Page 1 of 53 Diabetes Amylin/Calcitonin receptor-mediated signaling in POMC neurons influences energy balance and locomotor activity in chow-fed male mice Running title: POMC amylin signaling regulates energy homeostasis Bernd Coester1, Christina Koester-Hegmann1, Thomas A. Lutz1 and Christelle Le Foll1 1Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich (UZH), 8057 Zurich, Switzerland Corresponding author: Christelle Le Foll Institute of Veterinary Physiology University of Zurich Winterthurerstrasse 260 8057 Zurich Switzerland Tel. +41 44 635 88 36 Fax +41 44 635 89 32 email [email protected] Word Count: 5883 Number of tables: 1 Number of figures: 8 Online Supplemental Material: 2 tables and 6 figures 1 Diabetes Publish Ahead of Print, published online March 9, 2020 Diabetes Page 2 of 53 Abstract Amylin, a pancreatic hormone and neuropeptide, acts principally in the hindbrain to decrease food intake and has been recently shown to act as a neurotrophic factor to control the development of APNTS and ARCPVN axonal fiber outgrowth. Amylin is also able to activate ERK signaling specifically in POMC neurons independently of leptin. To investigate the physiological role of amylin signaling in POMC neurons, the core component of the amylin receptor, calcitonin receptor (CTR) was depleted from POMC neurons using an inducible mouse model. The loss of CTR in POMC neurons leads to increased body weight gain, increased adiposity, and glucose intolerance in male knockout mice, characterized by decreased energy expenditure (EE) and decreased expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). Furthermore, a decreased spontaneous locomotor activity and absent thermogenic reaction to the application of the amylin receptor agonist were observed in male and female mice. Together, these results show a significant physiological impact of amylin/calcitonin signaling in CTR-POMC neurons on energy metabolism and demonstrate the need for sex-specific approaches in obesity research and potentially treatment. Keywords: amylin, hypothalamus, POMC, CTR, CALCR, BAT, thermogenesis, energy expenditure, adiposity 2 Page 3 of 53 Diabetes INTRODUCTION Amylin is a pancreatic gut hormone that is co-released with insulin by ß-cells in response to meals (1). The main central effect of amylin is an acute reduction of food intake that occurs after amylin binding to neurons in the area postrema (AP) (2) . The amylin receptor consists of a core calcitonin receptor (CTRA/B) which is coupled to receptor activity-modifying proteins (RAMP1-3) (3; 4). These receptor components are expressed in single neurons of the AP (5), but amylin also has other binding sites in the CNS that have not been thoroughly studied yet, such as the nucleus of the solitary tract (NTS), the lateral hypothalamic area (LHA), and the ventromedial (VMN) and arcuate hypothalamic nucleus (ARC) (6) (7; 8). Recent data of whole brain imaging with fluorescently labelled rat amylin in vivo confirmed its binding in the ARC and AP (9). The ARC has come into focus as a mediator of amylin’s effects on energy expenditure, its interactions with leptin signaling pathways and its developmental effects on axonal fiber outgrowth (6; 10; 11). The effects of amylin in adults are of interest because chronic amylin treatment has a sizable effect on weight loss that cannot be solely explained by a reduction in food intake (12) and hypothalamic actions might contribute to the “leptin- sensitizing” effect of amylin (13; 14). Our current hypothetical model of amylin signaling in the ARC includes a direct effect of amylin on POMC neurons through ERK1/2-phosphorylation, and an indirect effect on AgRP- neurons through microglial IL-6 secretion (15). This study aims to investigate the direct physiological effect of endogenous amylin signaling on POMC neurons with a conditional genetic knockout model. The depletion of CTR specifically in POMC neurons with tamoxifen (Tx) induction after weaning aims to avoid early developmental effects of disturbed amylin signaling (15) and allows us to test the hypothesis that amylin signaling in POMC neurons is critical for the control of energy expenditure. 