Metabolic sensing in AgRP regulates sucrose preference and dopamine release in the nucleus accumbens.

Hunger increases the motivation for calorie consumption, often at the expense of low-taste appeal. However, the neural mechanisms integrating calorie-sensing with increased motivation for calorie consumption remain unknown. Agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus sense hunger, and the ingestion of caloric solutions promotes dopamine release in the absence of sweet taste perception. Therefore, we hypothesised that metabolic-sensing of hunger by AgRP neurons would be essential to promote dopamine release in the nucleus accumbens in response to caloric, but not non-caloric solutions. Moreover, we examined whether metabolic sensing in AgRP neurons affected taste preference for bitter solutions under conditions of energy need. Here we show that impaired metabolic sensing in AgRP neurons attenuated nucleus accumbens dopamine release in response to sucrose, but not saccharin, consumption. Furthermore, metabolic sensing in AgRP neurons was essential to distinguish nucleus accumbens dopamine response to sucrose consumption when compared with saccharin. Under conditions of hunger, metabolic sensing in AgRP neurons increased the preference for sucrose solutions laced with the bitter tastant, quinine, to ensure calorie consumption, whereas mice with impaired metabolic sensing in AgRP neurons maintained a strong aversion to sucrose/quinine solutions despite ongoing hunger. In conclusion, we demonstrate normal metabolic sensing in AgRP neurons drives the preference for calorie consumption, primarily when needed, by engaging dopamine release in the nucleus accumbens.

Ghrelin signalling in AgRP neurons links metabolic state to the sensory regulation of AgRP neural activity.

OBJECTIVE: The sensory detection of food and food cues suppresses Agouti related peptide (AgRP) neuronal activity prior to consumption with greatest suppression occurring in response to highly caloric food or interoceptive energy need. However, the interoceptive mechanisms priming an appropriate AgRP neural response to external sensory information of food availability remain unexplored. Since hunger increases plasma ghrelin, we hypothesized that ghrelin receptor (GHSR) signalling on AgRP neurons is a key interoceptive mechanism integrating energy need with external sensory cues predicting caloric availability. METHODS: We used in vivo photometry to measure the effects of ghrelin administration or fasting on AgRP neural activity with GCaMP6s and dopamine release in the nucleus accumbens with GRAB-DA in mice lacking ghrelin receptors in AgRP neurons. RESULTS: The deletion of GHSR on AgRP neurons prevented ghrelin-induced food intake, motivation and AgRP activity. The presentation of food (peanut butter pellet) or a wooden dowel suppressed AgRP activity in fasted WT but not mice lacking GHSRs in AgRP neurons. Similarly, peanut butter and a wooden dowel increased dopamine release in the nucleus accumbens after ip ghrelin injection in WT but not mice lacking GHSRs in AgRP neurons. No difference in dopamine release was observed in fasted mice. Finally, ip ghrelin administration did not directly increase dopamine neural activity in the ventral tegmental area. CONCLUSIONS: Our results suggest that AgRP GHSRs integrate an interoceptive state of energy need with external sensory information to produce an optimal change in AgRP neural activity. Thus, ghrelin signalling on AgRP neurons is more than just a feedback signal to increase AgRP activity during hunger.

Acute inhibition of hunger-sensing AgRP neurons promotes context-specific learning in mice.

