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Amylin/-mediated signaling in POMC neurons influences energy

balance and locomotor activity in chow-fed male mice

Running title: POMC signaling regulates energy

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 and , 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 APNTS and ARCPVN axonal fiber outgrowth. Amylin is also able to activate ERK signaling specifically in POMC neurons independently of . 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 (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 and demonstrate the need for sex-specific approaches in obesity research and potentially treatment.

Keywords: amylin, , POMC, CTR, CALCR, BAT, thermogenesis, energy expenditure, adiposity

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INTRODUCTION

Amylin is a pancreatic gut hormone that is co-released with 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.

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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;

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/J(#007914)] were bred with CTRfl/fl POMC-CreERT2 mice. Each mouse was genotyped using previously published primers (17).

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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%).

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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%

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H2O2 in KPBS and incubated in 0.3% 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. Sections were then placed in Cy3 goat anti-rabbit secondary antibody at 1:100

concentration for 2h at room temperature. Sections were again blocked before being incubated

with primary POMC antibody (1:1000, H-029-30, Phoenix Pharmaceuticals, Karlsruhe,

Germany) for 48h at 4C followed by Alexa fluor 488 donkey anti-rabbit (1:100, 711-545-152,

Jackson ImmunoResearch, Switzerland) secondary antibody for 2h. Sections were

counterstained with DAPI (0.5mg/l) and the slides cover slipped with Vectashield (Vectorlabs,

CA, USA) (15).

POMC-pSTAT3 double staining: IHC was performed as previously described (23). Briefly,

brain sections were demasked in NaOH and H2O2 followed by glycine and SDS. Sections were

blocked for 1h in 4% NGS, 0.4% triton and 1% BSA in KPBS and were then incubated in

rabbit anti-pSTAT3 (1:1000, 9145, Cell signaling) for 48h at 4C. Section were then incubated

in Cy3 goat anti-rabbit for 2h. Section were blocked again and incubated with rabbit-anti

POMC as above.

CTR staining: For pretreatment, sections were demasked in 0.01M sodium-citrate pH 6 at 90°C

in a steamer for 20 min. After cooling, sections were rinsed, blocked and incubated CTR

antibody (1:400, ab11042, Abcam, Netherlands) in 2.5% NGS-0.3% Triton-1% BSA in PBS

for 48h at 4C. Sections were incubated with a biotinylated goat-anti rabbit (Vectorlabs, CA,

USA) at 1:500 for 2h followed by streptavidin-conjugated 647 secondary antibody (Invitrogen)

at 1:1000 concentration in PBS-0.3% triton for 2h. Sections were counterstained with DAPI

(0.5mg/l) and the slides cover slipped with Vectashield.

Orexin A in LHA: Sections were blocked in 0.3% Triton-2% BSA-3% NGS in KPBS before

incubation with primary rabbit anti- A (1:1000; H003-30, Phoenix Pharmaceuticals) for

48h at 4C followed by Alexa fluor 488 goat anti-rabbit (1:100, Jackson ImmunoResearch,

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Switzerland) for 2h. Sections were then counterstained with DAPI and cover slipped with

Vectashield.

αMSH: IHC was performed as previously described (15), using the same series of sections as for the POMC-pERK staining.

RNA scope of the ARC and AP/NTS regions

Fresh frozen brain from two POMC-WT x CTRfl/fl (POMC-WTCTR) male mice (n=2) were cut in 14 µm section onto superfrost plus slides. After postfixing in 4% PFA for 15 min, dehydration in ethanol and ISH target retrieval (30 minutes of ACD enzyme protease IV

(Advanced Cell Diagnostics, Newark, CA, USA)), the mRNA signal of interest was detected using the RNAscope® Multiplex Fluorescent Reagent Kit (Cat No. 323130, Advanced Cell

Diagnostics). Specific probes targeting mouse CTR, POMC, RAMP1 or RAMP3 (Probe-Mm-

Calcr, Cat No. 494071-C3; Probe-Mm-Pomc-C2, Cat No. 314081-C2; Probe-Mm-RAMP1-

C1, Cat No. 532681-C1 and Probe-Mm-RAMP3-C1, Cat No. 497131-C1, Advanced Cell

Diagnostics) were used according to the manufacturer’s instructions (Advanced Cell

Diagnostics). The slides were counterstained with DAPI (Advanced Cell Diagnostics) and coverslipped using hardset fluorescent mounting medium before being scanned using a confocal microscope (Zeiss SP8 confocal system equipped with a 63X/1.40 objective (POMC:

HD1 555 laser 10%, RAMP1: HD1 488 laser 20%, RAMP3: HD1 488 laser 10%, CTR: HD2

647 laser 10%, DAPI: HD2 405 laser 5% with 10% gain, Z-stack 10 µm, and step size 0.5 µm).

The ARC, AP and NTS were scanned and for each images, 9 to 12 tiles of 63X images were merged.

iBAT tissue histology

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iBAT was fixed in 4% PB-PFA for 48h at 4°C. The tissue was embedded in paraffin and cut

into 5µm sections (Laboratory for Animal Model Pathology, Vetsuisse Faculty, University of

Zurich) and processed for hematoxylin/eosin (HE) (24). These HE-stained slides were scanned

and analyzed with the Visiopharm software (Hoersholm, Denmark) by classifying different

areas for vacuoles, membranes and nuclei and quantifying two ROIs per scan, with exclusion

of vessels or white adipose tissue (24). UCP1 immunostaining: paraffin-embedded tissue was

slide cut to 5µm sections and de-paraffinized in an oven at 60°C for 20min. Slides were then

immersed 2 times in xylene for 10min and rehydrated with decreasing ethanol steps. After

antigen retrieval at 95°C for 20min in sodium citrate buffer, slides were blocked in PBS with

0.3% Triton-3%NDS and incubated with rabbit anti-UCP1 (1:500, ab10983, Abcam) in

blocking buffer at 4°C for 48h. Slides then were placed in secondary antibody (1:100, donkey

anti rabbit, AF488, Jackson) in the same buffer and counter-stained with DAPI (25).

Imaging and quantitative analysis

Cells expressing pERK-POMC and pSTAT3-POMC in the ARC and orexin in the LH were

imaged on a 20X objective and three sections of the ARC and LH per animal were acquired

using an Axio Imager 2 microscope (Zeiss Germany), blinded, quantified and averaged (15).

UCP1 staining was acquired with a 100X oil-immersed objective and 3 ROIs were quantified

per mouse and averaged. Quantitative analysis of CTR immunopositive POMC::tDTomato

neurons was performed with a Zeiss SP8 confocal system equipped with a 20X/0.75 objective

(HD1 555 laser 5%, HD2 647, laser 7% with 10% gain, Z-stack 21 mm, and step of 1.5 mm).

Quantitative analysis of αMSH fiber density was performed as above (HD1 488, laser 2% with

10% gain, zoom 1, pinhole 1, Z-stack 21 mm, and step of 1.5 mm) (15; 23).To ensure similar

imaging conditions for all images, the same microscope set-up and acquisition settings were

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used to acquire all images within the same experiment. All quantifications were blinded to the experimenter.

Statistics

Statistical comparisons among variables were made by one- or two-way ANOVA, as appropriate, with Tukey post hoc analysis (GraphPad Prism, La Jolla, CA). When appropriate unpaired T-test was performed. All data are expressed as mean ± SEM.

