GHSR in a Subset of GABA Neurons Controls Food Deprivation-Induced Hyperphagia in Male Mice.

The growth hormone secretagogue receptor (GHSR), primarily known as the receptor for the hunger hormone ghrelin, potently controls food intake, yet the specific Ghsr-expressing cells mediating the orexigenic effects of this receptor remain incompletely characterized. Since Ghsr is expressed in gamma-aminobutyric acid (GABA)-producing neurons, we sought to investigate whether the selective expression of Ghsr in a subset of GABA neurons is sufficient to mediate GHSR's effects on feeding. First, we crossed mice that express a tamoxifen-dependent Cre recombinase in the subset of GABA neurons that express glutamic acid decarboxylase 2 (Gad2) enzyme (Gad2-CreER mice) with reporter mice, and found that ghrelin mainly targets a subset of Gad2-expressing neurons located in the hypothalamic arcuate nucleus (ARH) and that is predominantly segregated from Agouti-related protein (AgRP)-expressing neurons. Analysis of various single-cell RNA-sequencing datasets further corroborated that the primary subset of cells coexpressing Gad2 and Ghsr in the mouse brain are non-AgRP ARH neurons. Next, we crossed Gad2-CreER mice with reactivable GHSR-deficient mice to generate mice expressing Ghsr only in Gad2-expressing neurons (Gad2-GHSR mice). We found that ghrelin treatment induced the expression of the marker of transcriptional activation c-Fos in the ARH of Gad2-GHSR mice, yet failed to induce food intake. In contrast, food deprivation-induced refeeding was higher in Gad2-GHSR mice than in GHSR-deficient mice and similar to wild-type mice, suggesting that ghrelin-independent roles of GHSR in a subset of GABA neurons is sufficient for eliciting full compensatory hyperphagia in mice.

Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons.

BACKGROUND: SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) monogenic mutations or deficiency leads to excessive stereotypic behavior and impaired sociability, which frequently occur in autism cases. To date, the underlying mechanisms by which Shank3 mutation or deletion causes autism and the part of the brain in which Shank3 mutation leads to the autistic phenotypes are understudied. The hypothalamus is associated with stereotypic behavior and sociability. p38α, a mediator of inflammatory responses in the brain, has been postulated as a potential gene for certain cases of autism occurrence. However, it is unclear whether hypothalamus and p38α are involved in the development of autism caused by Shank3 mutations or deficiency. METHODS: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and immunoblotting were used to assess alternated signaling pathways in the hypothalamus of Shank3 knockout (Shank3-/-) mice. Home-Cage real-time monitoring test was performed to record stereotypic behavior and three-chamber test was used to monitor the sociability of mice. Adeno-associated viruses 9 (AAV9) were used to express p38α in the arcuate nucleus (ARC) or agouti-related peptide (AgRP) neurons. D176A and F327S mutations expressed constitutively active p38α. T180A and Y182F mutations expressed inactive p38α. RESULTS: We found that Shank3 controls stereotypic behavior and sociability by regulating p38α activity in AgRP neurons. Phosphorylated p38 level in hypothalamus is significantly enhanced in Shank3-/- mice. Consistently, overexpression of p38α in ARC or AgRP neurons elicits excessive stereotypic behavior and impairs sociability in wild-type (WT) mice. Notably, activated p38α in AgRP neurons increases stereotypic behavior and impairs sociability. Conversely, inactivated p38α in AgRP neurons significantly ameliorates autistic behaviors of Shank3-/- mice. In contrast, activated p38α in pro-opiomelanocortin (POMC) neurons does not affect stereotypic behavior and sociability in mice. LIMITATIONS: We demonstrated that SHANK3 regulates the phosphorylated p38 level in the hypothalamus and inactivated p38α in AgRP neurons significantly ameliorates autistic behaviors of Shank3-/- mice. However, we did not clarify the biochemical mechanism of SHANK3 inhibiting p38α in AgRP neurons. CONCLUSIONS: These results demonstrate that the Shank3 deficiency caused autistic-like behaviors by activating p38α signaling in AgRP neurons, suggesting that p38α signaling in AgRP neurons is a potential therapeutic target for Shank3 mutant-related autism.

Iron overload in hypothalamic AgRP neurons contributes to obesity and related metabolic disorders.

