Identification of Hypothalamic Glucoregulatory Neurons That Sense and Respond to Changes in Glycemia.

To investigate whether glucoregulatory neurons in the hypothalamus can sense and respond to physiological variation in the blood glucose (BG) level, we combined continuous arterial glucose monitoring with continuous measures of the activity of a specific subset of neurons located in the hypothalamic ventromedial nucleus that express pituitary adenylate cyclase activating peptide (VMNPACAP neurons) obtained using fiber photometry. Data were collected in conscious, free-living mice during a 1-h baseline monitoring period and a subsequent 2-h intervention period during which the BG level was raised either by consuming a chow or a high-sucrose meal or by intraperitoneal glucose injection. Cross-correlation analysis revealed that, following a 60- to 90-s delay, interventions that raise the BG level reliably associate with reduced VMNPACAP neuron activity (P < 0.01). In addition, a strong positive correlation between BG and spontaneous VMNPACAP neuron activity was observed under basal conditions but with a much longer (∼25 min) temporal offset, consistent with published evidence that VMNPACAP neuron activation raises the BG level. Together, these findings are suggestive of a closed-loop system whereby VMNPACAP neuron activation increases the BG level; detection of a rising BG level, in turn, feeds back to inhibit these neurons. To our knowledge, these findings constitute the first evidence of a role in glucose homeostasis for glucoregulatory neurocircuits that, like pancreatic ß-cells, sense and respond to physiological variation in glycemia. ARTICLE HIGHLIGHTS: By combining continuous arterial glucose monitoring with fiber photometry, studies investigated whether neurons in the murine ventromedial nucleus that express pituitary adenylate cyclase activating peptide (VMNPACAP neurons) detect and respond to changes in glycemia in vivo. VMNPACAP neuron activity rapidly decreases (within <2 min) when the blood glucose level is raised by either food consumption or glucose administration. Spontaneous VMNPACAP neuron activity also correlates positively with glycemia, but with a longer temporal offset, consistent with reports that hyperglycemia is induced by experimental activation of these neurons. Like pancreatic ß-cells, neurons in the hypothalamic ventromedial nucleus appear to sense and respond to physiological variation in glycemia.

Glucagon-like peptide-1 receptors in nucleus accumbens, ventral hippocampus, and lateral septum reduce alcohol reinforcement in mice.

Glucagon-like peptide 1 (GLP-1) receptor agonists can decrease alcohol intake by central mechanisms that are still poorly understood. The lateral septum (LS) and the ventral/caudal part of the hippocampus are enriched in GLP-1 receptors, and activity in these regions was shown to modulate reward-related behaviors. Using microinfusions of the GLP-1 receptor agonist exendin-4 in mice trained to self-administer oral alcohol in an operant assay, we tested whether pharmacological stimulation of GLP-1 receptors in hippocampus and LS decrease alcohol self-administration. We report that infusion of exendin-4 in the ventral hippocampus or LS was sufficient to reduce alcohol self-administration with as large effect sizes as we previously reported with systemic exendin-4 administration. Infusion of exendin-4 into the nucleus accumbens also reduced alcohol self-administration, as anticipated based on earlier reports, while infusion of exendin-4 into the caudate-putamen (dorsal striatum) had little effect, consistent with lack of GLP-1 receptor expression in this region. The distribution of exendin-4 after infusion into the LS or caudate putamen was visualized using a fluorescently labeled ligand. These findings add to our understanding of the circuit-level mechanisms underlying the ability of GLP-1 receptor agonists to reduce alcohol self-administration. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Acute changes in systemic glycemia gate access and action of GLP-1R agonist on brain structures controlling energy homeostasis.

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

Screening for type 2 diabetes and hypertension in seafarers' medical examinations.

