Identification of AgRP cells in the murine hindbrain that drive feeding.

OBJECTIVE: The central melanocortin system is essential for the regulation of food intake and body weight. Agouti-related protein (AgRP) is the sole orexigenic component of the central melanocortin system and is conserved across mammalian species. AgRP is currently known to be expressed exclusively in the mediobasal hypothalamus, and hypothalamic AgRP-expressing neurons are essential for feeding. Here we characterized a previously unknown population of AgRP cells in the mouse hindbrain. METHODS: Expression of AgRP in the hindbrain was investigated using gene expression analysis, single-cell RNA sequencing, immunofluorescent analysis and multiple transgenic mice with reporter expressions. Activation of AgRP neurons was achieved by Designer Receptors Exclusively Activated by Designer Drugs (DREADD) and by transcranial focal photo-stimulation using a step-function opsin with ultra-high light sensitivity (SOUL). RESULTS: AgRP expressing cells were present in the area postrema (AP) and the adjacent subpostrema area (SubP) and commissural nucleus of the solitary tract (cNTS) of the mouse hindbrain (termed AgRPHind herein). AgRPHind cells consisted of locally projecting neurons as well as tanycyte-like cells. Food deprivation stimulated hindbrain Agrp expression as well as neuronal activity of subsets of AgRPHind cells. In adult mice that lacked hypothalamic AgRP neurons, chemogenetic activation of AgRP neurons resulted in hyperphagia and weight gain. In addition, transcranial focal photo-stimulation of hindbrain AgRP cells increased food intake in adult mice with or without hypothalamic AgRP neurons. CONCLUSIONS: Our study indicates that the central melanocortin system in the hindbrain possesses an orexigenic component, and that AgRPHind neurons stimulate feeding independently of hypothalamic AgRP neurons.

Modulation of foraging-like behaviors by cholesterol-FGF19 axis.

BACKGROUND: Foraging for food precedes food consumption and is an important component of the overall metabolic programming that regulates feeding. Foraging is governed by central nervous system neuronal circuits but how it is influenced by diet and hormonal signals is still not well understood. RESULTS: In this study, we show that dietary cholesterol exerted suppressive effects on locomotor activity and that these effects were partially mediated by the neuropeptide Agouti-related protein (AgRP). High dietary cholesterol stimulated intestinal expression of fibroblast growth factor 15 (Fgf15), an ortholog of the human fibroblast growth factor 19 (FGF19). Intracerebroventricular infusion of FGF19 peptide reduced exploratory activity in the open field test paradigm. On the other hand, the lack of dietary cholesterol enhanced exploratory activity in the open field test, but this effect was abolished by central administration of FGF19. CONCLUSIONS: Experiments in this study show that dietary cholesterol suppresses locomotor activity and foraging-like behaviors, and this regulation is in part mediated by AgRP neurons. Dietary cholesterol or the central action of FGF19 suppresses exploratory behaviors, and the anxiogenic effects of dietary cholesterol may be mediated by the effect of FGF19 in the mouse brain. This study suggests that dietary cholesterol and intestinal hormone FGF15/19 signal a satiating state to the brain, thereby suppressing foraging-like behaviors.

ASB4 modulates central melanocortinergic neurons and calcitonin signaling to control satiety and glucose homeostasis.

Variants in the gene encoding ankyrin repeat and SOCS box-containing 4 (ASB4) are linked to human obesity. Here, we characterized the pathways underlying the metabolic functions of ASB4. Hypothalamic Asb4 expression was suppressed by fasting in wild-type mice but not in mice deficient in AgRP, which encodes Agouti-related protein (AgRP), an appetite-stimulating hormone, suggesting that ASB4 is a negative target of AgRP. Many ASB4 neurons in the brain were adjacent to AgRP terminals, and feeding induced by AgRP neuronal activation was disrupted in Asb4-deficient mice. Acute knockdown of Asb4 in the brain caused marked hyperphagia due to increased meal size, and Asb4 deficiency led to increased meal size and food intake at the onset of refeeding, when very large meals were consumed. Asb4-deficient mice were resistant to the meal-terminating effects of exogenously administered calcitonin and showed decreased neuronal expression of Calcr, which encodes the calcitonin receptor. Pro-opiomelanocortin (POMC) neurons in the arcuate nucleus in mice are involved in glucose homeostasis, and Asb4 deficiency specifically in POMC neurons resulted in glucose intolerance that was independent of obesity. Furthermore, individuals with type 2 diabetes showed reduced ASB4 abundance in the infundibular nuclei, the human equivalent of the arcuate nucleus. Together, our results indicate that ASB4 acts in the brain to improve glucose homeostasis and to induce satiety after substantial meals, particularly those after food deprivation.

