Melanocortin MC4R receptor is required for energy expenditure but not blood pressure effects of angiotensin II within the mouse brain.

The brain renin-angiotensin system (RAS) is implicated in control of blood pressure (BP), fluid intake, and energy expenditure (EE). Angiotensin II (ANG II) within the arcuate nucleus of the hypothalamus contributes to control of resting metabolic rate (RMR) and thereby EE through its actions on Agouti-related peptide (AgRP) neurons, which also contribute to EE control by leptin. First, we determined that although leptin stimulates EE in control littermates, mice with transgenic activation of the brain RAS (sRA) exhibit increased EE and leptin has no additive effect to exaggerate EE in these mice. These findings led us to hypothesize that leptin and ANG II in the brain stimulate EE through a shared mechanism. Because AgRP signaling to the melanocortin MC4R receptor contributes to the metabolic effects of leptin, we performed a series of studies examining RMR, fluid intake, and BP responses to ANG II in mice rendered deficient for expression of MC4R via a transcriptional block (Mc4r-TB). These mice were resistant to stimulation of RMR in response to activation of the endogenous brain RAS via chronic deoxycorticosterone acetate (DOCA)-salt treatment, whereas fluid and electrolyte effects remained intact. These mice were also resistant to stimulation of RMR via acute intracerebroventricular (ICV) injection of ANG II, whereas BP responses to ICV ANG II remained intact. Collectively, these data demonstrate that the effects of ANG II within the brain to control RMR and EE are dependent on MC4R signaling, whereas fluid homeostasis and BP responses are independent of MC4R signaling.

NPY derived from AGRP neurons controls feeding via Y1 and energy expenditure and food foraging behaviour via Y2 signalling.

OBJECTIVE: Aguti-related protein (AGRP) neurons in the arcuate nucleus of the hypothalamus (ARC), which co-express neuropeptide Y (NPY), are key regulators of feeding and energy homeostasis. However, the precise role NPY has within these neurons and the specific pathways that it control are still unclear. In this article, we aimed to determine what aspects of feeding behaviour and energy homeostasis are controlled by NPY originating from AGRP neurons and which Y-receptor pathways are utilised to fulfil this function. METHODS: Novel conditional Agrpcre/+;Npylox/lox knockout mice were generated and comprehensively phenotyped, both under standard chow as well as high-fat-diet conditions. Designer receptor exclusively activated by designer drugs (DREADD) technology was used to assess the altered responses on feeding and energy homeostasis control in the absence of NPY in these neurons. Rescue experiments utilising Npy1r- and Npy2r-selective NPY ligands were performed to assess which component of the energy homeostasis control is dependent by which specific Y-receptor pathway. RESULTS: We show that the specific deletion of Npy only in AGRP neurons leads to a paradoxical mild obese phenotype associated with reduced locomotion and energy expenditure and increased feeding and Respiratory Quotient (RQ) that remain elevated under a positive energy balance. The activation of Npy-deficient AGRP neurons via DREADD's is still able to drive feeding, yet with a delayed onset. Additionally, Clozapine-N-oxide (CNO) treatment reduces locomotion without impacting on energy expenditure. Rescue experiments re-introducing Npy1r- and Npy2r-selective NPY ligands revealed that the increased feeding and RQ are mostly driven by Npy1r, whereas energy expenditure and locomotion are controlled by Npy2r signalling. CONCLUSION: Together, these results demonstrate that NPY originating from AGRP neurons is not only critical to initiate but also for continuously driving feeding, and we for the first time identify which Y-receptor controls which pathway.

AgRP neurons: Regulators of feeding, energy expenditure, and behavior.

Neurons in the hypothalamic arcuate nucleus (ARC) that express agouti-related peptide (AgRP) govern a critical aspect of survival: the drive to eat. Equally important to survival is the timing at which food is consumed-seeking or eating food to alleviate hunger in the face of a more pressing threat, like the risk of predation, is clearly maladaptive. To ensure optimal prioritization of behaviors within a given environment, therefore, AgRP neurons must integrate signals of internal need states with contextual environmental cues. In this state-of-the-art review, we highlight recent advances that extend our understanding of AgRP neurons, including the neural circuits they engage to regulate feeding, energy expenditure, and behavior. We also discuss key findings that illustrate how both classical feedback and anticipatory feedforward signals regulate this neuronal population and how the integration of these signals may be disrupted in states of energy excess. Finally, we examine both technical and conceptual challenges facing the field moving forward.

