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1110 Diabetes Volume 69, June 2020

Amylin/–Mediated Signaling in POMC Neurons Influences Energy Balance and Locomotor Activity in Chow-Fed Male Mice

Bernd Coester, Christina Koester-Hegmann, Thomas A. Lutz, and Christelle Le Foll

Diabetes 2020;69:1110–1125 | https://doi.org/10.2337/db19-0849

Amylin, a pancreatic and , acts but amylin also has other binding sites in the central ner- principally in the hindbrain to decrease food intake and vous system that have not been thoroughly studied yet, has recently been shown to act as a neurotrophic factor such as the nucleus of the solitary tract (NTS), the lateral to control the development of area postrema → nucleus hypothalamic area (LHA), and the ventromedial (VMN) of the solitary tract and arcuate hypothalamic nucleus → and arcuate (ARC) hypothalamic nucleus (6–8). Recent data paraventricular nucleus axonal fiber outgrowth. Amylin is of whole brain imaging with fluorescently labeled rat amylin fi also able to activate ERK signaling speci cally in POMC in vivo confirmed its binding in the ARC and AP (9). The neurons independently of . For investigation of the ARC has come into focus as a mediator of amylin’s effects physiological role of amylin signaling in POMC neurons, on energy expenditure (EE), its interactions with leptin the core component of the amylin receptor, calcitonin signaling pathways, and its developmental effects on ax- receptor (CTR), was depleted from POMC neurons using onal fiber outgrowth (6,10,11). The effects of amylin in an inducible mouse model. The loss of CTR in POMC adults are of interest because chronic amylin treatment has neurons leads to increased body weight gain, increased adiposity, and glucose intolerance in male knockout mice, a sizable effect on weight loss that cannot be solely explained characterized by decreased energy expenditure (EE) and by a reduction in food intake (12) and hypothalamic actions “ ” decreased expression of uncoupling protein 1 (UCP1) in might contribute to the leptin-sensitizing effect of amylin brown . Furthermore, a decreased spon- (13,14). taneous locomotor activity and absent thermogenic re- Our current hypothetical model of amylin signaling in action to the application of the amylin receptor agonist the ARC includes a direct effect of amylin on POMC neurons were observed in male and female mice. Together, these through ERK1/2 phosphorylation and an indirect effect on results show a significant physiological impact of amylin/ AgRP neurons through microglial IL-6 secretion (15). This calcitonin signaling in CTR-POMC neurons on energy study aims to investigate the direct physiological effect of metabolism and demonstrate the need for sex-specific endogenous amylin signaling on POMC neurons with a approaches in obesity research and potentially treatment. conditional genetic knockout (KO) model. The depletion of CTR specifically in POMC neurons with tamoxifen (Tx) induction after weaning aims to avoid early developmental Amylin is a pancreatic gut hormone that is co-released with effects of disturbed amylin signaling (15) and allows us to by b-cells in response to meals (1). The main central test the hypothesis that amylin signaling in POMC neu- effect of amylin is an acute reduction of food intake that rons is critical for the control of EE. occurs after amylin binding to neurons in the area post- rema (AP) (2). The amylin receptor consists of a core cal- RESEARCH DESIGN AND METHODS citonin receptor (CTRA/B), which is coupled to receptor Animal Husbandry and Diet activity–modifying proteins (RAMP1–3) (3,4). These receptor Animals were kept in a temperature-controlled environ- components are expressed in single neurons of the AP (5), ment (21 6 2°C) on a 12:12 h light cycle with lights off at

Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Zurich, © 2020 by the American Diabetes Association. Readers may use this article as Switzerland long as the work is properly cited, the use is educational and not for profit, and the Corresponding author: Christelle Le Foll, [email protected] work is not altered. More information is available at https://www.diabetesjournals .org/content/license. Received 27 August 2019 and accepted 28 February 2020 This article contains supplementary material online at https://diabetes.diabetesjournals .org/lookup/suppl/doi:10.2337/db19-0849/-/DC1. diabetes.diabetesjournals.org Coester and Associates 1111