OBJECTIVE: An environmental context, which reliably predicts food availability, can increase the appetitive food drive within the same environment context. However, hunger is required for the development of such a context-induced feeding (CIF) response, suggesting the neural circuits sensitive to hunger link an internal energy state with a particular environment context. Since Agouti related peptide (AgRP) neurons are activated by energy deficit, we hypothesised that AgRP neurons are both necessary and sufficient to drive CIF. METHODS: To examine the role of AgRP neurons in the CIF process, we used fibre photometry with GCaMP7f, chemogenetic activation of AgRP neurons, as well as optogenetic control of AgRP neurons to facilitate acute temporal control not permitted with chemogenetics. RESULTS: A CIF response at test was only observed when mice were fasted during context training and AgRP population activity at test showed an attenuated inhibitory response to food, suggesting increased food-seeking and/or decreased satiety signalling drives the increased feeding response at test. Intriguingly, chemogenetic activation of AgRP neurons during context training did not increase CIF, suggesting precise temporal firing properties may be required. Indeed, termination of AgRP neuronal photostimulation during context training (ON-OFF in context), in the presence or absence of food, increased CIF. Moreover, photoinhibition of AgRP neurons during context training in fasted mice was sufficient to drive a subsequent CIF in the absence of food. CONCLUSIONS: Our results suggest that AgRP neurons regulate the acquisition of CIF when the acute inhibition of AgRP activity is temporally matched to context exposure. These results establish acute AgRP inhibition as a salient neural event underscoring the effect of hunger on associative learning.

Identification of a Stress-Sensitive Anorexigenic Neurocircuit From Medial Prefrontal Cortex to Lateral Hypothalamus.

BACKGROUND: A greater understanding of how the brain controls appetite is fundamental to developing new approaches for treating diseases characterized by dysfunctional feeding behavior, such as obesity and anorexia nervosa. METHODS: By modeling neural network dynamics related to homeostatic state and body mass index, we identified a novel pathway projecting from the medial prefrontal cortex (mPFC) to the lateral hypothalamus (LH) in humans (n = 53). We then assessed the physiological role and dissected the function of this mPFC-LH circuit in mice. RESULTS: In vivo recordings of population calcium activity revealed that this glutamatergic mPFC-LH pathway is activated in response to acute stressors and inhibited during food consumption, suggesting a role in stress-related control over food intake. Consistent with this role, inhibition of this circuit increased feeding and sucrose seeking during mild stressors, but not under nonstressful conditions. Finally, chemogenetic or optogenetic activation of the mPFC-LH pathway is sufficient to suppress food intake and sucrose seeking in mice. CONCLUSIONS: These studies identify a glutamatergic mPFC-LH circuit as a novel stress-sensitive anorexigenic neural pathway involved in the cortical control of food intake.

In Vivo Photometry Reveals Insulin and 2-Deoxyglucose Maintain Prolonged Inhibition of VMH Vglut2 Neurons in Male Mice.

The ventromedial hypothalamic (VMH) nucleus is a well-established hub for energy and glucose homeostasis. In particular, VMH neurons are thought to be important for initiating the counterregulatory response to hypoglycemia, and ex vivo electrophysiology and immunohistochemistry data indicate a clear role for VMH neurons in sensing glucose concentration. However, the temporal response of VMH neurons to physiologically relevant changes in glucose availability in vivo has been hampered by a lack of available tools for measuring neuronal activity over time. Since the majority of neurons within the VMH are glutamatergic and can be targeted using the vesicular glutamate transporter Vglut2, we expressed cre-dependent GCaMP7s in Vglut2 cre mice and examined the response profile of VMH to intraperitoneal injections of glucose, insulin, and 2-deoxyglucose (2DG). We show that reduced available glucose via insulin-induced hypoglycemia and 2DG-induced glucoprivation, but not hyperglycemia induced by glucose injection, inhibits VMH Vglut2 neuronal population activity in vivo. Surprisingly, this inhibition was maintained for at least 45 minutes despite prolonged hypoglycemia and initiation of a counterregulatory response. Thus, although VMH stimulation, via pharmacological, electrical, or optogenetic approaches, is sufficient to drive a counterregulatory response, our data suggest VMH Vglut2 neurons are not the main drivers required to do so, since VMH Vglut2 neuronal population activity remains suppressed during hypoglycemia and glucoprivation.

Metabolic sensing in AgRP neurons integrates homeostatic state with dopamine signalling in the striatum.

