressed G protein-coupled receptor-10 (GPR10) causes obesity in animals. Here, we identify multiple rare variants in GPR10 in people with severe obesity and in normal weight controls. These variants impair ligand binding and G protein-dependent signalling in cells. Transgenic mice harbouring a loss of function GPR10 variant found in an individual with obesity, gain excessive weight due to decreased energy expenditure rather than increased food intake. This evidence supports a role for GPR10 in human energy homeostasis. Therapeutic targeting of GPR10 may represent an effective weight-loss strategy.

The hypothalamic RFamide, QRFP, increases feeding and locomotor activity: The role of Gpr103 and orexin receptors.

Here we show that central administration of pyroglutamylated arginine-phenylamine-amide peptide (QRFP/26RFa) increases both food intake and locomotor activity, without any significant effect on energy expenditure, thermogenesis or reward. Germline knock out of either of the mouse QRFP receptor orthologs, Gpr103a and Gpr103b, did not produce a metabolic phenotype. However, both receptors are required for the effect of centrally administered QRFP to increase feeding and locomotor activity. As central injection of QRFP activated orexin/hypocretin neurons in the lateral hypothalamus, we compared the action of QRFP and orexin on behaviour. Both peptides increased arousal and locomotor activity. However, while orexin increased consummatory behaviour, QRFP also affected other appetitive behaviours. Furthermore, the feeding but not the locomotor response to QRFP, was blocked by co-administration of an orexin receptor 1 antagonist. These results suggest that QRFP agonism induces both appetitive and consummatory behaviour, but only the latter is dependent on orexin/hypocretin receptor signalling.

Hypothalamic AgRP neurons exert top-down control on systemic TNF-α release during endotoxemia.

Loss of appetite and negative energy balance are common features of endotoxemia in all animals and are thought to have protective roles by reducing nutrient availability to host and pathogen metabolism. Accordingly, fasting and caloric restriction have well-established anti-inflammatory properties. However, in response to reduced nutrient availability at the cellular and organ levels, negative energy balance also recruits distinct energy-sensing brain circuits, but it is not known whether these neuronal systems have a role in its anti-inflammatory effects. Here, we report that hypothalamic AgRP neurons-a critical neuronal population for the central representation of negative energy balance-have parallel immunoregulatory functions. We found that when endotoxemia occurs in fasted mice, the activity of AgRP neurons remains sustained, but this activity does not influence feeding behavior and endotoxemic anorexia. Furthermore, we found that endotoxemia acutely desensitizes AgRP neurons, which also become refractory to inhibitory signals. Mimicking this sustained AgRP neuron activity in fed mice by chemogenetic activation-a manipulation known to recapitulate core behavioral features of fasting-results in reduced acute tumor necrosis factor alpha (TNF-α) release during endotoxemia. Mechanistically, we found that endogenous glucocorticoids play an important role: glucocorticoid receptor deletion from AgRP neurons prevents their endotoxemia-induced desensitization, and importantly, it counteracts the fasting-induced suppression of TNF-α release, resulting in prolonged sickness. Together, these findings provide evidence directly linking AgRP neuron activity to the acute response during endotoxemia, suggesting that these neurons are a functional component of the immunoregulatory effects associated with negative energy balance and catabolic metabolism.

Anorectic and aversive effects of GLP-1 receptor agonism are mediated by brainstem cholecystokinin neurons, and modulated by GIP receptor activation.