1000 h. After weaning, they were separated by genotype Care, Basel, Switzerland) at the tail was measured before and group-housed with littermates or single-housed for and 15, 30, 45, 60, 90, and 120 min after gavage or injection. the Tx injections (16). Tx (cat. no. T5648; Sigma-Aldrich, In addition, baseline blood samples after a 2-h fast during the Merck KGaA, Darmstadt, Germany) was dissolved in pure study were obtained by tongue bleeding during a brief ethanol and mixed with corn oil (cat. no. C8267; Sigma- 30-s isoflurane anesthesia (2%). Insulin and leptin were Aldrich) for a final concentration of 100 mg/mL. Mice were measured (Meso Scale Discovery, Rockville, MD). injected at 4 weeks old with a dose of 150 mg/g or corn oil Telemetric Sensors, Indirect Calorimetry, and Body for 5 days. A first cohort of mice was fed standard chow Composition Measurements (65% carbohydrate, 22% protein, and 12.5% fat as per- TA-F10 sensors for body temperature and activity mea- cent of total energy content, cat. no. 3430; Provimi Kliba, surements (Data Sciences International, New Brighton, MN) Kaiseraugst, Switzerland), and two separate cohorts were were implanted intraperitoneally under brief isoflurane fed a high-fat diet (HFD) (35% carbohydrate, 20% protein, anesthesia (2%). Treatment with nonsteroidal anti- and 45% fat as percent of total energy content, cat. no. inflammatory agents (2 mg/kg subcutaneous [s.c.] Metacam) D12451; Research Diets, New Brunswick, NJ) ad libitum. The and antibiotics (7.5 mg/kg s.c. Baytril 2.5%) before surgery animals were kept in an enriched environment in wood chip and during the following 5 days was provided while the mice bedding with cardboard houses and tissues as nesting ma- recovered.Subsequently,miceweresinglehousedina terial. The Veterinary Office of the Canton of Zurich, Zurich, 16-cage PhenoMaster indirect calorimetry system (TSE Switzerland, approved allanimalprocedures. Systems, Bad Homburg, Germany) (18). After 1 week of POMC-creCTR Mice adaptation, data were collected at baseline or following POMC-cre:ERT2 (C57BL/6J;129X1/SvJ-Tg(Pomc-cre/ERT2) a 12- h fast and injection with saline or sCT (10 mg/kg i.p.). #Jke; MGI:5569339) (16) (kindly provided by Joel Elmquist, From these values, EE and respiratory exchange ratio (RER) fl fl UT Southwestern), CTR / (Calcr,tm1(fl).; MGI:5751436) were calculated based on equations of Weir (20). Body mass (frozen sperm kindly provided by Drs. Jean-Pierre David composition from L1 to L4 was performed using a computed and Thorsten Shinke, University Medical Center Hamburg) tomography scan (Quantum GX microCT; PerkinElmer, (17). The crossing resulted in two groups of mice: POMC- Waltham, MA). Lean and fat mass (FM) were quantified as fl fl fl fl WT 3 CTR / (POMC-WTCTR) and POMC-Cre 3 CTR / previously validated (21). Analyze 12.0 software (Analyze- (POMC-CreCTR). For confirmation of the genetic model, Direct, Overland Park, KS) was used to quantify visceral Ai14 reporter mice [B6.Cg-Gt(ROSA)26Sor,tm14(CAG- and subcutaneous fat volume in the computed tomography fl fl tdTomato)Hze./J(#007914)]werebredwithCTR / POMC- images. EE data were corrected for individual lean body CreERT2 mice. Each mouse was genotyped using previously mass (LBM) (in grams) and FM (in grams) using the fol- published primers (17). lowing equation: LBM 1 0.2 FM, as recommended by Even and Nadkarni (22). Effect of Amylin, sCT, and Leptin on Food Intake Mice were single housed in BioDAQ cages (Research Diets), Mouse Perfusion and following 7 days of acclimation, mice were fasted for For assessment of phosphorylated (p)ERK signaling, mice 12 h during the light phase. At dark onset mice were were fasted for 12 h and at dark onset were injected with m injected intraperitoneally (i.p.) with amylin (50, 500 mg/kg) saline or amylin (50 g/kg i.p.) as previously described (cat. no. H-9475; Bachem, Bubendorf, Switzerland), salmon (15). For assessment of pSTAT3 signaling, mice were fasted calcitonin (sCT) (10 mg/kg) (cat. no. 4033011.0001; Bachem) for 2 h and at dark onset were injected with saline or leptin or saline (NaCl 0.9%) in a crossover design and food was (5 mg/kg i.p.) (23). The brains were frozen in hexane on 2 m returned. Using the same paradigm described above, dry ice, stored at 80°C, cut in 25 m sections (Leica a separate cohort of mice was also tested for anorectic Biosystems, Wetzlar, Germany), mounted on Superfrost fi response to leptin (5 mg/kg i.p.) (PeproTech, London, Plus slides (Thermo Fisher Scienti c, Reinach, Switzerland), U.K.). Food intake was recorded for the following 24 h, and stored in cryoprotectant (50% 0.02 mol/L potassium and the mice could rest for two more days before the next phosphate buffered saline (KPBS), 30% ethylene glycol, 2 injection. Baseline food intake was calculated by averag- 20% glycerol) at 20°C until staining. ing food intake over a 3-day period prior to injections. Immunohistochemistry Meal pattern criteria were an intermeal interval of 600 s and a minimal meal of 0.02 g (18). Male and female food POMC-pERK Double Staining intake data were pooled, since no differences were ob- For pretreatment, sections were demasked in 0.5% NaOH 1 served between sexes (19). 1% H2O2 in KPBS and incubated in 0.3% in KPBS. Sections were blocked and incubated with pERK Glucose and Insulin Tolerance Test and Blood antibody (1:1,000, cat. no. 9101; Cell Signaling Technol- Sampling ogy) in 2% normal goat serum (NGS)–0.3% Triton–1% Food was removed 2 h prior to lights off, and mice were BSA in KPBS for 48 h at 4°C. Sections were then placed in gavaged (2 g/kg glucose) or injected (0.5 units/kg insulin) Cy3 goat anti-rabbit secondary antibody at 1:100 concen- at lights off. Blood glucose (Contour XT; Ascensia Diabetes tration for 2 h at room temperature. Sections were again 1112 POMC Amylin Signaling Regulates Energy Diabetes Volume 69, June 2020 blocked before being incubated with primary POMC anti- Probe-Mm-RAMP1-C1, cat no. 532681-C1, and Probe-Mm- body (1:1,000, cat. no. H-029–30; Phoenix Pharmaceut- RAMP3-C1, cat no. 497131-C1; Advanced Cell Diagnostics) icals, Karlsruhe, Germany) for 48 h at 4°C followed by were used according to the manufacturer’s instructions. Alexa Fluor 488 donkey anti-rabbit (1:100, cat. no. 711- The slides were counterstained with DAPI (Advanced Cell 545-152; Jackson ImmunoResearch, Cambridgeshire, U.K.) Diagnostics) and cover slipped using hard-set fluorescent secondary antibody for 2 h. Sections were counterstained mounting medium before being scanned using a confocal with DAPI (0.5 mg/L) and the slides cover slipped with microscope (Zeiss SP8 confocal system equipped with a VECTASHIELD (Vector Laboratories, Burlingame, CA) (15). 363/1.40 objective (POMC: HD1 555 laser 10%, RAMP1: HD1 488 laser 20%, RAMP3: HD1 488 laser 10%, CTR: POMC-pSTAT3 Double Staining HD2 647 laser 10%, DAPI: HD2 405 laser 5% with 10% Immunohistochemistry (IHC) was performed as previously gain, Z stack 10 mm, and step size 0.5 mm). The ARC, AP, fl described (23). Brie y, brain sections were demasked in and NTS were scanned, and for each images, 9–12 tiles NaOH and H2O2 followed by glycine and SDS. Sections of 363 images were merged. were blocked for 1 h in 4% NGS, 0.4% triton, and 1% BSA in KPBS and were then incubated in rabbit anti-pSTAT3 Interscapular Brown Adipose Tissue Histology (1:1,000, cat. no. 9145; Cell Signaling Technology) for 48 h Interscapular brown adipose tissue (iBAT) was fixed in 4% at 4°C. Section were then incubated in Cy3 goat anti-rabbit phosphate buffer-paraformaldehyde for 48 h at 4°C. The for 2 h. Sections were blocked again and incubated with tissue was embedded in paraffin and cut into 5-mmsections rabbit anti-POMC as described above. (Laboratory for Animal Model Pathology, Vetsuisse Faculty, University of Zurich) and processed for hematoxylin-eosin CTR Staining (H-E) (24). These H-E–stained slides were scanned and For pretreatment, sections were demasked in 0.01 mol/L analyzed with the Visiopharm software (Hoersholm, Den- sodium citrate, pH 6, at 90°C in a steamer for 20 min. After mark) by classifying different areas for vacuoles, membranes, cooling, sections were rinsed, blocked, and incubated with and nuclei and quantifying two regions of interest per scan, CTR antibody (1:400, cat. no. ab11042; Abcam, Cambridge, with exclusion of vessels or white adipose tissue (24). U.K.) in 2.5% NGS–0.3% Triton–1% BSA in PBS for 48 h at 4°C. Sections were incubated with a biotinylated goat anti- UCP1 Immunostaining rabbit (Vector Laboratories) at 1:500 for 2 h followed by Paraffin-embedded