Iron overload is closely associated with metabolic dysfunction. However, the role of iron in the hypothalamus remains unclear. Here, we find that hypothalamic iron levels are increased, particularly in agouti-related peptide (AgRP)-expressing neurons in high-fat-diet-fed mice. Using pharmacological or genetic approaches, we reduce iron overload in AgRP neurons by central deferoxamine administration or transferrin receptor 1 (Tfrc) deletion, ameliorating diet-induced obesity and related metabolic dysfunction. Conversely, Tfrc-mediated iron overload in AgRP neurons leads to overeating and adiposity. Mechanistically, the reduction of iron overload in AgRP neurons inhibits AgRP neuron activity; improves insulin and leptin sensitivity; and inhibits iron-induced oxidative stress, endoplasmic reticulum stress, nuclear factor κB signaling, and suppression of cytokine signaling 3 expression. These results highlight the critical role of hypothalamic iron in obesity development and suggest targets for treating obesity and related metabolic disorders.

A brainstem-hypothalamus neuronal circuit reduces feeding upon heat exposure.

Empirical evidence suggests that heat exposure reduces food intake. However, the neurocircuit architecture and the signalling mechanisms that form an associative interface between sensory and metabolic modalities remain unknown, despite primary thermoceptive neurons in the pontine parabrachial nucleus becoming well characterized1. Tanycytes are a specialized cell type along the wall of the third ventricle2 that bidirectionally transport hormones and signalling molecules between the brain's parenchyma and ventricular system3-8. Here we show that tanycytes are activated upon acute thermal challenge and are necessary to reduce food intake afterwards. Virus-mediated gene manipulation and circuit mapping showed that thermosensing glutamatergic neurons of the parabrachial nucleus innervate tanycytes either directly or through second-order hypothalamic neurons. Heat-dependent Fos expression in tanycytes suggested their ability to produce signalling molecules, including vascular endothelial growth factor A (VEGFA). Instead of discharging VEGFA into the cerebrospinal fluid for a systemic effect, VEGFA was released along the parenchymal processes of tanycytes in the arcuate nucleus. VEGFA then increased the spike threshold of Flt1-expressing dopamine and agouti-related peptide (Agrp)-containing neurons, thus priming net anorexigenic output. Indeed, both acute heat and the chemogenetic activation of glutamatergic parabrachial neurons at thermoneutrality reduced food intake for hours, in a manner that is sensitive to both Vegfa loss-of-function and blockage of vesicle-associated membrane protein 2 (VAMP2)-dependent exocytosis from tanycytes. Overall, we define a multimodal neurocircuit in which tanycytes link parabrachial sensory relay to the long-term enforcement of a metabolic code.

Jinkui Shenqi pills ameliorate diabetes by regulating hypothalamic insulin resistance and POMC/AgRP expression and activity.

BACKGROUND: Research on the imbalance of proopiomelanocortin (POMC)/agouti-related protein (AgRP) neurons in the hypothalamus holds potential insights into the pathophysiology of diabetes. Jinkui Shenqi pills (JSP), a prevalent traditional Chinese medicine, regulate hypothalamic function and treat diabetes. PURPOSE: To investigate the hypoglycemic effect of JSP and explore the probable mechanism in treating diabetes. METHODS: A type 2 diabetes mouse model was used to investigate the pharmacodynamics of JSP. The glucose-lowering efficacy of JSP was assessed through various metrics including body weight, food consumption, fasting blood glucose (FBG), serum insulin levels, and an oral glucose tolerance test (OGTT). To elucidate the modulatory effects of JSP on hypothalamic mechanisms, we quantified the expression and activity of POMC and AgRP and assessed the insulin-mediated phosphoinositide 3-kinase (PI3K)/protein kinase A (AKT)/forkhead box O1 (FOXO1) pathway in diabetic mice via western blotting and immunohistochemistry. Additionally, primary hypothalamic neurons were exposed to high glucose and palmitic acid levels to induce insulin resistance, and the influence of JSP on POMC/AgRP protein expression and activation was evaluated by PI3K protein inhibition using western blotting and immunofluorescence. RESULTS: Medium- and high-dose JSP treatment effectively inhibited appetite, resulting in a steady declining trend in body weight, FBG, and OGTT results in diabetic mice (p < 0.05). These JSP groups also had significantly increased insulin levels (p < 0.05). Importantly, the medium-dose group exhibited notable protection of hypothalamic neuronal and synaptic structures, leading to augmentation of dendritic length and branching (p < 0.05). Furthermore, low-, medium-, and high-dose JSP groups exhibited increased phosphorylated (p) INSR, PI3K, pPI3K, AKT, and pAKT expression, as well as decreased FOXO1 and increased pFOXO1 expression, indicating improved hypothalamic insulin resistance in diabetic mice (p < 0.05). Treatment with 10% JSP-enriched serum produced a marked elevation of both expression and activation of POMC (p < 0.05), with a concurrent reduction in AgRP expression and activation within primary hypothalamic neurons (p < 0.05). Intriguingly, these effects could be attributed to the regulatory dynamics of PI3K activity. CONCLUSION: Our findings suggest that JSP can ameliorate diabetes by regulating POMC/AgRP expression and activity. The insulin-mediated PI3K/AKT/FOXO1 pathway plays an important regulatory role in this intricate process.