OBJECTIVE: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective medications to reduce appetite and body weight. These actions are centrally mediated; however, the neuronal substrates involved are poorly understood. METHODS: We employed a combination of neuroanatomical, genetic, and behavioral approaches in the mouse to investigate the involvement of caudal brainstem cholecystokinin-expressing neurons in the effect of the GLP-1RA exendin-4. We further confirmed key neuroanatomical findings in the non-human primate brain. RESULTS: We found that cholecystokinin-expressing neurons in the caudal brainstem are required for the anorectic and body weight-lowering effects of GLP-1RAs and for the induction of GLP-1RA-induced conditioned taste avoidance. We further show that, while cholecystokinin-expressing neurons are not a direct target for glucose-dependent insulinotropic peptide (GIP), GIP receptor activation results in a reduced recruitment of these GLP-1RA-responsive neurons and a selective reduction of conditioned taste avoidance. CONCLUSIONS: In addition to disclosing a neuronal population required for the full appetite- and body weight-lowering effect of GLP-1RAs, our data also provide a novel framework for understanding and ameliorating GLP-1RA-induced nausea - a major factor for withdrawal from treatment.

Do oxytocin neurones affect feeding?

There has been a long history of research on the effects of oxytocin on feeding behaviour. The classic-held view is that the neurohormone is anorexigenic at least in rodents, although the data for humans are not so clear cut. Likewise, a physiological role for oxytocin is disputed. Thus, although pharmacological, anatomical and physiological data suggest oxytocin may have a function in satiety signalling, this view is not supported by the latest research using the genetic recording and manipulation of oxytocin neurones. Here, we avoid a discussion of the pharmacological effects of oxytocin and examine evidence, from both sides of the argument, concerning whether the endogenous oxytocin system has a role in the regulation of normal feeding.

Ventromedial Nucleus of the Hypothalamus Neurons Under the Magnifying Glass.

The ventromedial nucleus of the hypothalamus (VMH) is a complex brain structure that is integral to many neuroendocrine functions, including glucose regulation, thermogenesis, and appetitive, social, and sexual behaviors. As such, it is of little surprise that the nucleus is under intensive investigation to decipher the mechanisms which underlie these diverse roles. Developments in genetic and investigative tools, for example the targeting of steroidogenic factor-1-expressing neurons, have allowed us to take a closer look at the VMH, its connections, and how it affects competing behaviors. In the current review, we aim to integrate recent findings into the literature and contemplate the conclusions that can be drawn.

Brain control of appetite during sickness.

Given the high-energy requirements to sustain immune responses and healing processes, it is intriguing that lack of appetite (i.e., anorexia) is a cardinal feature of sickness behaviour. While our understanding of the brain mechanisms that control appetite is rapidly growing, how inflammation affects these mechanisms is not fully understood. Here, we discuss advances in our understanding of discrete appetite controlling mechanisms and how inflammation influences their function. We further discuss the pathophysiological significance of anorexia and negative energy balance during the immune regulatory response. LINKED ARTICLES: This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc.

The cytokine GDF15 signals through a population of brainstem cholecystokinin neurons to mediate anorectic signalling.

The cytokine, GDF15, is produced in pathological states which cause cellular stress, including cancer. When over expressed, it causes dramatic weight reduction, suggesting a role in disease-related anorexia. Here, we demonstrate that the GDF15 receptor, GFRAL, is located in a subset of cholecystokinin neurons which span the area postrema and the nucleus of the tractus solitarius of the mouse. GDF15 activates GFRALAP/NTS neurons and supports conditioned taste and place aversions, while the anorexia it causes can be blocked by a monoclonal antibody directed at GFRAL or by disrupting CCK neuronal signalling. The cancer-therapeutic drug, cisplatin, induces the release of GDF15 and activates GFRALAP/NTS neurons, as well as causing significant reductions in food intake and body weight in mice. These metabolic effects of cisplatin are abolished by pre-treatment with the GFRAL monoclonal antibody. Our results suggest that GFRAL neutralising antibodies or antagonists may provide a co-treatment opportunity for patients undergoing chemotherapy.

Experimental Models of Impaired Hypoglycaemia-Associated Counter-Regulation.