A gene-diet interaction controlling relative intake of dietary carbohydrates and fats.

OBJECTIVE: Preference for dietary fat vs. carbohydrate varies markedly across free-living individuals. It is recognized that food choice is under genetic and physiological regulation, and that the central melanocortin system is involved. However, how genetic and dietary factors interact to regulate relative macronutrient intake is not well understood. METHODS: We investigated how the choice for food rich in carbohydrate vs. fat is influenced by dietary cholesterol availability and agouti-related protein (AGRP), the orexigenic component of the central melanocortin system. We assessed how macronutrient intake and different metabolic parameters correlate with plasma AGRP in a cohort of obese humans. We also examined how both dietary cholesterol levels and inhibiting de novo cholesterol synthesis affect carbohydrate and fat intake in mice, and how dietary cholesterol deficiency during the postnatal period impacts macronutrient intake patterns in adulthood. RESULTS: In obese human subjects, plasma levels of AGRP correlated inversely with consumption of carbohydrates over fats. Moreover, AgRP-deficient mice preferred to consume more calories from carbohydrates than fats, more so when each diet lacked cholesterol. Intriguingly, inhibiting cholesterol biosynthesis (simvastatin) promoted carbohydrate intake at the expense of fat without altering total caloric consumption, an effect that was remarkably absent in AgRP-deficient mice. Finally, feeding lactating C57BL/6 dams and pups a cholesterol-free diet prior to weaning led the offspring to prefer fats over carbohydrates as adults, indicating that altered cholesterol metabolism early in life programs adaptive changes to macronutrient intake. CONCLUSIONS: Together, our study illustrates a specific gene-diet interaction in modulating food choice.

Neuronal modulation of hepatic lipid accumulation induced by bingelike drinking.

Excessive alcohol consumption, including binge drinking, is a common cause of fatty liver disease. Binge drinking rapidly induces hepatic steatosis, an early step in the pathogenesis of chronic liver injury. Despite its prevalence, the process by which excessive alcohol consumption promotes hepatic lipid accumulation remains unclear. Alcohol exerts potent effects on the brain, including hypothalamic neurons crucial for metabolic regulation. However, whether or not the brain plays a role in alcohol-induced hepatic steatosis is unknown. In the brain, alcohol increases extracellular levels of adenosine, a potent neuromodulator, and previous work implicates adenosine signaling as being important for the development of alcoholic fatty liver disease. Acute alcohol exposure also increases both the activity of agouti-related protein (AgRP)-expressing neurons and AgRP immunoreactivity. Here, we show that adenosine receptor A2B signaling in the brain modulates the extent of alcohol-induced fatty liver in mice and that both the AgRP neuropeptide and the sympathetic nervous system are indispensable for hepatic steatosis induced by bingelike alcohol consumption. Together, these results indicate that the brain plays an integral role in alcohol-induced hepatic lipid accumulation and that central adenosine signaling, hypothalamic AgRP, and the sympathetic nervous system are crucial mediators of this process.

Regulation of Hepatic Lipid Accumulation and Distribution by Agouti-Related Protein in Male Mice.

