Acute Activation of GFRAL in the Area Postrema Contributes to Glucose Regulation Independent of Weight.

GDF15 regulates energy balance and glucose homeostasis in rodents by activating its receptor GFRAL, expressed in the area postrema of the brain. However, whether GDF15-GFRAL signaling in the area postrema regulates glucose tolerance independent of changes in food intake and weight and contributes to the glucose-lowering effect of metformin remain unknown. Herein, we report that direct, acute GDF15 infusion into the area postrema of rats fed a high-fat diet increased intravenous glucose tolerance and insulin sensitivity to lower hepatic glucose production independent of changes in food intake, weight, and plasma insulin levels under conscious, unrestrained, and nonstressed conditions. In parallel, metformin infusion concurrently increased plasma GDF15 levels and glucose tolerance. Finally, a knockdown of GFRAL expression in the area postrema negated administration of GDF15, as well as metformin, to increase glucose tolerance independent of changes in food intake, weight, and plasma insulin levels. In summary, activation of GFRAL in the area postrema contributes to glucose regulation of GDF15 and metformin in vivo.

Metformin triggers a kidney GDF15-dependent area postrema axis to regulate food intake and body weight.

Metformin, the most widely prescribed medication for obesity-associated type 2 diabetes (T2D), lowers plasma glucose levels, food intake, and body weight in rodents and humans, but the mechanistic site(s) of action remain elusive. Metformin increases plasma growth/differentiation factor 15 (GDF15) levels to regulate energy balance, while GDF15 administration activates GDNF family receptor α-like (GFRAL) that is highly expressed in the area postrema (AP) and the nucleus of the solitary tract (NTS) of the hindbrain to lower food intake and body weight. However, the tissue-specific contribution of plasma GDF15 levels after metformin treatment is still under debate. Here, we found that metformin increased plasma GDF15 levels in high-fat (HF) fed male rats through the upregulation of GDF15 synthesis in the kidney. Importantly, the kidney-specific knockdown of GDF15 expression as well as the AP-specific knockdown of GFRAL expression negated the ability of metformin to lower food intake and body weight gain. Taken together, we unveil the kidney as a target of metformin to regulate energy homeostasis through a kidney GDF15-dependent AP axis.

A glucose-sensing mechanism with glucose transporter 1 and pyruvate kinase in the area postrema regulates hepatic glucose production in rats.

The area postrema (AP) of the brain is exposed to circulating metabolites and hormones. However, whether AP detects glucose changes to exert biological responses remains unknown. Its neighboring nuclei, the nucleus tractus solitarius (NTS), responds to acute glucose infusion by inhibiting hepatic glucose production, but the mechanism also remains elusive. Herein, we characterized AP and NTS glucose-sensing mechanisms. Infusion of glucose into the AP, like the NTS, of chow rats suppressed glucose production during the pancreatic (basal insulin)-euglycemic clamps. Glucose transporter 1 or pyruvate kinase lentiviral-mediated knockdown in the AP negated AP glucose infusion to lower glucose production, while the glucoregulatory effect of NTS glucose infusion was also negated by knocking down glucose transporter 1 or pyruvate kinase in the NTS. Furthermore, we determined that high-fat (HF) feeding disrupts glucose infusion to lower glucose production in association with a modest reduction in the expression of glucose transporter 1, but not pyruvate kinase, in the AP and NTS. However, pyruvate dehydrogenase activator dichloroacetate infusion into the AP or NTS that enhanced downstream pyruvate metabolism and recapitulated the glucoregulatory effect of glucose in chow rats still failed to lower glucose production in HF rats. We discovered that a glucose transporter 1- and pyruvate kinase-dependent glucose-sensing mechanism in the AP (as well as the NTS) lowers glucose production in chow rats and that HF disrupts the glucose-sensing mechanism that is downstream of pyruvate metabolism in the AP and NTS. These findings highlight the role of AP and NTS in mediating glucose to regulate hepatic glucose production.

Three-plane description of astroglial architecture and gliovascular connections of area postrema in rat: Long tanycyte connections to other parts of brainstem.

