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.This work was supported by the Medical Research Council New Blood Fellowship [MR/M501736/1] to CB, a NIDDK K99/R00 award to CB, the Medical Research Council Metabolic Disease Unit programme grant, the Disease Model Core facilities, and the Wellcome Trust Cambridge Mouse Biochemistry Laboratory. Animal procedures were performed under Tony Coll’s home office PPL 80/2497 and Toni Vidal-Puig PPL 80/2484

Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF[alfa]) blunt the response of Neuropeptide Y/Agouti-related peptide (NPY/AgRP) glucose inhibited (GI) neurons to decreased glucose

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Rapid sensing of l-leucine by human and murine hypothalamic neurons: Neurochemical and mechanistic insights.

OBJECTIVE: Dietary proteins are sensed by hypothalamic neurons and strongly influence multiple aspects of metabolic health, including appetite, weight gain, and adiposity. However, little is known about the mechanisms by which hypothalamic neural circuits controlling behavior and metabolism sense protein availability. The aim of this study is to characterize how neurons from the mediobasal hypothalamus respond to a signal of protein availability: the amino acid l-leucine. METHODS: We used primary cultures of post-weaning murine mediobasal hypothalamic neurons, hypothalamic neurons derived from human induced pluripotent stem cells, and calcium imaging to characterize rapid neuronal responses to physiological changes in extracellular l-Leucine concentration. RESULTS: A neurochemically diverse subset of both mouse and human hypothalamic neurons responded rapidly to l-leucine. Consistent with l-leucine's anorexigenic role, we found that 25% of mouse MBH POMC neurons were activated by l-leucine. 10% of MBH NPY neurons were inhibited by l-leucine, and leucine rapidly reduced AGRP secretion, providing a mechanism for the rapid leucine-induced inhibition of foraging behavior in rodents. Surprisingly, none of the candidate mechanisms previously implicated in hypothalamic leucine sensing (KATP channels, mTORC1 signaling, amino-acid decarboxylation) were involved in the acute activity changes produced by l-leucine. Instead, our data indicate that leucine-induced neuronal activation involves a plasma membrane Ca2+ channel, whereas leucine-induced neuronal inhibition is mediated by inhibition of a store-operated Ca2+ current. CONCLUSIONS: A subset of neurons in the mediobasal hypothalamus rapidly respond to physiological changes in extracellular leucine concentration. Leucine can produce both increases and decreases in neuronal Ca2+ concentrations in a neurochemically-diverse group of neurons, including some POMC and NPY/AGRP neurons. Our data reveal that leucine can signal through novel mechanisms to rapidly affect neuronal activity

Brainstem Raphe Pallidus and the Adjacent Area Contain a Novel Action Site in the Melanocortin Circuitry Regulating Energy Balance

The central melanocortin system plays a critical role in the regulation of energy balance in rodents and humans. The melanocortin signals in both the hypothalamus and brainstem contribute to this regulation. However, how the melanocortin signals of the hypothalamus interact with those intrinsic to the brainstem in the regulation of energy balance is poorly understood. The brainstem raphe pallidus (RPa) and adjacent areas contain melanocortin 4 receptor (MC4-R)-bearing neurons and sympathetic premotor neurons regulating thermogenesis. Here we report that α-melanocyte-stimulating hormone (α-MSH)-immunoreactive (IR) fibers are in close apposition to MC4-R neurons in the RPa. Retrograde tracing studies revealed a unique direct projection from hypothalamic proopiomelanocortin (POMC) neurons to the RPa and adjacent areas of the brainstem in mice and rats. Furthermore, microinjection of the MC3/4-R agonist MTII into the RPa area dose-dependently stimulated oxygen consumption and inhibited feeding, whereas microinjection of the antagonist, SHU9119, enhanced feeding. These data suggest a novel pathway of hypothalamic POMC neuronal efferents to brainstem RPa area MC4-R neurons in the melanocortin circuitry that contribute to coordinate regulation of energy balance

