Arcuate Nucleus Of The Hypothalamus Research Articles

Systemic metabolic benefits of 17α-estradiol are not exclusively mediated by ERα in glutamatergic or GABAergic neurons.

17α-Estradiol (17αE2), a less-feminising enantiomer of 17β-estradiol, has been shown to prolong lifespan and improve metabolic health in a sex-specific manner in male, but not in female mice. Recent studies have demonstrated the pivotal role of estrogen receptor α (ERα) in mediating the effects of 17αE2 on metabolic health. However, the specific tissues and/or neuronal signalling pathways that 17αE2 acts through remain to be elucidated. ERα expression in glutamatergic and GABAergic neurons (principal excitatory and inhibitory neurons respectively) in the hypothalamus is essential for estradiol signalling. Therefore, we hypothesised that knocking out ERα from one of these neuronal populations would attenuate the established beneficial metabolic effects of 17αE2 in male mice exposed to a high fat diet. To test this hypothesis we used two established brain specific ERα KO models, targeting either glutamatergic or GABAergic neurons (Vglut2/Vgat-ERαKO). We show that both of these ERα KO models exhibit a strong reduction in ERα expression in the arcuate nucleus of the hypothalamus, a control centre for metabolic regulation. Deletion of ERα from GABAergic neurons significantly diminished the effect of 17αE2 on body weight relative to controls, although these animals still show metabolic benefits with 17αE2 treatment. The response to 17αE2 was unaffected by ERα deletion in glutamatergic neurons. Our results support a benefit of 17αE2 treatment in protection against metabolic dysfunction, but these effects do not depend on exclusive ERα expression in glutamatergic and GABAergic neurons and persist when ERα expression is strongly reduced in the arcuate nucleus of the hypothalamus.

SerpinA3N in Leptin Receptor Neurons Regulates Metabolic and Glucose Homeostasis

Obesity and associated conditions such as type 2 diabetes are among the most concerning issues for health in the U.S. Leptin receptor signaling in the brain plays a pivotal role in the regulation of food intake and energy expenditure. High fat diet induces inflammation within the hypothalamus, leading to leptin insensitivity which contribute to obesity. Previous studies have shown that a novel gene SerpinA3N, a serine protease inhibitor, which is highly expressed in the arcuate nucleus of the hypothalamus, where the leptin receptor is also highly expressed, is upregulated in diet-induced obesity and by leptin stimulation. However, the role of SerpinA3N in the leptin receptor expressing cells in the control of body weight and glucose homeostasis remain unknown. RNAseq analysis showed that SerpinA3N mRNA levels were significantly upregulated in the hypothalamus of mice fed high fat high sucrose diet (HFHSD) for 12 weeks. Upregulation of hypothalamic SerpinA3N in HFHSD fed mice was confirmed by Western blot. This increase in hypothalamic SerpinA3N appears to be caused by excess of nutrient as we found that exposure to fatty acid significantly increased SerpeinA3N promoter activity in mouse hypothalamic N39 cells. Using immunofluorescence staining, we confirmed that SerpinA3N is expressed the leptin receptor positive cells of the hypothalamus. Next, we investigated whether loss of SerpinA3N in leptin receptor expressing neurons affects metabolic and glucose homeostasis. For this, we crossed mice harboring floxed alleles of the SerpinA3N gene (SerpinA3Nfl/fl) with mice expressing Cre recombinase in leptin receptor cells (LepRCre). We found that female, but not male, conditional knockout (CKO, LepRCre/SerpinA3Nfl/fl) mice have significantly lower body weight when fed normal chow or HFHSD. This lower body weight was due to decreased fat mass, measured by NMR. However, no alteration in food intake was observed between female CKO mice and controls, indicating that increased energy expenditure may account for the lower body weight in female CKO mice. Interestingly, CKO mice displayed exaggerated weight loss and anorectic response following leptin treatment (1 mg/g, twice daily, IP), indicating that loss of SerpinA3N enhances leptin sensitivity. Female CKO mice fed normal chow diet also exhibited enhanced insulin sensitivity compared to control animals. On the other hand, male CKO mice displayed significantly improved glucose handling, but not change in insulin sensitivity, only when fed HFHSD. Finally, insulin-induced phosphorylation of AKT was elevated in skeletal muscle, but not in liver and white adipose tissue, of CKO mice relative to controls. These results demonstrate that SerpinA3N in leptin receptor-expressing neurons regulate body weight and glucose homeostasis in a sex-dependent manner. NIH and VA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Angiotensin-(1-7) Mas Receptors are Highly Colocalized to GABAergic Proopiomelanocortin Neurons in the Arcuate Nucleus of the Hypothalamus