The study demonstrates the astroglial and gliovascular structures of the area postrema (AP) in three planes, and compares them to our former findings on the subfornical organ (SFO) and the organon vasculosum laminae terminalis (OVLT). The results revealed long glial processes interconnecting the AP with deeper areas of brain stem. The laminin and ß-dystroglycan immunolabeling altered along the vessels indicating alterations of the gliovascular relations. These and the distributions of glial markers displayed similarities to the SFO and OVLT. In every organ, there was a central area with vimentin- and nestin-immunopositive glia, whereas GFAP and the water-channel aquaporin 4 were found at the periphery. This separation supports different functions of the two regions. The presence of nestin may indicate stem cell capabilities, whereas aquaporin 4 has been suggested by other studies to be a possible participant of osmoperception. Numerous S100-immunopositive glial cells were found approximately evenly distributed in both parts of the AP. Frequency of glutamine synthetase-immunoreactive cells was similar in the surrounding brain tissue in contrast to that found in the OVLT and SFO. Our findings on the three sensory circumventricular organs (AP, OVLT, and SFO) are compared in parallel.

Activation of Calcium-Sensing Receptor in the Area Postrema Inhibits Food Intake via Glutamatergic and GABAergic Signaling Pathways.

SCOPE: A high-protein diet has become a popular way to lose weight. Calcium-sensing receptor (CaSR) is activated by amino acids in addition to calcium ions. CaSR shows dense expression in the area postrema (AP), which participates in feeding regulation. The effect of CaSR in the AP on food intake and the potential mechanism involved is investigated. METHODS AND RESULTS: Male C57BL/6 mice are used to observe the effect of R568 (agonist of CaSR) on food intake. Enzyme-linked immunosorbent assay, immunofluorescence staining, and chemogenetics are used to explore the neural signaling involved. CaSR activation in the AP inhibited acute feeding; R568 increases the content of glutamate and γ-aminobutyric acid (GABA) in the AP, whereas only glutamatergic neurons mediate the effect of R568. GABA-A receptor and ionic glutamate receptor (N-methyl-D-aspartate receptor [NMDAR]) in the paraventricular nucleus of hypothalamus (PVN) are involved in the effect of R568. Promotion of oxytocin (OT) synthesis in the PVN also participates in the effect of R568, and this mechanism is mediated by NMDAR in the PVN. CONCLUSION: CaSR activation in the AP suppresses feeding, and AP-PVN glutamatergic and GABAergic signaling pathways are involved.

Brainstem ADCYAP1+ neurons control multiple aspects of sickness behaviour.

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2-4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.

Autoimmune Glial Fibrillary Acidic Protein Astrocytopathy Presenting with Area Postrema Syndrome-Like Symptoms without Medulla Oblongata Lesions.

INTRODUCTION: Autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy is a recently described steroid-responsive meningoencephalomyelitis positive for cerebrospinal fluid (CSF) anti-GFAP antibody. Area postrema syndrome (APS) involves intractable hiccups, nausea, and vomiting, which is caused by medulla oblongata (MO) impairment. APS is a characteristic symptom of aquaporin-4 (AQP4) autoimmunity, and it helps to differentiate between AQP4 and GFAP autoimmunity. Conversely, although 6 cases of autoimmune GFAP astrocytopathy with APS and MO lesions have been reported, the association between GFAP autoimmunity and APS is unclear. We report the case of a patient with autoimmune GFAP astrocytopathy presenting with APS-like symptoms without MO lesions and discuss the mechanisms underlying the symptoms. METHODS: CSF anti-GFAP antibody was detected using cell-based assays and immunohistochemical assays. RESULTS: A 54-year-old Japanese man developed persistent hiccups, intermittent vomiting, fever, anorexia, and inattention. Brain magnetic resonance imaging (MRI) showed periventricular lesions with radial linear periventricular enhancement, suggesting autoimmune GFAP astrocytopathy. However, no obvious MO lesions were identified on thin-slice images. Spinal cord MRI revealed hazy lesions with patchy enhancement along the cervical and thoracic cord. CSF analysis demonstrated inflammation, with positive results for anti-GFAP antibodies. Anti-AQP4 antibodies in the serum and CSF were negative. Esophagogastroduodenoscopy revealed gastroparesis and gastroesophageal reflux disease, and vonoprazan, mosapride, and rikkunshito were effective only against persistent hiccups. Steroid therapy was initiated, allowing clinical and radiological improvements. Repeated MRIs demonstrated no obvious MO lesions. CONCLUSION: This report suggests that autoimmune GFAP astrocytopathy presents with APS-like symptoms without obvious MO lesions. The possible causes of hiccups were gastroparesis and cervical cord lesions. Gastroesophageal reflux disease was not considered a major cause of the hiccups. Intermittent vomiting appeared to be associated with gastroparesis, cervical cord lesions, and viral-like symptoms. Testing for anti-GFAP antibodies should be considered in patients with APS-like symptoms in the context of typical clinical-MRI features of autoimmune GFAP astrocytopathy.

