S Neda Mousavy Gharavy1, Bryn M Owen2, Steven J Millership1,3,4, Pauline Chabosseau1, Grazia Pizza1, Aida Martinez-Sanchez1, Emirhan Tasoez5, Eleni Georgiadou1, Ming Hu1, Nicholas H F Fine1, David A Jacobson6, Matthew T Dickerson6, Olof Idevall-Hagren7, Alex Montoya3, Holger Kramer3, Zenobia Mehta1, Dominic J Withers3,4, Nikolay Ninov5, Paul J Gadue8, Fabian L Cardenas-Diaz8, Céline Cruciani-Guglielmacci9, Christophe Magnan9, Mark Ibberson10, Isabelle Leclerc1, Marianne Voz11, Guy A Rutter12,13. 1. Section of Cell Biology and Functional Genomics, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital, London, UK. 2. Section of Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital, London, UK. 3. MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Campus, London, UK. 4. Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK. 5. DFG-Center for Regenerative Therapies, Technische Universität Dresden, Dresden, Germany. 6. Department of Molecular Physiology and Biophysics Vanderbilt University, Nashville, TN, USA. 7. Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. 8. Children's Hospital of Philadelphia, CTRB, Philadelphia, PA, USA. 9. Regulation of Glycemia by Central Nervous System, BFA, UMR 8251, CNRS Université de Paris, Paris, France. 10. Vital-IT Group, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland. 11. Laboratory of Zebrafish Development and Disease Models, University of Liège (ULg), Liège, Belgium. 12. Section of Cell Biology and Functional Genomics, Department of Metabolism, Digestion and Reproduction, Imperial College London, Hammersmith Hospital, London, UK. g.rutter@imperial.ac.uk. 13. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore. g.rutter@imperial.ac.uk.
Abstract
AIMS/HYPOTHESIS: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. METHODS: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. RESULTS: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. CONCLUSIONS/ INTERPRETATION: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. DATA AVAILABILITY: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.
AIMS/HYPOTHESIS: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. METHODS: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. RESULTS: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. CONCLUSIONS/ INTERPRETATION: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. DATA AVAILABILITY: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.
Entities:
Keywords:
C2CD4A/B; Follicle-stimulating hormone; Genome-wide association studies; Glucose homeostasis; Type 2 diabetes
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