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CaMK1D signalling in AgRP neurons promotes ghrelin-mediated food intake

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Abstract

Hypothalamic AgRP/NPY neurons are key players in the control of feeding behaviour. Ghrelin, a major orexigenic hormone, activates AgRP/NPY neurons to stimulate food intake and adiposity. However, cell-autonomous ghrelin-dependent signalling mechanisms in AgRP/NPY neurons remain poorly defined. Here we show that calcium/calmodulin-dependent protein kinase ID (CaMK1D), a genetic hot spot in type 2 diabetes, is activated upon ghrelin stimulation and acts in AgRP/NPY neurons to mediate ghrelin-dependent food intake. Global Camk1d-knockout male mice are resistant to ghrelin, gain less body weight and are protected against high-fat-diet-induced obesity. Deletion of Camk1d in AgRP/NPY, but not in POMC, neurons is sufficient to recapitulate above phenotypes. In response to ghrelin, lack of CaMK1D attenuates phosphorylation of CREB and CREB-dependent expression of the orexigenic neuropeptides AgRP/NPY in fibre projections to the paraventricular nucleus (PVN). Hence, CaMK1D links ghrelin action to transcriptional control of orexigenic neuropeptide availability in AgRP neurons.

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Fig. 1: Global deletion of Camk1d gene reduces diet- and leptin deficiency-induced obesity in mice.
Fig. 2: Deletion of Camk1d attenuates ghrelin-induced food intake.
Fig. 3: CaMK1D deletion in AgRP neurons leads to reduced body weight and food intake.
Fig. 4: Conditional deletion of Camk1d in AgRP neurons decreases energy expenditure.
Fig. 5: CaMK1D is dispensable for ghrelin-induced electrophysiological activation of AgRP/NPY neurons.
Fig. 6: Lack of CaMK1D reduces ghrelin-induced AgRP/NPY expression and abundance in AgRP neuron projections to the PVN.

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Acknowledgements

We thank T. Alquier at University of Montreal for helpful discussions and D. Dembele at the IGBMC for ANCOVA analysis. We thank the Imaging Center of the IGBMC (ICI), the IGBMC molecular biology and virus core facility, the Institut Clinique de la souris (ICS) core facilities and other core facilities for their support on this research. This work was supported by the Agence Nationale de la Recherche (ANR) (AAPG 2021 HypoCaMK)), by the Fondation de Recherche Médicale (FRM) – Program: Equipe FRM (EQU201903007859, Prix Roger PROPICE pour la recherche sur le cancer du pancréas), by the FHU-OMAGE of region Grand-Est, from the European Foundation for the Study of Diabetes (EFSD)/Novo Nordisk Diabetes Research Programme and by the ANR-10-LABX-0030-INRT grant as well as the ANR-11-INBS-0009-INGESTEM grant, both French State funds managed by the ANR under the frame program Investissements d’Avenir. K.V. was supported by an Individual Fellowship (798961 INSULYSOSOME) in the framework of the Marie-Sklodovska Curie actions of the European Commission and a research grant from Société Francophone du Diabète. G.Y. received financial doctoral support from DFG-233886668/RTG1960. M. Quiñones was funded by a research contract Miguel Servet (CP21/00108) from the ISCIII co-funded by the European Union.

Author information

Authors and Affiliations

  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France

    Karl Vivot, Gergö Meszaros, Evanthia Pangou, Zhirong Zhang, Mengdi Qu, Eric Erbs, Erwan Grandgirard, Anna Schneider, Izabela Sumara & Romeo Ricci

  2. Centre National de la Recherche Scientifique, Illkirch, France

    Karl Vivot, Gergö Meszaros, Evanthia Pangou, Zhirong Zhang, Mengdi Qu, Eric Erbs, Erwan Grandgirard, Anna Schneider, Izabela Sumara & Romeo Ricci

  3. Institut National de la Santé et de la Recherche Médicale, Illkirch, France

    Karl Vivot, Gergö Meszaros, Evanthia Pangou, Zhirong Zhang, Mengdi Qu, Eric Erbs, Erwan Grandgirard, Anna Schneider, Izabela Sumara & Romeo Ricci

  4. Université de Strasbourg, Strasbourg, France

    Karl Vivot, Gergö Meszaros, Evanthia Pangou, Zhirong Zhang, Mengdi Qu, Eric Erbs, Erwan Grandgirard, Anna Schneider, Etienne Clauss–Creusot, Alexandre Charlet, Izabela Sumara & Romeo Ricci

  5. Biocenter, Institute for Zoology, and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, (CECAD), University of Cologne, Cologne, Germany

    Gagik Yeghiazaryan & Peter Kloppenburg

  6. Instituto de Investigación Sanitaria de Santiago de Compostela, Complexo Hospitalario Universitario de Santiago (CHUS/SERGAS), Santiago de Compostela, Spain