tissue was slide cut to 5-mm sections streptavidin-conjugated 647 secondary antibody (Invitro- and deparaffinized in an oven at 60°C for 20 min. Slides were – gen) at 1:1,000 concentration in PBS 0.3% Triton for 2 h. then immersed two times in xylene for 10 min and rehy- Sections were counterstained with DAPI (0.5 mg/L) and the drated with decreasing ethanol steps. After antigen retrieval slides cover slipped with VECTASHIELD. at 95°C for 20 min in sodium citrate buffer, slides were – A in LHA blocked in PBS with 0.3% Triton 3% normal donkey serum Sections were blocked in 0.3% Triton–2% BSA–3% NGS in (NDS) and incubated with rabbit anti-UCP1 (1:500, cat. no. KPBS before incubation with primary rabbit anti–orexin A ab10983; Abcam) in blocking buffer at 4°C for 48 h. Slides (1:1,000; cat. no. H003-30; Phoenix Pharmaceuticals) for then were placed in secondary antibody (1:100, Alexa Fluor 48 h at 4°C followed by Alexa Fluor 488 goat anti-rabbit 488 donkey anti-rabbit; Jackson Immunoresearch) in the (1:100; Jackson ImmunoResearch) for 2 h. Sections were same buffer and counterstained with DAPI (25). then counterstained with DAPI and cover slipped with VECTASHIELD. Imaging and Quantitative Analysis Cells expressing pERK-POMC and pSTAT3-POMC in the a -Melanocyte-Stimulating Hormone ARC and orexin in the lateral were imaged IHC was performed as previously described (15), using the on a 320 objective, and three sections of the ARC and same series of sections as for the POMC-pERK staining. lateral hypothalamus per animal were acquired with use of an Axio Imager 2 microscope (Zeiss, Oberkochen, Germany), RNA Scope of the ARC and AP/NTS Regions blinded, and quantifiedandaveraged(15).UCP1staining fl fl Fresh frozen brain from two POMC-WT 3 CTR / (POMC- was acquired with a 3100 oil-immersed objective, and three WTCTR) male mice (n 5 2) was cut in 14-mm sections onto regions of interest were quantified per mouse and aver- Superfrost Plus slides. After postfixing in 4% paraformal- aged. Quantitative analysis of CTR-immunopositive POMC: dehyde for 15 min, dehydration in ethanol, and in situ tDTomato neurons was performed with a Zeiss SP8 con- hybridization target retrieval (30 min of ACD enzyme pro- focal system equipped with a 320/0.75 objective (HD1 tease IV [Advanced Cell Diagnostics, Newark, CA]), the 555, laser 5%, and HD2 647, laser 7%, with 10% gain, Z mRNA signal of interest was detected using the RNAscope stack 21 mm, and step of 1.5 mm). Quantitative analysis of Multiplex Fluorescent Reagent Kit (cat no. 323130; Ad- a-melanocyte-stimulating hormone (a-MSH) fiber density vanced Cell Diagnostics). Specific probes targeting mouse was performed as described above (HD1 488, laser 2%, CTR, POMC, RAMP1, or RAMP3 (Probe-Mm-Calcr, cat with 10% gain, zoom 1, pinhole 1, Z stack 21 mm, and step no. 494071-C3; Probe-Mm-Pomc-C2, cat no. 314081-C2; of 1.5 mm) (15,23).To ensure similar imaging conditions diabetes.diabetesjournals.org Coester and Associates 1113 for all images, we used the same microscope setup and 2C and D). These results confirm the functional depletion acquisition settings to acquire all images within the of amylin signaling in the POMC-CTR KO model and that same experiment. The experimenter was blinded for all the induced KO at 4 weeks does not affect the early-life quantifications. development of POMC neuronal projections to other nu- clei such as the PVN (15). Statistics Statistical comparisons among variables were made by one- Male POMC-CTR KO Mice Present an Increased Body or two-way ANOVA, as appropriate, with Tukey post hoc Weight and Adiposity on Chow Diet analysis (GraphPad Prism, La Jolla, CA). When appropri- Starting at 9 weeks of age, male KO mice showed a 44% ate, the unpaired t test was performed. All data are expressed increase in body weight gain and 22% increase in cumu- as means 6 SEM. lative food intake compared with control groups (Fig. 3A– C). Overall, male KO mice tended to have smaller and Data and Resource Availability shorter meals, but this effect was overcompensated by a The data sets generated during or analyzed during the significant increase in meal number (Supplementary Table current study are available from the corresponding author 1). Meanwhile, female mice showed a 30% lower body upon reasonable request. weight gain after Tx treatment independent of genotype (Fig. 3D–F). Body composition analysis on chow diet RESULTS revealed that the excess body weight of male KO chow-fed fi CTR, POMC, RAMP1, or RAMP3 Localization in the mice was fat speci c and mostly driven by a 45% increase ARC, AP, and NTS of WT and POMC-CTR KO Mice in visceral FM (Fig. 3G). This effect was not found in chow- The depletion of CTR specifically in POMC neurons was fed female mice (Fig. 3H). The increase in visceral fat on assessed in POMC-WTCTR and POMC-CreCTR using tdTomato chow diet was not sufficient to induce an increase in blood reporter mice and CTR IHC. POMC-Cre:ERT2:tdTomato-CTR leptin levels, probably because subcutaneous fat, which floxed mice treated with Tx (i.e., KO) showed that POMC has a higher expression of leptin (27), was similar between neurons did not colocalize with CTR in the KO mice, which the groups (Table 1). CTR was clearly different from the control oil-treated floxed On an HFD (45% fat), male POMC-Cre mice had mice (Fig. 1A and B). It should be noted that ;20% of a 20% increase in body weight regardless of Tx or oil POMC neurons coexpressed CTR in the ARC (Fig. 1A). treatment, but there was no difference in body weight gain With use of in situ hybridization, POMC-WTCTR mice or food intake among all groups (Supplementary Fig. 1A– were further characterized to assess the colocalization of C). An almost opposite effect was observed in HFD-fed CTR POMC and CTRA with RAMP1 or RAMP3 in the ARC, AP, females, where oil- or Tx-treated POMC-Cre mice showed and NTS (Fig. 1C–H), but as seen at the protein level (Fig. a 15% decrease in body weight–associated with a 15% 1A), the colocalization of CTR and RAMP with POMC was decrease in cumulative food intake (Supplementary Fig. 1 – not detected in all POMC cells. We observed that CTRA is 1D F). Body composition analysis showed no consistent expressed with or without RAMP in the ARC, AP, and NTS difference in FM in HFD-fed male or female KO versus (Fig. 1C–H). RAMP3 expression was higher than RAMP1 control mice (Supplementary Fig. 1G and H). After 6 and in both the ARC and hindbrain (Fig. 1C–H). In the AP, 12 weeks on HFD, leptin levels were elevated by ;10- to RAMP3 was mostly colocalized with CTRA, while this was 20-fold compared with chow-fed mice and were similar not the case for RAMP1 (Fig. 1D and G). In the NTS, RAMP across all groups (Table 1). and CTR were found to be less colocalized than in the AP (Fig. 1E and H), suggesting that the AP might be the main Glucose Tolerance Is Partly Reduced in Male POMC- site of action for amylin signaling. A few POMC neurons CTR KO Mice Independent of Diet were also detected in the POMC-WTCTR mice in the rostral Eleven-week-old chow-fed male KO mice had the highest part of the NTS, and few colocalized with CTR (Supple- glucose levels at 15 and 30 min postgavage compared with mentary Fig. 5C and F). Thus, these results suggest that the control groups, which is reflected by a significantly in- 1 formation of AMY1 and AMY3 is present in POMC and creased area under the curve (AUC) (148% vs. Tx control) 2 POMC neurons. (Fig. 4A). Similarly, male KO mice displayed an increase of the AUC (142% vs. Tx control) and peak glucose levels on CTR-Depleted POMC Neurons Show Less Amylin- 45% HFD (Fig. 4E). Interestingly, HFD did not worsen Induced pERK, While a-MSH ARC–Paraventricular glucose tolerance compared with chow diet–fed KO mice, Nucleus Axon Fiber Development Is Unaffected although baseline fasting glucose levels were higher on Twelve-week-old POMC-CreCTR-Tx (KO) mice show no sig- HFD (124%, P 5 0.002) (Fig. 4A and E). There was no nificant increase in pERK-POMC–positive neurons in the difference in females among all groups fed chow or HFD ARC after amylin injection versus saline injection—contrary except for a lower glucose peak (220%) in WT controls to the three control groups (15) (Fig. 2A and B). The den- (Fig. 4C and G). When injected with insulin, male and sity of a-MSH fibers in the paraventricular nucleus (PVN) female mice displayed similar glucose excursions on chow (23,26) was similar between KO and control groups (Fig. diet, suggesting a similar insulin sensitivity and/or 1114 POMC Amylin Signaling Regulates Energy Homeostasis Diabetes Volume 69, June 2020