Previous studies from our laboratory have shown that circulating levels of angiotensin (Ang)-(1-7), a protective hormone of the renin-angiotensin system, are greatly reduced in obese hypertensive mice. Additionally, restoration of this hormone reduces cardiovascular sympathetic tone and lowers blood pressure in this model suggesting a central mechanism of action. In support of this, our recent data show that the sympatho-inhibitory and blood pressure lowering effects of Ang-(1-7) involve activation of mas receptors (masR) within the arcuate nucleus of the hypothalamus (ARC). More specifically, we have found that masR are highly colocalized to proopiomelanocortin (POMC) neurons in the ARC. Due to the heterogeneous nature of POMC neuron subtypes, we wanted to explore the neurotransmitter phenotype of these POMCmasR neurons. We hypothesized that due to the blood pressure lowering effects of Ang-(1-7), POMCmasR are likely colocalized to inhibitory GABAergic neurons. To test this, we performed RNAscope in-situ hybridization in male C57Bl/6J mice after twelve weeks of either a 60% high fat diet (HFD) or matched control diet (n=12-18/group). We used RNAscope to colocalize expression of POMC, masR, and the vesicular GABA transporter, Vgat, mRNA transcripts within the ARC. Colocalization of POMCmasR and Vgat was quantified and percent-colocalization compared between control and HFD groups. We found that POMCmasR are highly colocalized with the GABAergic marker, Vgat, to a similar extent in control and HFD groups (84±4 vs. 90±2% of neurons, respectively; p=0.294 unpaired t-test). The population of POMC neurons that did not express masR also did not colocalize with Vgat. As an initial step to determine if masR modulates GABAergic signaling in the ARC, we assessed gene expression of glutamate decarboxylase 2 (Gad2; a GABA synthetic enzyme) in the ARC following chronic six-week treatment with systemic Ang-(1-7) versus saline infusion in male mice fed a control diet versus HFD (n=9-13/group). We found that Ang-(1-7) increases ARC Gad2 mRNA in HFD mice (1.03±0.09 control diet vs. 0.93±0.07 HFD vs. 1.23±0.07 HFD plus Ang-(1-7); p=0.023 one-way ANOVA). Supporting specificity of these actions, Ang-(1-7) did not alter POMC mRNA in the ARC. These overall data suggest that masR primarily localize to GABA-expressing POMC neurons in the ARC, and Ang-(1-7) may directly modulate GABA synthesis pathways in this brain region. Further studies are needed to elucidate the functional importance of these ARC POMC GABAergic neurocircuits to the depressor effects of Ang-(1-7) in obesity. Funding: NIH R01 HL156986. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Metabolic Function of Leucine-Rich Repeat Transmembrane Neuronal 4 Expressed by Hypothalamic AgRP Neurons

It is well known that the agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus regulate energy balance and glucose homeostasis. While it was previously shown that excitatory and inhibitory synaptic input onto the AgRP neurons influence the activity and metabolic function of AgRP neurons, little is known about the role of synaptic adhesion molecules therein. In this study, we focused on a synaptic adhesion molecule, leucine-rich repeat transmembrane neuronal 4 (LRRTM4), which is expressed by post-synaptic part of excitatory synapses to be involved in synapse formation and synaptic transmission. We generated conditional knockout mice which lacks LRRTM4 specifically in the AgRP neurons, and assessed the electrophysiological and metabolic phenotypes. Our results should provide insight how synaptic machinery of hypothalamic AgRP neurons can shape in vivo metabolic function. This work was supported by the National Research Foundation of Korea (NRF-2022R1A2C3005613 to Jong-Woo Sohn) funded by the Korean Ministry of Science and ICT. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypothalamic GABAergic neurons expressing Cellular Retinoic Acid Binding Protein 1 (CRABP1) are sensitive to metabolic status and liraglutide in male mice.

Owing to their privileged anatomical location, neurons of the arcuate nucleus of the hypothalamus (ARC) play critical roles in sensing and responding to metabolic signals such as leptin and glucagon-like peptide 1 (GLP-1). In addition to the well-known proopiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons, subpopulations of GABAergic neurons are emerging as key regulators of energy balance. However, the precise identity of these metabolic neurons is still elusive. Here, we identified and characterized the molecular signature of a novel population of GABAergic neurons of the ARC expressing Cellular retinoic acid binding protein 1 (Crabp1). Using a combination of immunohistochemistry and in situ hybridization techniques, we investigated the expression of Crabp1 across the mouse brain and characterized the molecular identity of Crabp1ARC neurons. We also determined whether Crabp1ARC neurons are sensitive to fasting, leptin and GLP1R agonism by assessing cFOS immunoreactivity as a marker of neuronal activity. Crabp1ARC neurons represent a novel GABAergic neuronal population robustly enriched in the ARC and distinct from the prototypical melanocortin neurons. Crabp1ARC neurons overlap with three subpopulations of yet uncharacterized ARC neurons expressing Htr3b, Tbx19 and Tmem215, respectively. Notably, Crabp1ARC neurons express receptors for metabolic hormones and their activity is modulated by the nutritional state and GLP1R agonism. Crabp1ARC neurons represent a novel heterogenous population of GABAergic neurons sensitive to metabolic status.

Hypothalamic POMC neuron-specific knockout of MC4R affects insulin sensitivity by regulating Kir2.1

