High sugar-containing foods are readily consumed, even after meals and beyond fullness sensation (e.g., as desserts). Although reward-driven processing of palatable foods can promote overeating, the neurobiological mechanisms that underlie the selective appetite for sugar in states of satiety remain unclear. Hypothalamic pro-opiomelanocortin (POMC) neurons are principal regulators of satiety because they decrease food intake through excitatory melanocortin neuropeptides. We discovered that POMC neurons not only promote satiety in fed conditions but concomitantly switch on sugar appetite, which drives overconsumption. POMC neuron projections to the paraventricular thalamus selectively inhibited postsynaptic neurons through mu-opioid receptor signaling. This opioid circuit was strongly activated during sugar consumption, which was most notable in satiety states. Correspondingly, inhibiting its activity diminished high-sugar diet intake in sated mice.

The hypothalamus is a brain region that plays a key role in coordinating fundamental biological functions1. However, our understanding of the underlying cellular components and neurocircuitries have, until recently, emerged primarily from rodent studies2,3. Here we combine single-nucleus sequencing of 433,369 human hypothalamic cells with spatial transcriptomics, generating a comprehensive spatio-cellular transcriptional map of the hypothalamus, the 'HYPOMAP'. Although conservation of neuronal cell types between humans and mice, as based on transcriptomic identity, is generally high, there are notable exceptions. Specifically, there are significant disparities in the identity of pro-opiomelanocortin neurons and in the expression levels of G-protein-coupled receptors between the two species that carry direct implications for currently approved obesity treatments. Out of the 452 hypothalamic cell types, we find that 291 neuronal clusters are significantly enriched for expression of body mass index (BMI) genome-wide association study genes. This enrichment is driven by 426 'effector' genes. Rare deleterious variants in six of these (MC4R, PCSK1, POMC, CALCR, BSN and CORO1A) associate with BMI at population level, and CORO1A has not been linked previously to BMI. Thus, HYPOMAP provides a detailed atlas of the human hypothalamus in a spatial context and serves as an important resource to identify new druggable targets for treating a wide range of conditions, including reproductive, circadian and metabolic disorders.

Amylin analogs, including potential anti-obesity therapies like cagrilintide, act on neurons in the brainstem dorsal vagal complex (DVC) that express calcitonin receptors (CALCR). These receptors, often combined with receptor activity-modifying proteins (RAMPs), mediate the suppression of food intake and body weight. To understand the molecular and neural mechanisms of cagrilintide action, we used single-nucleus RNA sequencing to define 89 cell populations across the rat, mouse, and non-human primate caudal brainstem. We then integrated spatial profiling to reveal neuron distribution in the rat DVC. Furthermore, we compared the acute and long-term transcriptional responses to cagrilintide across DVC neurons of rats, which exhibit strong cagrilintide responsiveness, and mice, which respond poorly to cagrilintide over the long term. We found that cagrilintide promoted long-term transcriptional changes, including increased prolactin releasing hormone (Prlh) expression, in the nucleus of the solitary tract (NTS) Calcr/Prlh cells in rats, but not in mice, suggesting the importance of NTS Calcr/Prlh cells for sustained weight loss. Indeed, activating rat area postrema Calcr cells briefly reduced food intake but failed to decrease food intake or body weight over the long term. Overall, these results not only provide a cross-species and spatial atlas of DVC cell populations but also define the molecular and neural mediators of acute and long-term cagrilintide action.

Glucagon-like peptide-1 (GLP-1) and leptin signal recent feeding and long-term energy stores, respectively, and play complementary roles in the modulation of energy balance. Previous work using single-cell techniques in mice revealed the existence of a population of leptin receptor (Lepr)-containing dorsomedial hypothalamus (DMH) neurons marked by the expression of GLP-1 receptor (Glp1r; LepRGlp1r neurons) that play important roles in the control of feeding and body weight by leptin. Here, we demonstrate the existence of a population of LepRGlp1r neurons in the DMHs of nonhuman primates (NHPs), suggesting the potential translational relevance of these neurons. Consequently, we developed a GLP-1R/LepR dual agonist and demonstrated the physiological activity of both components in vivo using leptin-deficient and Lepr-deficient murine models. We further found roles for LepRGlp1r neurons in mediating the dual agonist's efficacy on food intake and body weight loss. Ablating Lepr in Glp1r-expressing neurons (LeprGlp1rKO mice) abrogated the suppression of food intake by the dual agonist. Furthermore, reactivation of Glp1r expression in Lepr neurons on an otherwise Glp1r-null background (Glp1rLeprRe mice) was sufficient to permit the suppression of food intake and body weight by the dual agonist. Hence, LepRGlp1r neurons represent targets for a GLP-1R/LepR dual agonist that potently reduces food intake and body weight.

Heat acclimation is an adaptive process that improves physiological performance and supports survival in the face of increasing environmental temperatures, but the underlying mechanisms are not well understood. Here we identified a discrete group of neurons in the mouse hypothalamic preoptic area (POA) that rheostatically increase their activity over the course of heat acclimation, a property required for mice to become heat tolerant. In non-acclimated mice, peripheral thermoafferent pathways via the parabrachial nucleus activate POA neurons and mediate acute heat-defense mechanisms. However, long-term heat exposure promotes the POA neurons to gain intrinsically warm-sensitive activity, independent of thermoafferent parabrachial input. This newly gained cell-autonomous warm sensitivity is required to recruit peripheral heat tolerance mechanisms in acclimated animals. This pacemaker-like, warm-sensitive activity is driven by a combination of increased sodium leak current and enhanced utilization of the NaV1.3 ion channel. We propose that this salient neuronal plasticity mechanism adaptively drives acclimation to promote heat tolerance. Ambroziak, Nencini, Pohle and colleagues identify a slowly emerging plasticity mechanism in a discrete set of hypothalamic preoptic neurons that is triggered by long-term heat exposure and that drives thermal acclimation to promote heat tolerance in mice.