Androgen receptor actions on AgRP neurons are not a major cause of reproductive and metabolic impairments in peripubertally androgenized mice.

Excess levels of circulating androgens during prenatal or peripubertal development are an important cause of polycystic ovary syndrome (PCOS), with the brain being a key target. Approximately half of the women diagnosed with PCOS also experience metabolic syndrome; common features including obesity, insulin resistance and hyperinsulinemia. Although a large amount of clinical and preclinical evidence has confirmed this relationship between androgens and the reproductive and metabolic features of PCOS, the mechanisms by which androgens cause this dysregulation are unknown. Neuron-specific androgen receptor knockout alleviates some PCOS-like features in a peripubertal dihydrotestosterone (DHT) mouse model, but the specific neuronal populations mediating these effects are undefined. A candidate population is the agouti-related peptide (AgRP)-expressing neurons, which are important for both reproductive and metabolic function. We used a well-characterised peripubertal androgenized mouse model and Cre-loxP transgenics to investigate whether deleting androgen receptors specifically from AgRP neurons can alleviate the induced reproductive and metabolic dysregulation. Androgen receptors were co-expressed in 66% of AgRP neurons in control mice, but only in <2% of AgRP neurons in knockout mice. The number of AgRP neurons was not altered by the treatments. Only 20% of androgen receptor knockout mice showed rescue of DHT-induced androgen-induced anovulation and acyclicity. Furthermore, androgen receptor knockout did not rescue metabolic dysfunction (body weight, adiposity or glucose and insulin tolerance). While we cannot rule out developmental compensation in our model, these results suggest peripubertal androgen excess does not markedly influence Agrp expression and does not dysregulate reproductive and metabolic function through direct actions of androgens onto AgRP neurons.

Intergenerational inheritance induced by a high-fat diet causes hyperphagia and reduced hypothalamic sensitivity to insulin and leptin in the second-generation of rats.

OBJECTIVE: The aim was to investigate the intergenerational inheritance induced by a high-fat diet on sensitivity to insulin and leptin in the hypothalamic control of satiety in second-generation offspring, which were fed a control diet. METHODS: Progenitor rats were fed a high-fat or a control diet for 59 d until weaning. The first-generation and second-generation offspring were fed the control diet until 90 d of age. Body mass and adiposity index of the progenitors fed the high-fat diet and the second-generation offspring from progenitors fed the high-fat diet were evaluated as were the gene expression of DNA methyltransferase 3a, angiotensin-converting enzyme type 2, angiotensin II type 2 receptor, insulin and leptin signaling pathway (insulin receptor, leptin receptor, insulin receptor substrate 2, protein kinase B, signal transducer and transcriptional activator 3, pro-opiomelanocortin, and neuropeptide Agouti-related protein), superoxide dismutase activity, and the concentration of carbonyl protein and satiety-regulating neuropeptides, pro-opiomelanocortin and neuropeptide Agouti-related protein, in the hypothalamus. RESULTS: The progenitor group fed a high-fat diet showed increased insulin resistance and reduced insulin-secreting beta-cell function and reduced food intake, without changes in caloric intake. The second-generation offspring from progenitors fed a high-fat diet, compared with second-generation offspring from progenitors fed a control diet group, had decreased insulin-secreting beta-cell function and increased food and caloric intake, insulin resistance, body mass, and adiposity index. Furthermore, second-generation offspring from progenitors fed a high-fat diet had increased DNA methyltransferase 3a, neuropeptide Agouti-related protein, angiotensin II type 1 receptor, and nicotinamide adenine dinucleotide phosphate oxidase p47phox gene expression, superoxide dismutase activity, and neuropeptide Agouti-related protein concentration in the hypothalamus. In addition, there were reduced in gene expression of the insulin receptor, leptin receptor, insulin receptor substrate 2, pro-opiomelanocortin, angiotensin II type 2 receptor, angiotensin-converting enzyme type 2, and angiotensin-(1-7) receptor and pro-opiomelanocortin concentration in the second-generation offspring from progenitors fed the high-fat diet. CONCLUSIONS: Overall, progenitors fed a high-fat diet induced changes in the hypothalamic control of satiety of the second-generation offspring from progenitors fed the high-fat diet through intergenerational inheritance. These changes led to hyperphagia, alterations in the hypothalamic pathways of insulin, and leptin and adiposity index increase, favoring the occurrence of different cardiometabolic disorders in the second-generation offspring from progenitors fed the high-fat diet fed only with the control diet.