AgRP neuron activity is required for acute exercise-induced feeding

While much is known about the role of neuropeptide Y/agouti-regulated peptide (NPY/AgRP) and pro-opiomelanocortin (POMC) neurons to regulate energy homeostasis, little is known about how forced energy expenditure, such as exercise, modulates these neurons and how this relates to energy intake. Therefore, we investigated the effects of acute exercise on neuronal activity in the arcuate nucleus (ARC) of the hypothalamus. To accomplish this, we utilized immunohistochemistry and patch-clamp electrophysiology experiments on NPY-GFP transgenic mice immediately after an acute bout of treadmill exercise. Due to the ability of NPY/AgRP and POMC neurons to mediate energy homeostasis, food intake studies were also performed immediately after an acute bout of treadmill exercise. AgRP-Ires-cre transgenic mice were used to induce loss in AgRP neuronal activation by bilaterally injecting an inhibitory cre-recombinase–dependent Adeno Associated Virus (AAV-hM4Di-mCherry) to assess AgRP neurons in food intake post-exercise. While we observed no difference in activation in POMC neurons, immediately after exercise, activation in ARC NPY/AgRP neurons is significantly increased compared to the sedentary control group; further confirmed by electrophysiology recording showing a significant increase in firing rate in NPY/AgRP neurons after acute exercise. Food intake was significantly increased immediately after an acute bout of exercise. This exercise-induced food intake was abolished when AgRP neuron activation was inhibited. Neuronal inhibition of AgRP neurons had no effect of hypothalamic paraventricular nucleus (PVN) activation immediately after a bout of acute exercise. Our results demonstrate NPY/AgRP activation is critical for acute exercise induced food intake in mice, thus providing insight into the subtle exercise induced response to facilitate energy replacement

Nutrient sensing in the nucleus of the solitary tract mediates non-aversive suppression of feeding via inhibition of AgRP neurons.

UNLABELLED: The nucleus of the solitary tract (NTS) is emerging as a major site of action for the appetite-suppressive effects of leading pharmacotherapies currently investigated to treat obesity. However, our understanding of how NTS neurons regulate appetite remains incomplete. OBJECTIVES: In this study, we used NTS nutrient sensing as an entry point to characterize stimulus-defined neuronal ensembles engaged by the NTS to produce physiological satiety. METHODS: We combined histological analysis, neuroanatomical assessment using inducible viral tracing tools, and functional tests to characterize hindbrain-forebrain circuits engaged by NTS leucine sensing to suppress hunger. RESULTS: We found that NTS detection of leucine engages NTS prolactin-releasing peptide (PrRP) neurons to inhibit AgRP neurons via a population of leptin receptor-expressing neurons in the dorsomedial hypothalamus. This circuit is necessary for the anorectic response to NTS leucine, the appetite-suppressive effect of high-protein diets, and the long-term control of energy balance. CONCLUSIONS: These results extend the integrative capability of AgRP neurons to include brainstem nutrient sensing inputs

Perineuronal Net Formation and the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus

In leptin-deficient ob/ob mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC)1, a critical brain area for energy and glucose homoeostasis2,3. Because this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period for leptin-dependent development of ARC neurocircuits has been proposed4. In other brain areas, critical-period closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh5. Here we report that in humans and rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed in PNN-like structures. In mice, these neurons are densely packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both γ-aminobutyric acid-ergic and leptin-receptor positive, including a majority of Agouti-related-peptide neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the critical period for maturation of Agouti-related-peptide neurons and is dependent on input from circulating leptin, because postnatal ob/ob mice have reduced ARC PNN-like material that is restored by leptin administration during the critical period. We conclude that neurons crucial to metabolic homoeostasis are enmeshed in PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC–median eminence junction, where metabolically relevant humoral signals are sensed

