AgRP Neuronal Suppression by Spontaneous Movement Encodes Exercise Reinforcement

Anorexia nervosa (AN) is a psychiatric illness with the highest mortality. Current treatment options have been limited to psychotherapy and nutritional support, with low efficacy and high relapse rates. Hypothalamic AgRP (agouti-related peptide) neurons that coexpress AGRP and neuropeptide Y (NPY) play a critical role in driving feeding while also modulating other complex behaviors. We have previously reported that genetic ablation of Tet3, which encodes a member of the TET family dioxygenases, specifically in AgRP neurons in mice, activates these neurons and increases the expression of AGRP, NPY, and the vesicular GABA transporter (VGAT), leading to hyperphagia and anxiolytic effects. Bobcat339 is a synthetic small molecule predicted to bind to the catalytic pockets of TET proteins. Here, we report that Bobcat339 is effective in mitigating AN and anxiety/depressive-like behaviors using a well-established mouse model of activity-based anorexia (ABA). We show that treating mice with Bobcat339 decreases TET3 expression in AgRP neurons and activates these neurons leading to increased feeding, decreased compulsive running, and diminished lethality in the ABA model. Mechanistically, Bobcat339 induces TET3 protein degradation while simultaneously stimulating the expression of AGRP, NPY, and VGAT in a TET3-dependent manner both in mouse and human neuronal cells, demonstrating a conserved, previously unsuspected mode of action of Bobcat339. Our findings suggest that Bobcat339 may potentially be a therapeutic for anorexia nervosa and stress-related disorders.

The sense of smell has potent effects on appetite, but the underlying neural pathways remain undefined. Here, we investigated how olfactory signals reach two subsets of appetite-linked ("appetite") neurons in the hypothalamic arcuate nucleus: Agouti-related peptide (AgRP) neurons, which stimulate appetite, and POMC (pro-opiomelanocortin) neurons, which suppress it. Using polysynaptic viral tracing, we show that AgRP and POMC neurons receive indirect input from partially overlapping but distinct areas of the olfactory cortex, indicating that they process different sets of olfactory information. We also identify different complements of neurons more directly upstream of AgRP and POMC neurons that could relay olfactory cortical signals to the appetite neurons. Single-cell transcriptomics shows heterogeneous expression of neuromodulator receptors among AgRP neurons, suggesting variations in the signals they receive. Integrated viral tracing and RNA localization further reveals selected brain areas where upstream neurons express cognate receptor ligands. Together, these findings outline multiple pathways by which distinct olfactory and modulatory signals are differentially routed to neurons that promote versus inhibit appetite.

Hypothalamic AgRP neurons regulate the hyperphagia of lactation

The sense of smell has potent effects on appetite, but the underlying neural pathways remain undefined. Here we investigated how olfactory signals reach two subsets of appetite-linked neurons in the hypothalamic arcuate nucleus: AgRP (agouti-related peptide) neurons, which stimulate appetite, and POMC (pro-opiomelanocortin) neurons, which suppress it. Using polysynaptic viral tracing, we show that AgRP and POMC neurons receive indirect input from partially overlapping but distinct areas of the olfactory cortex, indicating that they process different sets of olfactory information. We also identify different complements of neurons directly upstream of AgRP and POMC neurons that can relay olfactory cortical signals to the appetite neurons. Single cell transcriptomics shows heterogeneous expression of neuromodulator receptors among AgRP neurons, suggesting variations in the signals they receive. Integrated viral tracing and RNA localization further reveals selected brain areas where upstream neurons express cognate receptor ligands. Together, these findings outline multiple pathways by which distinct olfactory and modulatory signals are differentially routed to neurons that promote versus inhibit appetite.

Agouti-related peptide (AgRP) neurons show opposite pairings of anatomy and output across caloric states. In mice, 24-h caloric restriction (CR) increases AgRP-to-paraventricular hypothalamus (PVH) fiber density without increasing stimulation-evoked feeding, whereas 5-day high-fat diet (HFD) reduces fiber density yet increases stimulation-evoked feeding, including during PVH terminal stimulation. PVH Bdnf rises acutely under both challenges and remains elevated with sustained HFD. Receptor expression shifts with state: in AgRP neurons, CR increases TrkB transcripts, whereas HFD increases p75NTR transcripts. AgRP-specific deletions define directional roles: TrkB loss reduces AgRP-to-PVH fiber density and yields larger stimulation-evoked feeding, whereas p75NTR loss increases fiber density and limits responses. Thus, a TrkB/p75NTR balance in AgRP neurons provides rheostat-like control over the relationship between circuit structure and stimulation-evoked feeding across caloric states, identifying TrkB and p75NTR as tractable molecular handles for probing state-dependent control of AgRP circuit output.