BackgroundImbalance in energy regulation is a major cause of insulin resistance and diabetes. Melanocortin-4 receptor (MC4R) signaling at specific sites in the central nervous system has synergistic but non-overlapping functions. However, the mechanism by which MC4R in the arcuate nucleus (ARC) region regulates energy balance and insulin resistance remains unclear.MethodsThe MC4Rflox/flox mice with proopiomelanocortin (POMC) -Cre mice were crossed to generate the POMC-MC4Rflox/+ mice. Then POMC-MC4Rflox/+ mice were further mated with MC4Rflox/flox mice to generate the POMC-MC4Rflox/flox mice in which MC4R is selectively deleted in POMC neurons. Bilateral injections of 200 nl of AAV-sh-Kir2.1 (AAV-sh-NC was used as control) were made into the ARC of the hypothalamus. Oxygen consumption, carbon dioxide production, respiratory exchange ratio and energy expenditure were measured by using the CLAMS; Total, visceral and subcutaneous fat was analyzed using micro-CT. Co-immunoprecipitation assays (Co-IP) were used to analyze the interaction between MC4R and Kir2.1 in GT1-7 cells.ResultsPOMC neuron-specific ablation of MC4R in the ARC region promoted food intake, impaired energy expenditure, leading to increased weight gain and impaired systemic glucose homeostasis. Additionally, MC4R ablation reduced the activation of POMC neuron, and is not tissue-specific for peripheral regulation, suggesting the importance of its central regulation. Mechanistically, sequencing analysis and Co-IP assay demonstrated a direct interaction of MC4R with Kir2.1. Knockdown of Kir2.1 in POMC neuron-specific ablation of MC4R restored the effect of MC4R ablation on energy expenditure and systemic glucose homeostasis, indicating by reduced body weight and ameliorated insulin resistance.ConclusionHypothalamic POMC neuron-specific knockout of MC4R affects energy balance and insulin sensitivity by regulating Kir2.1. Kir2.1 represents a new target and pathway that could be targeted in obesity.

Physiological Appetite Regulation and Bariatric Surgery.

Obesity remains a common metabolic disorder and a threat to health as it is associated with numerous complications. Lifestyle modifications and caloric restriction can achieve limited weight loss. Bariatric surgery is an effective way of achieving substantial weight loss as well as glycemic control secondary to weight-related type 2 diabetes mellitus. It has been suggested that an anorexigenic gut hormone response following bariatric surgery contributes to weight loss. Understanding the changes in gut hormones and their contribution to weight loss physiology can lead to new therapeutic treatments for weight loss. Two distinct types of neurons in the arcuate hypothalamic nuclei control food intake: proopiomelanocortin neurons activated by the anorexigenic (satiety) hormones and neurons activated by the orexigenic peptides that release neuropeptide Y and agouti-related peptide (hunger centre). The arcuate nucleus of the hypothalamus integrates hormonal inputs from the gut and adipose tissue (the anorexigenic hormones cholecystokinin, polypeptide YY, glucagon-like peptide-1, oxyntomodulin, leptin, and others) and orexigeneic peptides (ghrelin). Replicating the endocrine response to bariatric surgery through pharmacological mimicry holds promise for medical treatment. Obesity has genetic and environmental factors. New advances in genetic testing have identified both monogenic and polygenic obesity-related genes. Understanding the function of genes contributing to obesity will increase insights into the biology of obesity. This review includes the physiology of appetite control, the influence of genetics on obesity, and the changes that occur following bariatric surgery. This has the potential to lead to the development of more subtle, individualised, treatments for obesity.

Perinatal Western-style diet exposure associated with decreased microglial counts throughout the arcuate nucleus of the hypothalamus in Japanese macaques.

Perinatal exposure to a high-fat, high-sugar Western-style diet (WSD) is associated with altered neural circuitry in the melanocortin system. This association may have an underlying inflammatory component, as consumption of a WSD during pregnancy can lead to an elevated inflammatory environment. Our group previously demonstrated that prenatal WSD exposure was associated with increased markers of inflammation in the placenta and fetal hypothalamus in Japanese macaques. In this follow-up study, we sought to determine whether this heightened inflammatory state persisted into the postnatal period, as prenatal exposure to inflammation has been shown to reprogram offspring immune function and long-term neuroinflammation would present a potential means for prolonged disruptions to microglia-mediated neuronal circuit formation. Neuroinflammation was approximated in 1-yr-old offspring by counting resident microglia and peripherally derived macrophages in the region of the hypothalamus examined in the fetal study, the arcuate nucleus (ARC). Microglia and macrophages were immunofluorescently stained with their shared marker, ionized calcium-binding adapter molecule 1 (Iba1), and quantified in 11 regions along the rostral-caudal axis of the ARC. A mixed-effects model revealed main effects of perinatal diet (P = 0.011) and spatial location (P = 0.003) on Iba1-stained cell count. Perinatal WSD exposure was associated with a slight decrease in the number of Iba1-stained cells, and cells were more densely located in the center of the ARC. These findings suggest that the heightened inflammatory state experienced in utero does not persist postnatally. This inflammatory response trajectory could have important implications for understanding how neurodevelopmental disorders progress.NEW & NOTEWORTHY Prenatal Western-style diet exposure is associated with increased microglial activity in utero. However, we found a potentially neuroprotective reduction in microglia count during early postnatal development. This trajectory could inform the timing of disruptions to microglia-mediated neuronal circuit formation. Additionally, this is the first study in juvenile macaques to characterize the distribution of microglia along the rostral-caudal axis of the arcuate nucleus of the hypothalamus. Nearby neuronal populations may be greater targets during inflammatory insults.