RESULTS

CTR, POMC, RAMP1 or RAMP3 localization in the ARC, AP and NTS of WT and

POMC-CTR KO mice

The depletion of CTR specifically in POMC neurons was assessed in POMC-WTCTR and

POMC-CreCTR using tdTomato reporter mice and CTR immunohistochemistry. POMC- cre:ERT2:tdTomato-CTR floxed mice treated with Tx (=KO) showed that POMC neurons did not co-localize with CTR in the KO mice, which was clearly different from the control oil- treated floxed mice (Fig. 1A, B). It is to be noticed that around 20% of POMC neurons co- expressed CTR in the ARC (Fig. 1A).

Using in situ hybridization, POMC-WTCTR mice were further characterized to assess the co- localization of POMC, CTRA with RAMP1 or RAMP3 in the ARC, AP and NTS (Fig. 1C-H) but as seen at the protein level (Fig. 1A), the co-localization of CTR and RAMP with POMC was not detected in all POMC+ cells. We observed that CTRA is expressed with or without

RAMP in the ARC, AP and NTS (Fig. 1C-H). RAMP3 expression was higher than RAMP1 in both the ARC and hindbrain (Fig. 1C-H). In the AP, RAMP3 was mostly co-localized with

CTRA while this was not the case for RAMP1 (Fig. 1D, G). In the NTS, RAMP and CTR were

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found to be less colocalized than in the AP (Fig. 1E, H), suggesting that the AP might be the

main site of action for amylin signaling. A few POMC neurons were also detected in the

POMC-WTCTR mice in the rostral part of the NTS and few co-localized with CTR (Suppl. Fig.

5C, F). Thus, these results suggest that the formation of AMY1 and AMY3 is present in

POMC+ and POMC- neurons.

CTR-depleted POMC neurons show less amylin-induced pERK, while -MSH ARC-

PVN axon fiber development is unaffected

12-week-old POMC-CreCTR-Tx (=KO) mice show no significant increase in pERK-POMC-

positive neurons in the ARC after amylin injection vs. saline injection, contrary to the three

control groups (Fig. 2A, B) (15). The density of -MSH fibers in the PVN (23; 26) was similar

between knockout and control groups (Fig. 2C, D). These results confirm the functional

depletion of amylin signaling in the POMC-CTR KO model and that the induced KO at 4

weeks does not affect the early-life development of POMC neuronal projections to other nuclei

such as the PVN (15) .

Male POMC-CTR KO mice present an increased body weight and adiposity on chow diet

Starting at 9 weeks of age, male KO mice showed a 44% increase in body weight gain and 22%

increase in cumulative food intake compared to control groups (Fig. 3A-C). Overall, male KO

mice tended to have smaller and shorter meals, but this effect was overcompensated by a

significant increase in meal number (Table S1). Meanwhile, female mice showed a 30% lower

body weight gain after Tx treatment independent of genotype (Fig. 3D-F). Body composition

analysis on chow diet revealed that the excess body weight of male KO chow-fed mice was fat

specific and mostly driven by a 45% increase in visceral fat mass (Fig. 3G). This effect was

not found in chow-fed female mice (Fig. 3H). The increase in visceral fat on chow diet was not

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sufficient to induce an increase in blood leptin levels, probably because subcutaneous fat, which has a higher expression of leptin (27), was similar between the groups (Table 1).

On a 45% high fat diet (HFD), male POMC-CreCTR mice had a 20% increase in body weight regardless of Tx or oil treatment, but there was no difference in body weight gain or food intake among all groups (Fig. S1A-C). An almost opposite effect was observed in HFD-fed females, where oil or Tx-treated POMC-CreCTR mice showed a 15% decrease in body weight associated with a 15% decrease in cumulative food intake (Fig. S1D-F). Body composition analysis showed no consistent difference in fat mass in HFD-fed male or female KO vs. control mice

(Fig. S1G, H). After 6 and 12 weeks on HFD, leptin levels were elevated by ~10 to 20-fold compared to chow-fed mice and were similar across all groups (Table 1).

Glucose tolerance is partly reduced in male POMC-CTR KO mice independent of diet

11-week-old chow-fed male KO mice had the highest glucose levels at 15 and 30 minutes post- gavage compared to control groups, which is reflected by a significantly increased area under the curve (AUC; +48% vs. Tx control, Fig. 4A). Similarly, male KO mice displayed an increase of the AUC (+42% vs. Tx control) and peak glucose levels under 45% HFD (Fig. 4E).

Interestingly, HFD did not worsen glucose tolerance compared to chow diet-fed KO mice, although baseline fasting glucose levels were higher under HFD (+24%, p=0.002; Fig. 4A, E).

There was no difference in females among all groups fed chow or HFD except for a lower glucose peak (-20%) in WT controls (Fig. 4C, G). When injected with insulin, male and female mice displayed similar glucose excursions on chow diet, suggesting a similar insulin sensitivity and/or counter-regulatory response to hypoglycemia. During HFD feeding, male POMC-

CreCTR+oil mice showed a reduced insulin tolerance compared to KO mice (Fig. 4F) and overall, all groups fed HFD were less responsive to insulin compared to chow-diet fed male and female mice (Fig. 4B, D, F, H).

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No significant difference in fasting insulin was observed among treatment groups within each

diet at any time point (Table 1). Mice that were fed HFD for 6 and 12 weeks showed increased

baseline levels of insulin by 2-fold and 5-fold respectively, compared to chow diet mice.

Male POMC-CTR KO mice have a reduced energy expenditure (EE), while their female

counterparts have increased baseline EE

In a three-day baseline measurement, male KO mice on chow diet reduced their EE by 11%

compared to the Tx control group (Fig. 5A, B, G), but there was no difference on HFD (Fig.

S2A-C). Further, in female KO mice, EE was significantly increased by 25% compared to KO

males (Fig. 5A) but while it only showed a trend to increase EE on chow (Fig. 5C, G), a

significant increase in EE on HFD (Fig. S2A-C) compared to their Tx controls was observed.

RER was similar between control and KO groups (Fig. 5D, E, F, H), although female KO mice

on chow diet decreased their RER by 5% during the light phase compared to male mice (Fig.

5H).

The loss of CTR signaling in POMC neurons alters interscapular brown adipose tissue

(iBAT) morphology and decreases uncoupling protein 1 (UCP1) density

Relative to Tx controls, male KO mice had a significantly lower number of cells (-42%) and

membrane surface (-33%) (Fig. 6A-B).This change in morphology was reflected by a 20%

decrease in UCP1 immunoreactivity in male KO mice (Fig. 6D, E). On the other hand, no

consistent difference between KO and control in iBAT histology and UCP1 immunoreactivity

was observed in female mice (Fig. 6B-E).

Male and female mice showed no difference in baseline body temperature (Fig. 6F-G). Male

and female KO mice did not increase their body temperature after sCT vs. saline injection,

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while Tx control mice on chow had an average 0.47°C increase of body temperature during the dark phase (Fig. 6H, I) and of 0.49°C on HFD (data not shown).

Baseline locomotor activity of POMC-CTR KO mice is reduced, but orexin A expression in LHA and corticosterone release are not impaired

Male and female KO mice were less active than Tx controls during dark and light phase, which resulted in a 30% reduction in cumulative 3d activity (Fig. 7A-C). On HFD, the baseline activity was also reduced by 40% in KO mice vs. controls (Fig. 7D-F). However, this baseline decrease in locomotor activity did not seem to directly affect EE, since opposite effects on EE were observed in male and female mice while locomotor activity was decreased in both sexes.

To investigate potential reasons for the decrease in spontaneous locomotor activity, plasma corticosterone and ACTH levels were measured from 11-week-old chow-fed mice, 2h after light onset and after dark onset, representing circadian nadir and peak of circulating corticosterone, respectively (28) (Table S2). Corticosterone was increased ~3-fold at peak secretion (p<0.0001) compared to baseline, while ACTH was only increased by 12% on average (p=0.028). However, there was no significant difference between groups at any time point. Because POMC neurons project to the LHA, a potential involvement of the LHA in altered locomotor activity was investigated but no difference in the number of Orexin A- positive LHA neurons in 12- to 15-week-old control and KO chow-fed mice (Fig. 7I-J) was detected.