Metabolic insights from a GHSR-A203E mutant mouse model

Objective: Binding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR), stimulates GH release, induces eating, and increases blood glucose. These processes may also be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by findings in short people with naturally occurring GHSR-A204E mutations and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. In this study, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. Methods: We generated mice with a GHSR mutation that replaces alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the previously described human GHSR-A204E mutation, and used them to conduct ex vivo neuronal electrophysiology and in vivo metabolic assessments. We also measured signaling within COS-7 and HEK293T cells transfected with wild-type GHSR (GHSR-WT) or GHSR-A203E constructs. Results: In COS-7 cells, GHSR-A203E resulted in lower baseline IP3 accumulation than GHSR-WT; ghrelin-induced IP3 accumulation was observed in both constructs. In HEK293T cells co-transfected with voltage-gated CaV2.2 calcium channel complex, GHSR-A203E had no effect on basal CaV2.2 current density while GHSR-WT did; both GHSR-A203E and GHSR-WT inhibited CaV2.2 current in the presence of ghrelin. In cultured hypothalamic neurons from GHSR-A203E and GHSR-deficient mice, native calcium currents were greater than those in neurons from wild-type mice; ghrelin inhibited calcium currents in cultured hypothalamic neurons from both GHSR-A203E and wild-type mice. In brain slices, resting membrane potentials of arcuate NPY neurons from GHSR-A203E mice were hyperpolarized compared to those from wild-type mice; the same percentage of arcuate NPY neurons from GHSR-A203E and wild-type mice depolarized upon ghrelin exposure. The GHSR-A203E mutation did not significantly affect body weight, body length, or femur length in the first ∼6 months of life, yet these parameters were lower in GHSR-A203E mice after 1 year of age. During a 7-d 60% caloric restriction regimen, GHSR-A203E mice lacked the usual marked rise in plasma GH and demonstrated an exaggerated drop in blood glucose. Administered ghrelin also exhibited reduced orexigenic and GH secretagogue efficacies in GHSR-A203E mice. Conclusions: Our data suggest that the A203E mutation ablates constitutive GHSR activity and that constitutive GHSR activity contributes to the native depolarizing conductance of GHSR-expressing arcuate NPY neurons. Although the A203E mutation does not block ghrelin-evoked signaling as assessed using in vitro and ex vivo models, GHSR-A203E mice lack the usual acute food intake response to administered ghrelin in vivo. The GHSR-A203E mutation also blunts GH release, and in aged mice leads to reduced body length and femur length, which are consistent with the short stature of human carriers of the GHSR-A204E mutation.Fil: Torz, Lola J.. Universidad de Copenhagen; DinamarcaFil: Osborne Lawrence, Sherri. Ut Southwestern Medical Center; Estados UnidosFil: Rodriguez, Juan. Ut Southwestern Medical Center; Estados UnidosFil: He, Zhenyan. Ut Southwestern Medical Center; Estados UnidosFil: Cornejo, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Mustafá, Emilio Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Jin, Chunyu. Universidad de Copenhagen; DinamarcaFil: Petersen, Natalia. Universidad de Copenhagen; DinamarcaFil: Hedegaard, Morten A.. Universidad de Copenhagen; DinamarcaFil: Nybo, Maja. Universidad de Copenhagen; DinamarcaFil: Martínez Damonte, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Metzger, Nathan P.. Ut Southwestern Medical Center; Estados UnidosFil: Mani, Bharath K.. Ut Southwestern Medical Center; Estados UnidosFil: Williams, Kevin W.. Ut Southwestern Medical Center; Estados UnidosFil: Raingo, Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Holst, Birgitte. Universidad de Copenhagen; DinamarcaFil: Zigman, Jeffrey M.. Ut Southwestern Medical Center; Estados Unido

Characterisation of distinct inhibitory interneuron populations in the spinal dorsal horn