Rôle de l’axe microbiote-intestin-cerveau dans la dérégulation du comportement alimentaire au cours de l’obésité et de l’hyperphagie boulimique : les mécanismes: Implication of the microbiota-gut-brain axis in the changes of eating behavior in obesity and binge eating disorder (BED): the mecanisms

Eating disorders (ED), particularly compulsive ED such as binge eating disorder (BED), play an important role in the pathophysiology of obesity. Recent data have highlighted the involvement of the microbiotagut-brain axis in the regulation of eating behavior; its deregulation could therefore contribute to the onset of obesity via the development or perpetuation of ED, in particular BED. This review highlights the various peripheral and central pathophysiological mechanisms linking intestinal dysbiosis and the disruption of eating behavior leading to obesity and BED. The main peripheral regulatory signals involved are peptides associated with food intake (ghrelin, GLP-1), short-chain fatty acids and bacterial components. The brain integration of peripheral and central signals modulating food intake, involving at homeostatic (arcuate nucleus of the hypothalamus) and hedonic (reward system) levels, will be described during BED and obesity. This better understanding of the role of the microbiota-gut-brain axis in the control of eating behavior in relation to the pathophysiology of obesity opens up prospects for innovative therapeutic strategies targeting gut microbiota reshaping.© 2023 Société francophone nutrition clinique et métabolisme (SFNCM).Published by Elsevier Masson SAS. All rights reserved.

Hypothalamic free fatty acid receptor-1 regulates whole-body energy balance

ObjectiveFree fatty acid receptor-1 (FFAR1) is a medium- and long-chain fatty acid sensing G protein-coupled receptor that is highly expressed in the hypothalamus. Here, we investigated the central role of FFAR1 on energy balance. MethodsCentral FFAR1 agonism and virogenic knockdown were performed in mice. Energy balance studies, infrared thermographic analysis of brown adipose tissue (BAT) and molecular analysis of the hypothalamus, BAT, white adipose tissue (WAT) and liver were carried out. ResultsPharmacological stimulation of FFAR1, using central administration of its agonist TUG-905 in diet-induced obese mice, decreases body weight and is associated with increased energy expenditure, BAT thermogenesis and browning of subcutaneous WAT (sWAT), as well as reduced AMP-activated protein kinase (AMPK) levels, reduced inflammation, and decreased endoplasmic reticulum (ER) stress in the hypothalamus. As FFAR1 is expressed in distinct hypothalamic neuronal subpopulations, we used an AAV vector expressing a shRNA to specifically knockdown Ffar1 in proopiomelanocortin (POMC) neurons of the arcuate nucleus of the hypothalamus (ARC) of obese mice. Our data showed that knockdown of Ffar1 in POMC neurons promoted hyperphagia and body weight gain. In parallel, these mice developed hepatic insulin resistance and steatosis. ConclusionsFFAR1 emerges as a new hypothalamic nutrient sensor regulating whole body energy balance. Moreover, pharmacological activation of FFAR1 could provide a therapeutic advance in the management of obesity and its associated metabolic disorders.

405 The Effects of Disease-Induced Inflammation on Reproductive Neuroendocrinology: Evidence from Sheep

Abstract Reproduction is dictated by the cohesive functioning of the organs of the hypothalamic-pituitary-gonadal (HPG) axis. As the name suggests, reproduction starts within the hypothalamus through the production and pulsatile secretion of gonadotropin releasing hormone (GnRH) followed by subsequent secretion of luteinizing hormone (LH) from the anterior pituitary and then steroidogenesis and gametogenesis in the gonads. Additionally, the HPG axis is tightly regulated by internal and external cues and, over the last twenty years, kisspeptin has been identified as a key modulator for reproduction through its stimulatory actions on GnRH neurons to secrete GnRH and subsequently LH. Growing evidence indicates that these kisspeptin expressing neurons are negatively affected by stressors (e.g., inflammation, social stress, metabolic stress) resulting in impaired reproduction. Inflammation caused by lipopolysaccharide (LPS, endotoxin), a component of the cell membrane of Gram-negative bacteria (e.g., Escherichia coli), is of particular interest due to its relationship with common livestock ailments, such as mastitis and metritis, and its suppression of GnRH and LH secretions. However, there is limited information about the influence disease-induced inflammation has on kisspeptin expressing neurons. In rats, acute endotoxin inflammation will cause about a 50% decrease in the number of kisspeptin immunopositive cells in the arcuate nucleus along with a suppression in LH secretion. In ewes, an acute dose will decrease the proportion of activated kisspeptin neurons in the ARC and POA. However, the mechanisms by which inflammation affects reproduction are not completely understood, especially in domestic livestock. Through a series of experiments, we aim to elucidate these mechanisms and effects of endotoxinemia in both an acute and a chronic model. To accomplish these goals, we administered endotoxin acutely to ewes. Overall, the suppression of LH secretion at the level of hypothalamus is possible, however, more research is required to further elucidate the mechanism. To evaluate a chronic model of systemic inflammation, we evaluated routes of endotoxin administration by randomly assigning twenty two castrated male sheep into one of five groups - control (CON), single acute IV dose (SAD; 400 ng/kg BW LPS) administered on Day 1, daily acute IV dose (DAD; 400 ng/kg BW LPS), daily increasing IV dose (DID; starting with 400 ng/kg BW LPS and increasing 20% each day), or steady subcutaneous dose (SSD; 20 µg/kg BW LPS over 24 hours), all administered during a one-week study period. Chronic inflammation, induced by a daily rising dose of IV administered LPS, suppresses LH secretion through effects on kisspeptin protein expression in the middle arcuate nucleus of the hypothalamus of sheep. Taken together, systemic inflammation associated with infection and disease likely impairs reproduction in part through suppression of kisspeptin-expressing neurons.