Impaired awareness of hypoglycaemia (IAH) affects around a quarter of patients with diabetes who receive insulin treatment. This condition is characterised by a progressive reduction in symptomatic and behavioural responses to hypoglycaemia, increasing risk of deeper drops in blood glucose, unconsciousness, and collapse. Thus, patients with IAH experience severe hypoglycaemic episodes more frequently, resulting in significant morbidity and mortality. IAH is thought to develop as a consequence of whole-body adaptations to repeated insulin-induced hypoglycaemia (RH), with widespread deficits in the hypoglycaemia counter-regulatory response (CRR). Despite this important insight, the precise pathophysiology by which RH leads to an attenuated CRR is unknown. Studies into the underlying mechanisms of IAH have employed a variety of protocols in humans and experimental species. The use of animal models has many investigational benefits, including the unprecedented increase in the availability of transgenic strains. However, modelling impaired hypoglycaemia-associated counter-regulation remains challenging and appropriate interpretation of findings across species and protocols even more so. Here, we review the experimental modelling of IAH and impaired hypoglycaemia-associated counter-regulation, with a focus on understanding species-specific variation in glucose homeostasis. This review will aid investigators in interpreting outputs from different studies in IAH and aid progress in the field.

Modified Peptide YY Molecule Attenuates the Activity of NPY/AgRP Neurons and Reduces Food Intake in Male Mice.

To study the effects of an analog of the gut-produced hormone peptide YY (PYY3-36), which has increased selectivity for the Y2 receptor; specifically, to record its effects on food intake and on hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neuron activity. NNC0165-1273, a modified form of the peptide hormone PYY3-36 with potent selectivity at Y2 receptor (>5000-fold over Y1, 1250-fold over Y4, and 650-fold over Y5 receptor), was tested in vivo and in vitro in mouse models. NNC0165-1273 has fivefold lower relative affinity for Y2 compared with PYY3-36, but >250-, 192-, and 400-fold higher selectivity, respectively, for the Y1, Y4, and Y5 receptors. NNC0165-1273 produced a reduction in nighttime feeding at a dose at which PYY3-36 loses efficacy. The normal behavioral satiety sequence observed suggests that NNC0165-1273 is not nauseating and, instead, reduces food intake by producing early satiety. Additionally, NNC0165-1273 blocked ghrelin-induced cFos expression in NPY/AgRP neurons. In vitro electrophysiological recordings showed that, opposite to ghrelin, NNC0165-1273 hyperpolarized NPY/AgRP neurons and reduced action potential frequency. Administration of NNC0165-1273 via subcutaneous osmotic minipump caused a dose-dependent decrease in body weight and fat mass in an obese mouse model. Finally, NNC0165-1273 attenuated the feeding response when NPY/AgRP neurons were activated using ghrelin or more selectively with designer receptors. NNC0165-1273 is nonnauseating and stimulates a satiety response through, at least in part, a direct action on hypothalamic NPY/AgRP neurons. Modification of PYY3-36 to produce compounds with increased affinity to Y2 receptors may be useful as antiobesity therapies in humans.

PACAP Neurons in the Ventromedial Hypothalamic Nucleus Are Glucose Inhibited and Their Selective Activation Induces Hyperglycaemia.

Background: Glucose-sensing neurons are located in several parts of the brain, but are concentrated in the ventromedial nucleus of the hypothalamus (VMH). The importance of these VMH neurons in glucose homeostasis is well-established, however, little is known about their individual identity. In the present study, we identified a distinct glucose-sensing population in the VMH and explored its place in the glucose-regulatory network. Methods: Using patch-clamp electrophysiology on Pacap-cre::EYFP cells, we explored the glucose-sensing ability of the pituitary adenylate cyclase-activating peptide (PACAP) neurons both inside and outside the VMH. We also mapped the efferent projections of these neurons using anterograde and retrograde tracing techniques. Finally, to test the functionality of PACAPVMH in vivo, we used DREADD technology and measured systemic responses. Results: We demonstrate that PACAP neurons inside (PACAPVMH), but not outside the VMH are intrinsically glucose inhibited (GI). Anatomical tracing techniques show that PACAPVMH neurons project to several areas that can influence autonomic output. In vivo, chemogenetic stimulation of these neurons inhibits insulin secretion leading to reduced glucose tolerance, implicating their role in systemic glucose regulation. Conclusion: These findings are important as they identify, for the first time, a specific VMH neuronal population involved in glucose homeostasis. Identifying the different glucose-sensing populations in the VMH will help piece together the different arms of glucose regulation providing vital information regarding central responses to glucose metabolic disorders including hypoglycaemia.