Agouti-related peptide (AgRP) neurons increase motivation for food, however, whether metabolic sensing of homeostatic state in AgRP neurons potentiates motivation by interacting with dopamine reward systems is unexplored. As a model of impaired metabolic-sensing, we used the AgRP-specific deletion of carnitine acetyltransferase (Crat) in mice. We hypothesised that metabolic sensing in AgRP neurons is required to increase motivation for food reward by modulating accumbal or striatal dopamine release. Studies confirmed that Crat deletion in AgRP neurons (KO) impaired ex vivo glucose-sensing, as well as in vivo responses to peripheral glucose injection or repeated palatable food presentation and consumption. Impaired metabolic-sensing in AgPP neurons reduced acute dopamine release (seconds) to palatable food consumption and during operant responding, as assessed by GRAB-DA photometry in the nucleus accumbens, but not the dorsal striatum. Impaired metabolic-sensing in AgRP neurons suppressed radiolabelled 18F-fDOPA accumulation after ~30 min in the dorsal striatum but not the nucleus accumbens. Impaired metabolic sensing in AgRP neurons suppressed motivated operant responding for sucrose rewards during fasting. Thus, metabolic-sensing in AgRP neurons is required for the appropriate temporal integration and transmission of homeostatic hunger-sensing to dopamine signalling in the striatum.

An open-source device for measuring food intake and operant behavior in rodent home-cages.

Feeding is critical for survival, and disruption in the mechanisms that govern food intake underlies disorders such as obesity and anorexia nervosa. It is important to understand both food intake and food motivation to reveal mechanisms underlying feeding disorders. Operant behavioral testing can be used to measure the motivational component to feeding, but most food intake monitoring systems do not measure operant behavior. Here, we present a new solution for monitoring both food intake and motivation in rodent home-cages: the Feeding Experimentation Device version 3 (FED3). FED3 measures food intake and operant behavior in rodent home-cages, enabling longitudinal studies of feeding behavior with minimal experimenter intervention. It has a programmable output for synchronizing behavior with optogenetic stimulation or neural recordings. Finally, FED3 design files are open-source and freely available, allowing researchers to modify FED3 to suit their needs.

Obesity and anorexia nervosa are two health conditions related to food intake. Researchers studying these disorders in animal models need to both measure food intake and assess behavioural factors: that is, why animals seek and consume food. Measuring an animal's food intake is usually done by weighing food containers. However, this can be inaccurate due to the small amount of food that rodents eat. As for studying feeding motivation, this can involve calculating the number of times an animal presses a lever to receive a food pellet. These tests are typically conducted in hour-long sessions in temporary testing cages, called operant boxes. Yet, these tests only measure a brief period of a rodent's life. In addition, it takes rodents time to adjust to these foreign environments, which can introduce stress and may alter their feeding behaviour. To address this, Matikainen-Ankney, Earnest, Ali et al. developed a device for monitoring food intake and feeding behaviours around the clock in rodent home cages with minimal experimenter intervention. This 'Feeding Experimentation Device' (FED3) features a pellet dispenser and two 'nose-poke' sensors to measure total food intake, as well as motivation for and learning about food rewards. The battery-powered, wire-free device fits in standard home cages, enabling long-term studies of feeding behaviour with minimal intervention from investigators and less stress on the animals. This means researchers can relate data to circadian rhythms and meal patterns, as Matikainen-Ankney did here. Moreover, the device software is open-source so researchers can customise it to suit their experimental needs. It can also be programmed to synchronise with other instruments used in animal experiments, or across labs running the same behavioural tasks for multi-site studies. Used in this way, it could help improve reproducibility and reliability of results from such studies. In summary, Matikainen-Ankney et al. have presented a new practical solution for studying food-related behaviours in mice and rats. Not only could the device be useful to researchers, it may also be suitable to use in educational settings such as teaching labs and classrooms.

Unacylated-Ghrelin Impairs Hippocampal Neurogenesis and Memory in Mice and Is Altered in Parkinson's Dementia in Humans.