Are decreased cocaine- and amphetamine regulated transcript and Agouti- related peptide levels associated Eating behavior in medication-free children with attention deficit and hyperactivity disorder?

This study aimed to investigate plasma levels of cocaine- and amphetamine-regulated transcript (CART), agouti-related protein (AgRP), cholecystokinin (CCK) and peptide YY (PYY) and their relationship with eating behaviors among children with attention deficit hyperactivity disorder (ADHD) and healthy controls. A total of 94 medication-free children with ADHD and 82 controls aged 8-14 years were included in this study. The Plasma levels of CART, AgRP, CCK and PYY were measured using enzyme-linked immunosorbent assay kits. The Children's Eating Behavior Questionnaire (CEBQ) was used to assess eating behaviors in children. CART and AgRP levels were found to be significantly lower in the ADHD group than in the control group, while CCK levels were found to be significantly higher in the ADHD group than in the control group. However, there was no significant difference in PYY levels between the groups. Compared to controls, those with ADHD demonstrated significantly higher scores on the CEBQ subscales of food responsiveness, emotional overeating, desire to drink, enjoyment of food, and food fussiness, and significantly lower scores on the slowness of eating subscale. CART was significantly correlated with emotional overeating and enjoyment of food scores, while AgRP was significantly correlated with emotional undereating scores. Covariance analysis was performed by controlling potential confounders such as body mass index, age and sex, and the results were found to be unchanged. It was concluded that CART, AgRP, and CCK may play a potential role in the pathogenesis of ADHD.

Dynamic, sex- and diet-specific pleiotropism in the PAC1 receptor-mediated regulation of arcuate proopiomelanocortin and Neuropeptide Y/Agouti related peptide neuronal excitability by anorexigenic ventromedial nucleus PACAP neurons.

This study furthers the investigation of how pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1 receptor (PAC1R) regulate the homeostatic energy balance circuitry. We hypothesized that apoptotic ablation of PACAP neurones in the hypothalamic ventromedial nucleus (VMN) would affect both energy intake and energy expenditure. We also hypothesized that selective PAC1R knockdown would impair the PACAP-induced excitation in anorexigenic proopiomelanocortin (POMC) neurones and inhibition of orexigenic neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurones in the hypothalamic arcuate nucleus (ARC). The results show CASPASE-3-induced ablation of VMN PACAP neurones leads to increased energy intake and meal frequency as well as decreased energy expenditure in lean animals. The effects were more robust in obese males, whereas we saw the opposite effects in obese females. We then utilized visualized whole-cell patch clamp recordings in hypothalamic slices. PAC1R knockdown in POMC neurones diminishes the PACAP-induced depolarization, increase in firing, decreases in energy intake and meal size, as well as increases in CO2 production and O2 consumption. Similarly, the lack of expression of the PAC1R in NPY/AgRP neurones greatly attenuates the PACAP-induced hyperpolarization, suppression of firing, decreases in energy intake and meal frequency, as well as increases in energy expenditure. The PACAP response in NPY/AgRP neurones switched from predominantly inhibitory to excitatory in fasted animals. Finally, the anorexigenic effect of PACAP was potentiated when oestradiol was injected into the ARC in ovariectomized females. This study demonstrates the critical role of anorexigenic VMN PACAP neurones and the PAC1R in exciting POMC and inhibiting NPY/AgRP neurons to control homeostatic feeding.