Central administration of ghrelin induces conditioned avoidance in rodents.

Feelings of hunger carry a negative-valence (emotion) signal that appears to be conveyed through agouti-related peptide (AgRP) neurons in the hypothalamic arcuate nucleus. The circulating hunger hormone, ghrelin, activates these neurons although it remains unclear whether it also carries a negative-valence signal. Given that ghrelin also activates pathways in the midbrain that are important for reward, it remains possible that ghrelin could act as a positive reinforcer and hence, carry a positive-valence signal. Here we used condition preference/avoidance tests to explore the reinforcing/aversive properties of ghrelin, delivered by intracerebroventricular (ICV) injection (2µg/injection once a day for 4 days). We found that ICV ghrelin produces conditioned avoidance, both in a conditioned place preference/avoidance test (CPP/CPA, in which the animals avoid a chamber previously paired to ghrelin injection) and in a conditioned flavor preference/avoidance test (CFP/CFA, in which the animals consume/avoid a taste previously paired to ghrelin injection). These effects of ghrelin to induce a CPA were observed when conditioning to ghrelin occurred in the absence or presence of food. We did not find evidence, however, that brain ghrelin delivery to rats induces malaise (in the pica test). Our data indicate that ICV ghrelin carries a negative-valence signal consistent with its role as a circulating hunger hormone and with its effects to activate AgRP neurones.

Sequential Exposure to Obesogenic Factors in Females Rats: From Physiological Changes to Lipid Metabolism in Liver and Mesenteric Adipose Tissue.

During their lifetime, females are subjected to different nutritional and hormonal factors that could increase the risk of obesity and associated comorbidities. From early postnatal periods until the postmenopausal phase, exposure to over nutrition, high-energy diet and oestrogen deficiency, are considered as significant obesity risk factors in women. In this study, we assessed how key transitional life events and exposure to different nutrition influence energy homeostasis in a rat model. Specifically, we assessed the sequential exposure to postnatal over nutrition, high-fat diet (HFD) after weaning, followed later by ovariectomy (OVX; as a model of menopause). Each obesity risk factor increased significantly body weight (BW) and adiposity, with additive effects after sequential exposure. Increased energy intake in both HFD and/or OVX groups, and decreased locomotor activity and energy expenditure after OVX can explain these metabolic changes. Our study also documents decreased lipogenic pathway in mesenteric adipose tissue after HFD and/or OVX, independent of previous postnatal programming, yet only HFD evoked this effect in liver. In addition, we report an increase in the expression of the hepatic PEPCK depending on previous metabolic status. Overall, our results identify the impact of different risk factors, which will help in understanding the development of obesity in females.

The thermogenic effect of leptin is dependent on a distinct population of prolactin-releasing peptide neurons in the dorsomedial hypothalamus.

Leptin is a critical regulator of metabolism, which acts on brain receptors (Lepr) to reduce energy intake and increase energy expenditure. Some of the cellular pathways mediating leptin's anorectic actions are identified, but those mediating the thermogenic effects have proven more difficult to decipher. We define a population of neurons in the dorsomedial hypothalamic nucleus (DMH) containing the RFamide PrRP, which is activated by leptin. Disruption of Lepr selectively in these cells blocks thermogenic responses to leptin and causes obesity. A separate population of leptin-insensitive PrRP neurons in the brainstem is required, instead, for the satiating actions of the gut-derived hormone cholecystokinin (CCK). Global deletion of PrRP (in a loxSTOPlox-PrRP mouse) results in obesity and attenuated responses to leptin and CCK. Cre-recombinase-mediated reactivation of PrRP in brainstem rescues the anorectic actions of CCK, but reactivation in the hypothalamus is required to re-establish the thermogenic effect of leptin.