BACKGROUND: The aims of the study are: 1) to replace the urine glucose test for diabetes with more than 50% false negatives, with an accurate screening for type 2 diabetes and hypertension in the mandatory biannual fit-for-duty medical examinations of seafarers; 2) to produce data driven "Green Ship" health pro-motion in the ships. A new health promotion and disease prevention public health intervention programme integrated in the fit-for-duty medical examinations for seafarers is being developed. MATERIALS AND METHODS: The lack of an accurate diagnosis of type 2 diabetes is replaced by accurate HbA1c and/or fasting glucose tests and the test for hypertension in various disease stages is based on the International Associations' Guidelines. A "Green Ship" health promotion programme is proposed for all on board, not only for diseased crew members. RESULTS: A protocol for an accurate biannual screening for diabetes and hypertension is presented. Educational programmes for medical doctors and seafarers on the management of hypertension and diabetes on board will be developed. Presuming that all crew members are potentially on their way to be pre-diseased or are diseased, the "Green Ship" health promotion programme is implemented for the whole crew. CONCLUSIONS: The International Labour Organization and the National Maritime Authorities are prompted to revise the International and the National Guidelines for Seafarers Medical Examinations, respectively. Con-certed actions are requested to implement public health promotion projects in shipping. Maritime medical doctors are prompted to use health dialogues and to report the clinical data in the Excel file. Sustainability is obtained by complying with the Sustainable Development Goals (3, 4, 8, 10, and 17).

Rethinking the use of urine dipstick for early diagnosis of Type 2 diabetes mellitus.

Transport workers, seafarers and fishers have biannual mandatory fit-for duty medical examinations. Urinedipstick is used for early diagnosis of Type 2 diabetes mellitus. Due to low sensitivity with more than 80% false negatives the method should be replaced by highly sensitive blood tests, Hb1Ac or similar for diagnosis of Type 2 diabetes mellitus to pursue the UN Global Sustainable Goals, especially Goal 3: Good health and well-being for all workers and Goal 8: Decent Work and Economic Growth.

Early diagnosis of T2DM using high sensitive tests in the mandatory medical examinations for fishers, seafarers and other transport workers.

Transport workers like seafarers, truck-, bus-, train- and taxi drivers and fishers have a known great inequity in health at work including high risk of developing type 2 diabetes. Their routine mandatory medical examinations use urine glucose for diabetes check with more than 50% false negatives, which should be replaced by high sensitive tests for diabetes-2, like A1C, Fasting Glucose (FPG) or Oral Glucose Tolerance Test (OGTT).

Insulin signalling in tanycytes gates hypothalamic insulin uptake and regulation of AgRP neuron activity.

Insulin acts on neurons and glial cells to regulate systemic glucose metabolism and feeding. However, the mechanisms of insulin access in discrete brain regions are incompletely defined. Here we show that insulin receptors in tanycytes, but not in brain endothelial cells, are required to regulate insulin access to the hypothalamic arcuate nucleus. Mice lacking insulin receptors in tanycytes (IR∆Tan mice) exhibit systemic insulin resistance, while displaying normal food intake and energy expenditure. Tanycytic insulin receptors are also necessary for the orexigenic effects of ghrelin, but not for the anorexic effects of leptin. IR∆Tan mice exhibit increased agouti-related peptide (AgRP) neuronal activity, while displaying blunted AgRP neuronal adaptations to feeding-related stimuli. Lastly, a highly palatable food decreases tanycytic and arcuate nucleus insulin signalling to levels comparable to those seen in IR∆Tan mice. These changes are rooted in modifications of cellular stress responses and of mitochondrial protein quality control in tanycytes. Conclusively, we reveal a critical role of tanycyte insulin receptors in gating feeding-state-dependent regulation of AgRP neurons and systemic insulin sensitivity, and show that insulin resistance in tanycytes contributes to the pleiotropic manifestations of obesity-associated insulin resistance.

Flash glucose monitoring and automated bolus calculation in type 1 diabetes treated with multiple daily insulin injections: a 26 week randomised, controlled, multicentre trial.