Single nuclei RNA sequencing of the rat AP and NTS following GDF15 treatment.

OBJECTIVE: Growth differentiation factor 15 (GDF15) is known to play a role in feeding, nausea, and body weight, with action through the GFRAL-RET receptor complex in the area postrema (AP) and nucleus tractus solitarius (NTS). To further elucidate the underlying cell type-specific molecular mechanisms downstream of GDF15 signaling, we used a single nuclei RNA sequencing (snRNAseq) approach to profile AP and NTS cellular subtype-specific transcriptomes after systemic GDF15 treatment. METHODS: AP and NTS micropunches were used for snRNAseq from Sprague Dawley rats 6 h following GDF15 or saline injection, and Seurat was used to identify cellular subtypes and cell type-specific alterations in gene expression that were due to the direct and secondary effects of systemic GDF15 treatment. RESULTS: Using the transcriptome profile of ∼35,000 individual AP/NTS nuclei, we identified 19 transcriptomically distinct cellular subtypes, including a single population Gfral and Ret positive excitatory neurons, representing the primary site of action for GDF15. A total of ∼600 cell type-specific differential expression events were identified in neurons and glia, including the identification of transcriptome alterations specific to the direct effects of GDF15 in the Gfral-Ret positive excitatory neurons and shared transcriptome alterations across neuronal and glial cell types. Downstream analyses identified shared and cell type-specific alterations in signaling pathways and upstream regulatory mechanisms of the observed transcriptome alterations. CONCLUSIONS: These data provide a considerable advance in our understanding of AP and NTS cell type-specific molecular mechanisms associated with GDF15 signaling. The identified cellular subtype-specific regulatory mechanism and signaling pathways likely represent important targets for future pharmacotherapies.

A survey of the mouse hindbrain in the fed and fasted states using single-nucleus RNA sequencing.

OBJECTIVE: The area postrema (AP) and nucleus tractus solitarius (NTS) located in the hindbrain are key nuclei that sense and integrate peripheral nutritional signals and consequently regulate feeding behaviour. While single-cell transcriptomics have been used in mice to reveal the gene expression profile and heterogeneity of key hypothalamic populations, similar in-depth studies have not yet been performed in the hindbrain. METHODS: Using single-nucleus RNA sequencing, we provide a detailed survey of 16,034 cells within the AP and NTS of mice in the fed and fasted states. RESULTS: Of these, 8,910 were neurons that group into 30 clusters, with 4,289 from mice fed ad libitum and 4,621 from overnight fasted mice. A total of 7,124 nuclei were from non-neuronal cells, including oligodendrocytes, astrocytes, and microglia. Interestingly, we identified that the oligodendrocyte population was particularly transcriptionally sensitive to an overnight fast. The receptors GLP1R, GIPR, GFRAL, and CALCR, which bind GLP1, GIP, GDF15, and amylin, respectively, are all expressed in the hindbrain and are major targets for anti-obesity therapeutics. We characterise the transcriptomes of these four populations and show that their gene expression profiles are not dramatically altered by an overnight fast. Notably, we find that roughly half of cells that express GIPR are oligodendrocytes. Additionally, we profile POMC-expressing neurons within the hindbrain and demonstrate that 84% of POMC neurons express either PCSK1, PSCK2, or both, implying that melanocortin peptides are likely produced by these neurons. CONCLUSION: We provide a detailed single-cell level characterisation of AP and NTS cells expressing receptors for key anti-obesity drugs that are either already approved for human use or in clinical trials. This resource will help delineate the mechanisms underlying the effectiveness of these compounds and also prove useful in the continued search for other novel therapeutic targets.