Proper regulation of energy metabolism requires neurons in the central nervous system to respond dynamically to signals that reflect the body's energy reserve, and one such signal is leptin. Agouti-related protein (AgRP) is a hypothalamic neuropeptide that is markedly upregulated in leptin deficiency, a condition that is associated with severe obesity, diabetes, and hepatic steatosis. Because deleting AgRP in mice does not alter energy balance, we sought to determine whether AgRP plays an indispensable role in regulating energy and hepatic lipid metabolism in the sensitized background of leptin deficiency. We generated male mice that are deficient for both leptin and AgRP [double-knockout (DKO)]. DKO mice and ob/ob littermates had similar body weights, food intake, energy expenditure, and plasma insulin levels, although DKO mice surprisingly developed heightened hyperglycemia with advancing age. Overall hepatic lipid content was reduced in young prediabetic DKO mice, but not in the older diabetic counterparts. Intriguingly, however, both young and older DKO mice had an altered zonal distribution of hepatic lipids with reduced periportal lipid deposition. Moreover, leptin stimulated, whereas AgRP inhibited, hepatic sympathetic activity. Ablating sympathetic nerves to the liver, which primarily innervate the portal regions, produced periportal lipid accumulation in wild-type mice. Collectively, our results highlight AgRP as a regulator of hepatic sympathetic activity and metabolic zonation.

Hypothalamic Sensing of Bile Acids, a Gut Feeling.

Bile acids facilitate dietary fat absorption upon release into the small intestine after a meal. A recent study by Liu and colleagues identifies a gut-brain axis wherein bile acids signal an energy-replete state to hypothalamic AgRP neurons via activation of neuronal FGF receptors, which orchestrate whole-body glucose metabolism.

Acute Lesioning and Rapid Repair of Hypothalamic Neurons outside the Blood-Brain Barrier.

Neurons expressing agouti-related protein (AgRP) are essential for feeding. The majority of these neurons are located outside the blood-brain barrier (BBB), allowing them to directly sense circulating metabolic factors. Here, we show that, in adult mice, AgRP neurons outside the BBB (AgRPOBBB) were rapidly ablated by peripheral administration of monosodium glutamate (MSG), whereas AgRP neurons inside the BBB and most proopiomelanocortin (POMC) neurons were spared. MSG treatment induced proliferation of tanycytes, the putative hypothalamic neural progenitor cells, but the newly proliferated tanycytes did not become neurons. Intriguingly, AgRPOBBB neuronal number increased within a week after MSG treatment, and newly emerging AgRP neurons were derived from post-mitotic cells, including some from the Pomc-expressing cell lineage. Our study reveals that the lack of protection by the BBB renders AgRPOBBB vulnerable to lesioning by circulating toxins but that the rapid re-emergence of AgRPOBBB is part of a reparative process to maintain energy balance.

Leptin potentiates astrogenesis in the developing hypothalamus.

BACKGROUND: The proper establishment of hypothalamic feeding circuits during early development has a profound influence on energy homeostasis, and perturbing this process could predispose individuals to obesity and its associated consequences later in life. The maturation of hypothalamic neuronal circuitry in rodents takes place during the initial postnatal weeks, and this coincides with a dramatic surge in the circulating level of leptin, which is known to regulate the outgrowth of key neuronal projections in the maturing hypothalamus. Coincidently, this early postnatal period also marks the rapid proliferation and expansion of astrocytes in the brain. METHODS: Here we examined the effects of leptin on the proliferative capacity of astrocytes in the developing hypothalamus by treating postnatal mice with leptin. Mutant mice were also generated to conditionally remove leptin receptors from glial fibrillary acidic protein (GFAP)-expressing cells in the postnatal period. RESULTS AND CONCLUSIONS: We show that GFAP-expressing cells in the periventricular zone of the 3rd ventricle were responsive to leptin during the initial postnatal week. Leptin enhanced the proliferation of astrocytes in the postnatal hypothalamus and conditional removal of leptin receptors from GFAP-expressing cells during early postnatal period limited astrocyte proliferation. While increasing evidence demonstrates a direct role of leptin in regulating astrocytes in the adult brain, and given the essential function of astrocytes in modulating neuronal function and connectivity, our study indicates that leptin may exert its metabolic effects, in part, by promoting hypothalamic astrogenesis during early postnatal development.

Hypothalamic inflammation in the control of metabolic function.