POMC neurons are not involved in acute reduction of food intake after exogenous amylin application

Amylin injections resulted in a dose-dependent anorectic effect that persisted for up to 4 hours in KO and control mice (Fig. S3A-E). Compared to baseline food intake after 12h overnight

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fasting and refeeding, amylin had a longer lasting effect in POMC-CreCTR –oil and POMC-

CreCTR –Tx mice than in POMC-WTCTR mice (Fig. S3F-J). sCT injections resulted in a food

intake reduction that lasted up to 12 hours (Fig. S3K-O). Compared to baseline food intake

after 12h fast, POMC-WTCTR –oil showed a weaker response to sCT injections than the other

groups (Fig. S3P-T). Overall, the depletion of amylin signaling in POMC neurons did not affect

the acute, AP-mediated effect of amylin or sCT on food intake.

POMC-CTR KO mice on chow diet show a delayed response to leptin injections on food

intake, but show similar levels of STAT3 phosphorylation

Leptin and leptin+amylin combination had a significantly stronger effect on food intake 1h

after injection in control mice than in KO mice, although this difference did not persist after 4h

(Fig. S4A-C). When the results are expressed as a percent of baseline, control and KO mice

responded similarly to the anorectic effect of leptin and leptin+amylin (Fig. S4D-F).

Furthermore, leptin induced-pSTAT3 in POMC neurons in the ARC was also similar in control

and KO mice, suggesting that the depletion of CTR in POMC neurons does not interfere with

leptin signaling in these mice (Fig. 8A-B) (10). Leptin-induced pSTAT3 was assessed as a

readout of leptin resistance in mice after 9 weeks on HFD. KO mice showed a 30% reduced

STAT3 phosphorylation in the ARC compared to Tx controls (p=0.01), which was not specific

for POMC neurons (Fig. 8C-D). Nevertheless, the overall pSTAT3- and POMC

immunoreactivity was 80% lower in the HFD-fed cohort compared to chow-fed mice (Fig. 8B,

D), which could explain the blunted phenotypic effects of HFD-fed KO mice.

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DISCUSSION

The goal of this study was to identify the physiological mechanisms controlled by amylin signaling in POMC neurons. We previously showed that amylin selectively activates ERK signaling in POMC neurons to enhance ARCPVN -MSH fiber outgrowth (15), but whether amylin influences energy balance through this pathway during adulthood remained to be investigated. To achieve this aim, Tx-inducible POMC-cre:ERT2 mice were crossed with CTR- floxed mice. After Tx induction, only male POMC-CTR KO mice increased their body weight and fat mass which was accompanied with a decrease in EE and UCP1 density in the iBAT.

Both sexes were affected by a loss of sCT-sensitivity for body temperature regulation and a remarkable decrease in locomotor activity. Surprisingly, this phenotype was not exacerbated by HFD, which could be due to a general reduction of POMC expression. Further, the depletion of CTR in POMC neurons did not affect amylin’s acute anorectic action.

The selective loss of CTR in POMC neurons through cre:ERT2 induction allows a normal axonal outgrowth of -MSH fibers ARCPVN, contrary to our previous KO models (15). We previously showed that amylin specifically activates ERK signaling in 60% of POMC neurons in the ARC and that amylin-induced pERK in POMC neurons is blunted in the ARC of

RAMP1 and RAMP3 mice (15). These previous studies pointed out for a crucial role of CTR- and amylin-mediated signaling in ARC POMC neurons. POMC neurons are highly pleomorphic in their physiological functions, connections and transmitter expression (29-31).

Thus, it is likely that only a subset of POMC neurons express CTR. In the POMC-CTR KO mice used in this study, ERK phosphorylation was impaired after exogenous amylin application, the density of CTR in the ARC of KO mice was reduced and CTR was shown to be depleted specifically from POMC neurons in the ARC. The presence of CTR+POMC+

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neurons was also detected in the NTS, however these neurons are sparse, which is in line with

a previous study comparing different POMC reporter mice (32). Some of the CTR+POMC+

neurons are co-localized with RAMP1 or RAMP3 but whether such few number of neurons

play a metabolic role our model is questionable. Thus, the metabolic effects seen in the POMC-

CTR KO mice could result from the loss of CTR in POMC neurons in the ARC and the NTS.

A recent study by Cheng et al. showed that CTR in the NTS is involved in the control energy

balance through the parabrachial nucleus and that CTRNTS neurons also send indirect signals

NTS to ARC neurons (33). In that study, CTR depleted mice failed to respond to the anorectic

effect of sCT and activation of these neurons acutely decreased food intake. After 5 weeks on

NTS chow diet, CTR depleted mice displayed higher food intake compared to control similar to

NTS what we observed but while chow diet-fed CTR depleted and control mice had a similar fat

mass, POMC-CTR KO mice displayed an increase in fat ratio suggesting a different role for

ARC and NTS CTR-mediated signaling. Since ~20% of POMC neurons were CTR positive

in the ARC, we can hypothesize that the decrease in pERK-POMC ARC signaling and the

metabolic alterations seen in the KO mice could also result from indirect signaling via synaptic

communication.

The response to acute exogenous amylin administration was not affected in our model,

suggesting that acute central amylin effect on meal pattern may solely depend on the caudal

hindbrain (34; 35) . Thus, the CTR knockout seems to be sufficient to impair amylin signaling

in this POMC subpopulation, confirming our hypothesis that amylin exerts a direct and AP-

independent (15) effect on POMC neurons through its core receptor component. However, a

recent study assessing the binding of fluorescent sCT to ARC neurons did not show binding to

POMC neurons but fluorescent sCT was internalized into NPY neurons (9). The binding of

fluorescent amylin was also assessed but we could not see any binding at the cell level in neither

cell type while whole-brain imaging showed binding in ARC and median eminence (9). This

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finding was surprising given the fact exogenous amylin did not activate ERK signaling in NPY neurons (15). Since it has been recently demonstrated that amylin can be produced by ARC neurons (36), we may therefore hypothesize that the effects observed in our study may be caused by brain-produced amylin instead of pancreatic amylin; the neuronal subtype producing amylin is currently under investigation in our laboratory. Further, since we showed that AMY1 is present in the ARC and NTS albeit at a lower amount, we could also hypothesize that the effects observed in POMC-CTR KO may result from an alteration in the CGRP pathways since

CGRP, which is a widespread , can bind to its secondary receptor AMY1 in the ARC but not in the NTS (37; 38). The in situ hybridization revealed that ARC neurons can be CTR+, POMC+CTR+, CTR+RAMP+ or POMC+CTR+RAMP+ highlighting the complexity of the study of amylin signaling. Thus, the depletion of CTR in our model can either affect the calcitonin-mediated or the amylin-mediated signaling pathway.