Figure 1—CTR and RAMP1 or RAMP3 are colocalized with POMC neurons in mice. Quantification of POMC- and CTR-immunopositive cells in the ARC (A) and original magnification 320 representative image and original magnification 340 orthogonal views of Tx-treated POMC- Cre:tDTomato-CTRWT/WT and POMC-Cre:tDTomato-CTRfl/fl ARC brain section immunostained for CTR (B). C–H: Representative ARC and NTS images of in situ hybridization of CTR (Calcr1A), POMC, and RAMP1 (C–E) or RAMP3 (F–H).The empty arrows indicate no colocalization with POMC, and the filled arrows indicate POMC-CTR colocalization. Values are means 6 SEM. n 5 4–5 per group. ***P , 0.001 after t test. Avg, average.

counterregulatory response to hypoglycemia. During HFD group (Fig. 5A, B, and G), but there was no difference in feeding, male POMC-CreCTR1oil mice showed a reduced HFD (Supplementary Fig. 2A–C). Further, in female KO insulin tolerance compared with KO mice (Fig. 4F), and mice, EE was significantly increased by 25% compared with overall, all groups fed HFD were less responsive to insulin KO males (Fig. 5A), but while it only showed a trend to compared with chow diet–fed male and female mice (Fig. increase EE on chow (Fig. 5C and G), a significant increase in 4B, D, F, and H). EE on HFD (Supplementary Fig. 2A–C) compared with their No significant difference in fasting insulin was observed Tx controls was observed. RER was similar between control among treatment groups within each diet at any time point and KO groups (Fig. 5D–F and H), although female KO mice (Table 1). Mice that were fed HFD for 6 and 12 weeks on chow diet decreased their RER by 5% during the light showed increased baseline levels of insulin by twofold and phase compared with male mice (Fig. 5H). fivefold, respectively, compared with chow diet mice. Loss of CTR Signaling in POMC Neurons Alters iBAT Male POMC-CTR KO Mice Have a Reduced EE, While Morphology and Decreases Uncoupling Protein 1 Their Female Counterparts Have Increased Baseline EE Density In a 3-day baseline measurement, male KO mice on chow Relative to Tx controls, male KO mice had a significantly diet reduced their EE by 11% compared with the Tx control lower number of cells (242%) and membrane surface diabetes.diabetesjournals.org Coester and Associates 1115