Blood-borne factors regulate adult hippocampal neurogenesis and cognition in mammals. We report that elevating circulating unacylated-ghrelin (UAG), using both pharmacological and genetic methods, reduced hippocampal neurogenesis and plasticity in mice. Spatial memory impairments observed in ghrelin-O-acyl transferase-null (GOAT-/-) mice that lack acyl-ghrelin (AG) but have high levels of UAG were rescued by acyl-ghrelin. Acyl-ghrelin-mediated neurogenesis in vitro was dependent on non-cell-autonomous BDNF signaling that was inhibited by UAG. These findings suggest that post-translational acylation of ghrelin is important to neurogenesis and memory in mice. To determine relevance in humans, we analyzed circulating AG:UAG in Parkinson disease (PD) patients diagnosed with dementia (PDD), cognitively intact PD patients, and controls. Notably, plasma AG:UAG was only reduced in PDD. Hippocampal ghrelin-receptor expression remained unchanged; however, GOAT+ cell number was reduced in PDD. We identify UAG as a regulator of hippocampal-dependent plasticity and spatial memory and AG:UAG as a putative circulating diagnostic biomarker of dementia.

Carnitine acetyltransferase (Crat) in hunger-sensing AgRP neurons permits adaptation to calorie restriction.

Hunger-sensing agouti-related peptide (AgRP) neurons ensure survival by adapting metabolism and behavior to low caloric environments. This adaption is accomplished by consolidating food intake, suppressing energy expenditure, and maximizing fat storage (nutrient partitioning) for energy preservation. The intracellular mechanisms responsible are unknown. Here we report that AgRP carnitine acetyltransferase (Crat) knockout (KO) mice exhibited increased fatty acid utilization and greater fat loss after 9 d of calorie restriction (CR). No differences were seen in mice with ad libitum food intake. Eleven days ad libitum feeding after CR resulted in greater food intake, rebound weight gain, and adiposity in AgRP Crat KO mice compared with wild-type controls, as KO mice act to restore pre-CR fat mass. Collectively, this study highlights the importance of Crat in AgRP neurons to regulate nutrient partitioning and fat mass during chronically reduced caloric intake. The increased food intake, body weight gain, and adiposity in KO mice after CR also highlights the detrimental and persistent metabolic consequence of impaired substrate utilization associated with CR. This finding may have significant implications for postdieting weight management in patients with metabolic diseases.-Reichenbach, A., Stark, R., Mequinion, M., Lockie, S. H., Lemus, M. B., Mynatt, R. L., Luquet, S., Andrews, Z. B. Carnitine acetyltransferase (Crat) in hunger-sensing AgRP neurons permits adaptation to calorie restriction.

Carnitine Acetyltransferase in AgRP Neurons Is Required for the Homeostatic Adaptation to Restricted Feeding in Male Mice.

Behavioral adaptation to periods of varying food availability is crucial for survival, and agouti-related protein (AgRP) neurons have been associated with entrainment to temporal restricted feeding. We have shown that carnitine acetyltransferase (Crat) in AgRP neurons enables metabolic flexibility and appropriate nutrient partitioning. In this study, by restricting food availability to 3 h/d during the light phase, we examined whether Crat is a component of a food-entrainable oscillator (FEO) that helps link behavior to food availability. AgRP Crat knockout (KO) mice consumed less food and regained less body weight but maintained blood glucose levels during the 25-day restricted feeding protocol. Importantly, we observed no difference in meal latency, food anticipatory activity (FAA), or brown adipose tissue temperature during the first 13 days of restricted feeding. However, as the restricted feeding paradigm progressed, we noticed an increased FAA in AgRP Crat KO mice. The delayed increase in FAA, which developed during the last 12 days of restricted feeding, corresponded with elevated plasma levels of corticosterone and nonesterified fatty acids, indicating it resulted from greater energy debt incurred by KO mice over the course of the experiment. These experiments highlight the importance of Crat in AgRP neurons in regulating feeding behavior and body weight gain during restricted feeding but not in synchronizing behavior to food availability. Thus, Crat within AgRP neurons forms a component of the homeostatic response to restricted feeding but is not likely to be a molecular component of FEO.