Modulation of GABA release by 5-HT1B receptors: An interplay with AMPA-receptors and voltage-gated Ca2+ channels

Obesity has become a worldwide health challenge and commonly results from the intake of more calories than the body requires. The brain represents the master controller of food intake and as such has been the target of obesity medications. However, key mechanisms of druggable targets remain to be defined. Neurons within the arcuate nucleus of the hypothalamus co-expressing neuropeptide Y (NPY), agouti-related protein (AgRP) and GABA (NAG) are fundamental stimulators of hunger and food intake. NAG neurons also inhibit local satiety-promoting pro-opiomelanocortin (POMC) neurons. Agonists of the 1B subtype of metabotropic serotonin receptor (5-HT1BR) reduce food intake in part through the inhibition of hunger-promoting NAG neurons. We first confirmed that 5-HT1BR activation suppressed intake of a palatable Western diet in a mouse model of common dietary-induced obesity and genetically prone obesity. Next, we combined several electrophysiological approaches to analyse the effect of 5-HT1BRs in NAG neuron cell activity and GABA release. 5-HT1BR activation reduced NAG neuron action potential frequency and neurotransmitter release. We found that 5-HT1BR impact on GABA release from NAG neurons is mediated through voltage-gated Ca2+ channels with a critical input from glutamate receptors of AMPA subtype (AMPARs). As a fundamental outcome, this type of interplay provides an uncommon example of metabotropic action of AMPARs which regulates inhibitory signalling due to modulation of GABA release. As a translational outcome, our results provide a key mechanism through which 5-HT1BR drugs inhibit appetite-stimulating neurons within the brain to suppress food intake.This article is part of the Special Issue on “Ukrainian Neuroscience”.

THU128 Transcription Factor ETV5 In POMC Neurons Regulate Energy And Glucose Homeostasis

Abstract Disclosure: S. Gahlot: None. A. Caron: None. S. Lee: None. J.K. Elmquist: None. Pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) are critical for the regulation of energy balance and glucose homeostasis. Pomc gene transcription is regulated by the Erythrocyte Transformation Specific (ETS)-domain transcription factors, including Etv1, Etv2, Etv4, and Etv5. Large-scale genome-wide association studies have identified ETV5 genetic variants linked with human obesity and body mass index. Histological studies in adult mouse pituitaries also show the co-localization of ETV5 with POMC and POMC-derived peptides: adrenocorticotropic hormone (ACTH) and alpha-melanocyte stimulating hormone (α-MSH). Furthermore, whole-body Etv5 knockout mice are lean, severely glucose intolerant and hypoinsulinaemic. While these findings suggest a potential role for Etv5 in the control of energy and glucose balance, the role of Etv5 remains unknown. This study aims to determine whether ETV5 binds to the Pomc promoter to alter its expression and regulate energy balance and glucose homeostasis. To assess whether ETV5 binds to the Pomc promoter, we performed chromatin immunoprecipitation with quantitative real-time PCR (ChIP-qPCR) in C57BL/6J mice. ChIP-qPCR determined that Etv5 directly binds to the Pomc promoter. We further performed luciferase reporter assay in mouse neuroblastoma N2a cell lysates to measure the Pomc promoter activity after Etv5 knockdown or overexpression. Luciferase assay revealed that Etv5 knockdown increased, while Etv5 overexpression reduced the Pomc promoter activity in N2a cells. Also, Etv5 overexpression blocked the leptin-induced POMC activation. Further, to determine the role of Etv5 in POMC neurons to regulate energy balance and glucose homeostasis, we used the Cre/loxP system to selectively delete Etv5 from POMC neurons both prenatally and postnatally. Mice were fed either a standard chow or a 60% high-fat diet (HFD), followed by serial body weight, food intake, body composition, metabolic assessment, glucose tolerance and insulin tolerance test measurements. We found that mice with a prenatal deletion of Etv5 in POMC neurons had reduced body weight, reduced lean mass, and were protected from HFD-induced insulin resistance. Similarly, postnatal deletion of Etv5 in POMC neurons resulted in reduced body weight, lean mass and fat mass, and increased insulin sensitivity and energy expenditure in mice of 8 weeks age. We also performed electrophysiological recordings in hypothalamic slices from these mice to assess POMC neuron excitability. We found that loss of Etv5 increased the activity and electrical excitability of POMC neurons. These findings highlight a previously unrecognized role of Etv5 in POMC neurons in the regulation of energy balance and glucose homeostasis. These results also point to the potential therapeutic applications for Etv5 against obesity. Presentation: Thursday, June 15, 2023

SAT592 Sexually Dimorphic Modulation Of Arcuate Kisspeptin Neurons Activity In Response To NPY