Figure 2—CTR depletion in POMC neurons of male and female postweaning mice fed chow diet decreases amylin-induced pERK in POMC neurons, but it does not affect ARC a-MSH fiber outgrowth. Amylin-induced (50 mg/kg i.p.) pERK IHC (red) in the ARC and VMN (A and B)of Tx-treated male and female POMC-WTCTR and POMC-CreCTR mice on chow diet. The sections were then double-stained for POMC (green), and the number of single- and double-labeled neurons was quantified in three separate sections (A and B), as indicated by the filled arrows. Male and female data are pooled in this figure, as no sex difference was observed for pERK-POMC using two-way ANOVA (P 5 0.54; F(1, 48) 5 0.363). Quantification of a-MSH immunoreactive (IR) fiber density in the PVN (C) and original magnification 320 representative image of a-MSH immunostaining in the PVN (D). Values are means 6 SEM. n 5 4–8 per group. Parameters with differing letters (a, b) differ from each other by P , 0.05 after two-way ANOVA (genotype, treatment [saline vs. amylin]) followed by Tukey post hoc test. Avg, average. 1116 POMC Amylin Signaling Regulates Energy Homeostasis Diabetes Volume 69, June 2020

Figure 3—CTR depletion in POMC neurons of male mice increased body weight, food intake, and FM on chow diet. Body weight (A and D), body weight gain after Tx injection (B and E), cumulative food intake (C and F) in male (A–C) and female (D–F) mice aged 4–14 weeks fed ad libitum chow diet. Body composition was assessed between lumbar vertebrae L1 and L4 in 16-week-old male (G) and female (H) mice fed ad libitum chow diet. Values are means 6 SEM. n 5 7–9 male mice and 7–10 female mice per group. A–F: Parameters with differing letters (a, b) differ from each other by P , 0.05 after two-way ANOVA (genotype, time) followed by Tukey post hoc test. G and H:*P , 0.05 after unpaired t test. Subcut., subcutaneous.

(233%) (Fig. 6A and B). This change in morphology was control in iBAT histology and UCP1 immunoreactivity was reflected by a 20% decrease in uncoupling protein 1 (UCP1) observed in female mice (Fig. 6B–E). immunoreactivity in male KO mice (Fig. 6D and E). On the Male and female mice showed no difference in baseline other hand, no consistent difference between KO and body temperature (Fig. 6F and G). Male and female KO diabetes.diabetesjournals.org Coester and Associates 1117

Table 1—Leptin and insulin blood levels in male and female mice 8 weeks post–Tx treatment on chow diet and after 6 and 12 weeks on 45% HFD Male POMC- Male POMC- Female POMC- Female POMC- WTCTR1Tx CreCTR1Tx WTCTR1Tx CreCTR1Tx Two-way ANOVA 8 weeks chow (n 5 8–10/group)

Insulin (pg/mL) 358 6 52 522 6 99 207 6 34 391 6 147 Pgenotype 5 0.13; Psex 5 0.04* Leptin (pg/mL) 1,007 6 239 872 6 146 522 6 79 467 6 82 Pgenotype 5 0.54; Psex 5 0.006** 6 weeks 45% HFD (n 5 6–10/group)

Insulin (pg/mL) 763 6 69 1,087 6 289 687 6 189 421 6 68 Pgenotype 5 0.70; Psex 5 0.24 Leptin (pg/mL) 13,815 6 1,959 16,779 6 4,155 10,573 6 4,347 17,198 6 6,382 Pgenotype 5 0.32; Psex 5 0.76 12 weeks 45% HFD (n 5 8–12/group)

Insulin (pg/mL) 1,094 6 165 1,828 6 423 1,080 6 281 1,553 6 453 Pgenotype 5 0.12; Psex 5 0.71 Leptin (pg/mL) 24,583 6 4,338 12,343 6 5,668 18,918 6 5,110 7,676 6 3,175 Pgenotype 5 0.30; Psex 5 0.02* Values are assessed by two-way ANOVA (genotype, sex) and Tukey post hoc test. All data are expressed as mean 6 SEM (pg/mL). *P , 0.05, **P , 0.01. mice did not have increased body temperature after sCT had a longer-lasting effect in POMC-CreCTR1oil and POMC- versus saline injection, while Tx control mice on chow had an CreCTR1Tx mice than in POMC-WTCTR mice (Supplemen- average of 0.47°C increase of body temperature during the tary Fig. 3F–J). sCT injections resulted in a food intake dark phase (Fig. 6H and I) and 0.49°C on HFD (data not shown). reduction that lasted up to 12 h (Supplementary Fig. 3K– O). Compared with baseline food intake after a 12-h fast, Baseline Locomotor Activity of POMC-CTR KO Mice Is POMC-WTCTR1oil showed a weaker response to sCT in- Reduced, but Orexin A Expression in LHA and jection than the other groups (Supplementary Fig. 3P–T). Corticosterone Release Are Not Impaired Overall, the depletion of amylin signaling in POMC neu- Male and female KO mice were less active than Tx controls rons did not affect the acute, AP-mediated effect of amylin during dark and light phase, which resulted in a 30% re- or sCT on food intake. duction in cumulative 3-day activity (Fig. 7A–C). On HFD, the baseline activity was also reduced by 40% in KO mice POMC-CTR KO Mice on Chow Diet Show a Delayed versus controls (Fig. 7D–F). However, this baseline decrease Response to Leptin Injections on Food Intake but Show in locomotor activity did not seem to directly affect EE, since Similar Levels of STAT3 Phosphorylation 1 fi opposite effects on EE were observed in male and female Leptin and leptin amylin combination had a signi cantly mice, while locomotor activity was decreased in both sexes. stronger effect on food intake 1 h after injection in control For investigation of potential reasons for the decrease mice than in KO mice, although this difference did not – in spontaneous locomotor activity, plasma corticosterone persist after 4 h (Supplementary Fig. 4A C). When the and ACTH levels were measured from 11-week-old chow- results are expressed as a percent of baseline, control and fedmice,2hafterlightonsetandafterdarkonset, KO mice responded similarly to the anorectic effect of 1 – representing circadian nadir and peak of circulating cor- leptin and leptin amylin (Supplementary Fig. 4D F). ticosterone, respectively (28) (Supplementary Table 2). Furthermore, leptin-induced pSTAT3 in POMC neurons in Corticosterone was increased approximately threefold at the ARC was also similar in control and KO mice, suggest- peak secretion (P , 0.0001) compared with baseline, while ing that the depletion of CTR in POMC neurons does not ACTH was only increased by 12% on average (P 5 0.028). interfere with leptin signaling in these mice (10) (Fig. 8A However, there was no significant difference between and B). Leptin-induced pSTAT3 was assessed as a readout groups at any time point. Because POMC neurons project of leptin resistance in mice after 9 weeks on HFD. KO mice to the LHA, a potential involvement of the LHA in altered showed a 30% reduced STAT3 phosphorylation in the ARC 5 locomotor activity was investigated, but no difference in compared with Tx controls (P 0.01), which was not fi the number of orexin A–positive LHA neurons in 12- to speci c for POMC neurons (Fig. 8C and D). Nevertheless, 15-week-old control and KO chow-fed mice (Fig. 7G and H) the overall pSTAT3 and POMC immunoreactivity was 80% was detected. lower in the HFD-fed cohort compared with chow-fed mice (Fig. 8B and D), which could explain the blunted pheno- POMC Neurons Are Not Involved in Acute Reduction of typic effects of HFD-fed KO mice. Food Intake After Exogenous Amylin Application Amylin injections resulted in a dose-dependent anorectic DISCUSSION effect that persisted for up to 4 h in KO and control mice The goal of this study was to identify the physiological (Supplementary Fig. 3A–E). Compared with baseline food mechanisms controlled by amylin signaling in POMC neu- intake after 12-h overnight fasting and refeeding, amylin rons. We have previously shown that amylin selectively 1118 POMC Amylin Signaling Regulates Energy Homeostasis Diabetes Volume 69, June 2020