Diet-induced obesity leads to devastating and common chronic diseases, fueling ongoing interest in determining new mechanisms underlying both obesity and its consequences. It is now well known that chronic overnutrition produces a unique form of inflammation in peripheral insulin target tissues, and efforts to limit this inflammation have met with some success in preserving insulin sensitivity in obese individuals. Recently, the activation of inflammatory pathways by dietary excess has also been observed among cells located in the mediobasal hypothalamus, a brain area that exerts central control over peripheral glucose, fat, and energy metabolism. Here we review progress in the field of diet-induced hypothalamic inflammation, drawing key distinctions between metabolic inflammation in the hypothalamus and that occurring in peripheral tissues. We focus on specific stimuli of the inflammatory response, the roles of individual hypothalamic cell types, and the links between hypothalamic inflammation and metabolic function under normal and pathophysiological circumstances. Finally, we explore the concept of controlling hypothalamic inflammation to mitigate metabolic disease.

Loss of Atg12, but not Atg5, in pro-opiomelanocortin neurons exacerbates diet-induced obesity.

The autophagy-related proteins ATG12 and ATG5 form a covalent complex essential for autophagy. Here, we demonstrate that ATG12 has distinct functions from ATG5 in pro-opiomelanocortin (POMC)-expressing neurons. Upon high-fat diet (HFD) consumption, mice lacking Atg12 in POMC-positive neurons exhibit accelerated weight gain, adiposity, and glucose intolerance, which is associated with increased food intake, reduced ambulation, and decreased LEP/leptin sensitivity. Importantly, although genetic deletion of either Atg12 or Atg5 renders POMC neurons autophagy-deficient, mice lacking Atg5 in POMC neurons do not exhibit these phenotypes. Hence, we propose nonautophagic functions for ATG12 in POMC neurons that counteract excessive weight gain in response to HFD consumption.

An estrogen-responsive module in the ventromedial hypothalamus selectively drives sex-specific activity in females.

Estrogen-receptor alpha (ERα) neurons in the ventrolateral region of the ventromedial hypothalamus (VMHVL) control an array of sex-specific responses to maximize reproductive success. In females, these VMHVL neurons are believed to coordinate metabolism and reproduction. However, it remains unknown whether specific neuronal populations control distinct components of this physiological repertoire. Here, we identify a subset of ERα VMHVL neurons that promotes hormone-dependent female locomotion. Activating Nkx2-1-expressing VMHVL neurons via pharmacogenetics elicits a female-specific burst of spontaneous movement, which requires ERα and Tac1 signaling. Disrupting the development of Nkx2-1(+) VMHVL neurons results in female-specific obesity, inactivity, and loss of VMHVL neurons coexpressing ERα and Tac1. Unexpectedly, two responses controlled by ERα(+) neurons, fertility and brown adipose tissue thermogenesis, are unaffected. We conclude that a dedicated subset of VMHVL neurons marked by ERα, NKX2-1, and Tac1 regulates estrogen-dependent fluctuations in physical activity and constitutes one of several neuroendocrine modules that drive sex-specific responses.

Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function.

Diets rich in saturated fat produce inflammation, gliosis, and neuronal stress in the mediobasal hypothalamus (MBH). Here, we show that microglia mediate this process and its functional impact. Although microglia and astrocytes accumulate in the MBH of mice fed a diet rich in saturated fatty acids (SFAs), only the microglia undergo inflammatory activation, along with a buildup of hypothalamic SFAs. Enteric gavage specifically with SFAs reproduces microglial activation and neuronal stress in the MBH, and SFA treatment activates murine microglia, but not astrocytes, in culture. Moreover, depleting microglia abrogates SFA-induced inflammation in hypothalamic slices. Remarkably, depleting microglia from the MBH of mice abolishes inflammation and neuronal stress induced by excess SFA consumption, and in this context, microglial depletion enhances leptin signaling and reduces food intake. We thus show that microglia sense SFAs and orchestrate an inflammatory process in the MBH that alters neuronal function when SFA consumption is high.

Coordinated regulation of hepatic energy stores by leptin and hypothalamic agouti-related protein.