    Mar Quiñones & Ruben Nogueiras

  7. CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III, Santiago de Compostela, Spain

    Mar Quiñones

  8. Centre National de la Recherche Scientifique, Institute of Cellular and Integrative Neurosciences, Strasbourg, France

    Etienne Clauss–Creusot & Alexandre Charlet

  9. Université Paris Cité, CNRS, Unité de Biologie Fonctionnelle et Adaptative, Paris, France

    Maya Faour, Claire Martin & Serge Luquet

  10. Institute of Cancer and Genomic Sciences, The University of Birmingham, Birmingham, UK

    Fedor Berditchevski

  11. Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela, Spain

    Ruben Nogueiras

  12. Laboratoire de Biochimie et de Biologie Moléculaire, Nouvel Hôpital Civil, Strasbourg, France

    Romeo Ricci

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  1. Karl Vivot
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  2. Gergö Meszaros
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  3. Evanthia Pangou
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  4. Zhirong Zhang
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  5. Mengdi Qu
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  6. Eric Erbs
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  7. Gagik Yeghiazaryan
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  8. Mar Quiñones
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  9. Erwan Grandgirard
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  10. Anna Schneider
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  11. Etienne Clauss–Creusot
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  13. Maya Faour
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  14. Claire Martin
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  16. Izabela Sumara
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Contributions

Conceptualization: R.R., R.N, P.K and S.L. Software: E.G. Methodology: K.V., E.P., Z.Z, Ma.Q, G.Y., E.E., E.C.–C. and M. Qu. Validation: K.V. Formal Analysis: K.V., C.M. Investigation: K.V., G.M., E.P., Z.Z., M. Quiñones., E.E., G.Y., M. Qu., A.S., M.F, C.M. Resources: E.E., F.B. Writing — Original Draft: R.R. and K.V., Supervision: K.V., S.L., P.K., R.N., A.C., I.S. and R.R, Funding Acquisition: R.R. and S.L.

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Correspondence to Karl Vivot or Romeo Ricci.

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Nature Metabolism thanks Timo D. Müller, Dominic Withers and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Deletion of CaMK1D in mice does not affect body size and CaMK1D knockout mice are born without any gross abnormalities.

a, Schematic screen of targeting of the CaMK1D locus using Knockout-first targeting strategy and generation of floxed and global knockout mice. For further details see material and methods. b, Validation of targeted ES clone by southern blot. Genomic DNA was digested using BspHI, BstEII, ApalI or EcoRI. The table shows the expected size of bands corresponding to the restriction enzyme used. A probe recognizing the neoR cassette was used. c, Gender distribution of mice and d, Mendelian ratios of born mice with indicated genetoypes. e, Body length and f, Tibia Length measurements of 7-week-old mice with indicated genotypes (CaMK1D+/+ n = 5; CaMK1D−/− n = 7). Data are presented as mean values +/− SEM.

Source data

Extended Data Fig. 2 CaMK1D signaling is redundant in pancreatic beta cell and liver function.

a, Blood insulin levels during IPGTTs in mice on a High fat diet (HFD). (n = 11/group). b, Glucose-stimulated insulin secretion (GSIS) in pancreatic islets isolated from mice. (CaMK1D+/+ n = 6; CaMK1D−/− n = 5). c, Expression of CaMK1D protein in pancreas and brain from mice with indicated genotypes. d, Body weight gain of mice on a Chow diet (CD) or on a High Fat Diet (HFD). (CD - CaMK1Dflox/flox n = 13; CD - AgRPCre/+; CaMK1Dflox/flox n = 13; HFD - CaMK1Dflox/flox n = 12; CD - AgRPCre/+; CaMK1Dflox/flox n = 10). e-f, Blood glucose levels during IPGTTs in mice on a CD (e), or on a HFD (f). (CD - CaMK1Dflox/flox n = 7; CD - AgRPCre/+; CaMK1Dflox/flox n = 6; HFD - CaMK1Dflox/flox n = 7; CD - AgRPCre/+; CaMK1Dflox/flox n = 8). g, Expression of CaMK1D mRNA in liver from mice. (CaMK1Dflox/flox n = 3; CD - AlbCre/+; CaMK1Dflox/flox n = 3) h, Body weight gain of mice on a CD or on a HFD. (CD - CaMK1Dflox/flox n = 8; CD - AlbCre/+; CaMK1Dflox/flox n = 9; HFD - CaMK1Dflox/flox n = 8; HFD - AlbCre/+; CaMK1Dflox/flox n = 10). Blood glucose levels during an IPGTT in mice on a CD (i) or on a HFD (j). (CD - CaMK1Dflox/flox n = 6; CD - AlbCre/+; CaMK1Dflox/flox n = 7; HFD - CaMK1Dflox/flox n = 8; HFD - AlbCre/+; CaMK1Dflox/flox n = 5). Blood glucose levels during an ITT in mice on a CD (k) or on a HFD (l). (CD - CaMK1Dflox/flox n = 5; CD - AlbCre/+; CaMK1Dflox/flox n = 6; HFD - CaMK1Dflox/flox n = 12; HFD - AlbCre/+; CaMK1Dflox/flox n = 14). Data are presented as mean values +/− SEM.