The depletion of CTR in POMC neurons of male mice on chow diet increased weight gain and adiposity, which was characterized by an increased visceral fat mass and resulted in glucose intolerance. Baseline insulin levels and insulin tolerance were unchanged in this model, and circulating leptin levels corresponded as expected to the stored fat deposits. EE was significantly decreased in male KO mice, which could explain why the development of adiposity preempts a significant change in food intake. Amylin has been shown to increase EE by increasing sympathetic nerve activity, and nestin-human RAMP1 overexpressing transgenic mice also increased their EE (39; 40). At least part of this effect could be attributed to amylin increasing POMC mRNA and activating the MC4R pathway that promotes satiety and thermogenesis (12; 41), or a thermogenic effect through ERK phosphorylation, as it has been shown in respect to leptin (42). The overall effect is relatively small, which makes sense considering that only a small subpopulation of neurons was altered in this mouse model and

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the hindbrain pathway seems to be unaffected. While baseline body temperature of our KO

mice was normal, these mice did not respond to sCT injection by increasing thermogenesis as

it has been shown previously in intact rats and mice (39; 43). Furthermore, iBAT morphology

was altered in male KO mice with a decreased membrane surface and cell number. These

findings support the idea that reduced activation of iBAT may contribute to the EE phenotype

and this is reflected with lower UCP1 immunoreactivity in iBAT of male KO mice. Conversely,

female KO mice did not present any alteration in their EE, iBAT histology, and UCP1 activity

and in consequence did not develop adiposity.

Sexual dimorphism has not yet been studied extensively regarding amylin signaling. However,

the hypothalamic POMC system has been shown to differ between sexes, which has been

attributed to a lower number, lower activity and/or lower mRNA expression of POMC neurons

in the male ARC, a development that is induced by exposure (44; 45).

Furthermore, -deficient DIO rats seem to have a better response to chronic exogenous

amylin application regarding body weight and energy expenditure (46). Notably, the

inactivation of STAT3 signaling in POMC neurons leads to a similar fat-specific weight gain

as we have seen in this study, albeit in this case only in female mice (47). We did not see any

difference in the number of POMC neurons between sexes, contrary to the study by Wang et

al. using the same POMC-cre:ERT2 mice induced at 11 weeks of age (44). Therefore, we believe

that the absolute number of POMC neurons may be less important for the different phenotypes

than their properties. A possible confounding factor in this regard is the tamoxifen induction at

four weeks, which at least in females possibly interferes with sexual maturation. Additionally,

even low doses of tamoxifen in adult female mice have been linked to substantial adipose tissue

browning and increased thermogenesis (48), which may have masked the effects we attempted

to measure.

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Both male and female KO mice showed a reduced locomotor activity, which however did not seem to be responsible for the changes in EE discussed earlier. For instance, the activity pattern does not match the EE data time-wise in a 24h cycle, and in females it even contradicts their increased EE. Since POMC neurons project to the LHA (49; 50) and since MC3R-expressing orexin neurons in the LHA modulate locomotor activity (51), we hypothesized that the decrease in amylin POMC signaling could affect LHA orexin neurons and hence contribute to the decrease in locomotor activity. However, when looking at orexin expression in the LHA, we could not find any differences between groups or sex, neither did the activity pattern in metabolic cages point to a fragmented sleep pattern as it has been shown with ablation of orexin neurons or knockout studies (52; 53). Nevertheless, whether the depletion of amylin signaling in POMC neurons decreases orexin neurons’ activity was not assessed. POMC neurons are also prominent in the , but we could not find marked differences in baseline corticosterone and ACTH release. Furthermore, this decrease in locomotor activity did not correspond to decreased food foraging behavior, since male KO mice ate more overall and tended to have smaller and more frequent meals than Tx controls.

Therefore, the underlying mechanism of this consistently observed change in locomotor activity is still unclear.

Given the increase in adiposity of chow-fed male POMC-CTR KO mice, we expected an exacerbation of this phenotype under HFD. However, the KO-specific effect was rather lost, which could be due to a marked reduction of POMC expression that prevented clear effects of the defective amylin signaling in POMC neurons on HFD. Indeed, recent pharmacological studies by Li et al. showed that amylin directly uses the system to increase thermogenesis (41). While leptin sensitivity was similar in chow- and HFD-fed KO mice,

20 Page 21 of 53 Diabetes

represented by a comparable pSTAT3 signaling, the HFD cohort had a lower overall pSTAT3

and POMC expression. However, when CTR is depleted in rat ARC and ventromedial nucleus

(VMN) using an AAV shRNA (which targets more cells than specifically POMC neurons),

VMN leptin binding and leptin-induced pSTAT3 is decreased (6). We previously demonstrated

that amylin activates the secretion of IL-6 by microglia which then binds to its gp130 neuronal

receptor to further enhance leptin-induced pSTAT3 (10; 23). These studies suggest that

amylin’s enhancing effect on leptin pSTAT3 signaling takes place mostly in NPY neurons,

while direct pERK induction in POMC neurons is responsible for alterations in energy balance

(10; 15; 23).

HFD-fed mice gradually become leptin resistant and have higher circulating levels of leptin in

their blood. However, reports on the effect of various high fat diets on POMC expression are

ambiguous. A 12-week study with C57BL6 mice showed an upregulation of POMC mRNA

(54), but other studies in mice and rats showed a relative downregulation of POMC in the

obesity-prone phenotype in response to HFD (55; 56). The time-course study by Souza et al.

also demonstrated that NPY and POMC expression do not uniformly change, but rather

fluctuate during the first days and weeks of HFD exposure compared to chow diet. They

highlight that an early reduction of POMC in response to HFD characterizes an obesity-prone

phenotype (55).

In conclusion, we demonstrated that amylin action in POMC neurons is more involved in

energy homeostasis of male than female mice and may promotes energy expenditure by

affecting iBAT thermogenesis. Notably, this influence persists into adulthood and goes beyond

the previously established neurotrophic influence of amylin on fiber outgrowth in the

development of hypothalamic feeding circuit (15; 23). However, the direct action of amylin on

POMC neurons seems to play a diminished role in a leptin-resistant state of HFD fed mice and

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leaves the question of how amylin improves leptin signaling in the hypothalamus. Given that only a fraction of pERK or pSTAT3 expression overlaps with POMC neurons in the ARC, other distinct cell populations are worth exploring. Since we have previously shown the crucial role of microglial amylin signaling during the early post-natal period in regards to the hypothalamic development (10), we hypothesize that these cells could also play a major role in the regulation of energy balance as microglia seem to play a more prominent role in a state of dietary excess (57) and have been shown to play a crucial role in improving leptin signaling

(10). Moreover, the phenotype observed in POMC-CTR KO mice could result from the contribution of ARC and NTS signaling and it will be worth exploring the contribution of each brain nucleus. Finally, our research underlines the importance to consider both sexes in studies on energy homeostasis, as the therapeutic benefit from interventions in hypothalamic circuits could vary greatly between sexes.

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ACKNOWLEDGMENTS

This work was funded by the Swiss National Science Foundation (TAL SNF 31003A_175458),

and UZH Forschungskredit (CLF UZH FK 17-066). We would like to acknowledge the

technical contributions of Josep Monné Rodríguez, Institute of Veterinary Pathology,

Vetsuisse Faculty; the Center for Microscopy and Image Analysis, University of Zurich; Petra

Seebeck, Zurich Integrative Rodent Physiology (ZIRP) and Pr Joel Elmquist and Dr Thorsten

Schinke for providing us the POMC-cre:ERT2 and CTR-floxed mice, respectively. We also

thank the technical assistance of Christina N. Boyle, Fabienne O. Villars, Salome Gamakharia

and Justyna B. Koczwara.

AUTHOR CONTRIBUTIONS

Conceptualization, C.L.F. and T.A.L.; Methodology, C.L.F; Investigation, B.C. and C.L.F.;

Writing – Original Draft, B.C.; Writing – Review & Editing, C.L.F. and T.A.L. Resources,

T.A.L.; Funding Acquisition, T.A.L. and C.L.F. C.L.F. is the guarantor of this work and, as

such, had full access to all the data in the study and take responsibility for the integrity of the

data and the accuracy of the data analysis.

PRIOR PRESENTATION

Parts of this study were presented in abstract form at the Society for the Study of Ingestive

Behavior, Utrecht, Netherland, July 2019.