Cotadutide (GLP-1/Glucagon dual receptor agonist) modulates hypothalamic orexigenic and anorexigenic neuropeptides in obese mice.

The hypothalamic neuropeptides linked to appetite and satiety were investigated in obese mice treated with cotadutide (a dual receptor agonist of glucagon-like peptide 1 (GLP-1R)/Glucagon (GCGR)). Twelve-week-old male C57BL/6 mice were fed a control diet (C group, n = 20) or a high-fat diet (HF group, n = 20) for ten weeks. Each group was further divided, adding cotadutide treatment and forming groups C, CC, HF, and HFC for four additional weeks. The hypothalamic arcuate neurons were labeled by immunofluorescence, and protein expressions (Western blotting) for neuropeptide Y (NPY), proopiomelanocortin (POMC), agouti-related protein (AgRP), and cocaine- and amphetamine-regulated transcript (CART). Cotadutide enhanced POMC and CART neuropeptides and depressed NPY and AGRP neuropeptides. In addition, gene expressions (RT-qPCR) determined that Lepr (leptin receptor) and Calcr (calcitonin receptor) were diminished in HF compared to C but enhanced in CC compared to C and HFC compared to HF. Besides, Socs3 (suppressor of cytokine signaling 3) was decreased in HFC compared to HF, while Sst (somatostatin) was higher in HFC compared to HF; Tac1 (tachykinin 1) and Mc4r (melanocortin-4-receptor) were lower in HF compared to C but increased in HFC compared to HF. Also, Glp1r and Gcgr were higher in HFC compared to HF. In conclusion, the findings are compelling, demonstrating the effects of cotadutide on hypothalamic neuropeptides and hormone receptors of obese mice. Cotadutide modulates energy balance through the gut-brain axis and its associated signaling pathways. The study provides insights into the mechanisms underlying cotadutide's anti-obesity effects and its possible implications for obesity treatment.

High Intensity Interval Training can Ameliorate Hypothalamic Appetite Regulation in Male Rats with Type 2 Diabetes: The Role of Leptin.

Disruption of leptin (LEP) signaling in the hypothalamus caused by type 2 diabetes (T2D) can impair appetite regulation. The aim of this study was to investigate whether the improvement in appetite regulation induced by high-intensity interval training (HIIT) in rats with T2D can be mediated by LEP signaling. In this study, 20 male Wister rats were randomly assigned to one of four groups: CO (non-type 2 diabetes control), T2D (type 2 diabetes), EX (non-type 2 diabetes exercise), and T2D + EX (type 2 diabetes + exercise).To induce T2D, a combination of a high-fat diet for 2 months and a single dose of streptozotocin (35 mg/kg) was administered. Rats in the EX and T2D + EX groups performed 4-10 intervals of treadmill running at 80-100% of their maximum velocity (Vmax). Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), serum levels of insulin (INS) and LEP (LEPS) as well as hypothalamic expression of LEP receptors (LEP-R), Janus kinase 2 (JAK-2), signal transducer and activator of transcription 3 (STAT-3), neuropeptide Y (NPY), agouti-related protein (AGRP), pro-opiomelanocortin cocaine (POMC), amphetamine-related transcript (CART), suppressor of cytokine signaling (SOCS3), forkhead box protein O1 (FOXO1) were assessed. ANOVA and Tukey post hoc tests were used to compare the results between the groups. The levels of LEPS and INS, as well as the levels of LEP-R, JAK-2, STAT-3, POMC, and CART in the hypothalamus were found to be higher in the T2D + EX group compared to the T2D group. On the other hand, the levels of HOMA-IR, NPY, AGRP, SOCS3, and FOXO1 were lower in the T2D + EX group compared to the T2D group (P < 0.0001). The findings of this study suggest that HIIT may improve appetite regulation in rats with T2D, and LEP signaling may play a crucial role in this improvement. Graphical abstract (leptin signaling in the hypothalamus), Leptin (LEP), Leptin receptor (LEP-R), Janus kinase 2 (JAK2), Signal transducer and activator of transcription 3 (STAT3), expressing Neuropeptide Y (NPY), Agouti-related protein (AGRP), anorexigenic neurons (expressing pro-opiomelanocortin cocaine (POMC), Amphetamine-related transcript (CART), suppressor of cytokine signaling (SOCS3), forkhead box protein O1 (FOXO1).