Hypoglycemia attenuates acute amylin-induced reduction of food intake in male rats.

The ability of amylin to inhibit gastric emptying and glucagon secretion in rats is reduced under hypoglycemic conditions. These effects are considered part of a fail-safe mechanism that prevents amylin from further decreasing nutrient supply when blood glucose levels are low. Because these actions and amylin-induced satiation are mediated by the area postrema (AP), it is plausible that these phenomena are based on the co-sensitivity of AP neurons to amylin and glucose. Using hyperinsulinemic glucose clamps in unrestrained and freely-feeding rats, we investigated whether amylin's ability to inhibit food intake is also reduced by hypoglycemia (HYPO). Following an 18 h fast, rats were infused with insulin and glucose for 45 min to clamp blood glucose at baseline levels (between 90 and 100 mg/dL). HYPO (approximately 55 mg/dL) was induced between 45 and 60 min and then maintained for the remainder of the clamp. Rats were injected with amylin (20 µg/kg) or saline and offered normal chow at 85 min. Food intake was measured at 30 and 60 min after amylin. Control hyperinsulinemic/euglycemic (EU) rats were maintained at approximately 150 mg/dL (which is a physiological periprandial glucose level) before and after amylin injection. Terminal experiments tested the effect of amylin to induce the phosphorylation of ERK, a marker of amylin action in the AP, in EU and HYPO conditions. Amylin significantly reduced 30- and 60-min food intake in EU rats, but the effect at 60-min was attenuated in HYPO rats. Interestingly, glucose infusion rate had to be dramatically reduced at meal onset in saline-treated, but not in amylin-treated, EU or HYPO rats; this suggests that meal-related glucose appearance in the blood was inhibited by amylin under both EU and HYPO. Finally, amylin induced a similar pERK response in the AP in EU and HYPO rats. We conclude that amylin's action to decrease eating is blunted in hypoglycemia, and this effect seems to be downstream from amylin-induced pERK in AP neurons. These data allow us to extend the idea of a hypoglycemic brake on amylin's actions to its food intake-reducing effect, but also demonstrate that amylin can buffer meal-induced glucose appearance at EU and HYPO levels.

Fibroblast Growth Factor-1 Activates Neurons in the Arcuate Nucleus and Dorsal Vagal Complex.

Central administration of fibroblast growth factor-1 (FGF1) results in long-lasting resolution of hyperglycemia in various rodent models, but the pre- and postsynaptic mechanisms mediating the central effects of FGF1 are unknown. Here we utilize electrophysiology recordings from neuronal populations in the arcuate nucleus of the hypothalamus (ARH), nucleus of the solitary tract (NTS), and area postrema (AP) to investigate the mechanisms underlying FGF1 actions. While FGF1 did not alter membrane potential in ARH-NPY-GFP neurons, it reversibly depolarized 83% of ARH-POMC-EGFP neurons and decreased the frequency of inhibitory inputs onto ARH-POMC-EGFP neurons. This depolarizing effect persisted in the presence of FGF receptor (R) blocker FIIN1, but was blocked by pretreatment with the voltage-gated sodium channel (VGSC) blocker tetrodotoxin (TTX). Non-FGF1 subfamilies can activate vascular endothelial growth factor receptors (VEGFR). Surprisingly, the VEGFR inhibitors axitinib and BMS605541 blocked FGF1 effects on ARH-POMC-EGFP neurons. We also demonstrate that FGF1 induces c-Fos in the dorsal vagal complex, activates NTS-NPY-GFP neurons through a FGFR mediated pathway, and requires VGSCs to activate AP neurons. We conclude that FGF1 acts in multiple brain regions independent of FGFRs. These studies present anatomical and mechanistic pathways for the future investigation of the pharmacological and physiological role of FGF1 in metabolic processes.

Area Postrema Cell Types that Mediate Nausea-Associated Behaviors.

Nausea, the unpleasant sensation of visceral malaise, remains a mysterious process. The area postrema is implicated in some nausea responses and is anatomically privileged to detect blood-borne signals. To investigate nausea mechanisms, we built an area postrema cell atlas through single-nucleus RNA sequencing, revealing a few neuron types. Using mouse genetic tools for cell-specific manipulation, we discovered excitatory neurons that induce nausea-related behaviors, with one neuron type mediating aversion imposed by multiple poisons. Nausea-associated responses to agonists of identified area postrema receptors were observed and suppressed by targeted cell ablation and/or gene knockout. Anatomical mapping revealed a distributed network of long-range excitatory but not inhibitory projections with subtype-specific patterning. These studies reveal the basic organization of area postrema nausea circuitry and provide a framework toward understanding and therapeutically controlling nausea.