AgRP Neurons Require Carnitine Acetyltransferase to Regulate Metabolic Flexibility and Peripheral Nutrient Partitioning.

AgRP neurons control peripheral substrate utilization and nutrient partitioning during conditions of energy deficit and nutrient replenishment, although the molecular mechanism is unknown. We examined whether carnitine acetyltransferase (Crat) in AgRP neurons affects peripheral nutrient partitioning. Crat deletion in AgRP neurons reduced food intake and feeding behavior and increased glycerol supply to the liver during fasting, as a gluconeogenic substrate, which was mediated by changes to sympathetic output and peripheral fatty acid metabolism in the liver. Crat deletion in AgRP neurons increased peripheral fatty acid substrate utilization and attenuated the switch to glucose utilization after refeeding, indicating altered nutrient partitioning. Proteomic analysis in AgRP neurons shows that Crat regulates protein acetylation and metabolic processing. Collectively, our studies highlight that AgRP neurons require Crat to provide the metabolic flexibility to optimize nutrient partitioning and regulate peripheral substrate utilization, particularly during fasting and refeeding.

Des-Acyl Ghrelin and Ghrelin O-Acyltransferase Regulate Hypothalamic-Pituitary-Adrenal Axis Activation and Anxiety in Response to Acute Stress.

Ghrelin exists in two forms in circulation, acyl ghrelin and des-acyl ghrelin, both of which have distinct and fundamental roles in a variety of physiological functions. Despite this fact, a large proportion of papers simply measure and refer to plasma ghrelin without specifying the acylation status. It is therefore critical to assess and state the acylation status of plasma ghrelin in all studies. In this study we tested the effect of des-acyl ghrelin administration on the hypothalamic-pituitary-adrenal axis and on anxiety-like behavior of mice lacking endogenous ghrelin and in ghrelin-O-acyltransferase (GOAT) knockout (KO) mice that have no endogenous acyl ghrelin and high endogenous des-acyl ghrelin. Our results show des-acyl ghrelin produces an anxiogenic effect under nonstressed conditions, but this switches to an anxiolytic effect under stress. Des-acyl ghrelin influences plasma corticosterone under both nonstressed and stressed conditions, although c-fos activation in the paraventricular nucleus of the hypothalamus is not different. By contrast, GOAT KO are anxious under both nonstressed and stressed conditions, although this is not due to corticosterone release from the adrenals but rather from impaired feedback actions in the paraventricular nucleus of the hypothalamus, as assessed by c-fos activation. These results reveal des-acyl ghrelin treatment and GOAT deletion have differential effects on the hypothalamic-pituitary-adrenal axis and anxiety-like behavior, suggesting that anxiety-like behavior in GOAT KO mice is not due to high plasma des-acyl ghrelin.

Central Administration of the Ciliary Neurotrophic Factor Analogue, Axokine, Does Not Play a Role in Long-Term Energy Homeostasis in Adult Mice.

BACKGROUND/AIMS: Ciliary neurotrophic factor (CNTF) exerts powerful anorectic effects and has been suggested to regulate long-term energy balance by inducing adult neurogenesis in the arcuate nucleus of the hypothalamus. METHODS: The CNTF analogue, Axokine, was infused into the lateral ventricle of high-fat-fed mice for 1 week. Food intake, energy expenditure, body mass, glucose metabolism, and neurogenesis in the arcuate nucleus (ARC) of the hypothalamus were assessed 3 weeks after cessation of Axokine treatment. RESULTS: Short-term administration of Axokine induced an anorexic response but did not promote sustained weight loss. Instead, a rapid rebound in food intake and body mass occurred immediately after cessation of Axokine treatment, and this tended to reduce insulin sensitivity. Immunolabeling of 5-bromo-2'-deoxyuridine revealed limited neurogenesis in the ARC 3 weeks after Axokine treatment. CONCLUSION: These findings suggest that Axokine/CNTF does not induce substantial or sustained ARC neurogenesis or contribute to the long-term regulation of energy balance in mice.