Figure 4—CTR depletion in POMC neurons of male mice decreased glucose tolerance on chow and 45% HFD. Oral glucose tolerance tests (A, C, E, and G) and insulin tolerance tests (B, D, F, and H) were performed in male (A, B, E, and F) and female (C, D, G, and H) mice on chow (A– D) and 45% HFD (E–H) in 11- and 12-week-old (chow diet) and 12- and 15-week-old (45% HFD) mice, respectively. AUC was calculated from baseline for each test. Values are mean 6 SEM. n 5 7–12 male mice and 5–7 female mice per group on chow diet and n 5 4–5 male mice and 4–6 female mice per group on 45% HFD. Parameters with differing letters (a, b, c) differ from each other by P , 0.05 after two-way ANOVA (genotype, time) followed by Tukey post hoc test. See also Supplementary Table 1. Gen, genotype; ns, not significant. *P , 0.05, ***P , 0.001, ****P , 0.0001. diabetes.diabetesjournals.org Coester and Associates 1119

Figure 5—CTR depletion in POMC neurons decreased EE in male but not in female mice on chow diet. Three-day (3d) EE (A) and RER (D), average of 3-day EE (B and C) and RER (E and F), and 12-h average in dark and light and total 24-h EE (G) and RER (H) in Tx-treated 15-week- old male and female POMC-WTCTR and POMC-CreCTR mice. EE was normalized to LBM 1 0.2 FM presented in Fig. 2G and H. Values are means 6 SEM. n 5 6 male mice and 5–6 female mice per group. Parameters with differing letters (a, b, c) differ from each other by P , 0.05 after two-way (genotype [gen], time) or three-way (genotype, sex, time) ANOVA followed by Tukey post hoc test. The gray area represents the dark phase. avg, average; Gen, genotype; ns, not significant. *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001. †P , 0.05, male vs. female POMC-CreCTR1Tx. activates ERK signaling in POMC neurons to enhance ARC (29–31). Thus, it is likely that only a subset of POMC → PVN a-MSH fiber outgrowth (15), but whether amylin neurons express CTR. In the POMC-CTR KO mice used in influences energy balance through this pathway during this study, ERK phosphorylation was impaired after ex- adulthood remained to be investigated. For achievement of ogenous amylin application, the density of CTR in the this aim, Tx-inducible POMC-cre:ERT2 mice were crossed ARC of KO mice was reduced, and CTR was shown to be with CTR-floxed mice. After Tx induction, only male POMC- depleted specifically from POMC neurons in the ARC. The 1 1 CTR KO mice had an increase in body weight and FM, presence of CTR POMC neurons was also detected in the which was accompanied by a decrease in EE and UCP1 NTS; however, these neurons are sparse, which is in line density in the iBAT. Both sexes were affected by a loss of with a previous study comparing different POMC reporter 1 1 sCT-sensitivity for body temperature regulation and a re- mice (32). Some of the CTR POMC neurons are colo- markable decrease in locomotor activity. Surprisingly, this calized with RAMP1 or RAMP3, but whether such a low phenotype was not exacerbated by HFD, which could be number of neurons play a metabolic role in our model due to a general reduction of POMC expression. Further, is questionable. Thus, the metabolic effects seen in the the depletion of CTR in POMC neurons did not affect POMC-CTR KO mice could result from the loss of CTR in amylin’s acute anorectic action. POMC neurons in the ARC and the NTS. A recent study by The selective loss of CTR in POMC neurons through Cheng et al. (33) showed that CTR in the NTS is involved in cre:ERT2 induction allows a normal axonal outgrowth of the control energy balance through the parabrachial nu- a-MSH fibers ARC → PVN, which is contrary to our pre- cleus and that CTRNTS neurons also send indirect signals to vious KO models (15). We previously showed that amylin ARC neurons. In that study, CTRNTS-depleted mice failed specifically activates ERK signaling in 60% of POMC neu- to respond to the anorectic effect of sCT and activation of rons in the ARC and that amylin-induced pERK in POMC these neurons acutely decreased food intake. After 5 weeks neurons is blunted in the ARC of RAMP1 and RAMP3 mice on chow diet, CTRNTS-depleted mice displayed higher food (15). These previous studies pointed out a crucial role of intake compared with control similar to what we observed, CTR- and amylin-mediated signaling in ARC POMC neu- but while chow diet–fed CTRNTS-depleted and control mice rons. POMC neurons are highly pleomorphic in their phys- had a similar FM, POMC-CTR KO mice displayed an in- iological functions, connections, and transmitter expression crease in fat ratio, suggesting a different role for ARC and 1120 POMC Amylin Signaling Regulates Energy Homeostasis Diabetes Volume 69, June 2020