Both NPY-Expressing and CART-Expressing Neurons Increase Energy Expenditure and Trabecular Bone Mass in Response to AP1 Antagonism, But Have Opposite Effects on Bone Resorption.

Energy metabolism and bone homeostasis share several neuronal regulatory pathways. Within the ventral hypothalamus (VHT), the orexigenic neurons co-express Agouti-related peptide (AgRP) and neuropeptide Y (NPY) and the anorexigenic neurons co-express, α-melanocyte stimulating hormone derived from proopiomelanocortin (POMC), and cocaine and amphetamine-regulated transcript (CART). These neurons regulate both processes, yet their relative contribution is unknown. Previously, using genetically targeted activator protein (AP1) alterations as a tool, we showed in adult mice that AgRP or POMC neurons are capable of inducing whole-body energy catabolism and bone accrual, with different effects on bone resorption. Here, we investigated whether co-residing neurons exert similar regulatory effects. We show that AP1 antagonists targeted to NPY-producing or CART-producing neurons in adult mice stimulate energy expenditure, reduce body weight gain and adiposity and promote trabecular bone formation and mass, yet again via different effects on bone resorption, as measured by serum level of carboxy-terminal collagen type I crosslinks (CTX). In addition, AP1 antagonists promote neurite expansion, increasing neurite number, length, and surface area in primary hypothalamic neuronal cultures. Overall, our data demonstrate that the orexigenic NPY and anorexigenic CART neurons both have the capacity to stimulate energy burning state and increase bone mass. © 2020 American Society for Bone and Mineral Research.

Control of Energy Expenditure by AgRP Neurons of the Arcuate Nucleus: Neurocircuitry, Signaling Pathways, and Angiotensin.

PURPOSE OF REVIEW: Here, we review the current understanding of the functional neuroanatomy of neurons expressing Agouti-related peptide (AgRP) and the angiotensin 1A receptor (AT1A) within the arcuate nucleus (ARC) in the control of energy balance. RECENT FINDINGS: The development and maintenance of obesity involves suppression of resting metabolic rate (RMR). RMR control is integrated via AgRP and proopiomelanocortin neurons within the ARC. Their projections to other hypothalamic and extrahypothalamic nuclei contribute to RMR control, though relatively little is known about the contributions of individual projections and the neurotransmitters involved. Recent studies highlight a role for AT1A, localized to AgRP neurons, but the specific function of AT1A within these cells remains unclear. AT1A functions within AgRP neurons to control RMR, but additional work is required to clarify its role within subpopulations of AgRP neurons projecting to distinct second-order nuclei, and the molecular mediators of its signaling within these cells.

AgRP Accountants Compute Caloric Cost.

The gut-brain communication underlying energy homeostasis has been a topic of interest for years. In two new papers, Beutler et al. (2017) and Su et al. (2017) delve into the mechanisms by which satiation is represented in a well-studied population of orexigenic neurons.

A Hypothalamic Phosphatase Switch Coordinates Energy Expenditure with Feeding.

Beige adipocytes can interconvert between white and brown-like states and switch between energy storage versus expenditure. Here we report that beige adipocyte plasticity is important for feeding-associated changes in energy expenditure and is coordinated by the hypothalamus and the phosphatase TCPTP. A fasting-induced and glucocorticoid-mediated induction of TCPTP, inhibited insulin signaling in AgRP/NPY neurons, repressed the browning of white fat and decreased energy expenditure. Conversely feeding reduced hypothalamic TCPTP, to increase AgRP/NPY neuronal insulin signaling, white adipose tissue browning and energy expenditure. The feeding-induced repression of hypothalamic TCPTP was defective in obesity. Mice lacking TCPTP in AgRP/NPY neurons were resistant to diet-induced obesity and had increased beige fat activity and energy expenditure. The deletion of hypothalamic TCPTP in obesity restored feeding-induced browning and increased energy expenditure to promote weight loss. Our studies define a hypothalamic switch that coordinates energy expenditure with feeding for the maintenance of energy balance.

mTORC1 in AGRP neurons integrates exteroceptive and interoceptive food-related cues in the modulation of adaptive energy expenditure in mice.