Short-term calorie restriction enhances adult hippocampal neurogenesis and remote fear memory in a Ghsr-dependent manner.

The beneficial effects of calorie restriction (CR) have been described at both organismal and cellular levels in multiple organs. However, our understanding of the causal mediators of such hormesis is poorly understood, particularly in the context of higher brain function. Here, we show that the receptor for the orexigenic hormone acyl-ghrelin, the growth hormone secretagogue receptor (Ghsr), is enriched in the neurogenic niche of the hippocampal dentate gyrus (DG). Acute elevation of acyl-ghrelin levels by injection or by overnight CR, increased DG levels of the neurogenic transcription factor, Egr-1. Two weeks of CR increased the subsequent number of mature newborn neurons in the DG of adult wild-type but not Ghsr(-/-) mice. CR wild-type mice also showed improved remote contextual fear memory. Our findings suggest that Ghsr mediates the beneficial effects of CR on enhancing adult hippocampal neurogenesis and memory.

Hypothalamic carnitine metabolism integrates nutrient and hormonal feedback to regulate energy homeostasis.

The maintenance of energy homeostasis requires the hypothalamic integration of nutrient feedback cues, such as glucose, fatty acids, amino acids, and metabolic hormones such as insulin, leptin and ghrelin. Although hypothalamic neurons are critical to maintain energy homeostasis research efforts have focused on feedback mechanisms in isolation, such as glucose alone, fatty acids alone or single hormones. However this seems rather too simplistic considering the range of nutrient and endocrine changes associated with different metabolic states, such as starvation (negative energy balance) or diet-induced obesity (positive energy balance). In order to understand how neurons integrate multiple nutrient or hormonal signals, we need to identify and examine potential intracellular convergence points or common molecular targets that have the ability to sense glucose, fatty acids, amino acids and hormones. In this review, we focus on the role of carnitine metabolism in neurons regulating energy homeostasis. Hypothalamic carnitine metabolism represents a novel means for neurons to facilitate and control both nutrient and hormonal feedback. In terms of nutrient regulation, carnitine metabolism regulates hypothalamic fatty acid sensing through the actions of CPT1 and has an underappreciated role in glucose sensing since carnitine metabolism also buffers mitochondrial matrix levels of acetyl-CoA, an allosteric inhibitor of pyruvate dehydrogenase and hence glucose metabolism. Studies also show that hypothalamic CPT1 activity also controls hormonal feedback. We hypothesis that hypothalamic carnitine metabolism represents a key molecular target that can concurrently integrate nutrient and hormonal information, which is critical to maintain energy homeostasis. We also suggest this is relevant to broader neuroendocrine research as it predicts that hormonal signaling in the brain varies depending on current nutrient status. Indeed, the metabolic action of ghrelin, leptin or insulin at POMC or NPY neurons may depend on appropriate nutrient-sensing in these neurons and we hypothesize carnitine metabolism is critical in the integrative processing. Future research is required to examine the neuron-specific effects of carnitine metabolism on concurrent nutrient- and hormonal-sensing in AgRP and POMC neurons.

A stereological analysis of NPY, POMC, Orexin, GFAP astrocyte, and Iba1 microglia cell number and volume in diet-induced obese male mice.