Viral depletion of calcitonin receptors in the area postrema: A proof-of-concept study.

The area postrema (AP), located in the caudal hindbrain, is one of the primary binding sites for the endocrine satiation hormone amylin. Amylin is co-secreted with insulin from pancreatic ß-cells and binds to heterodimeric receptors that consist of a calcitonin core receptor (CTR) paired with receptor-activity modifying protein (RAMP) 1 or 3. In this study, we aim to validate a CTR-floxed (CTRfl/fl) mouse model for the functional and site-specific depletion of amylin/CTR signaling in the AP and the nucleus tractus solitarius (NTS). CTRfl/fl mice were injected in the NTS with adeno-associated virus (AAV) containing a green fluorescent protein tag (GFP) and Cre recombinase to create a locally restricted knockout of CTR in the caudal hindbrain. KO mice showed a lack of c-Fos expression, a marker for neuronal activation, in the AP, NTS and LPBN after amylin injection. The effect of amylin and salmon calcitonin (sCT), an amylin receptor agonist, on food intake was blunted in KO mice, confirming a functional reduction of amylin signaling in the hindbrain.

Timekeeping in the hindbrain: a multi-oscillatory circadian centre in the mouse dorsal vagal complex.

Metabolic and cardiovascular processes controlled by the hindbrain exhibit 24 h rhythms, but the extent to which the hindbrain possesses endogenous circadian timekeeping is unresolved. Here we provide compelling evidence that genetic, neuronal, and vascular activities of the brainstem's dorsal vagal complex are subject to intrinsic circadian control with a crucial role for the connection between its components in regulating their rhythmic properties. Robust 24 h variation in clock gene expression in vivo and neuronal firing ex vivo were observed in the area postrema (AP) and nucleus of the solitary tract (NTS), together with enhanced nocturnal responsiveness to metabolic cues. Unexpectedly, we also find functional and molecular evidence for increased penetration of blood borne molecules into the NTS at night. Our findings reveal that the hindbrain houses a local network complex of neuronal and non-neuronal autonomous circadian oscillators, with clear implications for understanding local temporal control of physiology in the brainstem.

GDF15 mediates adiposity resistance through actions on GFRAL neurons in the hindbrain AP/NTS.

BACKGROUND: Elevated circulating levels of the divergent transforming growth factor-beta (TGFb) family cytokine, growth differentiation factor 15 (GDF15), acting through its CNS receptor, glial-derived neurotrophic factor receptor alpha-like (GFRAL), can cause anorexia and weight loss leading to anorexia/cachexia syndrome of cancer and other diseases. Preclinical studies suggest that administration of drugs based on recombinant GDF15 might be used to treat severe obesity. However, the role of the GDF15-GFRAL pathway in the physiological regulation of body weight and metabolism is unclear. The critical site of action of GFRAL in the CNS has also not been proven beyond doubt. To investigate these two aspects, we have inhibited the actions of GDF15 in mice started on high-fat diet (HFD). METHODS: The actions of GDF15 were inhibited using two methods: (1) Groups of 8 mice under HFD had their endogenous GDF15 neutralised by monoclonal antibody treatment, (2) Groups of 15 mice received AAV-shRNA to knockdown GFRAL at its hypothesised major sites of action, the hindbrain area postrema (AP) and the nucleus of the solitary tract (NTS). Metabolic measurements were determined during both experiments. CONCLUSIONS: Treating mice with monoclonal antibody to GDF15 shortly after commencing HFD results in more rapid gain of body weight, adiposity and hepatic lipid deposition than the control groups. This is accompanied by reduced glucose and insulin tolerance and greater expression of pro-inflammatory cytokines in adipose tissue. Localised AP and NTS shRNA-GFRAL knockdown in mice commencing HFD similarly caused an increase in body weight and adiposity. This effect was in proportion to the effectiveness of GFRAL knockdown, indicated by quantitative analysis of hindbrain GFRAL staining. We conclude that the GDF15-GFRAL axis plays an important role in resistance to obesity in HFD-fed mice and that the major site of action of GDF15 in the CNS is GFRAL-expressing neurons in the AP and NTS.