Abstract Disclosure: N.D. Mansano: None. R. Frazao: None. Kisspeptin neurons are part of an intricate brain circuit that receives neuronal inputs related tonutritional status from at least three neuronal populations, including NPY/AgRP and POMC neurons of the arcuate nucleus of the hypothalamus (ARH) and cells in the ventral premammillary nucleus. Its well-known action on the HPG axis, hypothalamic kisspeptin neurons also modulate the activity of specific neurons of the paraventricular and the dorsomedial nuclei of the hypothalamus, regions involved in the regulation of appetite and energy expenditure. However, it is not completely elucidated if NPY can influence the HPG axis through the modulation of kisspeptin neuron activity. To determine the NPY effect in the hypothalamic kisspeptin neurons we performed whole-cell patch-clamp recordings. The hypothalamic slices were obtained from the adult Kiss1/hrGFP female (diestrus-stage) or male mice. Kisspeptin cells located in the arcuate nucleus of the hypothalamus (ARHkisspeptin) and at the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeNkisspeptin) were recorded. On female mice, ARHkisspeptin neurons exhibited a rest membrane potential (RMP) average of -52.5±2.5 mV (-40 to -62 mV) and 1.3±0.2 GΩ input resistance (IR). NPY induced the hyperpolarization of 45% of the ARHkisspeptin neurons (n= 9 of 20 cells, out of 9 mice). ARHkisspeptin neuron’s RMP hyperpolarization was followed by a significant decrease in IR and frequency of action potentials (fAPS). In the AVPV/PeNkisspeptin neurons showed RMP average of -69.6±1.9 mV (-51 to - 88 mV) and IR of 0.8±0.09 GΩ (n=18 cells out of 9 mice). No significant NPY-induced effect was noted when recordings were performed on AVPV/PeNkisspeptin cells from female mice. To elucidate if the ARHkisspeptin neuron’s hyperpolarization depends on action potentials (APs) mediated by synaptic transmission we further evaluated NPY effects in the presence of TTX and amino acid receptor antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10μM), DL-2-amino-5-phosphonopentanoic acid (AP5, 50μM), and picrotoxin (50μM). NPY induced no effect on ARHkisspeptin neuron’s RMP, and IR in the presence of TTX and amino acid receptor antagonists. Suggesting that NPY-induced hyperpolarization of ARHkisspeptin neurons is an indirect effect. To further verify whether sex must be considered as a biological variable to study the NPY effect, we recorded ARHkisspeptin cells from male mice. ARHkisspeptin neurons recorded from male mice exhibited RPM average of -55.6±1.5 (-45 to -66 mV) and IR of 1.5±0.15 GΩ (n= 13 cells out of 10 mice). Interestingly, NPY induced no effect on RMP, IR, or fAPs in male animals. In conclusion, our results suggest that NPY-induced hyperpolarization of ARHkisspeptin cells depends on AP’s mediate synaptic transmission, as a sex-specific effect. Presentation: Saturday, June 17, 2023

FRI007 Kisspeptin Does Not Affect Food Intake In Women With Overweight Or Obesity

Abstract Disclosure: C. Izzi-Engbeaya: None. M. Choudhury: None. B. Patel: None. B. Muzi: None. A. Qayum: None. E.G. Mills: None. M. Ahsan: None. M. Phylactou: None. S. Clarke: None. L. Aslett: None. A.N. Comninos: None. A. Abbara: None. T. Tan: None. W.S. Dhillo: None. Background: Kisspeptin neurons in the hypothalamus are believed to play a role in mediating the interaction between metabolic and reproductive health. Functional neuroanatomical connections exist between kisspeptin neurons and appetite-regulating neurons in the arcuate nucleus of the hypothalamus. Female (but not male) global kisspeptin receptor knockout mice have reduced food intake compared to controls. However, studies of kisspeptin administration have yielded conflicting results, with either decreased, or unchanged food intake reported in rodent studies. Consistent with some of the literature, kisspeptin administration does not influence food intake in men. However, the effects of kisspeptin on food intake have not previously been studied in women. Research Question: What is the effect of kisspeptin administration on food intake in women? Methodology: A randomized controlled crossover study was performed in women with overweight or obesity (BMI >25 kg/m2) who were not in receipt of exogenous oral and/or transdermal estrogens or progestins. Each woman attended two study visits in random order following an overnight fast. During each study visit they received either a 2-hour intravenous infusion of kisspeptin-54 at a rate of 1.0 nmol/kg/hr, or a rate-matched vehicle infusion. The study was single-blinded, with participants unaware of the infusion identity. Participants were given an ad libitum meal 45 minutes after the start of each infusion. Blood samples were taken for reproductive and metabolic profiling, and visual analogue scales (VAS) to assess hunger were completed at regular intervals throughout both study visits. Data are presented as mean±SD. Results: 17 women (age 49±12 years, BMI 34±7 kg/m2) completed both study visits. As expected, LH levels were higher during kisspeptin than during vehicle infusion (p<0.01), confirming bioactivity of the kisspeptin dose. Pre-meal estradiol levels were similar during kisspeptin and vehicle infusions (p=0.34). There were no differences in pre-meal hunger VAS scores 30 mins after the start of the infusion (kisspeptin 5.2±2.7 vs vehicle 5.9±2.3, p=0.33) or bodyweight-adjusted food intake (kisspeptin 6.8±3.7 kcal/kg vs vehicle 6.2±2.8 kcal/kg, p=0.33). There were also no differences in glucose (p=0.97) or insulin (p=0.68) levels between kisspeptin and vehicle infusions. Conclusions: Acute intravenous administration of a biologically active dose of kisspeptin did not affect food intake or hunger in women with overweight or obesity. Furthermore, kisspeptin administration did not alter fasting or post-prandial glucose levels. This is the first study to examine the effects of kisspeptin on hunger and food intake in women and provides reassuring safety data for the ongoing development of kisspeptin-based therapeutics. Presentation: Friday, June 16, 2023

FRI284 The GnRH Pulse Generator Activity In Mouse Models Of Polycystic Ovary Syndrome