The hypothalamic arcuate nucleus (ARC) contains 2 key neural populations, neuropeptide Y (NPY) and proopiomelanocortin (POMC), and, together with orexin neurons in the lateral hypothalamus, plays an integral role in energy homeostasis. However, no studies have examined total neuronal number and volume after high-fat diet (HFD) exposure using sophisticated stereology. We used design-based stereology to estimate NPY and POMC neuronal number and volume, as well as glial fibrillary acidic protein (astrocyte marker) and ionized calcium-binding adapter molecule 1 (microglia marker) cell number in the ARC; as well as orexin neurons in the lateral hypothalamus. Stereological analysis indicated approximately 8000 NPY and approximately 9000 POMC neurons in the ARC, and approximately 7500 orexin neurons in the lateral hypothalamus. HFD exposure did not affect total neuronal number in any population. However, HFD significantly increased average NPY cell volume and affected NPY and POMC cell volume distribution. HFD reduced orexin cell volume but had a bimodal effect on volume distribution with increased cells at relatively small volumes and decreased cells with relatively large volumes. ARC glial fibrillary acidic protein cells increased after 2 months on a HFD, although no significant difference after 6 months on chow diet or HFD was observed. No differences in ARC ionized calcium-binding adapter molecule 1 cell number were observed in any group. Thus, HFD affects ARC NPY or POMC neuronal cell volume number not cell number. Our results demonstrate the importance of stereology to perform robust unbiased analysis of cell number and volume. These data should be an empirical baseline reference to which future studies are compared.

Ghrelin-related peptides exert protective effects in the cerebral circulation of male mice through a nonclassical ghrelin receptor(s).

The ghrelin-related peptides, acylated ghrelin, des-acylated ghrelin, and obestatin, are novel gastrointestinal hormones. We firstly investigated whether the ghrelin gene, ghrelin O-acyltransferase, and the ghrelin receptor (GH secretagogue receptor 1a [GHSR1a]) are expressed in mouse cerebral arteries. Secondly, we assessed the cerebrovascular actions of ghrelin-related peptides by examining their effects on vasodilator nitric oxide (NO) and superoxide production. Using RT-PCR, we found the ghrelin gene and ghrelin O-acyltransferase to be expressed at negligible levels in cerebral arteries from male wild-type mice. mRNA expression of GHSR1a was also found to be low in cerebral arteries, and GHSR protein was undetectable in GHSR-enhanced green fluorescent protein mice. We next found that exogenous acylated ghrelin had no effect on the tone of perfused cerebral arteries or superoxide production. By contrast, exogenous des-acylated ghrelin or obestatin elicited powerful vasodilator responses (EC50 < 10 pmol/L) that were abolished by the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester. Furthermore, exogenous des-acylated ghrelin suppressed superoxide production in cerebral arteries. Consistent with our GHSR expression data, vasodilator effects of des-acylated ghrelin or obestatin were sustained in the presence of YIL-781 (GHSR1a antagonist) and in arteries from Ghsr-deficient mice. Using ghrelin-deficient (Ghrl(-/-)) mice, we also found that endogenous production of ghrelin-related peptides regulates NO bioactivity and superoxide levels in the cerebral circulation. Specifically, we show that NO bioactivity was markedly reduced in Ghrl(-/-) vs wild-type mice, and superoxide levels were elevated. These findings reveal protective actions of exogenous and endogenous ghrelin-related peptides in the cerebral circulation and show the existence of a novel ghrelin receptor(s) in the cerebral endothelium.

Acyl ghrelin acts in the brain to control liver function and peripheral glucose homeostasis in male mice.

Recent evidence suggests that peripheral ghrelin regulates glucose metabolism. Here, we designed experiments to examine how central acyl ghrelin infusion affects peripheral glucose metabolism under pair-fed or ad libitum feeding conditions. Mice received intracerebroventricular (icv) infusion of artificial cerebrospinal fluid (aCSF), ghrelin, and allowed to eat ad libitum (icv ghrelin ad lib) or ghrelin and pair-fed to the aCSF group (icv ghrelin pf). Minipumps delivered acyl ghrelin at a dose of 0.25 µg/h at 0.5 µL/h for 7 days. There was no difference in daily blood glucose, insulin, glucagon, triglycerides, or nonesterified fatty acids. Body weight gain and food intake was significantly higher in icv ghrelin ad lib mice. However, both icv ghrelin ad lib and icv ghrelin pf groups exhibited heavier white adipose mass. Icv ghrelin pf mice exhibited better glucose tolerance than aCSF or icv ghrelin ad lib mice during a glucose tolerance test, although both icv ghrelin ad lib and icv ghrelin pf increased insulin release during the glucose tolerance test. Central acyl ghrelin infusion and pair feeding also increased breakdown of liver glycogen and triglyceride, and regulated genes involved in hepatic lipid and glucose metabolism. Icv ghrelin pf mice had an increase in plasma blood glucose during a pyruvate tolerance test relative to icv ghrelin ad lib or aCSF mice. Our results suggest that under conditions of negative energy (icv ghrelin pf), central acyl ghrelin engages a neural circuit that influences hepatic glucose function. Metabolic status affects the ability of central acyl ghrelin to regulate peripheral glucose homeostasis.