AIMS/HYPOTHESIS: We aimed to compare the effects of intermittently scanned continuous glucose monitoring (isCGM) and carbohydrate counting with automated bolus calculation (ABC) with usual care. METHODS: In a randomised, controlled, open-label trial carried out at five diabetes clinics in the Capital Region of Denmark, 170 adults with type 1 diabetes for ≥1 year, multiple daily insulin injections and HbA1c > 53 mmol/mol (7.0%) were randomly assigned 1:1:1:1 with centrally prepared envelopes to usual care (n = 42), ABC (n = 41), isCGM (n = 48) or ABC+isCGM (n = 39). Blinded continuous glucose monitoring data, HbA1c and patient-reported outcomes were recorded at baseline and after 26 weeks. The primary outcome was change in time in range using isCGM vs usual care. RESULTS: Baseline characteristics were comparable across arms: mean age 47 (SD 13.7) years, median (IQR) diabetes duration 18 (10-28) years and HbA1c 65 (61-72) mmol/mol (8.1% [7.7-8.7%]). Change in time in range using isCGM was comparable to usual care (% difference of 3.9 [-12-23], p = 0.660). The same was true for the ABC and ABC+isCGM arms and for hypo- and hyperglycaemia. Also compared with usual care, using ABC+isCGM reduced HbA1c (4 [95% CI 1, 8] mmol/mol) (0.4 [0.1, 0.7] %-point) and glucose CV (11% [4%, 17%]) and improved treatment satisfaction, psychosocial self-efficacy and present life quality. Treatment satisfaction also improved by using isCGM alone vs usual care. Statistical significance was maintained after multiple testing adjustment concerning glucose CV and treatment satisfaction with ABC+isCGM, and treatment satisfaction with isCGM. Discontinuation was most common among ABC only users, and among completers the ABC was used 4 (2-5) times/day and the number of daily isCGM scans was 5 (1-7) at study end. CONCLUSIONS/INTERPRETATION: isCGM alone did not improve time in range, but treatment satisfaction increased in technology-naive people with type 1 diabetes and suboptimal HbA1c. The combination of ABC+isCGM appears advantageous regarding glycaemic variables and patient-reported outcomes, but many showed resistance towards ABC. TRIAL REGISTRATION: ClinicalTrials.gov NCT03682237. FUNDING: The study is investigator initiated and financed by the Capital Region of Denmark.

Role of hypothalamic MAPK/ERK signaling and central action of FGF1 in diabetes remission.

The capacity of the brain to elicit sustained remission of hyperglycemia in rodent models of type 2 diabetes following intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) is well established. Here, we show that following icv FGF1 injection, hypothalamic signaling by extracellular signal-regulated kinases 1 and 2 (ERK1/2), members of the mitogen-activated protein kinase (MAPK) family, is induced for at least 24 h. Further, we show that this prolonged response is required for the sustained antidiabetic action of FGF1 since it is abolished by sustained (but not acute) pharmacologic blockade of hypothalamic MAPK/ERK signaling. We also demonstrate that FGF1 R50E, a FGF1 mutant that activates FGF receptors but induces only transient hypothalamic MAPK/ERK signaling, fails to mimic the sustained glucose lowering induced by FGF1. These data identify sustained activation of hypothalamic MAPK/ERK signaling as playing an essential role in the mechanism underlying diabetes remission induced by icv FGF1 administration.

A genetic map of the mouse dorsal vagal complex and its role in obesity.

The brainstem dorsal vagal complex (DVC) is known to regulate energy balance and is the target of appetite-suppressing hormones, such as glucagon-like peptide 1 (GLP-1). Here we provide a comprehensive genetic map of the DVC and identify neuronal populations that control feeding. Combining bulk and single-nucleus gene expression and chromatin profiling of DVC cells, we reveal 25 neuronal populations with unique transcriptional and chromatin accessibility landscapes and peptide receptor expression profiles. GLP-1 receptor (GLP-1R) agonist administration induces gene expression alterations specific to two distinct sets of Glp1r neurons-one population in the area postrema and one in the nucleus of the solitary tract that also expresses calcitonin receptor (Calcr). Transcripts and regions of accessible chromatin near obesity-associated genetic variants are enriched in the area postrema and the nucleus of the solitary tract neurons that express Glp1r and/or Calcr, and activating several of these neuronal populations decreases feeding in rodents. Thus, DVC neuronal populations associated with obesity predisposition suppress feeding and may represent therapeutic targets for obesity.

Transcriptomic analysis links diverse hypothalamic cell types to fibroblast growth factor 1-induced sustained diabetes remission.

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.

Study protocol for optimising glycaemic control in type 1 diabetes treated with multiple daily insulin injections: intermittently scanned continuous glucose monitoring, carbohydrate counting with automated bolus calculation, or both? A randomised controlled trial.