The dorsal horn of the spinal cord is the first node in the somatosensory pathway, and is an area essential for controlling the flow of sensory information sent to the brain. Interneurons constitute the vast majority of neurons in this area, and between 25-40% of those in laminae I-III are inhibitory. These inhibitory interneurons are critical for normal somatosensation, for example, by suppressing pain in the absence of noxious stimuli. Interneurons of the dorsal horn are poorly understood due to their morphological and functional diversity, and this is a major factor limiting our understanding of the neuronal circuitry of the dorsal horn. In order to better understand sensory processing in the dorsal horn it is first necessary to characterise the neurons in this area, and to determine the neuronal circuits in which they are integrated. To address this issue, two separate and non-overlapping populations of inhibitory interneurons in the dorsal horn were thoroughly characterised in terms of their morphological and physiological properties. To achieve this, whole-cell recordings were taken from neurons labelled with green fluorescent protein (GFP) under the control of the Prion promoter (PrP) and the neuropeptide Y (NPY) promoter in spinal cord slices from mice. The recording electrodes contained Neurobiotin, which filled the cells during recording and was revealed with fluorescent molecules, enabling three-dimensional reconstruction of cell bodies and dendrites and axons of neurons. Slices containing these labelled neurons were then resectioned for immunocytochemical reactions to determine their neurochemical content and their synaptic inputs and outputs. This study demonstrated that both PrP- and NPY-GFP cells were morphologically heterogeneous although neither group contained islet cells, which are a distinct morphological class of interneuron. PrP- and NPY-GFP cells in lamina II could not be distinguished from each other by using hierarchical cluster analysis with measures of somatodendritic morphology. This suggests that morphological properties may not be useful in distinguishing these populations of interneurons. The vast majority of PrP- and NPY-GFP cells either displayed tonic or initial burst firing of action potentials. However, these groups of cells showed significant differences in some of their active and passive membrane properties, such as membrane resistance, spike frequency adaptation and mV drop in action potential height. When hierarchical cluster analysis was used to group these cells in lamina II based on physiological parameters, PrP- and NPY-GFP cells could be distinguished with some accuracy. This suggests that some physiological differences may exist between these two groups. Within the PrP-GFP group there was a subset that included lamina I among its synaptic outputs, and these cells could provide inhibition to the projection neurons located in this lamina, since GFP boutons from this mouse line can form synapses with giant cells and neurokinin-1 receptor (NK1r)-expressing lamina I neurons. Some PrP-GFP cells showed immunoreactivity for neuronal nitric oxide synthase (nNOS) or galanin, and these two groups had slight morphological differences, which included their laminar location and the spread of their processes. Several experimental approaches, such as electrophysiological, pharmacological and anatomical techniques, indicated that PrP-GFP cells received input from many different types of primary afferent fibre, including peptidergic and non-peptidergic C-afferents, as well as low-threshold mechanosensory fibres. Taken together these findings establish the PrP-GFP cells as a much more functionally heterogeneous group than previously reported. NPY-GFP cells were located in laminae II and III, but were preferentially found in lamina III. The lamina III cells had dendrites with a greater dorsoventral extent than the lamina II cells, and this extent was seen be more dorsal from the soma than ventral. Many NPY-GFP cells received synaptic input from C-fibres, and a subset of those tested lacked TRPV1. Since the TRPV1-lacking C-fibres mostly correspond to the non-peptidergic C-fibres, including non-peptidergic nociceptors and C-low threshold mechanoreceptors, this suggests that NPY-GFP cells could receive input from these fibres. Dorsal root stimulation experiments showed that labelled NPY-GFP cells with somata located in lamina III often received synaptic input from unmyelinated C-fibres, and NPY-expressing neurons in lamina III could respond to noxious mechanical stimuli. A select group of NPY-GFP cells were seen to innervate putative anterolateral tract (ALT) neurons located in lamina III, which could be identified by their dense innervation by bundles of axons containing either NPY or calcitonin gene related peptide (CGRP). Taken together these data suggest that the PrP- and NPY-GFP neurons are distinct populations based on their primary afferent input and post-synaptic targets, and that more than one functional population exists within each of these groups. Despite their many differences, morphological parameters do not appear to be useful in distinguishing the PrP- and NPY-GFP cells, or detecting different functional populations within these groups. The PrP-GFP cells are more morphologically heterogeneous than previous reports suggested, and due to similar features with cells that require the transcription factor Bhlhb5 to develop, they may include a population that are involved in suppressing itch-related signals. NPY-GFP cells could play a role in limiting the spread and intensity of noxious stimuli due to their input from C-fibres, and a small subset of these could inhibit ALT neurons in lamina III. These results further support the view that different neurochemical populations of inhibitory neurons have distinct functional roles, and also highlight the complexity of the neuronal circuitry in the superficial dorsal horn

Heterogeneity of hypothalamic pro-opiomelanocortin-expressing neurons revealed by single-cell RNA sequencing

$\textbf{Objective}$ Arcuate proopiomelanocortin (POMC) neurons are critical nodes in the control of body weight. Often characterized simply as direct targets for leptin, recent data suggest a more complex architecture. $\textbf{Methods}$ Using single cell RNA sequencing, we have generated an atlas of gene expression in murine POMC neurons. $\textbf{Results}$ Of 163 neurons, 118 expressed high levels of $\textit{Pomc}$ with little/no Agrp expression and were considered “canonical” POMC neurons (P$^{+}$). The other 45/163 expressed low levels of $\textit{Pomc}$ and high levels of $\textit{Agrp}$ (A$^{+}$P$_{+}$). Unbiased clustering analysis of P$^{+}$ neurons revealed four different classes, each with distinct cell surface receptor gene expression profiles. Further, only 12% (14/118) of P$^{+}$ neurons expressed the leptin receptor ($\textit{Lepr}$) compared with 58% (26/45) of A$^{+}$P$_{+}$ neurons. In contrast, the insulin receptor ($\textit{Insr}$) was expressed at similar frequency on P$^{+}$ and A$^{+}$P$_{+}$ neurons (64% and 55%, respectively). $\textbf{Conclusion}$ These data reveal arcuate POMC neurons to be a highly heterogeneous population. Accession Numbers: GSE92707.This work was supported by the UK Medical Research Council (MRC) Metabolic Disease Unit (MRC_MC_UU_12012/1 & MRC_MC_UU_12012/5), a Wellcome Trust Strategic Award (100574/Z/12/Z), and the Helmholtz Alliance ICEMED