Hypothalamic neurogenesis is not required for the improved insulin sensitivity following exercise training.

Neurons within the hypothalamic arcuate nucleus (ARC) are important regulators of energy balance. Recent studies suggest that neurogenesis in the ARC is an important regulator of body mass in response to pharmacological stressors. Regular exercise training improves insulin action, and is a primary treatment modality for obesity and type 2 diabetes. We examined whether exercise training causes hypothalamic neurogenesis and whether this contributes to exercise-induced improvements in insulin action. Short-term exercise in adult mice induced a proneurogenic transcriptional program involving growth factors, cell proliferation, and neurogenic regulators in the hypothalamus. Daily exercise training for 7 days increased hypothalamic cell proliferation 3.5-fold above that of sedentary mice, and exercise-induced cell proliferation was maintained in diet-induced obese mice. Colocalization studies indicated negligible neurogenesis in the ARC of sedentary or exercise-trained mice. Blocking cell proliferation via administration of the mitotic blocker arabinosylcytosine (AraC) did not affect food intake or body mass in obese mice. While 4 weeks of exercise training improved whole-body insulin sensitivity compared with sedentary mice, insulin action was not affected by AraC administration. These data suggest that regular exercise training induces significant non-neuronal cell proliferation in the hypothalamus of obese mice, but this proliferation is not required for enhanced insulin action.

Evidence that diet-induced hyperleptinemia, but not hypothalamic gliosis, causes ghrelin resistance in NPY/AgRP neurons of male mice.

High-fat diet (HFD) feeding causes ghrelin resistance in arcuate neuropeptide Y (NPY)/Agouti-related peptide neurons. In the current study, we investigated the time course over which this occurs and the mechanisms responsible for ghrelin resistance. After 3 weeks of HFD feeding, neither peripheral nor central ghrelin increased food intake and or activated NPY neurons as demonstrated by a lack of Fos immunoreactivity or whole-cell patch-clamp electrophysiology. Pair-feeding studies that matched HFD calorie intake with chow calorie intake show that HFD exposure does not cause ghrelin resistance independent of body weight gain. We observed increased plasma leptin in mice fed a HFD for 3 weeks and show that leptin-deficient obese ob/ob mice are still ghrelin sensitive but become ghrelin resistant when central leptin is coadministered. Moreover, ob/ob mice fed a HFD for 3 weeks remain ghrelin sensitive, and the ability of ghrelin to induce action potential firing in NPY neurons was blocked by leptin. We also examined hypothalamic gliosis in mice fed a chow diet or HFD, as well as in ob/ob mice fed a chow diet or HFD and lean controls. HFD-fed mice exhibited increased glial fibrillary acidic protein-positive cells compared with chow-fed mice, suggesting that hypothalamic gliosis may underlie ghrelin resistance. However, we also observed an increase in hypothalamic gliosis in ob/ob mice fed a HFD compared with chow-fed ob/ob and lean control mice. Because ob/ob mice fed a HFD remain ghrelin sensitive, our results suggest that hypothalamic gliosis does not underlie ghrelin resistance. Further, pair-feeding a HFD to match the calorie intake of chow-fed controls did not increase body weight gain or cause central ghrelin resistance; thus, our evidence suggests that diet-induced hyperleptinemia, rather than diet-induced hypothalamic gliosis or HFD exposure, causes ghrelin resistance.