Energy dissipation through interscapular brown adipose tissue (iBAT) thermogenesis is an important contributor to adaptive energy expenditure. However, it remains unresolved how acute and chronic changes in energy availability are detected by the brain to adjust iBAT activity and maintain energy homeostasis. Here, we provide evidence that AGRP inhibitory tone to iBAT represents an energy-sparing circuit that integrates environmental food cues and internal signals of energy availability. We establish a role for the nutrient-sensing mTORC1 signaling pathway within AGRP neurons in the detection of environmental food cues and internal signals of energy availability, and in the bi-directional control of iBAT thermogenesis during nutrient deficiency and excess. Collectively, our findings provide insights into how mTORC1 signaling within AGRP neurons surveys energy availability to engage iBAT thermogenesis, and identify AGRP neurons as a neuronal substrate for the coordination of energy intake and adaptive expenditure under varying physiological and environmental contexts.

Deletion of ATF4 in AgRP Neurons Promotes Fat Loss Mainly via Increasing Energy Expenditure.

Although many functions of activating transcription factor 4 (ATF4) are identified, a role of ATF4 in the hypothalamus in regulating energy homeostasis is unknown. Here, we generated adult-onset agouti-related peptide neuron-specific ATF4 knockout (AgRP-ATF4 KO) mice and found that these mice were lean, with improved insulin and leptin sensitivity and decreased hepatic lipid accumulation. Furthermore, AgRP-ATF4 KO mice showed reduced food intake and increased energy expenditure, mainly because of enhanced thermogenesis in brown adipose tissue. Moreover, AgRP-ATF4 KO mice were resistant to high-fat diet-induced obesity, insulin resistance, and liver steatosis and maintained at a higher body temperature under cold stress. Interestingly, the expression of FOXO1 was directly regulated by ATF4 via binding to the cAMP-responsive element site on its promoter in hypothalamic GT1-7 cells. Finally, Foxo1 expression was reduced in the arcuate nucleus (ARC) of the hypothalamus of AgRP-ATF4 KO mice, and adenovirus-mediated overexpression of FOXO1 in ARC increased the fat mass in AgRP-ATF4 KO mice. Collectively, our data demonstrate a novel function of ATF4 in AgRP neurons of the hypothalamus in energy balance and lipid metabolism and suggest hypothalamic ATF4 as a potential drug target for treating obesity and its related metabolic disorders.

AgRP Neuron-Specific Deletion of Glucocorticoid Receptor Leads to Increased Energy Expenditure and Decreased Body Weight in Female Mice on a High-Fat Diet.

Agouti-related protein (AgRP) expressed in the arcuate nucleus is a potent orexigenic neuropeptide, which increases food intake and reduces energy expenditure resulting in increases in body weight (BW). Glucocorticoids, key hormones that regulate energy balance, have been shown in rodents to regulate the expression of AgRP. In this study, we generated AgRP-specific glucocorticoid receptor (GR)-deficient (knockout [KO]) mice. Female and male KO mice on a high-fat diet (HFD) showed decreases in BW at the age of 6 weeks compared with wild-type mice, and the differences remained significant until 16 weeks old. The degree of resistance to diet-induced obesity was more robust in female than in male mice. On a chow diet, the female KO mice showed slightly but significantly attenuated weight gain compared with wild-type mice after 11 weeks, whereas there were no significant differences in BW in males between genotypes. Visceral fat pad mass was significantly decreased in female KO mice on HFD, whereas there were no significant differences in lean body mass between genotypes. Although food intake was similar between genotypes, oxygen consumption was significantly increased in female KO mice on HFD. In addition, the uncoupling protein-1 expression in the brown adipose tissues was increased in KO mice. These data demonstrate that the absence of GR signaling in AgRP neurons resulted in increases in energy expenditure accompanied by decreases in adiposity in mice fed HFD, indicating that GR signaling in AgRP neurons suppresses energy expenditure under HFD conditions.