Figure 6—CTR depletion in POMC neurons decreased UCP1 iBAT content in male mice on chow diet and altered the effect of sCT on body temperature in male and female mice. iBAT was stained for H-E (A–C) in 16-week-old male (A and B)andfemale(B and C) Tx-treated male and female POMC-WTCTR and POMC-CreCTR mice, and UCP1 was detected by immunofluorescence (D and E). Original magnification 320 representative image of the H-E staining (B); original magnification 3100 representative image of the UCP1 staining (E). Core body temperature over 3 days (F), average of 3 days (3d) during dark and light cycle (G), Dbody temperature between saline and sCT (5 mg/kg i.p.) injection over 24 h (H) (three-way ANOVA, Psex 5 0.29; F(1, 11) 5 0.775), and average Dtemperature during dark and light phase (I) (three-way ANOVA, Psex 5 0.25; F(1, 11) 5 1.471) in Tx-treated 15-week-old male and female POMC-WTCTR and POMC-CreCTR mice. Male and female data are pooled in F, G, H, and I. Values are means 6 SEM. n 5 7–12 per group on chow diet. *P , 0.05 or parameters with differing letters (a, b) differ from each other by P , 0.05 after two-way ANOVA followed by Tukey post hoc test. The gray area represents the dark phase. A.U., arbitrary units; Gen, genotype; ns, not significant. *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001. diabetes.diabetesjournals.org Coester and Associates 1121

Figure 7—CTR depletion in POMC neurons of male and female mice fed chow and 45% HFD had decreased locomotor activity, while it did not affect the number of orexin neurons in the lateral hypothalamus (LH). Locomotor activity over 3 days (A and D), average of 3 days (3d) of locomotor activity during dark and light cycle and total 24-h period (B and E), and cumulative locomotor activity over 3 days (C and F) on chow CTR CTR (A–C) and 45% HFD (D–F) in Tx-treated male and female POMC-WT and POMC-Cre mice. C: Chow, two-way ANOVA, Psex 5 0.76; F(1, 10) 5 0.091. F: HFD, two-way ANOVA, Psex 5 0.32; F(1, 17) 5 1.025). H: Lateral hypothalamus brain sections were stained for orexin (green) and counterstained with DAPI (blue), and the number of neurons was quantified in three separate sections in chow-fed mice; three- way ANOVA, Psex 5 0.61; F(1, 7) 5 0.2782. G: Original magnification 320 representative image of the mediobasal hypothalamus including the ARC, VMN, and LHA. Male and female data are pooled in this figure, as no sex effect was observed. Values are means 6 SEM. n 5 7–12 per group on chow diet and 45% HFD. Parameters with differing letters (a, b, c) differ from each other by P , 0.05 after two-way ANOVA (genotype, time) followed by Tukey post hoc test. A.U., arbitrary units; Gen, genotype; ns, not significant. *P , 0.05 after unpaired t test. ****P , 0.0001.

NTS CTR-mediated signaling. Since ;20% of POMC neu- depend on the caudal hindbrain (34,35). Thus, the CTR KO rons were CTR positive in the ARC, we can hypothesize seems to be sufficient to impair amylin signaling in this that the decrease in pERK-POMC ARC signaling and the POMC subpopulation, confirming our hypothesis that amy- metabolic alterations seen in the KO mice could also result lin exerts a direct and AP-independent (15) effect on POMC from indirect signaling via synaptic communication. neurons through its core receptor component. However, The response to acute exogenous amylin admin- a recent study assessing the binding of fluorescent sCT to istration was not affected in our model, suggesting that ARC neurons did not show binding to POMC neurons but acute central amylin effect on meal pattern may solely fluorescent sCT was internalized into NPY neurons (9). 1122 POMC Amylin Signaling Regulates Energy Homeostasis Diabetes Volume 69, June 2020

Figure 8—CTR depletion in POMC neurons of male and female mice fed chow diet does not affect leptin signaling in the ARC and VMN, but 45% HFD decreases the number of POMC neurons and the number of leptin-induced pSTAT3-positive neurons. Leptin-induced (5 mg/kg i.p.) pSTAT3 immunofluorescence (red) in the ARC and VMN (A) of Tx-treated male and female POMC-WTCTR and POMC-CreCTR mice. The sections were then double-stained for POMC (green), and the number of single- and double-labeled neurons was quantified in three separate sections in chow-fed mice (A and B) and 45% HFD-fed mice (C and D). Male and female data are pooled in this figure. n was not high enough to run a three-way ANOVA. Values are means 6 SEM. n 5 4–8 per group on chow diet. Parameters with differing letters (a, b) differ from each other by P , 0.05 after two-way ANOVA (genotype, treatment [saline vs. leptin]) followed by Tukey post hoc test. Avg, average. diabetes.diabetesjournals.org Coester and Associates 1123