3 Diabetes Page 4 of 53 MATERIALS AND METHODS Animal husbandry and diet Animals were kept in a temperature-controlled environment (21±2°C) on a 12:12h light cycle with lights off at 1000h. After weaning, they were separated by genotype and group-housed with littermates or single-housed for the tamoxifen (Tx) injections (16). Tamoxifen (Sigma T5648, Merck KGaA, Darmstadt, Germany) was dissolved in pure ethanol and mixed with corn oil (Sigma C8267) for a final concentration of 100mg/ml. Mice were injected at 4-week- old with a dose of 150µg/g or corn oil for 5 days. A first cohort of mice was fed standard chow (65% carbohydrate, 22% protein and 12.5% fat as percent of total energy content, 3430 Provimi Kliba, Kaiseraugst, Switzerland) and two separate cohorts were fed 45% high fat diet (35% carbohydrate, 20% protein and 45% fat as percent of total energy content, D12451, Research Diets, New Brunswick, NJ) ad libitum. The animals were kept in an enriched environment in wood chip bedding with cardboard houses and tissues as nesting material. The Veterinary Office of the Canton Zurich, Switzerland approved all animal procedures. POMC-creCTR mice POMC-cre:ERT2 (C57BL/6J;129X1/SvJ-Tg(Pomc-cre/ERT2)#Jke; MGI:5569339) (16) (kindly provided by Pr. Joel Elmquist UT Southwestern), CTRfl/fl (Calcr<tm1(fl)>; MGI:5751436) (frozen sperm was kindly provided by Dr. Jean-Pierre David and Dr Thorsten Shinke, University Medical Center Hamburg) (17). The crossing resulted in 2 groups of mice: POMC-WT x CTRfl/fl (POMC-WTCTR) and POMC-Cre x CTRfl/fl (POMC-CreCTR). To confirm the genetic model, Ai14 reporter mice [B6.Cg-Gt(ROSA)26Sor<tm14(CAG- tdTomato)Hze>/J(#007914)] were bred with CTRfl/fl POMC-CreERT2 mice. Each mouse was genotyped using previously published primers (17). 4 Page 5 of 53 Diabetes Effect of amylin, sCT and leptin on food intake Mice were singled-housed in BioDAQ cages (Research Diets, New Brunswick, NJ) and following 7 days of acclimation, mice were fasted for 12h during the light phase. At dark onset mice were injected intraperitoneally (i.p) with amylin (50, 500µg/kg; H-9475 Bachem, Bubendorf, CH), sCT (10µg/kg; 4033011.0001 Bachem) or saline (NaCl 0.9%) in a crossover design and food was returned. Using the same paradigm as above, a separate cohort of mice was also tested for their anorectic response to leptin (5 mg/kg i.p.; Peprotech, UK). Food intake was recorded for the following 24h and the mice could rest for two more days before the next injection. Baseline food intake was calculated by averaging food intake over a 3-day period prior to injections. Meal pattern criteria were an inter-meal-interval (IMI) of 600 s and a minimal meal of 0.02g (18). Male and female data were pooled since no difference was observed between sexes (19). Glucose and insulin tolerance test, blood sampling Food was removed 2 h prior to lights off and mice were gavaged (glucose; 2g/kg) or injected (insulin; 0.5U/kg) at lights off. Blood glucose (ContourXT, Bayer, Germany) at the tail was measured before and 15, 30, 45, 60, 90 and 120 min after gavage or injection. In addition, baseline blood samples after a 2h fast during the study were obtained by tongue bleeding during a brief 30 sec isoflurane anesthesia (2%). Insulin and leptin were measured (Mesoscale Discovery, MD, USA). Telemetric sensors, indirect calorimetry and body composition measurements TA-F10 sensors for body temperature and activity measurements (Data Sciences International, MN, USA) were implanted intraperitoneally under brief isoflurane anaesthesia (2%). 5 Diabetes Page 6 of 53 Treatment with non-steroidal anti-inflammatory agents (Metacam 2mg/kg s.c.) and antibiotics (Baytril 2.5%, 7.5mg/kg s.c.) before surgery and during the following 5 days was provided while the mice recovered. Subsequently, mice were single-housed in a 16-cage PhenoMaster indirect calorimetry system (TSE Systems; Bad Homburg, Germany) (18). After one week of adaptation, data were collected at baseline or following a 12h fast and injection with saline or sCT (10µg/kg i.p.). From these values, energy expenditure (EE) and respiratory exchange ratio (RER) were calculated based on equations from Weir (20). Body mass composition from L1 to L4 was performed using a CT scan (Quantum GX micro CT, PerkinElmer, Waltham, USA). Lean and fat mass were quantified as previously validated (21). Analyze 12.0 software (AnalyzeDirect, Overland Park, USA) was used to quantify visceral and subcutaneous fat volume in the CT images. EE data were corrected for individual lean body mass (LBM in g) and fat mass (FM in g) using the following equation: LBM + 0.2FM, as recommended by Even and Nadkarni (22). Mouse perfusion To assess pERK signaling, mice were fasted for 12 hours and at dark onset were injected with saline or amylin (50µg/kg i.p.) as previously published (15). To assess pSTAT3 signaling, mice were fasted for 2 hours and at dark onset were injected with saline or leptin (5mg/kg i.p.) (23). The brains were frozen in hexane on dry ice, stored at -80°C, cut in 25µm sections (Leica Biosystems, Germany), mounted on Superfrost Plus slides (Thermo Fisher Scientific, Reinach, Switzerland) and stored in cryoprotectant (50% 0.02M KPBS, 30% ethylene glycol, 20% glycerol) at -20°C until staining. Immunohistochemistry POMC-pERK double staining: For pretreatment, sections were demasked in 0.5% NaOH + 1% 6 Page 7 of 53 Diabetes H2O2 in KPBS and incubated in 0.3% glycine in KPBS. Sections were blocked and incubated pERK antibody (1:1000, 9101, Cell signaling) in 2% NGS-0.3% Triton-1% BSA in KPBS for 48h at 4C.