Source data

Extended Data Fig. 3 Deletion of CaMK1D does not result in anxiety-like behavior and stress-induced anhedonia.

a,Distance travelled each 5 min, and (b) in total. (c) Rear number each 5 min, and (d) in total. e, Number of entries in the center or periphery of the arena. f, Time spent in the center or periphery of the arena. g, Average speed during an open field test. (n = 8/group). h, Amount of sucrose and water consumed over 3 day in while sucrose and water were both available in the same time. (CaMK1D+/+ n = 8; CaMK1D−/− n = 7). Data are presented as mean values +/− SEM.

Source data

Extended Data Fig. 4 Deletion of CaMK1D in the Nervous Tissue reproduces phenotypes in whole-body knockout mice.

a, Expression of CaMK1D protein in mice. b, Body weight gain on a Chow diet (CD) or on a High Fat Diet (HFD). (CD - CaMK1Dflox/flox n = 12; CD - NestinCre/+ n = 11 and CD - NestinCre/+; CaMK1Dflox/flox n = 10; HFD - CaMK1Dflox/flox n = 12; HFD - NestinCre/+ n = 12 HFD - NestinCre/+; CaMK1Dflox/flox n = 11). Cumulative food intake on (c) CD or on (d) HFD. (CD - CaMK1Dflox/flox n = 12; CD - NestinCre/+ n = 11 and CD - NestinCre/+; CaMK1Dflox/flox n = 10; HFD - CaMK1Dflox/flox n = 12; HFD - NestinCre/+ n = 12 HFD - NestinCre/+; CaMK1Dflox/flox n = 11) (*p ≤ 0.05). (e) Cumulative food intake. (CaMK1Dflox/flox n = 10; NestinCre/+ n = 12 and NestinCre/+; CaMK1Dflox/flox n = 8). (f) Cumulative food intake after ghrelin injection (30 µg / day) on a CD. (CaMK1Dflox/flox n = 7; NestinCre/+ n = 10 and NestinCre/+; CaMK1Dflox/flox n = 9). Data are presented as mean values +/− SEM. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. Statistical tests included two-way ANOVA bonferroni post hoc test (b,c,d,e,f).

Source data

Extended Data Fig. 5 Deletion of CaMK1D in AgRP neurons.

a, Verification of recombination in the CaMK1D locus in hypothalamus (Hyp), cortex (Crtx), liver, tail and white blood cells (WBC) from mice. b, Expression of CaMK1D mRNA in arcuate nucleus, cortex and liver from mice (n = 4 / group).Statistical tests included unpaired t-test (b). Data are presented as mean values +/− SEM.

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Extended Data Fig. 6 Deletion of CaMK1D in POMC neurons does not affect energy metabolism in mice.

a, Verification of recombination in the CaMK1D locus in hypothalamus (Hyp), cortex (Crtx), liver, tail and white blood cells (WBC) from mice. b, Body weight gain on a Chow diet (CD) or on a High Fat Diet (HFD). (CD - CaMK1Dflox/flox n = 11; CD -POMCCre/+ n = 8; CD -POMCCre/+; CaMK1Dflox/flox n = 8; HFD - CaMK1Dflox/flox n = 10; HFD -POMCCre/+ n = 10 and HFD -POMCCre/+; CaMK1Dflox/flox n = 13).c, Cumulative food intake on a CD. (CD - CaMK1Dflox/flox n = 10; CD -POMCCre/+ n = 8; CD -POMCCre/+; CaMK1Dflox/flox n = 8). d, Cumulative food intake on a HFD. (HFD - CaMK1Dflox/flox n = 10; HFD -POMCCre/+ n = 8; HFD -POMCCre/+; CaMK1Dflox/flox n = 10). e, Cumulative food intake. Food intake was determined 24 h after fasting. (CaMK1Dflox/flox n = 10; POMCCre/+ n = 8; POMCCre/+; CaMK1Dflox/flox n = 10). f, Cumulative food intake after ghrelin injection. (CaMK1Dflox/flox n = 8; POMCCre/+ n = 7; POMCCre/+; CaMK1Dflox/flox n = 8). Data are presented as mean values +/− SEM. ∗∗p < 0.01 and ∗∗∗p < 0.001. Statistical tests included two-way ANOVA bonferroni post hoc test (f).

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Vivot, K., Meszaros, G., Pangou, E. et al. CaMK1D signalling in AgRP neurons promotes ghrelin-mediated food intake. Nat Metab 5, 1045–1058 (2023). https://doi.org/10.1038/s42255-023-00814-x

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