The binding of fluorescent amylin was also assessed, but POMC system has been shown to differ between sexes, we could not see any binding at the cell level in either cell which has been attributed to a lower number, lower ac- type, while whole brain imaging showed binding in ARC tivity, and/or lower mRNA expression of POMC neurons in and median eminence (9). This finding was surprising given the male ARC, a development that is induced by testos- the fact that exogenous amylin did not activate ERK sig- terone exposure (44,45). Furthermore, -deficient naling in NPY neurons (15). Since it has recently been DIO rats seem to have a better response to chronic exog- demonstrated that amylin can be produced by ARC neu- enous amylin application regarding body weight and EE rons (36), we may therefore hypothesize that the effects (46). Notably, the inactivation of STAT3 signaling in POMC observed in our study may be caused by brain-produced neurons leads to a fat-specific weight gain similar to that amylin instead of pancreatic amylin; the neuronal subtype seen in this study, albeit in this case only in female mice producing amylin is currently under investigation in our (47). We did not see any difference in the number of POMC laboratory. Further, since we showed that AMY1 is present neurons between sexes—contrary to the study by Wang in the ARC and NTS, albeit at a lower amount, we could et al. (44) using the same POMC-cre:ERT2 mice induced at also hypothesize that the effects observed in POMC-CTR 11 weeks of age. Therefore, we believe that the absolute KO may result from an alteration in the CGRP pathways, number of POMC neurons may be less important for the since CGRP, which is a widespread , can different phenotypes than their properties. A possible con- bind to its secondary receptor AMY1 in the ARC but not in founding factor in this regard is the Tx induction at 4 the NTS (37,38). The in situ hybridization revealed that weeks, which, at least in females, possibly interferes with 1 1 1 1 1 ARC neurons can be CTR , POMC CTR , CTR RAMP , sexual maturation. Additionally, even low doses of Tx in 1 1 1 or POMC CTR RAMP , highlighting the complexity of adult female mice have been linked to substantial adipose the study of amylin signaling. Thus, the depletion of CTR tissue browning and increased thermogenesis (48), which in our model can affect either the calcitonin-mediated or may have masked the effects that we attempted to measure. the amylin-mediated signaling pathway. Both male and female KO mice showed a reduced lo- The depletion of CTR in POMC neurons of male mice on comotor activity, which, however, did not seem to be chow diet increased weight gain and adiposity, which was responsible for the changes in EE discussed earlier. For characterized by an increased visceral FM and resulted instance, the activity pattern does not match the EE data in glucose intolerance. Baseline insulin levels and insulin time-wise in a 24 h cycle, and in females it even contradicts tolerance were unchanged in this model, and circulating their increased EE. Since POMC neurons project to the leptin levels corresponded as expected to the stored fat LHA (49,50) and since MC3R-expressing orexin neurons in deposits. EE was significantly decreased in male KO mice, the LHA modulate locomotor activity (51), we hypothe- which could explain why the development of adiposity sized that the decrease in amylin POMC signaling could preempts a significant change in food intake. Amylin has affect LHA orexin neurons and, hence, contribute to the been shown to increase EE by increasing sympathetic nerve decrease in locomotor activity. However, when looking at activity, and nestin–human RAMP1–overexpressing trans- orexin expression in the LHA, we could not find any differ- genic mice also increased their EE (39,40). At least part of ences between groups or sexes, and the activity pattern in this effect could be attributed to amylin increasing POMC metabolic cages also did not point to a fragmented sleep mRNA and activating the MC4R pathway that promotes pattern as has been shown with ablation of orexin neurons satiety and thermogenesis (12,41), or a thermogenic effect or KO studies (52,53). Nevertheless, whether through ERK phosphorylation, as has been shown with the depletion of amylin signaling in POMC neurons decreases respect to leptin (42). The overall effect is relatively small, orexin neurons’ activity was not assessed. POMC neurons which makes sense considering that only a small sub- are also prominent in the , but we could not population of neurons was altered in this mouse model find marked differences in baseline corticosterone and and the hindbrain pathway seems to be unaffected. While ACTH release. Furthermore, this decrease in locomotor baseline body temperature of our KO mice was normal, activity did not correspond to decreased food foraging these mice did not respond to sCT injection by increasing behavior, since male KO mice ate more overall and tended thermogenesis as has previously been shown in intact rats to have smaller and more frequent meals than Tx controls. and mice (39,43). Furthermore, iBAT morphology was al- Therefore, the underlying mechanism of this consistently tered in male KO mice with a decreased membrane surface observed change in locomotor activity is still unclear. and cell number. These findings support the idea that Given the increase in adiposity of chow-fed male POMC- reduced activation of iBAT may contribute to the EE phe- CTR KO mice, we expected an exacerbation of this pheno- notype, and this is reflected with lower UCP1 immunore- type under HFD. However, the KO-specific effect was rather activity in iBAT of male KO mice. Conversely, female KO lost, which could be due to a marked reduction of POMC mice did not present any alteration in their EE, iBAT expression that prevented clear effects of the defective histology, and UCP1 activity and in consequence did not amylin signaling in POMC neurons on HFD. Indeed, recent develop adiposity. pharmacological studies by Li et al. (41) showed that amylin Sexual dimorphism has not yet been studied extensively directly uses the system to increase thermo- regarding amylin signaling. However, the hypothalamic genesis. While leptin sensitivity was similar in chow- and 1124 POMC Amylin Signaling Regulates Energy Homeostasis Diabetes Volume 69, June 2020

HFD-fed KO mice, represented by a comparable pSTAT3 Acknowledgments. The authors acknowledge the technical contributions signaling, the HFD cohort had a lower overall pSTAT3 and of Josep Monné Rodríguez (Center for Microscopy and Image Analysis, Vetsuisse POMC expression. However, when CTR is depleted in rat Faculty, Institute of Veterinary Pathology, University of Zurich), Petra Seebeck ARC and VMN using an AAV shRNA (which targets more (Zurich Integrative Rodent Physiology), and Joel Elmquist (UT Southwestern, Dallas, TX) and Dr. Thorsten Schinke (University Medical Center Hamburg-Eppendorf, cells than specifically POMC neurons), VMN leptin binding Hamburg, Germany) for providing the POMC-cre:ERT2 and CTR-floxed mice, re- and leptin-induced pSTAT3 are decreased (6). We previously spectively. The authors also thank Christina N. Boyle (Vetphysiologie, UZH), Fabienne demonstrated that amylin activates the secretion of IL-6 by O. Villars (Vetphysiologie, UZH), Salome Gamakharia (Vetphysiologie, UZH), and microglia, which then binds to its gp130 neuronal receptor Justyna B. Koczwara (Sapienza University of Rome) for technical assistance. to further enhance leptin-induced pSTAT3 (10,23). These Funding. This work was funded by the Swiss National Science Foundation studies suggest that amylin’s enhancing effect on leptin (grant SNF 31003A_175458 to T.A.L.) and UZH Forschungskredit (grant FK pSTAT3 signaling takes place mostly in NPY neurons, while 17-066 to C.L.F.). direct pERK induction in POMC neurons is responsible for Duality of Interest. No potential conflicts of interest relevant to this article alterations in energy balance (10,15,23). were reported. HFD-fed mice gradually become leptin resistant and Author Contributions. C.L.F. and T.A.L. contributed to study conceptual- have higher circulating levels of leptin in their blood. How- ization. C.L.F. designed the experiments. B.C., C.K.-H., and C.L.F. performed the experiments. B.C. wrote the original draft of the manuscript. C.L.F. and T.A.L. ever, reports on the effect of various high-fat diets on reviewed and edited the manuscript. T.A.L. and C.L.F. acquired funding. C.L.F. is the POMC expression are ambiguous. A 12-week study with guarantor of this work and, as such, had full access to all the data in the study and C57BL6 mice showed an upregulation of POMC mRNA takes responsibility for the integrity of the data and the accuracy of the data analysis. (54), but other studies in mice and rats showed a relative Prior Presentation. Parts of this study were presented in abstract form at downregulation of POMC in the obesity-prone phenotype the 27th Annual Meeting of the Society for the Study of Ingestive Behavior, Utrecht, in response to HFD (55,56). The time course study by the Netherlands, 9–13 July 2019. Souza et al. 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