Autophagy in Hypothalamic AgRP Neurons Regulates Food Intake and Energy Balance

A high-fat diet (HFD) is linked with cytokines production by non-neuronal cells within the hypothalamus, which mediates metabolic inflammation. These cytokines then activate different inflammatory mediators in the arcuate nucleus of the hypothalamus (ARC), a primary hypothalamic area accommodating proopiomelanocortin (POMC) and agouti-related peptide (AGRP) neurons, first-order neurons that sense and integrate peripheral metabolic signals and then respond accordingly. These mediators, such as inhibitor of κB kinase-β (IKKβ), suppression of cytokine signaling 3 (SOCS3), c-Jun N-terminal kinases (JNKs), protein kinase C (PKC), etc., cause insulin and leptin resistance in POMC and AGRP neurons and support obesity and related metabolic complications. On the other hand, inhibition of these mediators has been shown to counteract the impaired metabolism. Therefore, it is important to discuss the contribution of neuronal and non-neuronal cells in HFD-induced hypothalamic inflammation. Furthermore, understanding few other questions, such as the diets causing hypothalamic inflammation, the gender disparity in response to HFD feeding, and how hypothalamic inflammation affects ARC neurons to cause impaired metabolism, will be helpful for the development of therapeutic approaches to prevent or treat HFD-induced obesity.

Metabolic feedback from the periphery to the brain results from a dynamic physiologic fluctuation of nutrients and hormones, including glucose and fatty acids, ghrelin, leptin, and insulin. The specific interactions between humoral factors and how they influence feeding is largely unknown. We hypothesized that acute glucose availability may alter how the brain responds to ghrelin, a hormonal signal of energy availability. Acute glucose administration suppressed a range of ghrelin-induced behaviors as well as gene expression changes in hypothalamic neuropeptide Y (NPY) and agouti-related peptide (AgRP) neurons after ghrelin administration. Knockdown of the energy-sensing molecule AMP-activated protein kinase (AMPK) in AgRP neurons resulted in loss of the glucose effect, and mice responded as though pretreated with saline. Conversely, 2-deoxyglucose (2-DG), which decreases glucose availability, potentiated ghrelin-induced feeding and increased hypothalamic NPY mRNA levels. AMPK knockdown did not alter the additive effect of 2-DG and ghrelin on feeding. Our findings support the idea that computation of energy status is dynamic, is informed by multiple signals, and responds to acute fluctuations in metabolic state. These observations are broadly relevant to the investigation of neuroendocrine control of feeding and highlight the underappreciated complexity of control within these systems.

The BBSome, a complex of eight Bardet-Biedl syndrome (BBS) proteins involved in cilia function, has emerged as an important regulator of energy balance, but the underlying cellular and molecular mechanisms are not fully understood. Here, we show that the control of energy homeostasis by the anorexigenic proopiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AgRP) neurons require intact BBSome. Targeted disruption of the BBSome by Bbs1 gene deletion in POMC or AgRP neurons increases body weight and adiposity. We demonstrate that obesity in mice lacking the Bbs1 gene in POMC neurons is associated with hyperphagia. Mechanistically, we present evidence implicating the BBSome in the trafficking of G protein-coupled neuropeptide Y Y2 receptor (NPY2R) and serotonin 5-hydroxytryptamine (HT)2C receptor (5-HT2CR) to cilia and plasma membrane, respectively. Consistent with this, loss of the BBSome reduced cell surface expression of the 5-HT2CR, interfered with serotonin-evoked increase in intracellular calcium and membrane potential, and blunted the anorectic and weight-reducing responses evoked by the 5-HT2cR agonist, lorcaserin. Finally, we show that disruption of the BBSome causes the 5-HT2CR to be stalled in the late endosome. Our results demonstrate the significance of the hypothalamic BBSome for the control of energy balance through regulation of trafficking of important metabolic receptors.

Currently available inducible Cre/loxP systems, despite their considerable utility in gene manipulation, have pitfalls in certain scenarios, such as unsatisfactory recombination rates and deleterious effects on physiology and behavior. To overcome these limitations, we designed a new, inducible gene-targeting system by introducing an in-frame nonsense mutation into the coding sequence of Cre recombinase (nsCre). Mutant mRNAs transcribed from nsCre transgene can be efficiently translated into full-length, functional Cre recombinase in the presence of nonsense suppressors such as aminoglycosides. In a proof-of-concept model, GABA signaling from hypothalamic neurons expressing agouti-related peptide (AgRP) was genetically inactivated within 4 d after treatment with a synthetic aminoglycoside. Disruption of GABA synthesis in AgRP neurons in young adult mice led to a dramatic loss of body weight due to reduced food intake and elevated energy expenditure; they also manifested glucose intolerance. In contrast, older mice with genetic inactivation of GABA signaling by AgRP neurons had only transient reduction of feeding and body weight; their energy expenditure and glucose tolerance were unaffected. These results indicate that GABAergic signaling from AgRP neurons plays a key role in the control of feeding and metabolism through an age-dependent mechanism. This new genetic technique will augment current tools used to elucidate mechanisms underlying many physiological and neurological processes.