Endogenous amylin contributes to birth of microglial cells in arcuate nucleus of hypothalamus and area postrema during fetal development.

Amylin acts in the area postrema (AP) and arcuate nucleus (ARC) to control food intake. Amylin also increases axonal fiber outgrowth from the AP→nucleus tractus solitarius and from ARC→hypothalamic paraventricular nucleus. More recently, exogenous amylin infusion for 4 wk was shown to increase neurogenesis in adult rats in the AP. Furthermore, amylin has been shown to enhance leptin signaling in the ARC and ventromedial nucleus of the hypothalamus (VMN). Thus, we hypothesized that endogenous amylin could be a critical factor in regulating cell birth in the ARC and AP and that amylin could also be involved in the birth of leptin-sensitive neurons. Amylin+/- dams were injected with BrdU at embryonic day 12 and at postnatalday 2; BrdU+ cells were quantified in wild-type (WT) and amylin knockout (KO) mice. The number of BrdU+HuC/D+ neurons was similar in ARC and AP, but the number of BrdU+Iba1+ microglia was significantly decreased in both nuclei. Five-week-old WT and KO littermates were injected with leptin to test whether amylin is involved in the birth of leptin-sensitive neurons. Although there was no difference in the number of BrdU+c-Fos+ neurons in the ARC and dorsomedial nucleus, an increase in BrdU+c-Fos+ neurons was seen in VMN and lateral hypothalamus (LH) in amylin KO mice. In conclusion, these data suggest that during fetal development, endogenous amylin favors the birth of microglial cells in the ARC and AP and that it decreases the birth of leptin-sensitive neurons in the VMN and LH.

GDF15 acts synergistically with liraglutide but is not necessary for the weight loss induced by bariatric surgery in mice.

OBJECTIVE: Analogues of GDF15 (Growth Differentiation Factor 15) are promising new anti-obesity therapies as pharmacological treatment with GDF15 results in dramatic reductions of food intake and body weight. GDF15 exerts its central anorexic effects by binding to the GFRAL receptor exclusively expressed in the Area Postrema (AP) and the Nucleus of the Solitary Tract (NTS) of the hindbrain. We sought to determine if GDF15 is an indispensable factor for other interventions that cause weight loss and which are also known to act via these hindbrain regions. METHODS: To explore the role of GDF15 on food choice we performed macronutrient intake studies in mice treated pharmacologically with GDF15 and in mice having either GDF15 or GFRAL deleted. Next we performed vertical sleeve gastrectomy (VSG) surgeries in a cohort of diet-induced obese Gdf15-null and control mice. To explore the anatomical co-localization of neurons in the hindbrain responding to GLP-1 and/or GDF15 we used GLP-1R reporter mice treated with GDF15, as well as naïve mouse brain and human brain stained by ISH and IHC, respectively, for GLP-1R and GFRAL. Lastly we performed a series of food intake experiments where we treated mice with targeted genetic disruption of either Gdf15 or Gfral with liraglutide; Glp1r-null mice with GDF15; or combined liraglutide and GDF15 treatment in wild-type mice. RESULTS: We found that GDF15 treatment significantly lowered the preference for fat intake in mice, whereas no changes in fat intake were observed after genetic deletion of Gdf15 or Gfral. In addition, deletion of Gdf15 did not alter the food intake or bodyweight after sleeve gastrectomy. Lack of GDF15 or GFRAL signaling did not alter the ability of the GLP-1R agonist liraglutide to reduce food intake. Similarly lack of GLP-1R signaling did not reduce GDF15's anorexic effect. Interestingly, there was a significant synergistic effect on weight loss when treating wild-type mice with both GDF15 and liraglutide. CONCLUSION: These data suggest that while GDF15 does not play a role in the potent effects of VSG in mice there seems to be a potential therapeutic benefit of activating GFRAL and GLP-1R systems simultaneously.

In renovascular hypertension, TNF-α type-1 receptors in the area postrema mediate increases in cardiac and renal sympathetic nerve activity and blood pressure.