Weight loss and regain are associated with profound cytoskeleton changes in collapsing and re-expanding adipocytes. These drastic changes in the adipose tissue may drive proinflammatory signalling and fibrotic processes during weight regain. It remains unclear how adipose tissue re-expansion affects adipose tissue function and thereby the comorbidities related to obesity. The aim of this study was to characterize structural and metabolic changes in subcutaneous adipose tissue after weight loss and regain in diet-induced obesity (DIO) mice. DIO mice underwent calorie restriction to achieve a 25% weight loss and then returned to ad libitum high fat diet feeding to regain weight. Inguinal adipose tissue was collected and analysed from five groups of mice during the experiment: before high fat diet feeding (Lean) , before weight loss (DIO 1) , after weight loss (WL) , after 3 weeks of weight regain (WR) and age-matched ad libitum DIO mice (DIO 2) . We found that WR group had lower weight and fat mass compared to DIO2 group (31.9±1.6 vs. 23.9±1.9 %) and lower basal lipolysis measured ex-vivo. We detected a larger population of small adipocytes in WR mice compared to DIO2 group, resulting in smaller average adipocyte size (81.6±1.4 vs. 52.5±0.9 µm) and increased expression of cell renewal markers Cidea, Antrx1 and Prkaa in adipose tissue, indicating activation of adipogenesis. Expression of cytokines Il2, Ccl19, Cdc3 in adipose tissue in WR compared to DIO was reduced as well as plasma levels of cytokines Il2, KC/GRO and Inf-γ. In conclusion, our study does not support that increased adipogenesis in weight regain results in accelerated fat accumulation in mice. Weight regain is accompanied by reduced systemic and adipose inflammation. This suggests in general a beneficial effect of regain-induced adipogenesis resulting in potentially healthier adipose tissue in mice. Weather these findings represent the human situation with weight loss and regain is unknown and is currently under investigation. Disclosure M. K. Gerstenberg: Employee; Novo Nordisk. R. E. Kuhre: Employee; Novo Nordisk. N. Petersen: Employee; Novo Nordisk A/S. A. Bookout: Employee; Novo Nordisk. C. M. Castorena: Employee; Novo Nordisk. L. Torz: Other Relationship; Novo Nordisk A/S. T. H. Dovmark: None. S. Lundh: Employee; Novo Nordisk A/S. S. H. Madsen: None. B. S. Andersen: Employee; Novo Nordisk A/S. T. A. Pedersen: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S.

Therapies based on glucagon-like peptide-1 (GLP-1) long-acting analogs and insulin are often used in the treat-ment of metabolic diseases. Both insulin and GLP-1 receptors are expressed in metabolically relevant brain regions, suggesting a cooperative action. However, the mechanisms underlying the synergistic actions of in-sulin and GLP-1R agonists remain elusive. In this study, we show that insulin-induced hypoglycemia enhances GLP-1R agonists entry in hypothalamic and area, leading to enhanced whole-body fat oxidation. Mechanisti-cally, this phenomenon relies on the release of tanycyctic vascular endothelial growth factor A, which is selec-tively impaired after calorie-rich diet exposure. In humans, low blood glucose also correlates with enhanced blood-to-brain passage of insulin, suggesting that blood glucose gates the passage other energy-related sig-nals in the brain. This study implies that the preventing hyperglycemia is important to harnessing the full benefit of GLP-1R agonist entry in the brain and action onto lipid mobilization and body weight loss.

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

Raw data prism files for the manuscript "A selective role for receptor activity-modifying protein in sub-chronic action of the amylin selective receptor agonist NN1213 compared to salmon calcitonin on body weight and food intake in male mice"

The amylin receptor (AMY) and calcitonin receptor (CTR) agonists induce acute suppression of food intake in rodents by binding to receptors in the area postrema (AP) and potentially by targeting arcuate (ARC) neurons directly. Salmon calcitonin (sCT) induces more potent, longer lasting anorectic effects compared to amylin. We thus aimed to investigate whether AMY/CTR agonists target key neuronal populations in the ARC, and whether differing brain distribution patterns could mediate the observed differences in efficacy with sCT and amylin treatment. Brains were examined by whole brain 3D imaging and confocal microscopy following subcutaneous administration of fluorescently labelled peptides to mice. We found that sCT, but not amylin, internalizes into a subset of ARC NPY neurons, along with an unknown subset of ARC, AP and dorsal vagal motor nucleus cells. ARC POMC neurons were not targeted. Furthermore, amylin and sCT displayed similar distribution patterns binding to receptors in the AP, the organum vasculosum of the lamina terminalis (OVLT) and the ARC. Amylin distributed within the median eminence with only specs of sCT being present in this region, however amylin was only detectable 10 minutes after injection while sCT displayed a residence time of up to 2 hours post injection. We conclude that AMY/CTR agonists bind to receptors in a subset of ARC NPY neurons and in circumventricular organs. Furthermore, the more sustained and greater anorectic efficacy of sCT compared to rat amylin is not attributable to differences in brain distribution patterns but may more likely be explained by greater potency at both the CTR and AMY.