Like obesity, prolonged food deprivation induces severe hepatic steatosis; however, the functional significance of this phenomenon is not well understood. In this study, we show that the fall in plasma leptin concentration during fasting is required for the development of hepatic steatosis in mice. Removal of leptin receptors from AGRP neurons diminishes fasting-induced hepatic steatosis. Furthermore, the suppressive effects of leptin on fasting-induced hepatic steatosis are absent in mice lacking the gene encoding agouti-related protein (Agrp), suggesting that this function of leptin is mediated by AGRP. Prolonged fasting leads to suppression of hepatic sympathetic activity, increased expression of acyl CoA:diacylglycerol acyltransferase-2 in the liver, and elevation of hepatic triglyceride content and all of these effects are blunted in the absence of AGRP. AGRP deficiency, despite having no effects on feeding or body adiposity in the free-fed state, impairs triglyceride and ketone body release from the liver during prolonged fasting. Furthermore, reducing CNS Agrp expression in wild-type mice by RNAi protected against the development of hepatic steatosis not only during starvation, but also in response to consumption of a high-fat diet. These findings identify the leptin-AGRP circuit as a critical modulator of hepatic triglyceride stores in starvation and suggest a vital role for this circuit in sustaining the supply of energy from the liver to extrahepatic tissues during periods of prolonged food deprivation.

Modulation of AgRP-neuronal function by SOCS3 as an initiating event in diet-induced hypothalamic leptin resistance.

Chronic consumption of a fat-rich diet leads to attenuation of leptin signaling in hypothalamic neurons, a hallmark feature of cellular leptin resistance. To date, little is known about the temporal and spatial dysregulation of neuronal function under conditions of nutrient excess. We show that agouti-related protein (AgRP)-expressing neurons precede proopiomelanocortin neurons in developing diet-induced cellular leptin resistance. High-fat diet-induced up-regulation of suppressor of cytokine signaling-3 (SOCS3) occurs in AgRP neurons before proopiomelanocortin and other hypothalamic neurons. SOCS3 expression in AgRP neurons increases after 2 d of high-fat feeding, but reduces after switching to a low-fat diet for 1 d. Consistently, transgenic overexpression of SOCS3 in AgRP neurons produces metabolic phenotypes resembling those observed after short-term high-fat feeding. We further show that AgRP neurons are the predominant cell type situated outside the blood-brain barrier in the mediobasal hypothalamus. AgRP neurons are more responsive to low levels of circulating leptin, but they are also more prone to development of leptin resistance in response to a small increase in blood leptin concentrations. Collectively, these results suggest that AgRP neurons are able to sense slight changes in plasma metabolic signals, allowing them to serve as first-line responders to fluctuation of energy intake. Furthermore, modulation of SOCS3 expression in AgRP neurons may play a dynamic and physiological role in metabolic fine tuning in response to short-term changes of nutritional status.

AgRP innervation onto POMC neurons increases with age and is accelerated with chronic high-fat feeding in male mice.

In many mammals, body weight increases continuously throughout adulthood until late middle age. The hormone leptin is necessary for maintaining body weight, in that high levels of leptin promote negative energy balance. As animals age, however, their increase in body weight is accompanied by a steady rise in circulating leptin levels, indicating the progressive development of counterregulatory mechanisms to antagonize leptin's anorexigenic effects. Hypothalamic neurons coexpressing agouti-related peptide (AgRP) and neuropeptide Y are direct leptin targets. These neurons promote positive energy balance, and they inhibit anorexigenic proopiomelanocortin (POMC) neurons via direct neuropeptide action and release of γ-aminobutyric acid. We show here that AgRP and neuropeptide Y innvervation onto POMC neurons increases dramatically with age in male mice. This is associated with progressive increase of inhibitory postsynaptic currents and decrease of POMC firing rate with age. Neuronal activity is significantly attenuated in POMC neurons that receive a high density of AgRP puncta. These high-density AgRP inputs correlate with leptin levels in normal mice and are nearly absent in mice lacking leptin. The progression of increased AgRP innervation onto POMC somas is accelerated in hyperleptinemic, diet-induced obese mice. Together our study suggests that modulation of hypothalamic AgRP innervation constitutes one mechanism to counter the effects of the age-associated rise in leptin levels, thus sustaining body weight and fat mass at an elevated level in adulthood.

Metabolic transceivers: in tune with the central melanocortin system.

The central melanocortin system plays an essential role in the regulation of energy metabolism. Key to this regulation are the responses of neurons expressing proopiomelanocortin (POMC) and agouti-related protein (AgRP) to blood-borne metabolic signals. Recent evidence has demonstrated that POMC and AgRP neurons are not simply mirror opposites of each other in function and responsiveness to metabolic signals, nor are they exclusively first-order neurons. These neurons act as central transceivers, integrating both hormonal and neural signals, and then transmitting this information to peripheral tissues via the autonomic nervous system to coordinate whole-body energy metabolism. This review focuses on most recent developments obtained from rodent studies on the function, metabolic regulation, and circuitry of the central melanocortin system.