GIRK2 potassium channels expressed by the AgRP neurons decrease adiposity and body weight in mice.

It is well known that the neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons increase appetite and decrease thermogenesis. Previous studies demonstrated that optogenetic and/or chemogenetic manipulations of NPY/AgRP neuronal activity alter food intake and/or energy expenditure (EE). However, little is known about intrinsic molecules regulating NPY/AgRP neuronal excitability to affect long-term metabolic function. Here, we found that the G protein-gated inwardly rectifying K+ (GIRK) channels are key to stabilize NPY/AgRP neurons and that NPY/AgRP neuron-selective deletion of the GIRK2 subunit results in a persistently increased excitability of the NPY/AgRP neurons. Interestingly, increased body weight and adiposity observed in the NPY/AgRP neuron-selective GIRK2 knockout mice were due to decreased sympathetic activity and EE, while food intake remained unchanged. The conditional knockout mice also showed compromised adaptation to coldness. In summary, our study identified GIRK2 as a key determinant of NPY/AgRP neuronal excitability and driver of EE in physiological and stress conditions.

Bridging Energy Need and Feeding Behavior: The Impact of eIF2α Phosphorylation in AgRP Neurons.

Eukaryotic translation initiation factor 2α (eIF2α) is a key mediator of the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR). In mammals, eIF2α is phosphorylated by overnutrition-induced ER stress and is related to the development of obesity. Here, we studied the function of phosphorylated eIF2α (p-eIF2α) in agouti-related peptide (AgRP) neurons using a mouse model (AgRPeIF2αA/A) with an AgRP neuron-specific substitution from Ser 51 to Ala in eIF2α, which impairs eIF2α phosphorylation in AgRP neurons. These AgRPeIF2αA/A mice had decreases in starvation-induced AgRP neuronal activity and food intake and an increased responsiveness to leptin. Intriguingly, impairment of eIF2α phosphorylation produced decreases in the starvation-induced expression of UPR and autophagy genes in AgRP neurons. Collectively, these findings suggest that eIF2α phosphorylation regulates AgRP neuronal activity by affecting intracellular responses such as the UPR and autophagy during starvation, thereby participating in the homeostatic control of whole-body energy metabolism. ARTICLE HIGHLIGHTS: This study examines the impact of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, triggered by an energy deficit, on hypothalamic AgRP neurons and its subsequent influence on whole-body energy homeostasis. Impaired eIF2α phosphorylation diminishes the unfolded protein response and autophagy, both of which are crucial for energy deficit-induced activation of AgRP neurons. This study highlights the significance of eIF2α phosphorylation as a cellular marker indicating the availability of energy in AgRP neurons and as a molecular switch that regulates homeostatic feeding behavior.

Spexin2 Is a Novel Food Regulator in Gallus gallus.

Spexin2 (SPX2), a paralog of SPX1, is a newly identified gene in non-mammalian vertebrates. Limited studies in fish have evidenced its important role in food intake and energy balance modulation. However, little is known about its biological functions in birds. Using the chicken (c-) as a model, we cloned the full-length cDNA of SPX2 by using RACE-PCR. It is 1189 base pair (bp) in length and predicted to generate a protein of 75 amino acids that contains a 14 amino acids mature peptide. Tissue distribution analysis showed that cSPX2 transcripts were detected in a wide array of tissues, with abundant expression in the pituitary, testis, and adrenal gland. cSPX2 was also observed to be ubiquitously expressed in chicken brain regions, with the highest expression in the hypothalamus. Its expression was significantly upregulated in the hypothalamus after 24 or 36 h of food deprivation, and the feeding behavior of chicks was obviously suppressed after peripheral injection with cSPX2. Mechanistically, further studies evidenced that cSPX2 acts as a satiety factor via upregulating cocaine and amphetamine regulated transcript (CART) and downregulating agouti-related neuropeptide (AGRP) in hypothalamus. Using a pGL4-SRE-luciferase reporter system, cSPX2 was demonstrated to effectively activate a chicken galanin II type receptor (cGALR2), a cGALR2-like receptor (cGALR2L), and a galanin III type receptor (cGALR3), with the highest binding affinity for cGALR2L. Collectively, we firstly identified that cSPX2 serves as a novel appetite monitor in chicken. Our findings will help clarify the physiological functions of SPX2 in birds as well as its functional evolution in vertebrates.

The gut signals to AGRP-expressing cells of the pituitary to control glucose homeostasis.