Abstract Disclosure: Z. Zhou: None. M. Pardo-Navarro: None. S.Y. Han: None. A.E. Herbison: None. Up to one in five women worldwide suffer from polycystic ovary syndrome (PCOS), a condition typified by problems with fertility, high androgen levels, and altered metabolism. Primarily due to the unknown aetiology of this disorder, limited strategies are available for PCOS treatment. The hypothalamic GnRH pulse generator is responsible for driving the pulsatile release of LH to control ovarian function. As PCOS patients show profound increase in LH pulse frequency, it is thought that over-activity of the GnRH pulse generator may have a major role in generating the sub-fertility of women with PCOS. Recent studies involving the real-time imaging of neural activity in genetic mouse models have revealed that a population of kisspeptin neurons located in the arcuate nucleus of the hypothalamus is the long-sought after “GnRH pulse generator”. These arcuate kisspeptin neurons exhibit synchronization events (SEs) that correlate perfectly with pulsatile LH secretion. With this technique, we aimed to characterize the activity pattern of the arcuate kisspeptin neurons in two most-widely used mouse models of PCOS: prenatal androgen (PNA) and peripubertal androgen models. In the first model, PNA female mice exhibited near-acyclic estrous cycles as previously reported, but surprisingly exhibited only a slightly (∼30%) increased frequency of arcuate kisspeptin neuron SEs over a 24-hour recording period compared to the controls. The profiles and dynamics of individual SEs were not different between PNA and control animals. Concurrent fiber photometry recording and serial blood sampling showed that each SE is tightly followed by an LH pulse in all animals. Three weeks following ovariectomy, the SE frequency and profile were not different between PNA and control mice. In the second peripubertal model, female mice implanted with dihydrotestosterone capsules at 3-weeks of age exhibited significant weight gain but decreased arcuate kisspeptin neuron activity over a 24-hour recording period also without changes in SE dynamics compared to the controls. In conclusion, it appears that neither of the two most-commonly used PCOS mouse models mirror the human condition in which many PCOS women exhibit robust increases in pulsatile LH secretion. Presentation: Friday, June 16, 2023

FRI275 Glutamate Receptor Agonists Stimulate Kisspeptin Neurons In The Arcuate Nucleus Of The Hypothalamus

Abstract Disclosure: R. Bearss: None. R. Piet: None. KNDy neurons co-express kisspeptin, neurokinin B (NKB), dynorphin A (Dyn A), and glutamate. These neurons, located in the arcuate nucleus of the hypothalamus, drive pulsatile gonadotropin releasing hormone (GnRH) secretion. This eventually promotes pulsatile gonadotropin hormone release from the anterior pituitary, important for fertility in both sexes. The current hypothesized mechanism for pulse generation involves KNDy neuron interconnectivity and reciprocal regulation through the combined actions of NKB, DynA and glutamate to coordinate KNDy neuron activity. There is evidence that KNDy neurons express NKB, Dyn A and glutamate receptors and that NKB stimulates KNDy neuron electrical activity while Dyn A suppresses it. There is also evidence that ionotropic glutamate receptor antagonists can inhibit KNDy neuron coordinated activity and pulsatile gonadotropin hormone release, indicating that glutamatergic neurotransmission plays a significant role in activity coordination. The purpose of the following experiments was to investigate the response of KNDy neurons to varied glutamate receptor activation. We used calcium imaging to monitor the activity of KNDy neurons in brain slices. We crossed Kiss1-Cre mice—expressing Cre recombinase (Cre) in kisspeptin neurons—with Cre-dependent GCaMP6f mice to generate Kiss1-Cre x GCaMP6f mice that express GCaMP6f in kisspeptin neurons. GCaMP6f fluorescence increases when binding to intracellular calcium and can, thus, be used as an inferential marker of electrical activity. Coronal brain slices (200 µm thickness) obtained from male Kiss1-cre x GCaMP6f mice and containing the arcuate nucleus were placed in a recording chamber under an epifluorescence microscope. KNDy neurons were exposed for 2 minutes to a bath application of either the ionotropic glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 10µM), the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA, 50µM), or the group 1 metabotropic glutamate receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 50µM). AMPA, NMDA, and DHPG increased KNDy neuron fluorescence by 7.8±0.5% (n=113 in 7 slices from 6 mice), 7.5±0.5% (n=154 in 6 slices from 5 mice), and 6.1±0.7% (n=159 in 7 slices from 5 mice) respectively. All cells that were excited by NMDA or DHPG were also excited by AMPA. These experiments provide evidence that activation of ionotropic AMPA and NMDA receptors as well as group 1 mGluRs stimulate KNDy neuron activity. Further investigation into the precise role of individual glutamate receptor subtypes on KNDy neuron coordinated activity is needed. Presentation: Friday, June 16, 2023

FRI064 The Role Of GLP-1 Receptor Agonists In The Treatment Of Pituitary And Hypothalamic Tumor Induced Obesity