INTRODUCTION: There are beneficial effects of advanced carbohydrate counting with an automatic bolus calculator (ABC) and intermittently scanned continuous glucose monitoring (isCGM) in persons with type 1 diabetes. We aim to compare the effects of isCGM, training in carbohydrate counting with ABC and the combination of the two concepts with standard care. METHODS AND ANALYSIS: A multi-centre randomised controlled trial with inclusion criteria: ≥18 years, type 1 diabetes ≥1 year, injection therapy, HbA1c >53 mmol/mol, whereas daily use of carbohydrate counting and/or CGM/isCGM wear are exclusion criteria. Inclusion was initiated in October 2018 and is ongoing. Eligible persons are randomised into four groups: standard care, ABC, isCGM or ABC+isCGM. Devices used are FreeStyle Libre Flash and smart phone diabetes application mySugr. Participants attend group courses according to treatment allocation with different educational contents. Participants are followed for 26 weeks with clinical visits and telephone consultations. At baseline and at study end, participants wear blinded CGM, have blood samples performed and fill in questionnaires on person-related outcomes, and at baseline also on personality traits and hypoglycaemia awareness. The primary outcome is the difference in time spent in normoglycaemia (4-10 mmol/L) at study end versus baseline between the isCGM group and the standard care group. Secondary outcomes will also be analysed. Results are expected in 2020. ETHICS AND DISSEMINATION: Regional Scientific Ethics Committee approval (H-17040573). Results will be sought disseminated at conferences and in high impact journals.Trial registration numberClinicalTrial.gov registry (NCT03682237).

Semaglutide lowers body weight in rodents via distributed neural pathways.

Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.

Salmon calcitonin distributes into the arcuate nucleus to a subset of NPY neurons in mice.

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

Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites.

Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.

The Hypothalamic Arcuate Nucleus-Median Eminence Is a Target for Sustained Diabetes Remission Induced by Fibroblast Growth Factor 1.

In rodent models of type 2 diabetes (T2D), sustained remission of diabetic hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1). To identify the brain areas responsible for this effect, we first used immunohistochemistry to map the hypothalamic distribution of phosphorylated extracellular signal-related kinase 1/2 (pERK1/2), a marker of mitogen-activated protein kinase-ERK signal transduction downstream of FGF receptor activation. Twenty minutes after icv FGF1 injection in adult male Wistar rats, pERK1/2 staining was detected primarily in two hypothalamic areas: the arcuate nucleus-median eminence (ARC-ME) and the paraventricular nucleus (PVN). To determine whether an action of FGF1 localized to either the ARC-ME or the PVN is capable of mimicking the sustained antidiabetic effect elicited by icv FGF1, we microinjected either saline vehicle or a low dose of FGF1 (0.3 µg/side) bilaterally into either the ARC-ME area or PVN of Zucker Diabetic Fatty rats, a model of T2D, and monitored daily food intake, body weight, and blood glucose levels over a 3-week period. Whereas bilateral intra-arcuate microinjection of saline vehicle was without effect, remission of hyperglycemia lasting >3 weeks was observed following bilateral microinjection of FGF1 into the ARC-ME. This antidiabetic effect cannot be attributed to leakage of FGF1 into cerebrospinal fluid and subsequent action on other brain areas, since icv injection of the same total dose was without effect. Combined with our finding that bilateral microinjection of the same dose of FGF1 into the PVN was without effect on glycemia or other parameters, we conclude that the ARC-ME area (but not the PVN) is a target for sustained remission of diabetic hyperglycemia induced by FGF1.

Breastfeeding at night is rarely followed by hypoglycaemia in women with type 1 diabetes using carbohydrate counting and flexible insulin therapy.