AIMS: Neuroinflammation is a common feature in renovascular, obesity-related, and angiotensin II mediated hypertension. There is evidence that increased release of the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) contributes to the development of the hypertension, but the underlying neural mechanisms are unclear. Here, we investigated whether TNF-α stimulates neurons in the area postrema (AP), a circumventricular organ, to elicit sympathetic excitation, and increases in blood pressure (BP). METHODS AND RESULTS: In rats with renovascular hypertension, AP neurons that expressed TNF-α type-1 receptor (TNFR1) remained constantly activated (expressed c-Fos) and injection of TNFR1 neutralizing antibody into the AP returned BP (systolic: ∼151 mmHg) to normotensive levels (systolic: ∼108 mmHg). Nanoinjection of TNF-α (100 pg/50 nL) into the AP of anaesthetized normotensive rats increased BP (∼16 mmHg) and sympathetic nerve activity, predominantly to the heart (∼53%), but also to the kidneys (∼35%). These responses were abolished by prior injection of a TNFR1 neutralizing antibody (1 ng/50 nL) within the same site. TNFR1 were expressed in the somata of neurons activated by TNF-α that were retrogradely labelled from the rostral ventrolateral medulla (RVLM). CONCLUSION: These findings indicate that in renovascular hypertension, blocking TNFR1 receptors in the AP significantly reduces BP, while activation of TNFR1 expressing neurons in the AP by TNF-α increases BP in normotensive rats. This is mediated, in part, by projections to the RVLM and an increase in both cardiac and renal sympathetic nerve activity. These findings support the notion that proinflammatory cytokines and neuroinflammation are important pathological mechanisms in the development and maintenance of hypertension.

Rodent models of leptin receptor deficiency are less sensitive to amylin.

The pancreatic hormone amylin is released from beta cells following nutrient ingestion and contributes to the control of body weight and glucose homeostasis. Amylin reduces food intake by activating neurons in the area postrema (AP). Amylin was also shown to synergize with the adipokine leptin, with combination therapy producing greater weight loss and food intake reduction than either hormone alone. Although amylin and leptin were initially thought to interact downstream of the AP in the hypothalamus, recent findings show that the two hormones can act on the same AP neurons, suggesting a more direct relationship. The objective of this study was to determine whether amylin action depends on functional leptin signaling. We tested the ability of amylin to induce satiation and to activate its primary target neurons in the AP in two rodent models of LepR deficiency, the db/db mouse and the Zucker diabetic fatty (ZDF) rat. When compared with wild-type (WT) mice, db/db mice exhibited reduced amylin-induced satiation, reduced amylin-induced Fos in the AP, and a lower expression of calcitonin receptor (CTR) protein, the core component of all amylin receptors. ZDF rats also showed no reduction in food intake following amylin treatment; however, unlike the db/db mice, levels of amylin-induced Fos and CTR in the AP were no different than WT rats. Our results suggest that LepR expression is required for the full anorexic effect of amylin; however, the neuronal activation in the AP seems to depend on the type of LepR mutation.

Glucagon-like peptide-1 (GLP-1) action in the mouse area postrema neurons.

Glucagon-like peptide-1 (GLP-1) is a peptide hormone and member of the incretin family. GLP-1 related drugs, such as liraglutide, are widely used to treat diabetic patients and work by stimulating pancreatic ß cells to increase glucose-dependent insulin secretion. However, extrapancreatic effects, such as appetite suppression or emesis, are observed in response to GLP-1 receptor agonists. In this study we used the in vitro patch-clamp method in acute brainstem preparations of mice and demonstrated that GLP-1 acts directly on area postrema neurons. It is known that activation of the area postrema is related to the induction of homeostatic autonomic nervous systems, including nausea. Approximately,half of the neurons tested in the area postrema were excited by GLP-1 in the presence of tetrodotoxin, and is thought to be through adenylate cyclase-cAMP pathways. Excitation was not frequently observed in nucleus tractus solitaries neurons or in area postrema neurons from GLP-1 receptor knock-out mice. These results indicate that GLP-1 receptor agonists excite area postrema neurons and potentially leading to the expression of extra-pancreatic effects. This is the first study to show that GLP-1 directly activates area postrema neurons.