Abstract Disclosure: C.S. Botero Suarez: None. D. Broutin: None. H. Yau: None. Introduction: Glucagon-like peptide-1 receptor agonists (GLP1-RA) act by stimulating the GLP-1 receptor resulting in a reduction in food intake, increased energy expenditure, and improved glycemic control. The role of GLP1-RA in pituitary/suprasellar tumors causing hyperphagia and hypothalamic obesity is yet unknown. We present a case of a patient with a large non-functional pituitary mass resulting in severe hyperphagia and hypothalamic obesity. A trial of GLP-1RA showed significant improvement of hyperphagia. Case Description: A 30-year-old African American male is seen in the Endocrinology clinic. He was recently diagnosed with a non-functional pituitary mass 2 months prior, after a presentation of hyperphagia, significant weight gain, and altered personality changes causing significant social distress. He underwent transsphenoidal pituitary resection which resulted in postoperative hypopituitarism. His physical exam showed normal vital signs, a weight of 185 lbs, and a BMI of 25. His medications included hydrocortisone total of 15 mg daily, desmopressin 0.1mg daily, levothyroxine 137mcg daily, and risperidone 1mg daily. A brain MRI showed postoperative changes depicted by a T1 hyperintensity within the sella as well as T2 hyperintensity of the chiasm and hypothalamus indicating edema and a mass effect. Based on his presentation of marked hyperphagia and rapid weight gain with suggestive imaging, the clinical diagnosis of hypothalamic obesity was made. Further follow-up revealed a rapid weight gain of 45 lbs in 2 months, a worsening glycemic control, and a HbA1C of 7.2% compared to a HbA1C of 5.4% in his prior visit. Lab work showed a TSH of 0.017 mlU/mL, fT4 of 1.4ng/dL, prolactin 3.94 ng/mL, alpha subunit of 0.1 ng/mL (reference 0.1-0.5 ng/mL), ACTH of 15 pg/mL, morning cortisol of < 3 ug/dL. Given the significant metabolic consequences of his marked hyperphagia, related weight gain, and new onset of diabetes, the patient was started on semaglutide 0.5mg weekly and later up-titrated to 1mg weekly. There was marked improvement of hyperphagia, and his weight remained neutral at the 3-month follow-up. Discussion: Hyperphagia is a rare presentation of sellar and suprasellar tumors and can lead to hypothalamic obesity. Tumors can affect the arcuate nucleus of the hypothalamus, which is a key regulator of energy homeostasis and is regulated by orexigenic (AgRP/NPY) and anorexigenic neurons (POMC/CART). GLP-1R agonists, have shown to cause significant weight loss due to decreased gastric emptying and stimulating POMC/CART neurons, but they also inhibit neuropeptide Y (NPY) and agouti-related peptide (AgRP), leading to reduced hunger, energy intake, and food cravings. Although the clinical role of GLP1-RA in pituitary tumors with significant hyperphagia is unknown, we believe early initiation can help avoid a rapid progression of obesity and glycemic deterioration. Presentation: Friday, June 16, 2023

Fasting Modulates GABAergic Synaptic Transmission to Arcuate Kisspeptin Neurons in Female Mice.

It is well-established that the hypothalamic-pituitary-gonadal (HPG) axis is suppressed due to negative energy balance. However, less information is available on whether kisspeptin neuronal activity contributes to fasting-induced responses. In the present study, female and male mice were fasted for 24 hours or provided food ad libitum (fed group) to determine whether acute fasting is sufficient to modulate kisspeptin neuronal activity. In female mice, fasting attenuated luteinizing hormone (LH) and prolactin (PRL) serum levels and increased follicle-stimulating hormone levels compared with the fed group. In contrast, fasting did not affect gonadotropin or PRL secretion in male mice. By measuring genes related to LH pulse generation in micropunches obtained from the arcuate nucleus of the hypothalamus (ARH), we observed that fasting reduced Kiss1 mRNA levels in female and male mice. In contrast, Pdyn expression was upregulated only in fasted female mice, whereas no changes in the Tac2 mRNA levels were observed in both sexes. Interestingly, the frequency and amplitude of the GABAergic postsynaptic currents recorded from ARH kisspeptin neurons (ARHKisspeptin) were reduced in 24-hour fasted female mice but not in males. Additionally, neuropeptide Y induced a hyperpolarization in the resting membrane potential of ARHKisspeptin neurons of fed female mice but not in males. Thus, the response of ARHKisspeptin neurons to fasting is sexually dependent with a female bias, associated with changes in gonadotropins and PRL secretion. Our findings suggest that GABAergic transmission to ARHKisspeptin neurons modulates the activity of the HPG axis during situations of negative energy balance.

Electrophysiological Comparison of Definitive Pro-opiomelanocortin Neurons in the Arcuate Nucleus and the Retrochiasmatic Area of Male and Female Mice.

Pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC) are considered a major site of leptin action. Due to increasing evidence that POMC neurons are highly heterogeneous and indications that the conventional molecular tools to study their functions have important limitations, a reassessment of leptin's effects on definitive POMC neurons is needed. POMC neurons are also expressed in the retrochiasmatic area (RCA), where their function is poorly understood. Furthermore, the response of POMC neurons to leptin in females is largely unknown. Therefore, the present study aimed to determine the differences in leptin responsiveness of POMC neurons in the ARC and the RCA using a mouse model allowing adult-inducible fluorescent labeling. We performed whole-cell patch clamp electrophysiology on 154 POMC neurons from male and female mice. We confirmed and extended the model by which leptin depolarizes POMC neurons, in both the ARC and the RCA. Furthermore, we characterized the electrophysiological properties of an underappreciated subpopulation representing ∼10% of hypothalamic POMC neurons that are inhibited by leptin. We also provide evidence that sex does not appear to be a major determinant of basal properties and leptin responsiveness of POMC neurons, but that females are overall less responsive to leptin compared to males.