Central functional response to the novel peptide cannabinoid, hemopressin.

Hemopressin is the first peptide ligand to be described for the CB1 cannabinoid receptor. Hemopressin acts as an inverse agonist in vivo and can cross the blood-brain barrier to both inhibit appetite and induce antinociception. Despite being highly effective, synthetic CB1 inverse agonists are limited therapeutically due to unwanted, over dampening of central reward pathways. However, hemopressin appears to have its effect on appetite by affecting satiety rather than reward, suggesting an alternative mode of action which might avoid adverse side effects. Here, to resolve the neuronal circuitry mediating hemopressin's actions, we have combined blood-oxygen-level-dependent, pharmacological-challenge magnetic resonance imaging with c-Fos functional activity mapping to compare brain regions responsive to systemic administration of hemopressin and the synthetic CB1 inverse agonist, AM251. Using these complementary methods, we demonstrate that hemopressin activates distinct neuronal substrates within the brain, focused mainly on the feeding-related circuits of the mediobasal hypothalamus and in nociceptive regions of the periaqueductal grey (PAG) and dorsal raphe (DR). In contrast to AM251, there is a distinct lack of activation of the brain reward centres, such as the ventral tegmental area, nucleus accumbens and orbitofrontal cortex, which normally form a functional activity signature for the central action of synthetic CB1 receptor inverse agonists. Thus, hemopressin modulates the function of key feeding-related brain nuclei of the mediobasal hypothalamus, and descending pain pathways of the PAG and DR, and not higher limbic structures. Thus, hemopressin may offer behaviourally selective effects on nociception and appetite, without engaging reward pathways.

Physiological Roles of GPR10 and PrRP Signaling.

Prolactin-releasing peptide (PrRP) was first isolated from bovine hypothalamus, and was found to act as an endogenous ligand at the G-protein-coupled receptor 10 (GPR10 or hGR3). Although originally named as it can affect the secretion of prolactin from anterior pituitary cells, the potential functions for this peptide have been greatly expanded over the past decade. Anatomical, pharmacological, and physiological studies indicate that PrRP, signaling via the GPR10 receptor, may have a wide range of roles in neuroendocrinology; such as in energy homeostasis, stress responses, cardiovascular regulation, and circadian function. This review will provide the current knowledge of the PrRP and GPR10 signaling system, its putative functions, implications for therapy, and future perspectives.

Hypermetabolism in a triple-transgenic mouse model of Alzheimer's disease.

A common feature of Alzheimer's disease (AD) is weight loss, even though there is often an increase in food intake in AD patients. The reasons for this weight loss are unknown, but may be due to increased energy expenditure (metabolic rate) or a reduction in energy intake. This was investigated in the present study, using a triple-transgenic (3xTgAD) mouse model of AD. Two-month-old 3xTgAD mice displayed greater food intake (17%) and body weight (34%) but no difference in metabolic rate, as compared with nontransgenic controls (non-Tg). At 12 months of age, 3xTgAD mice still consumed more food (30%), but their body weight was significantly lower (15%) than non-Tg controls. This reduction in body weight was accompanied by a significant rise in metabolic rate, indicated by greater oxygen consumption (24%) and carbon dioxide production (29%); the effects were also observed in 18-month-old 3xTgAD mice. These data demonstrate for the first time the existence of a hypermetabolic state in an experimental model of AD, but whether this can explain the weight loss observed in AD patients remains to be determined.

A role for the melatonin-related receptor GPR50 in leptin signaling, adaptive thermogenesis, and torpor.