Insulin ameliorating endotoxaemia-induced muscle wasting is associated with the alteration of hypothalamic neuropeptides and inflammation in rats.

OBJECTIVES: Septic patients always develop muscle wasting, which delays the rehabilitation and contributes to the increased complications and mortality. Previous studies have implied the crucial role of central inflammation and neuropeptides in the energy balance and muscle metabolism. Insulin has been confirmed to attenuate muscle degradation and inhibit inflammation. We tested the hypothesis whether insulin ameliorating muscle wasting was associated with modulating hypothalamic inflammation and neuropeptides. DESIGN AND SUBJECTS: Thirty-two adult male Sprague-Dawley rats were in intraperitoneally injected with lipopolysaccharide (LPS) (5 mg/kg) or saline, followed by subcutaneous injection of insulin (5 IU/kg) or saline. Twenty-four hours after injection, skeletal muscle and hypothalamus tissues were harvested. Muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle ring finger 1 (MuRF-1) and muscle atrophy F-box (MAFbx), as well as 3-methylhistidine (3-MH) and tyrosine release. Hypothalamic inflammatory markers and neuropeptides expression were also measured in four groups. RESULTS: LPS injection led to significant increase in hypothalamic inflammation as well as muscle wasting. Also, increased hypothalamic neuropeptides, proopiomelanocortin (POMC), cocaine and amphetamine-related transcript (CART) and neuropeptides Y (NPY) and decreased agouti-related protein (AgRP) were observed. Insulin treatment ameliorated endotoxaemia-induced muscle wasting and hypothalamic inflammation, and attenuated the alteration of neuropeptides, POMC, CART and AgRP. CONCLUSION: Hypothalamic inflammation and neuropeptides are involved in the endotoxaemia-induced muscle wasting. Insulin treatment can reduce muscle wasting, which is associated with reduced hypothalamic inflammation and alteration of hypothalamic neuropeptides.

ROCK1 in AgRP neurons regulates energy expenditure and locomotor activity in male mice.

Normal leptin signaling is essential for the maintenance of body weight homeostasis. Proopiomelanocortin- and agouti-related peptide (AgRP)-producing neurons play critical roles in regulating energy metabolism. Our recent work demonstrates that deletion of Rho-kinase 1 (ROCK1) in the AgRP neurons of mice increased body weight and adiposity. Here, we report that selective loss of ROCK1 in AgRP neurons caused a significant decrease in energy expenditure and locomotor activity of mice. These effects were independent of any change in food intake. Furthermore, AgRP neuron-specific ROCK1-deficient mice displayed central leptin resistance, as evidenced by impaired Signal Transducer and Activator of Transcription 3 activation in response to leptin administration. Leptin's ability to hyperpolarize and decrease firing rate of AgRP neurons was also abolished in the absence of ROCK1. Moreover, diet-induced and genetic forms of obesity resulted in reduced ROCK1 activity in murine arcuate nucleus. Of note, high-fat diet also impaired leptin-stimulated ROCK1 activity in arcuate nucleus, suggesting that a defect in hypothalamic ROCK1 activity may contribute to the pathogenesis of central leptin resistance in obesity. Together, these data demonstrate that ROCK1 activation in hypothalamic AgRP neurons is required for the homeostatic regulation of energy expenditure and adiposity. These results further support previous work identifying ROCK1 as a key regulator of energy balance and suggest that targeting ROCK1 in the hypothalamus may lead to development of antiobesity therapeutics.

TXNIP in Agrp neurons regulates adiposity, energy expenditure, and central leptin sensitivity.