Glucose homeostasis can be improved after bariatric surgery, which alters bile flow and stimulates gut hormone secretion, particularly FGF15/19. FGFR1 expression in AGRP-expressing cells is required for bile acids' ability to improve glucose control. We show that the mouse Agrp gene has 3 promoter/enhancer regions that direct transcription of each of their own AGRP transcripts. One of these Agrp promoters/enhancers, Agrp-B, is regulated by bile acids. We generated an Agrp-B knockin FLP/knockout allele. AGRP-B-expressing cells are found in endocrine cells of the pars tuberalis and coexpress diacylglycerol lipase B - an endocannabinoid biosynthetic enzyme - distinct from pars tuberalis thyrotropes. AGRP-B expression is also found in the folliculostellate cells of the pituitary's anterior lobe. Mice without AGRP-B were protected from glucose intolerance induced by high-fat feeding but not from excess weight gain. Chemogenetic inhibition of AGRP-B cells improved glucose tolerance by enhancing glucose-stimulated insulin secretion. Inhibition of the AGRP-B cells also caused weight loss. The improved glucose tolerance and reduced body weight persisted up to 6 weeks after cessation of the DREADD-mediated inhibition, suggesting the presence of a biological switch for glucose homeostasis that is regulated by long-term stability of food availability.

Nuclear receptor 5A2 regulation of Agrp underlies olanzapine-induced hyperphagia.

Antipsychotic (AP) drugs are efficacious treatments for various psychiatric disorders, but excessive weight gain and subsequent development of metabolic disease remain serious side effects of their use. Increased food intake leads to AP-induced weight gain, but the underlying molecular mechanisms remain unknown. In previous studies, we identified the neuropeptide Agrp and the transcription factor nuclear receptor subfamily 5 group A member 2 (Nr5a2) as significantly upregulated genes in the hypothalamus following AP-induced hyperphagia. While Agrp is expressed specifically in the arcuate nucleus of the hypothalamus and plays a critical role in appetite stimulation, Nr5a2 is expressed in both the CNS and periphery, but its role in food intake behaviors remains unknown. In this study, we investigated the role of hypothalamic Nr5a2 in AP-induced hyperphagia and weight gain. In hypothalamic cell lines, olanzapine treatment resulted in a dose-dependent increase in gene expression of Nr5a2 and Agrp. In mice, the pharmacological inhibition of NR5A2 decreased olanzapine-induced hyperphagia and weight gain, while the knockdown of Nr5a2 in the arcuate nucleus partially reversed olanzapine-induced hyperphagia. Chromatin-immunoprecipitation studies showed for the first time that NR5A2 directly binds to the Agrp promoter region. Lastly, the analysis of single-cell RNA seq data confirms that Nr5a2 and Agrp are co-expressed in a subset of neurons in the arcuate nucleus. In summary, we identify Nr5a2 as a key mechanistic driver of AP-induced food intake. These findings can inform future clinical development of APs that do not activate hyperphagia and weight gain.

The hypothalamic neuropeptide, QRFP, regulates high fat diet intake in female Long-Evans rats following ovariectomy.

Obesity rates in women continue to increase throughout the lifespan and obesity-related comorbidities are prevalent in women in estrogen deficiency. The hypothalamic neuropeptide, QRFP, is an orexigenic peptide that increases the intake of high fat diet (HFD) in female rats and is overexpressed following ovariectomy (OVX). Therefore, the goal of the current series of experiments was to elucidate the effect of QRFP on HFD intake following OVX and determine if QRFP-26 administration in ovariectomized females altered expression of prepro-neuropeptide Y (NPY), agouti-related peptide (AgRP) and proopiomelanocortin (POMC) mRNA in the mediobasal hypothalamus (MBH) and prepro-orexin in the lateral hypothalamus (LH). The intake of HFD was measured following acute administration of QRFP-26 prior to or following estradiol benzoate (EB) treatment in ovariectomized females. When administered prior to EB treatment, QRFP-26 increased HFD intake. EB treatment attenuated the effects of QRFP-26 on HFD intake. Sub-chronic, continuous administration of QRFP-26 increased HFD intake and weight gain following OVX. Subchronic, continuous administration of QRFP siRNA into the 3rd ventricle via osmotic pump decreased prepro-QRFP mRNA levels in the MBH by ∼75%, decreased HFD intake and decreased weight gain following OVX. QRFP-26administration did not alter the expression of prepro-NPY, AgRP or POMC mRNA in the MBH, but decreased prepro-orexin mRNA in the LH of ovariectomized females. Overall, results from these studies support the orexigenic neuropeptide, QRFP, as an important mediator of the ingestion of highly palatable foods and subsequent weight gain in females during estrogen deficiency.