The ability of mammals to maintain a constant body temperature has proven to be a profound evolutionary advantage, allowing members of this class to thrive in most environments on earth. Intriguingly, some mammals employ bouts of deep hypothermia (torpor) to cope with reduced food supply and harsh climates [1, 2]. During torpor, physiological processes such as respiration, cardiac function, and metabolic rate are severely depressed, yet the neural mechanisms that regulate torpor remain unclear [3]. Hypothalamic responses to energy signals, such as leptin, influence the expression of torpor [4-7]. We show that the orphan receptor GPR50 plays an important role in adaptive thermogenesis and torpor. Unlike wild-type mice, Gpr50(-/-) mice readily enter torpor in response to fasting and 2-deoxyglucose administration. Decreased thermogenesis in Gpr50(-/-) mice is not due to a deficit in brown adipose tissue, the principal site of nonshivering thermogenesis in mice [8]. GPR50 is highly expressed in the hypothalamus of several species, including man [9, 10]. In line with this, altered thermoregulation in Gpr50(-/-) mice is associated with attenuated responses to leptin and a suppression of thyrotropin-releasing hormone. Thus, our findings identify hypothalamic circuits involved in torpor and reveal GPR50 to be a novel component of adaptive thermogenesis in mammals.

The peptide hemopressin acts through CB1 cannabinoid receptors to reduce food intake in rats and mice.

Hemopressin is a short, nine amino acid peptide (H-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH) isolated from rat brain that behaves as an inverse agonist at the cannabinoid receptor CB(1), and is shown here to inhibit agonist-induced receptor internalization in a heterologous cell model. Since this peptide occurs naturally in the rodent brain, we determined its effect on appetite, an established central target of cannabinoid signaling. Hemopressin dose-dependently decreases night-time food intake in normal male rats and mice, as well as in obese ob/ob male mice, when administered centrally or systemically, without causing any obvious adverse side effects. The normal, behavioral satiety sequence is maintained in male mice fasted overnight, though refeeding is attenuated. The anorectic effect is absent in CB(1) receptor null mutant male mice, and hemopressin can block CB(1) agonist-induced hyperphagia in male rats, providing strong evidence for antagonism of the CB(1) receptor in vivo. We speculate that hemopressin may act as an endogenous functional antagonist at CB(1) receptors and modulate the activity of appetite pathways in the brain.

Functional magnetic resonance imaging and c-Fos mapping in rats following a glucoprivic dose of 2-deoxy-D-glucose.

The glucose analogue, 2-deoxy-D-glucose (2-DG) is an inhibitor of glycolysis and, when administered systemically or centrally, induces glucoprivation leading to counter-regulatory responses, including increased feeding behaviour. Investigations into how the brain responds to glucoprivation could have important therapeutic potential, as disruptions or defects in the defence of the brain's 'glucostatic' circuitry may be partly responsible for pathological conditions resulting from diabetes and obesity. To define the 'glucostat' brain circuitry further we have combined blood-oxygen-level-dependent pharmacological-challenge magnetic resonance imaging (phMRI) with whole-brain c-Fos functional activity mapping to characterise brain regions responsive to an orexigenic dose of 2-DG [200 mg/kg; subcutaneous (s.c.)]. For phMRI, rats were imaged using a T(2)*-weighted gradient echo in a 7T magnet for 60 min under alpha-chloralose anaesthesia, whereas animals for immunohistochemistry were unanaesthetised and freely behaving. These complementary methods demonstrated functional brain activity in a number of previously characterised glucose-sensing brain regions such as those in the hypothalamus and brainstem following administration of 2-DG compared with vehicle. As the study mapped whole-brain functional responses, it also identified the orbitofrontal cortex and striatum (nucleus accumbens and ventral pallidum) as novel 2-DG-responsive brain regions. These regions make up a corticostriatal connection with the hypothalamus, by which aspects of motivation, salience and reward can impinge on the hypothalamic control of feeding behaviour. This study, therefore, provides further evidence for a common integrated circuit involved in the induction of feeding behaviour, and illustrates the valuable potential of phMRI in investigating central pharmacological actions.