Thioredoxin interacting protein (TXNIP) has recently been described as a key regulator of energy metabolism through pleiotropic actions that include nutrient sensing in the mediobasal hypothalamus (MBH). However, the role of TXNIP in neurochemically specific hypothalamic subpopulations and the circuits downstream from MBH TXNIP engaged to regulate energy homeostasis remain unexplored. To evaluate the metabolic role of TXNIP activity specifically within arcuate Agrp neurons, we generated Agrp-specific TXNIP gain-of-function and loss-of-function mouse models using Agrp-Ires-cre mice, TXNIP (flox/flox) mice, and a lentivector expressing the human TXNIP isoform conditionally in the presence of Cre recombinase. Overexpression of TXNIP in Agrp neurons predisposed to diet-induced obesity and adipose tissue storage by decreasing energy expenditure and spontaneous locomotion, without affecting food intake. Conversely, Agrp neuronal TXNIP deletion protected against diet-induced obesity and adipose tissue storage by increasing energy expenditure and spontaneous locomotion, also without affecting food intake. TXNIP overexpression in Agrp neurons did not primarily affect glycemic control, whereas deletion of TXNIP in Agrp neurons improved fasting glucose levels and glucose tolerance independently of its effects on body weight and adiposity. Bidirectional manipulation of TXNIP expression induced reciprocal changes in central leptin sensitivity and the neural regulation of lipolysis. Together, these results identify a critical role for TXNIP in Agrp neurons in mediating diet-induced obesity through the regulation of energy expenditure and adipose tissue metabolism, independently of food intake. They also reveal a previously unidentified role for Agrp neurons in the brain-adipose axis.

PDK1-Foxo1 in agouti-related peptide neurons regulates energy homeostasis by modulating food intake and energy expenditure.

Insulin and leptin intracellular signaling pathways converge and act synergistically on the hypothalamic phosphatidylinositol-3-OH kinase/3-phosphoinositide-dependent protein kinase 1 (PDK1). However, little is known about whether PDK1 in agouti-related peptide (AGRP) neurons contributes to energy homeostasis. We generated AGRP neuron-specific PDK1 knockout (AGRPPdk1(-/-)) mice and mice with selective expression of transactivation-defective Foxo1 (Δ256Foxo1(AGRP)Pdk1(-/-)). The AGRPPdk1(-/-) mice showed reductions in food intake, body length, and body weight. The Δ256Foxo1(AGRP)Pdk1(-/-) mice showed increased body weight, food intake, and reduced locomotor activity. After four weeks of calorie-restricted feeding, oxygen consumption and locomotor activity were elevated in AGRPPdk1(-/-) mice and reduced in Δ256Foxo1(AGRP)Pdk1(-/-) mice. In vitro, ghrelin-induced changes in [Ca(2+)](i) and inhibition of ghrelin by leptin were significantly attenuated in AGRPPdk1(-/-) neurons compared to control neurons. However, ghrelin-induced [Ca(2+)](i) changes and leptin inhibition were restored in Δ256Foxo1(AGRP)Pdk1(-/-) mice. These results suggested that PDK1 and Foxo1 signaling pathways play important roles in the control of energy homeostasis through AGRP-independent mechanisms.

Neuropeptide Y and agouti-related peptide mediate complementary functions of hyperphagia and reduced energy expenditure in leptin receptor deficiency.

Neuropeptide Y (NPY) and agouti-related peptide (AGRP) can produce hyperphagia, reduce energy expenditure, and promote triglyceride deposition in adipose depots. As these two neuropeptides are coexpressed within the hypothalamic arcuate nucleus and mediate a major portion of the obesity caused by leptin signaling deficiency, we sought to determine whether the two neuropeptides mediated identical or complementary actions. Because of separate neuropeptide receptors and signal transduction mechanisms, there is a possibility of distinct encoding systems for the feeding and energy expenditure aspects of leptin-regulated metabolism. We have genetically added NPY deficiency and/or AGRP deficiency to LEPR deficiency isolated to AGRP cells. Our results indicate that the obesity of LEPR deficiency in AGRP/NPY neurons can produce obesity with either AGRP or NPY alone with AGRP producing hyperphagia while NPY promotes reduced energy expenditure. The absence of both NPY and AGRP prevents the development of obesity attributable to isolated LEPR deficiency in AGRP/NPY neurons. Operant behavioral testing indicated that there were no alterations in the reward for a food pellet from the AGRP-specific LEPR deficiency.

C-type natriuretic peptide as a new regulator of food intake and energy expenditure.