Rapamycin ameliorates age-dependent obesity associated with increased mTOR signaling in hypothalamic POMC neurons.

VIDEO ABSTRACT: The prevalence of obesity in older people is the leading cause of metabolic syndromes. Central neurons serving as homeostatic sensors for body-weight control include hypothalamic neurons that express pro-opiomelanocortin (POMC) or neuropeptide-Y (NPY) and agouti-related protein (AgRP). Here, we report an age-dependent increase of mammalian target of rapamycin (mTOR) signaling in POMC neurons that elevates the ATP-sensitive potassium (K(ATP)) channel activity cell-autonomously to silence POMC neurons. Systemic or intracerebral administration of the mTOR inhibitor rapamycin causes weight loss in old mice. Intracerebral rapamycin infusion into old mice enhances the excitability and neurite projection of POMC neurons, thereby causing a reduction of food intake and body weight. Conversely, young mice lacking the mTOR-negative regulator TSC1 in POMC neurons, but not those lacking TSC1 in NPY/AgRP neurons, were obese. Our study reveals that an increase in mTOR signaling in hypothalamic POMC neurons contributes to age-dependent obesity.

Impairment of central leptin-mediated PI3K signaling manifested as hepatic steatosis independent of hyperphagia and obesity.

Hepatic steatosis is generally thought to develop via peripheral mechanisms associated with obesity. We show that chronic central infusion of leptin suppresses hepatic lipogenic gene expression and reduces triglyceride content via stimulation of hepatic sympathetic activity. This leptin function is independent of feeding and body weight but requires phosphatidylinositol 3-kinase (PI3K) signaling. Attenuation of leptin-induced PI3K signaling, brought about by transgenic expression of phosphatase and tensin homolog (PTEN) in leptin receptor neurons, leads to decreased hepatic sympathetic tone and increased triglyceride levels without affecting adiposity or hepatic insulin signaling. Central leptin's effects on hepatic norepinephrine levels and triglyceride content are blunted in these mutant mice. Simultaneous downregulation of PI3K and signal transducer and activator of transcription-3 (Stat3) in leptin receptor neurons does not exacerbate obesity but causes more severe hepatic steatosis. Together, our results indicate that central cellular leptin resistance in PI3K signaling manifests as hepatic steatosis without causing obesity.

Agrp neurons mediate Sirt1's action on the melanocortin system and energy balance: roles for Sirt1 in neuronal firing and synaptic plasticity.

Sirt1 has been associated with various effects of calorie restriction, including an increase in lifespan. Here we show in mice that a central regulatory component in energy metabolism, the hypothalamic melanocortin system, is affected by Sirt1, which promotes the activity and connectivity of this system resulting in negative energy balance. In adult mice, the pharmacological inhibition of brain Sirt1 activity decreased Agrp neuronal activity and the inhibitory tone on the anorexigenic POMC neurons, as measured by the number of synaptic inputs to these neurons. When a Sirt1 inhibitor (EX-527) was injected either peripherally (i.p., 10 mg/kg) or directly into the brain (i.c.v., 1.5 nmol/mouse), it decreased both food intake during the dark cycle and ghrelin-induced food intake. This effect on feeding is mediated by upstream melanocortin receptors, because the MC4R antagonist, SHU9119, reversed Sirt1's effect on food intake. This action of Sirt1 required an appropriate shift in the mitochondrial redox state: in the absence of such an adaptation enabled by the mitochondrial protein, UCP2, Sirt1-induced cellular and behavioral responses were impaired. In accordance with the pharmacological results, the selective knock-out of Sirt1 in hypothalamic Agrp neurons through the use of Cre-Lox technology decreased electric responses of Agrp neurons to ghrelin and decreased food intake, leading to decreased lean mass, fat mass, and body weight. The present data indicate that Sirt1 has a central mode of action by acting on the NPY/Agrp neurons to affect body metabolism.