Modeling the effects of genetic- and diet-induced obesity on melanoma progression in zebrafish.

Obesity is a rising concern and associated with an increase in numerous cancers, often in a sex-specific manner. Preclinical models are needed to deconvolute the intersection between obesity, sex and melanoma. Here, we generated a zebrafish system that can be used as a platform for studying these factors. We studied how germline overexpression of Agrp along with a high-fat diet affects melanomas dependent on BRAFV600E and loss of p53. This revealed an increase in tumor incidence and area in male, but not female, obese fish, consistent with the clinical literature. We then determined whether this was further affected by additional somatic mutations in the clinically relevant genes rb1 or ptena/b. We found that the male obesogenic effect on melanoma was present with tumors generated with BRAF;p53;Rb1 but not BRAF;p53;Pten. These data indicate that both germline (Agrp) and somatic (BRAF, Rb1) mutations contribute to obesity-related effects in melanoma. Given the rapid genetic tools available in the zebrafish, this provides a high-throughput system to dissect the interactions of genetics, diet, sex and host factors in obesity-related cancers.

Hypothalamic AgRP neurons exert top-down control on systemic TNF-α release during endotoxemia.

Loss of appetite and negative energy balance are common features of endotoxemia in all animals and are thought to have protective roles by reducing nutrient availability to host and pathogen metabolism. Accordingly, fasting and caloric restriction have well-established anti-inflammatory properties. However, in response to reduced nutrient availability at the cellular and organ levels, negative energy balance also recruits distinct energy-sensing brain circuits, but it is not known whether these neuronal systems have a role in its anti-inflammatory effects. Here, we report that hypothalamic AgRP neurons-a critical neuronal population for the central representation of negative energy balance-have parallel immunoregulatory functions. We found that when endotoxemia occurs in fasted mice, the activity of AgRP neurons remains sustained, but this activity does not influence feeding behavior and endotoxemic anorexia. Furthermore, we found that endotoxemia acutely desensitizes AgRP neurons, which also become refractory to inhibitory signals. Mimicking this sustained AgRP neuron activity in fed mice by chemogenetic activation-a manipulation known to recapitulate core behavioral features of fasting-results in reduced acute tumor necrosis factor alpha (TNF-α) release during endotoxemia. Mechanistically, we found that endogenous glucocorticoids play an important role: glucocorticoid receptor deletion from AgRP neurons prevents their endotoxemia-induced desensitization, and importantly, it counteracts the fasting-induced suppression of TNF-α release, resulting in prolonged sickness. Together, these findings provide evidence directly linking AgRP neuron activity to the acute response during endotoxemia, suggesting that these neurons are a functional component of the immunoregulatory effects associated with negative energy balance and catabolic metabolism.

Lipid biosynthesis enzyme Agpat5 in AgRP-neurons is required for insulin-induced hypoglycemia sensing and glucagon secretion.

The counterregulatory response to hypoglycemia that restores normal blood glucose levels is an essential physiological function. It is initiated, in large part, by incompletely characterized brain hypoglycemia sensing neurons that trigger the secretion of counterregulatory hormones, in particular glucagon, to stimulate hepatic glucose production. In a genetic screen of recombinant inbred BXD mice we previously identified Agpat5 as a candidate regulator of hypoglycemia-induced glucagon secretion. Here, using genetic mouse models, we demonstrate that Agpat5 expressed in agouti-related peptide neurons is required for their activation by hypoglycemia, for hypoglycemia-induced vagal nerve activity, and glucagon secretion. We find that inactivation of Agpat5 leads to increased fatty acid oxidation and ATP production and that suppressing Cpt1a-dependent fatty acid import into mitochondria restores hypoglycemia sensing. Collectively, our data show that AgRP neurons are involved in the control of glucagon secretion and that Agpat5, by partitioning fatty acyl-CoAs away from mitochondrial fatty acid oxidation and ATP generation, ensures that the fall in intracellular ATP, which triggers neuronal firing, faithfully reflects changes in glycemia.