DECLARATION OF INTERESTS

The authors declare no conflict of interest.

MATERIAL AVAILABILITY

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The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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FIGURE LEGENDS

Figure 1. CTR and RAMP1 or RAMP3 are colocalized with POMC neurons in mice. (A)

Quantification of POMC and CTR immunopositive cells in the ARC and (B) 20X

representative image and 40X orthogonal views of Tx-treated POMC-Cre:tDTomato-CTRwt/wt

and POMC-Cre:tDTomato-CTRfl/fl ARC brain section immunostained for CTR. (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 arrow indicate no colocalization with POMC and

the filled arrow indicate POMC-CTR colocalization. Values are mean ± SEM; n = 4-5 per

group. ***P<0.001 after T-test.

Figure 2. CTR depletion in POMC neurons of male and female post-weaning mice fed

chow diet decreases amylin-induced pERK in POMC neurons but it does not affect ARC

–MSH fiber outgrowth. Amylin-induced (i.p.; 50 ug/kg) pERK immunohistochemistry (red)

in the ARC and VMN (A, B) of tamoxifen (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-labelled neurons was quantified in 3 separate sections (A,

B); Male and female data are pooled in this figure as no sex difference was observed for pERK-

POMC using 2-way ANOVA (P=0.54 F(1, 48)=0.363). (C) Quantification of -MSH

immunoreactive fiber density in the PVN and (D) 20X representative image of -MSH

immunostaining in the PVN. Values are mean ± SEM; n = 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.

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Figure 3. CTR depletion in POMC neurons of male mice increased body weight, food intake and fat mass on chow diet. Body weight (A, D), body weight gain after tamoxifen

(Tx) injection (B, E), cumulative food intake (C, F) in male (A-C) and female (D-F) mice aged from 4 to 14-week-old fed ad libitum chow diet and body composition assessed between lumbar vertebrae L1-L4 in 16-week-old male (G) and female (H) mice fed ad libitum chow diet. Values are mean ± SEM; n = 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 2-way ANOVA

(genotype, time) followed by Tukey post-hoc test. (G-H) *p<0.05 after unpaired T-test.

Figure 4. CTR depletion in POMC neurons of male mice decreased glucose tolerance on chow and 45% HFD. Oral glucose tolerance test (A, C, E, G) and insulin tolerance test (B,

D, F, H) were performed in male (A, B, E, F) and female (C, D, G, H) mice on chow (A, B,

C, D) and 45% HFD (E, F, G, H) at 11 and 12-week-old (chow diet) and 12 and 15-week-old mice (45% HFD) respectively. Area under the curve (AUC) was calculated from baseline for each test. Values are mean ± SEM; n = 7–12 male mice and 5–7 female mice per group on chow diet and n = 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 2-way ANOVA (genotype, time) followed by Tukey post-hoc test. See also Table S1.

Figure 5. CTR depletion in POMC neurons decreased EE in male but not in female mice on chow diet. 3 day EE (A) and RER (D), average of 3d EE (B, C) and RER (E, F) and 12h average in dark, light and total 24h EE (G) and RER (H) in tamoxifen (Tx)-treated 15-week- old male and female POMC-WTCTR and POMC-CreCTR mice. EE was normalized to

LBM*0.2FM presented in Fig. 2 G, H. Values are mean ± SEM; n = 6 male mice and 5-6 female mice per group. Parameters with differing letters (a, b, c) differ from each other by

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p<0.05 after 2-way (genotype (gen), time) or 3-way (genotype, sex, time) ANOVA followed

by Tukey post-hoc test. †P<0.05 Male vs. female POMC-CreCTR+Tx. The grey area represents

the dark phase.

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.

Interscapular brown adipose tissue (iBAT) was stained for H&E (A, B, C) in 16-week-old male

(A, B) and female (B, C) tamoxifen (Tx)-treated male and female POMC-WTCTR and POMC-

CreCTR mice and UCP1 was detected by immunofluorescence (D, E). 20X representative image

of the H&E staining (B); 100X representative image of the UCP1 staining (E). Core body

temperature over 3 days (F), average of 3 days during dark and light cycle (G), delta body

temperature between saline and sCT (i.p. 5 ug/kg) injection over 24h (H, 3-way ANOVA Sex

P=0.29 F(1, 11)=0.775) and average delta temperature during dark and light phase (I, 3-way

ANOVA Sex P=0.25 F(1, 11)=1.471) in Tx-treated 15-week-old male and female POMC-

WTCTR and POMC-CreCTR mice. Male and female data are pooled in figures H-I. Values are

mean ± SEM; n = 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 2-way ANOVA followed by Tukey post-hoc test.

The grey area represents the dark phase.

Figure 7. CTR depletion in POMC neurons of male and female mice fed chow and 45%

HFD decreased locomotor activity while it did not affect the number of orexin neurons in

the LH. Locomotor activity over 3 days (A, D), average of 3 days of locomotor activity during

dark, light cycle and total 24h period (B, E), cumulative locomotor activity over 3d (C, F) on

chow (A, B, C) and 45% HFD (D, E, F) in tamoxifen (Tx)-treated male and female POMC-

WTCTR and POMC-CreCTR mice (C-Chow: 2-way ANOVA Sex P=0.76 F(1, 10)=0.091; F-

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HFD: 2-way ANOVA Sex P=0.32 F(1, 17)=1.025). (H) Lateral hypothalamus (LH) brain sections were stained for orexin (green) and counterstained with DAPI (blue) and the number of neurons was quantified in 3 separate sections in chow-fed mice; 3-way ANOVA Sex P=0.61

F(1, 7)=0.2782. (G) 20X representative image of the mediobasal hypothalamus including the arcuate nucleus (ARC), ventromedial hypothalamus (VMN) and LH. Male and female data are pooled in this figure as no sex effect was observed. Values are mean ± SEM; n = 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 2-way ANOVA (genotype, time) followed by Tukey post-hoc test.

*p<0.05 after unpaired T-test.

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 (i.p.; 5 mg/kg) pSTAT3 immunofluorescence (red) in the ARC and VMN (A) of tamoxifen (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-labelled neurons was quantified in 3 separate sections in chow-fed mice (A, B) and 45% HFD-fed mice

(C, D). Male and female data are pooled in this figure. The n number was not high enough to run a 3-way ANOVA. Values are mean ± SEM; n = 4–8 per group on chow diet. Parameters with differing letters (a, b) differ from each other by P<0.05 after 2-way ANOVA (genotype, treatment (saline vs leptin)) followed by Tukey post-hoc test.

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Table 1. Leptin and insulin blood levels 8 weeks in male and female mice post tamoxifen (Tx) treatment on chow diet and after 6 and 12 weeks on 45% high fat diet (HFD). Values are assessed by 2-way ANOVA (Genotype, Sex) and Tukey post-hoc test. All data are expressed as mean [pg/ml] ± SEM.