The physiological implication of C-type natriuretic peptide (CNP) including energy metabolism has not been elucidated, because of markedly short stature in CNP-null mice. In the present study we analyzed food intake and energy expenditure of CNP-null mice with chondrocyte-targeted CNP expression (CNP-Tg/Nppc(-/-) mice), in which marked skeletal dysplasia was rescued, to investigate the significance of CNP under minimal influences of skeletal phenotypes. In CNP-Tg/Nppc(-/-) mice, body weight and body fat ratio were reduced by 24% and 32%, respectively, at 20 wk of age, and decreases of blood glucose levels during insulin tolerance tests were 2-fold exaggerated at 17 wk of age, as compared with CNP-Tg/Nppc(+/+) mice. Urinary noradrenalin excretion of CNP-Tg/Nppc(-/-) mice was greater than that of CNP-Tg/Nppc(+/+) mice by 28%. In CNP-Tg/Nppc(-/-) mice, rectal temperature at 1600 h was higher by 1.1 C, and uncoupling protein-1 mRNA expression in the brown adipose tissue was 2-fold increased, which was canceled by propranolol administration, as compared with CNP-Tg/Nppc(+/+) mice. Oxygen consumption was significantly increased in CNP-Tg/Nppc(-/-) mice compared with that in CNP-Tg/Nppc(+/+) mice. Food intake of CNP-Tg/Nppc(-/-) mice upon ad libitum feeding and refeeding after 48 h starvation were reduced by 21% and 61%, respectively, as compared with CNP-Tg/Nppc(+/+) mice. This study unveiled a new aspect of CNP as a molecule regulating food intake and energy expenditure. Further analyses on precise mechanisms of CNP actions would lead to the better understanding of the significance of the CNP/guanylyl cyclase-B system in food intake and energy expenditure.

Energy expenditure and aging.

The study of energy expenditure (EE) has deep roots in understanding aging and lifespan in all species. In humans, total EE decreases substantially in advanced age resulting from parallel changes in resting metabolic rate (RMR) and activity EE. For RMR, this reduction appears to be due to a reduction in organ mass and specific metabolic rates of individual tissues. However, these anatomical changes explain very little regarding the decline in activity EE, which is governed by both genetic and environmental sources. The biological control centers for activity EE are closely coupled with body mass fluctuations and seem to originate in the brain. Several candidate neuromodulators may be involved in the age-related reduction of activity EE that include: orexin, agouti-related proteins and dopaminergic pathways. Unfortunately, the existing body of research has primarily focused on how neuromodulators influence weight gain and only a few studies have been performed in aging models. Recent evidence suggests that activity EE has an important role in dictating lifespan and thus places emphasis on future research to uncover the underlying biological mechanisms. The study of EE continues to unlock clues to aging.

Tail suspension increases energy expenditure independently of the melanocortin system in mice.

Space travelers experience anorexia and body weight loss in a microgravity environment, and microgravity-like situations cause changes in hypothalamic activity. Hypothalamic melanocortins play a critical role in the regulation of metabolism. Therefore, we hypothesized that microgravity affects metabolism through alterations in specific hypothalamic signaling pathways, including melanocortin signaling. To address this hypothesis, the microgravity-like situation was produced by an antiorthostatic tail suspension in wild-type and agouti mice, and the effect of tail suspension on energy expenditure and hypothalamic gene expression was examined. Energy expenditure was measured using indirect calorimetry before and during the tail suspension protocol. Hypothalamic tissues were collected for gene expression analysis at the end of the 3 h tail suspension period. Tail suspension significantly increased oxygen consumption, carbon dioxide production, and heat production in wild-type mice. Tail suspension-induced increases in energy expenditure were not attenuated in agouti mice. Although tail suspension did not alter hypothalamic proopiomelanocortin (POMC) and agouti-related protein (AGRP) mRNA levels, it significantly increased hypothalamic interleukin 6 (Il-6) mRNA levels. These data are consistent with the hypothesis that microgravity increases energy expenditure and suggest that these effects are mediated through hypothalamic signaling pathways that are independent of melanocortins, but possibly used by Il-6.