Targeting Gβγ subunits in hypothalamic AgRP neurons to treat obesity.

Recent studies from our laboratory have demonstrated that within the arcuate nucleus of the hypothalamus (ARC), angiotensin II (ANG) type 1A receptors (AT1A) are expressed by the subset of neurons which express both the leptin receptor (LepR) and agouti‐related peptide (AgRP). AT1A expression in this subpopulation of AgRP neurons is critically involved in the integrative control of thermogenic adipose sympathetic nerve activity (SNA) and thereby resting metabolic rate (RMR) by various stimuli including leptin. It remains unclear, however, how LepR signaling results in AT1A activation within AgRP neurons. We hypothesize that activation of LepR (i) increases de novo synthesis of AGT and thus autocrine and/or paracrine ANG signaling within the ARC, and (ii) increases AT1A expression and trafficking to the cell membrane, increasing AgRP neuron sensitivity to ANG. Consistent with our first hypothesis, AGT is co‐localized within the ARC of C57BL/6J mice with cells expressing AgRP and LepR, as determined by fluorescent in situ hybridization and confocal microscopy. Studies of an immortalized mouse hypothalamic cell line (N43/5 cells, which exhibit a gene signature typical of AgRP cells) demonstrate that AGT mRNA is increased in response to leptin treatment. Selective genetic disruption of the AGT gene in LepR or AgRP neurons (AGTLepR‐KO and AGTAgRP‐KO mice) has no effect on energy flux under baseline chow‐fed conditions, though ongoing studies are focused on examining metabolic responses of these animals to high fat diet and leptin. Consistent with our second hypothesis, leptin application to N43/5 cells results in increased AT1A mRNA, and leptin injection in wildtype C57BL/6J mice increases AT1A mRNA in the ARC. Ongoing studies utilizing the new HiBiT‐tagging system (Promega) support cell surface localization of AT1A in the N43/5 cell line, which is increased by leptin treatment and reduced by ANG application. Ongoing studies are aimed at (i) examining the roles of pSTAT3, PI3K and ERK in the stimulation of AGT and AT1A expression by leptin, (ii) further characterizing AGTLepR‐KO and AGTAgRP‐KO mice by examining SNA and RMR in response to high fat diet and leptin, and (iii) using the HiBiT extracellular detection system to clarify the mechanism(s) by which leptin modulates cell surface localization of AT1A.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anorexia nervosa (AN) is an eating disorder observed primarily in girls and women, and is characterized by a low body mass index, hypophagia, and hyperactivity. The activity-based anorexia (ABA) paradigm models aspects of AN, and refers to the progressive weight loss, hypophagia, and hyperactivity developed by rodents exposed to time-restricted feeding and running wheel access. Recent studies identified white adipose tissue (WAT) as a primary location of the ‘metabolic memory’ of prior obesity, and implicated WAT-derived signals as drivers of recidivism to obesity following weight loss. Here, we tested whether an obese WAT transplant could attenuate ABA-induced weight loss in normal female mice. Recipient mice received a WAT transplant harvested from normal chow-fed, or HFD-fed obese mice; obese fat recipient (OFR) and control fat recipient (CFR) mice were then tested for ABA. During ABA, OFR mice survived longer than CFR mice, defined as maintaining 75% of their initial body weight. Next, we tested whether agouti-related peptide (AgRP) neurons, which regulate feeding behavior and metabolic sensing, mediate this effect of obese WAT transplant. CFR and OFR mice received either control or neonatal AgRP ablation, and were assessed for ABA. OFR intact mice maintained higher body weights longer than CFR intact mice, and this effect was abolished by neonatal AgRP ablation; further, ablation reduced survival in OFR, but not CFR mice. In summary, obese WAT transplant communicates with AgRP neurons to increase body weight maintenance during ABA. These findings encourage the examination of obese WAT-derived factors as potential treatments for AN.

Role of the Energy Sensor AMP-Activated Protein Kinase (AMPK) and Its Downstream Effector Uncoupling Protein 2 (UCP2) in the Orexigenic Effect of Endogenous Ghrelin

Angiotensin AT1R expressing cells within the ventral hypothalamus modulate integrative control of cardiometabolic functions