Male POMC-WTCTR Male POMC-CreCTR Female POMC-WTCTR Female POMC-CreCTR Two-way ANOVA +Tx +Tx +Tx +Tx 8wk Chow (n=8-10/group) Insulin (pg/ml) 358±52 522±99 207±34 391±147 Genotype p=0.13 Sex*p=0.04 Leptin (pg/ml) 1007±239 872±146 522±79 467±82 Genotype p=0.54 Sex**p=0.006 6wk 45%HFD (n=6-10/group) Insulin (pg/ml) 763±69 1087±289 687±189 421±68 Genotype p=0.70 Sex p=0.24 Leptin (pg/ml) 13815±1959 16779±4155 10573±4347 17198±6382 Genotype p=0.32 Sex p=0.76 12wk 45%HFD(n=8-12/group) Insulin (pg/ml) 1094±165 1828±423 1080±281 1553±453 Genotype p=0.12 Sex p=0.71 Leptin (pg/ml) 24583±4338 12343±5668 18918±5110 7676±3175 Genotype p=0.30 Sex* p=0.02

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37 A B Diabetes Page 38 of 53

100

75

50 ✱✱✱ ✱✱✱

(Avg/section) 25 Number of neurons 0 POMC POMC+CTR+ %CTR+ +POMC+

POMC-WTCTR+Tx+ Saline POMC-CreCTR+Tx+ Saline

C DAPI Calcr POMC RAMP1 D DAPI Calcr RAMP1 E DAPI Calcr RAMP1

ARC 30 µm AP 30 µm

3V

NTS CTR

100 µm 100 µm 100 µm CC F DAPI Calcr POMC RAMP3 G DAPI Calcr RAMP3 H DAPI Calcr POMC RAMP3 POMC - WT

ARC 30 µm

AP

3V NTS CC CC 100 µm 100 µm 100 µm Page 39 of 53 Diabetes A 35 B 15 Diabetes C 1000 Page 40b of 53 a b a b a a 30 b b a a b 750 b a,b a a,b a a 10 a a b a 25 b a b b b b 500 20

Body Weight (g) Body Weight 5

♂ 250 15 ♂ Cumulative food Intake (kcal) Tx gain after Tx (g) ♂ Body Weight 10 0 0 3.5 4 5 6 7 8 9 10 11 12 13 14 6 7 8 9 10 11 12 13 14 4 5 6 7 8 9 10 11 Age (weeks) Age (weeks) Age (weeks)

POMC-WTCTR+Oil POMC-WTCTR+Tx POMC-CreCTR+Oil POMC-CreCTR+Tx D E F 35 15 1000

30 a a 750 a a 10 a a a b b 25 a a a b b b b a,b b b b 500 20

Body Weight (g) Body Weight 5

♀ 250 15 Cumulative food Intake (kcal) ♀ Tx gain after Tx (g) ♀ Body Weight 10 0 0 3.5 4 5 6 7 8 9 10 11 12 13 14 6 7 8 9 10 11 12 13 14 4 5 6 7 8 9 10 11 Age (weeks) Age (weeks) Age (weeks) G ✱ 40

30 POMC-WTCTR+Tx 20 CTR 6 POMC-Cre +Tx 4 ✱ 2

Body composition L1-L4 0 Lean (g) Visceral (g) Subcut. (g) Total Fat Mass (g) % Fat Ratio LBM*0.2FM ♂

H 25 20 15 10 5

1

Body composition L1-L4 0 Lean (g) Visceral (g) Subcut. (g) Total Fat Mass (g) % Fat Ratio LBM*0.2FM ♀ Page 41 of 53 Chow Diabetes a a,b a a a a A 10000 a,b a,b a b B 25000 400 b 250 b 8000 Gen ns 20000 6000 Time **** 15000 a Time x Gen ns a 4000 10000 a,b 2000 5000 300 b 200 AUC (mg/dl/120min) ♂ 0 ♂ AUC (mg/dl/120min) 0

+ oil + oil + Tx + Tx + oil + oil + Tx + Tx CTR CTR CTR CTR CTR CTR CTR CTR

POMC-WTPOMC-CrePOMC-WTPOMC-Cre POMC-WTPOMC-CrePOMC-WTPOMC-Cre 200 150 Glucose (mg/dl) ♂ ♂ Glucose (mg/dl) Gen * Time **** Time x Gen * 100 100 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Minutes Minutes C D 15000 a a a a 25000 a a a a 400 250 a Gen ns 20000 a,b 10000 Time **** b 15000 Time x Gen * b 10000 5000 200 dl ) 300 5000 ♀ AUC (mg/dl/120min) ♀ AUC (mg/dl/120min) 0 0

+ oil + oil + Tx + Tx + oil + oil + Tx + Tx CTR CTR CTR CTR 150 CTR CTR CTR CTR

200 POMC-WTPOMC-CrePOMC-WTPOMC-Cre POMC-WTPOMC-CrePOMC-WTPOMC-Cre ♀ Glucose ( mg / ♀ Glucose (mg/dl) 100 Gen ns Time **** Time x Gen ns 100 50 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Minutes Minutes POMC-WTCTR + oil POMC-WTCTR + Tx POMC-CreCTR + oil POMC-CreCTR + Tx

45% HFD

a a a a a E a,c b,c c a F a a a,b a,b a a 10000 20000 a,b ab a 500 a a b a,b a a 8000 200 b a b b a 15000 a 6000 b 10000 4000 400 5000 2000 150 ♂ AUC (mg/dl/120min)

♂ AUC (mg/dl/120min) 0 0

+Oil +Oil +Tx +Tx + oil + oil + Tx + Tx CTR CTR 300 CTR CTR 100 CTR CTR CTR CTR

POMC-WTPOMC-Cre POMC-WTPOMC-Cre POMC-WTPOMC-CrePOMC-WTPOMC-Cre Glucose (mg/dl)

♂ Glucose (mg/dl) 200 ♂ 50 Gen ns Gen **** Time **** Time **** Time x Gen ns Time x Gen ns 100 0 0 20 40 60 80 100 120 0 20 40 60 80 Minutes Minutes G H a 15000 a a a a 200 15000 a a a a 500 a a 10000 10000 b 5000 150 5000 400 a a dl ) dl ) a a ♀ AUC (mg/dl/120min) a a,b ♀ AUC (mg/dl/120min) 0 0

b b +Oil +Oil +Tx +Tx + oil + oil + Tx + Tx CTR CTR 300 CTR CTR 100 CTR CTR CTR CTR

POMC-WT POMC-WTPOMC-Cre POMC-Cre POMC-WTPOMC-CrePOMC-WTPOMC-Cre

♀ Glucose ( mg / 50 ♀ Glucose ( mg / 200 Gen ns Gen ns Time **** Time **** Time x Gen *** Time x Gen ns 100 0 0 20 40 60 80 100 120 0 20 40 60 80 Minutes Minutes A B C 0.6 † † † 0.6 Diabetes 0.6 Page 42 of 53

** † * 0.4 † † † †† 0.4 * 0.4 *

Sex ** 0.2 0.2 0.2 Average 3d EE Gen ns Average 3d EE ♀ Sex x Gen * ♂ Gen ** Gen ns (kJ/h)/(LBM+0.2FM) (kJ/h)/(LBM+0.2FM)

EE (kJ/h)/(LBM*0.2FM) Time **** Time **** Time **** Time x Sex**** Time x Gen * Time x Gen ** 0.0 0.0 0.0 1 13 25 37 49 61 2 4 6 8 10 12 14 16 18 20 22 2 4 6 8 10 12 14 16 18 20 22 Hours Hours Hours

Male POMC-WTCTR+Tx Male POMC-CreCTR+Tx Female POMC-WTCTR+Tx Female POMC-CreCTR+Tx D E F 1.1 1.1 1.1

1.1 1.0

1.0 1.0 1.0 1.0 RER 0.9 0.9 Sex ** 0.9 Average 3d RER Gen ns Gen ns Average 3d RER

♀ Gen ns Sex x Gen *** ♂ 0.9 Time **** 0.9 Time **** Time **** Time x Gen ns Time x Sex** Time x Gen ** 0.8 0.8 0.8 0 12 24 36 48 60 2 4 6 8 10 12 14 16 18 20 22 2 4 6 8 10 12 14 16 18 20 22 Hours Hours Hours G H