AIMS/HYPOTHESIS: Hypoglycaemia in association with breastfeeding is a feared condition in mothers with type 1 diabetes. Thus, routine carbohydrate intake at each breastfeed, particularly at night, is often recommended despite lack of evidence. We aimed to evaluate glucose levels during breastfeeding, focusing on whether night-time breastfeeding induced hypoglycaemia in mothers with type 1 diabetes. METHODS: Of 43 consecutive mothers with type 1 diabetes, 33 (77%) were included prospectively 1 month after a singleton delivery. Twenty-six mothers (mean [SD] age 30.7 [5.8] years, mean [SD] duration of diabetes 18.6 [10.3] years) were breastfeeding and seven mothers (mean [SD] age 31.7 [5.6] years, mean [SD] duration of diabetes 20.4 [6.2] years) were bottle-feeding their infants with formula. All were experienced in carbohydrate counting using individually tailored insulin therapy with insulin analogues (45% on insulin pump, 55% on multiple daily injections). Thirty-two women with type 1 diabetes, matched for age ±1 year and BMI ±1 kg/m2, who had not given birth or breastfed in the previous year, served as a control group. Blinded continuous glucose monitoring (CGM) for 6 days was applied at 1, 2 and 6 months postpartum in the breastfeeding mothers who recorded breastfeeds and carbohydrate intake at each CGM period. CGM was applied at 1 month postpartum in the formula-feeding mothers and once in the control women. The insulin dose was individually tailored after each CGM period. RESULTS: The percentage of night-time spent with CGM <4.0 mmol/l was low (4.6%, 3.1% and 2.7% at each CGM period in the breastfeeding mothers vs 1.6% in the control women, p = 0.77), and the breastfeeding mothers spent a greater proportion of the night-time in the target range of 4.0-10.0 mmol/l (p = 0.01). Symptomatic hypoglycaemia occurred two or three times per week at 1, 2 and 6 months postpartum in both breastfeeding mothers and the control women. Severe hypoglycaemia was reported by one mother (3%) during the 6 month postpartum period and by one control woman (3%) in the previous year (p = 0.74). In breastfeeding mothers at 1 month, the insulin dose was 18% (-67% to +48%) lower than before pregnancy (p = 0.04). In total, carbohydrate was not consumed in relation to 438 recorded night-time breastfeeds, and CGM <4.0 mmol/l within 3 h occurred after 20 (4.6%) of these breastfeeds. CONCLUSIONS/INTERPRETATION: The percentage of night-time spent in hypoglycaemia was low in the breastfeeding mothers with type 1 diabetes and was similar in the control women. Breastfeeding at night-time rarely induced hypoglycaemia. The historical recommendation of routine carbohydrate intake at night-time breastfeeding may be obsolete in mothers with type 1 diabetes who have properly reduced insulin dose with sufficient carbohydrate intake. TRIAL REGISTRATION: ClinicalTrials.gov NCT02898428.

PYY(3-36) and exendin-4 reduce food intake and activate neuronal circuits in a synergistic manner in mice.

Peptide YY(3-36) ((PYY(3-36)) and glucagon like peptide 1 (GLP-1) in combination reduce food intake and body weight in an additive or synergistic manner in animal models and in humans. Nevertheless, the mechanisms behind are not completely understood. The present study aims to investigate the effect of combining PYY(3-36) and the GLP-1 receptor agonist exendin-4 (Ex4) by examining acute food intake and global neuronal activation as measured by c-fos in C57BL/6 J mice. An additive reduction in food intake was found 1.5 h after s.c dosing with the combination of PYY(3-36) (200 µg/kg) and Ex4 (2.5 µg/kg). This was associated with a synergistic enhancement of c-fos reactivity in central amygdalar nucleus (CeA), rostral part of the mediobasal arcuate nucleus (ARH), supratrigeminal nucleus (SUT), lateral parabrachial nucleus (PB), area postrema (AP) and nucleus tractus solitarius (NTS) compared to vehicle, PYY(3-36) and Ex4 individually dosed mice. The regions activated by Ex4 individually and PYY(3-36) and Ex4 in combination resembled each other, but the combination group had a significantly stronger c-fos response. Twenty-five brain areas were activated by PYY(3-36) and Ex4 in combination compared to vehicle versus nine brain areas by Ex4 individually. No significant increase in c-fos reactivity was found by PYY(3-36) compared to vehicle dosed mice. The neuronal activation of ARH and the AP/NTS to PB to CeA pathway is important for appetite regulation while SUT has not previously been reported in the regulation of energy balance. As PYY(3-36) and Ex4 act on different neurons leading to recruitment of different signalling pathways within and to the brain, an interaction of these pathways may contribute to their additive/synergistic action. Thus, PYY(3-36) boosts the effect of Ex4 possibly by inducing less inhibition of neuronal activity leading to an enhanced neuronal activity induced by Ex4.