10 a,b a a,b b a a a a a ba,c c 1.1 a a a a a,b a b b a a a a Male POMC-WTCTR+Tx 8 CTR 1.0 Male POMC-Cre +Tx CTR 6 Female POMC-WT +Tx 0.9 Female POMC-CreCTR+Tx 4

2 RER (avg/12h) 0.8 0.1 0 0.0

EE (kJ/12h) /LBM +0.2FM) Dark Light Total Dark Light Total POMC-WTCTR+Tx POMC-CreCTR+Tx PageA 43 of 53 ✱ Male DiabetesB 400000 a a,b b a a a,b b a,b 300000 200000 100000 ✱ a a b a 3V A.U. ♂ 4000 3000 Male 2000 1000 0 Membrane Number of cells Vacuoles surface 100 µm

C Female a b a,b a,b 450000 a b a,b a,b 300000

150000 Female a a a a A.U. 4000 ♀ 3000 2000 1000 CTR CTR 0 E POMC-WT +Tx POMC-Cre +Tx Membrane Number of cells Vacuoles surface

D ✱ 12500 a,b aa b a b b a,b Male Male 10000 20 µm 7500

5000

2500 UCP1 density (A.U.) 0 Male Female Female

F G 39 Gen ns Time *** Time x Gen ns 38 a a b b 38 37 37 36

Temperature36 (°C) 35 10 35 0 0 720 1440 2160 2880 3600 4320 Dark Light Minutes Average 3d Temperature (°C) POMC-WTCTR+Tx POMC-CreCTR+Tx H I 2.5 * Gen **** * POMC-WTCTR +Oil 2.0 Time **** 1.0 Time x Gen ** 1.5 * * POMC-CreCTR+Oil 1.0 0.5 CTR 0.5 POMC-WT +Tx 0.0 0.0 POMC-CreCTR+Tx -0.5 -0.5 -1.0 Temperature (°C)

Delta Body Temperature (°C) Delta Body Temperature -1.5 -1.0 0 2 4 6 8 10 12 14 16 18 20 22 24 Average Delta (sCT-Saline) Dark Light Hours A B Diabetes C Page 44 of 53

Gen ns 15 6000 Time **** a,c a,c b b a c 20 Time x Gen * * 10 4000

10 5 2000 Average 3d Activity Locomotor Activity (A.U.) Locomotor

0 0 Cumulative activity 3d 0 0 720 1440 2160 2880 3600 4320 Dark Light Total Minutes D E F Gen ns Time **** Time x Gen * 20 a b c c a b 8000 20 * 15 6000

10 10 4000

5 2000

Locomotor Activity (A.U.) Locomotor 0 0 0 0 720 1440 2160 2880 3600 4320 Activity (A.U.) Locomotor Dark Light Total Minutes Cumulative activity 3d (A.U)

G POMC-WTCTR POMC-CreCTR H Orexin POMC-CreWT+Tx 150 POMC-CreCTR+Tx LH F WT Oil POMC-Cre +Tx 3V VMN 100 POMC-CreCTR+Tx ARC 50 μm 50 neurons / section 0 Number of Orexin-A positive Number of Orexin-A +Oil +Oil +Tx +Tx CTR CTR

Tx CTR CTR

POMC-WTPOMC-CrePOMC-WTPOMC-Cre D C B A Page 45of53 Leptin Saline Number of neurons Leptin Saline Number of neurons POMC POMC 2 (Avg/section) 2 (Avg/section) 00 00 µm 100 150 100 200 300 400 50 µm 0 0 ARC 3V - - 3V WT WT SA3VNpTT-R OCpSTAT3-POMC POMC pSTAT3-ARC pSTAT3-VMN SA3VNpTT-R OCpSTAT3-POMC POMC pSTAT3-ARC pSTAT3-VMN ARC CTR CTR aab a ba aab a ba VMN +Tx +Tx VMN pSTAT3 pSTAT3 POMC POMC a a - - Cre Cre c aab a ba CTR CTR +Tx +Tx b 2 2 POMC POMC 00 00 µm µm aaa a aa aaa a aa - - WT WT CTR CTR Diabetes +Tx +Tx POMC POMC POMC POMC aaa a aa aaa a aa - - Cre Cre CTR CTR +Tx +Tx 50µm 50µm POMC POMC POMC-Cre POMC-WT POMC-WT POMC-Cre - - WT WT pSTAT3 pSTAT3 CTR CTR ARC ARC +Tx +Tx CTR CTR CTR CTR POMC POMC POMC POMC +Tx+ Leptin +Tx+ Saline +Tx+ Saline +Tx+ Leptin - - Cre Cre CTR CTR +Tx +Tx Diabetes Page 46 of 53

Table S1. Meal pattern of ad libitum-fed male and female mice for 24h, during the 12h light and dark phase. *p<0.05 after unpaired T-test between POMC-WTCTR + Tx and POMC-CreCTR + Tx groups.

POMC-WTCTR + POMC-CreCTR + POMC-WTCTR + POMC-CreCTR + POMC-WTCTR + POMC-CreCTR + Tx Tx Tx Tx Tx Tx 24h 12h light phase 12h dark phase Ad libitum (all mice) n=8/group Total Food intake (g) 2.88±0.28 3.33±0.41 0.35±0.13 0.51±0.13 2.53±0.30 2.82±0.37 Meal number 8.44±0.77 10.8±0.87* 1.7±0.48 3.00±0.29* 6.75±0.93 7.79±0.78 Meal size 0.34±0.04 0.30±0.06 0.21±0.04 0.17±0.04 0.44±0.08 0.42±0.10 Meal duration (Min) 41±6.5 31.5±4.22 18±4 21.5±5 60±13 41.5±7* Ad libitum (Male mice) n=4/group Total Food intake (g) 3.10±0.57 3.15±0.09 0.28±0.13 0.35±0.17 2.82±0.51 2.80±0.01 Meal number 7.88±0.13 11±0.87* 1.4±0.77 2.50±0.50 6.50±1.6 8.50±1.32 Meal size 0.39±0.07 0.24±0.04 0.25±0.04 0.13±0.04* 0.51±0.15 0.36±0.05 Meal duration (Min) 47.4±10 25.5±5.8 22.3±4 12.2±1.2* 70.11±23.5 38.7±10.5 Ad libitum (Female mice) n=4/group Total Food intake (g) 2.66±0.10 3.46±0.75 0.42±0.23 0.64±0.18 2.24±0.31 2.83±0.69 Meal number 9±0.91 10.62±1.01 2±0.65 3.38±0.24 7±1.2 7.25±1.01 Meal size 0.28±0.03 0.34±0.10 0.17±0.05 0.21±0.07 0.37±0.09 0.47±0.17 Meal duration (Min) 34.2±8.2 35.6±5.51 15.1±7.1 28.5±6.75 49.43±14.92 43.47±10.42 Page 47 of 53 Diabetes

Table S2. Corticosterone and ACTH plasma levels were measured 2h after lights on (circadian nadir) and lights off (secretion peak), respectively. Values are assessed by two-way ANOVA or a mixed model if values were missing. There was a significant difference for corticosterone and ACTH levels in the 12-hour cycle, but no significant differences between groups. All data are expressed as mean [pg/ml] ± SEM. n=8-10/group.

POMC-WTCTR POMC-WTCTR POMC-CreCTR POMC-CreCTR Mixed-Effect Analysis +Oil +Tx +Oil +Tx Corticosterone 2h after lights on 946±89 650±58 482±34 564±48 Group P=0.15 2h after lights off 2109±85 1899±123 1744±51 1888±47 Time*** P<0.0001 ACTH 2h after lights on 389±16 381±20 351±13 428±13 Group P=0.26 2h after lights off 400±15 394±16 434±16 514±12 Time* P=0.028 Diabetes Page 48 of 53

Figure S1. CTR depletion in POMC neurons of male and female mice body weight, food intake and fat mass on 45% HFD. Body weight (A, D), body weight gain after tamoxifen (Tx) injection (B, E), cumulative food intake (C, F) and body composition measured between lumbar vertebrae L1-L4 (G, H) in male (A, B, C, G) and female (D, E, F, H) mice aged from 5 to 15-week-old ad libitum-fed chow diet. Values are mean ± SEM; n =4-5 male mice and 4-6 female mice per group. 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. A B C 40 20 1250

a 35 1000 a 15

b 30 750 b 10 25 500 Body Weight (g)

♂ 5 20 250 Cumulative food Intake (kcal) Body Weight gain after Tx (g) ♂ TX ♂ 15 0 0 5 6 7 8 9 101112131415 7 8 9 10 11 12 13 14 15 7 8 9 10 11 12 13 14 15 16 Age (weeks) Age (weeks) Age (weeks)

POMC-WTCTR+Oil POMC-WTCTR+Tx POMC-CreCTR+Oil POMC-CreCTR+Tx D E F 40 20 1250

35 15 1000 a a 30 a a a,b 750 b a 10 b b c 25 b 500 c Body Weight (g)

♀ 5 20 250 Cumulative food Intake (kcal) Body Weight gain after Tx (g) ♀ ♀ 15 TX 0 0 5 6 7 8 9 101112131415 7 8 9 10 11 12 13 14 15 7 8 9 10 11 12 13 14 15 16 Age (weeks) Age (weeks) Age (weeks) G

60 a a,ba,b b 50 40 30 POMC-WTCTR+Oil 20 a aa a a aa a CTR 8 a aa a POMC-Cre +Oil CTR 6 a aa a a aa a POMC-WT +Tx 4 CTR 2 POMC-Cre +Tx

Body composition L1-L4 0

♂ Lean(g) Visceral(g) Subcut.(g) TotalFat Mass(g) %Fat Ratio LBM*0.2FM

H a a,ba,b b 50 40 30 20 8 a aa a a aa a aa a a 6 a aa a a aa a 4 2

Body composition L1-L4 0 Lean (g) Visceral (g) Subcut. (g) Total Fat Mass (g) % Fat Ratio LBM*0.2FM ♀ Page 49 of 53 Diabetes

Figure S2. CTR depletion in POMC neurons increases EE in female but not in male mice on 45% HFD. 3 day EE (A) and RER (D), average of 3d EE (B) and RER (E) and 12h average in dark, light and total 24h EE (C) and RER (F) in tamoxifen (Tx)-treated 15-week-old male and female POMC-WTCTR and POMC- CreCTR mice. EE was normalised to LBM*0.2FM (presented in Fig. S1G-H). Values are mean ± 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, time) or three-way (genotype, sex, time) ANOVA followed by Tukey post-hoc test. The grey area represents the dark phase. Diabetes Page 50 of 53

Figure S3. Effect of amylin and salmon calcitonin (sCT) on food intake in male and female mice on chow diet. Amylin at 2 different doses (i.p. 50 and 500 ug/kg) and sCT (5 ug/kg) were injected and tested against vehicle-treated mice in a randomised cross-over manner in 4 groups of male and female mice. Results were expressed in g (A, B, C, D, E, K, L, M, O, P) or as percent of baseline (F, G, H, I, J, P, Q, R, S, T) which represent food intake after a 12h fast. Food intake after 1h (A, F, K, P), 2h (B, G, L, Q), 4h (C, H, M, R), 12h (D, I, N, S) and 24h (E, J, O, T) was measured. Values are mean ± SEM; n = 4–7 per group. *p< 0.05, **p< 0.01 or parameters with differing letters (a, b) differ from each other by p<0.05 after two-way ANOVA (genotype (gen), treatment (RX)) followed by Tukey post-hoc test. Dotted lines represent baseline food intake after 12h fast and refeeding (100%; F-J, P-T). Page 51 of 53 Diabetes

Figure S4. Effect of leptin (5 mg/kg) and amylin (50 ug/kg) on food intake in male and female mice on chow diet. Amylin (i.p. 50 ug/kg) and leptin (5 mg/kg) were injected and tested against vehicle-treated mice in a randomised cross-over manner in tamoxifen (Tx)-treated male and female POMC-WTCTR and POMC- CreCTR mice. Results were expressed in g (A, B, C) or as percent of baseline (D, E, F) which represent food intake after a 12h fast. Food intake after 1h (A, D), 4h (B, E) and 12h (C, F) was measured. Values are mean ± SEM; n = 5–9 per group. Parameters with differing letters (a, b) differ from each other by p<0.05 after two- way ANOVA (genotype (gen), treatment (RX)) followed by Tukey post-hoc test. Dotted lines represent baseline food intake after 12h fast and refeeding (100%; D-F).

A Gen NS; RX P=0.12 B Gen NS; RX P=0.0057 C Gen P=0.08; RX P=0.08

a a,b b a a,b b a a a a a a 1.0 a a,b b a a a 2.0 5

0.8 4 1.5 0.6 3 1.0 0.4 2 0.5 0.2 1

0.0 0.0 0 h p p h p p h p p h p p h p p h p p 1h cumulative food intake (g) e e e e e e 4h cumulative food intake (g) e e e e e e e e e e e e

V L L V L L V L L V L L 12h cumulative food intake (g) V L L V L L + + + + + + y y y y y y m m m m m m A A A A A A POMC-WTCTR+Tx POMC-CreCTR+Tx D E F Gen NS; RX P=0.06 Gen NS; RX P=0.011 Gen NS; RX NS 400 a a a a a a 400 a a a a a a 400 a a a a aa

300 300 300

200 200 200 (%baseline) (%baseline) 100 100 (%baseline) 100

0 0 0 4h cumulative food intake 1h cumulative food intake

Veh Lep Amy+Lep Veh Lep Amy+Lep 12h cumulative food intake Veh Lep Amy+Lep Diabetes Page 52 of 53

Figure S5. POMC, CTR and RAMP1 or RAMP3 are colocalized in NTS neurons of POMC-WTCTR mice. Representative 20X and 63X NTS images of in situ hybridization of CTR (Calcr1A), POMC and RAMP1 (A) Caudal of the AP (-7.83 mm from bregma), (B) Level of the AP (-7.47 mm), (C) Rostral of the AP (-6.95 mm). Representative 20X and 63X NTS images of in situ hybridization of CTR (Calcr1A), POMC and RAMP3 (D) Caudal of the AP, (E) Level of the AP, (F) Rostral of the AP. The empty arrow indicates a single staining and the filled arrow indicates POMC-CTR or RAMP-CTR colocalization. A B C DAPI Calcr1A POMC 30 µm RAMP1

AP

4V 4V

NTS

NTS NTS CC CTR 100 µm CC D E F DAPIDAPICalcrCalcr1APOMCPOMCRAMP3 RAMP3 4V POMC-WT

NTS

AP

NTS NTS CC CC Page 53 of 53 Diabetes

Figure S6. Endogenous POMC expression in POMC:tDTomato-WTCTR and POMC-:tDTomatoCreCTR mice injected with tamoxifen in the AP/NTS region. Representative 20X (A, D) Caudal of the AP (-7.83 mm from bregma), (B, E) Level of the AP (-7.47 mm), (C, F) Rostral of the AP (-7.21 mm).

AP/NTS regions A DAPI POMC tDTomato B C WT AP NTS 4V NTS NTS CC CC POMC:tDTomato-CTR

D E F KO

4V NTS AP NTS CC NTS CC

POMC:tDTomato-CTR 50 μm