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Functional screening and rational design of compounds targeting GPR132 to treat diabetes

Nature Metabolism volume 5, pages 1726–1746 (2023)Cite this article

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Abstract

Chronic inflammation due to islet-residing macrophages plays key roles in the development of type 2 diabetes mellitus. By systematically profiling intra-islet lipid–transmembrane receptor signalling in islet-resident macrophages, we identified endogenous 9(S)-hydroxy-10,12-octadecadienoic acid–G-protein-coupled receptor 132 (GPR132)–Gi signalling as a significant contributor to islet macrophage reprogramming and found that GPR132 deficiency in macrophages reversed metabolic disorders in mice fed a high-fat diet. The cryo-electron microscopy structures of GPR132 bound with two endogenous agonists, N‐palmitoylglycine and 9(S)-hydroxy-10,12-octadecadienoic acid, enabled us to rationally design both GPR132 agonists and antagonists with high potency and selectivity through stepwise translational approaches. We ultimately identified a selective GPR132 antagonist, NOX-6-18, that modulates macrophage reprogramming within pancreatic islets, decreases weight gain and enhances glucose metabolism in mice fed a high-fat diet. Our study not only illustrates that intra-islet lipid signalling contributes to islet macrophage reprogramming but also provides a broadly applicable strategy for the identification of important G-protein-coupled receptor targets in pathophysiological processes, followed by the rational design of therapeutic leads for refractory diseases such as diabetes.

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Fig. 1: Upregulation of the GPR132 signalling circuit during islet-resident macrophage reprogramming.
Fig. 2: Reversed islet inflammation and improved glucose homeostasis in Lyz2-cre+/−Gpr132fl/fl mice under HFD conditions.
Fig. 3: Cryo-EM structures of GPR132–Gi.
Fig. 4: Structural basis of the constitutive activity and NPGLY-induced activation of GPR132 and binding and activation of GPR132 by 9(S)-HODE.
Fig. 5: Structure-based development of a Gi-biased GPR132 agonist.
Fig. 6: Structural basis of binding and activation of GPR132 by NOX-6-7.
Fig. 7: Development of a selective GPR132 antagonist.
Fig. 8: Gpr132 antagonist improved glucose metabolism in HFD-fed mice.

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Data availability

Raw data are provided in the figures, extended data and tables. There is no restriction on data availability. The cryo-EM density maps for the Apo-GPR132–Gi, NPGLY–GPR132–Gi, 9(S)-HODE–GPR132–Gi and NOX-6-7–GPR132–Gi complexes have been deposited in the Electron Microscopy Data Bank under accession codes EMD-34948, EMD-34950, EMD-34951 and EMD-35044, respectively. The coordinates for the model of Apo-GPR132–Gi, NPGLY-GPR132–Gi, 9(S)-HODE–GPR132–Gi and NOX-6-7–GPR132–Gi complexes have been deposited in the PDB under accession numbers PDB 8HQE, PDB 8HQM, PDB 8HQN and PDB 8HVI, respectively. Source data are provided with this paper. All other data are available upon request to the corresponding authors. RNA-sequencing data have been deposited in the Gene Expression Omnibus under accession numbers GSE239708 and GSE239831.

References

  1. Taylor, R., Al-Mrabeh, A. & Sattar, N. Understanding the mechanisms of reversal of type 2 diabetes. Lancet Diabetes Endocrinol. 7, 726–736 (2019).

    Article  CAS  PubMed  Google Scholar 

  2. Siehler, J., Blochinger, A. K., Meier, M. & Lickert, H. Engineering islets from stem cells for advanced therapies of diabetes. Nat. Rev. Drug Discov. 20, 920–940 (2021).

    Article  CAS  PubMed  Google Scholar 

  3. Talchai, C., Xuan, S., Lin, H. V., Sussel, L. & Accili, D. Pancreatic beta cell dedifferentiation as a mechanism of diabetic beta cell failure. Cell 150, 1223–1234 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ying, W. et al. Expansion of islet-resident macrophages leads to inflammation affecting beta cell proliferation and function in obesity. Cell Metab. 29, 457–474 (2019).

    Article  CAS  PubMed  Google Scholar 

  5. Ying, W., Fu, W., Lee, Y. S. & Olefsky, J. M. The role of macrophages in obesity-associated islet inflammation and β-cell abnormalities. Nat. Rev. Endocrinol. 16, 81–90 (2020).

    Article  PubMed  Google Scholar 

  6. Cheng, J. et al. Autonomous sensing of the insulin peptide by an olfactory G protein-coupled receptor modulates glucose metabolism. Cell Metab. 34, 240–255 (2022).

    Article  CAS  PubMed  Google Scholar 

  7. Weitz, J. R. et al. Mouse pancreatic islet macrophages use locally released ATP to monitor beta cell activity. Diabetologia 61, 182–192 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Ferris, S. T. et al. The islet-resident macrophage is in an inflammatory state and senses microbial products in blood. J. Exp. Med. 214, 2369–2385 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Scarpellini, E. et al. International consensus on the diagnosis and management of dumping syndrome. Nat. Rev. Endocrinol. 16, 448–466 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Yan, J. J. et al. Therapeutic effects of lysophosphatidylcholine in experimental sepsis. Nat. Med. 10, 161–167 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Lahvic, J. L. et al. Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132. Proc. Natl Acad. Sci. USA 115, 9252–9257 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chen, X. et al. Mouse placenta fetal macrophages arise from endothelial cells outside the placenta. Dev. Cell 57, 2652–2660 (2022).

    Article  CAS  PubMed  Google Scholar 

  13. McCubbrey, A. L., Allison, K. C., Lee-Sherick, A. B., Jakubzick, C. V. & Janssen, W. J. Promoter specificity and efficacy in conditional and inducible transgenic targeting of lung macrophages. Front. Immunol. 8, 1618 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Bhattacharyya, A. et al. IL10 trains macrophage profibrotic function after lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 322, L495–l502 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ramanujam, D. et al. MicroRNA-21-dependent macrophage-to-fibroblast signaling determines the cardiac response to pressure overload. Circulation 143, 1513–1525 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ying, W. et al. Expansion of islet-resident macrophages leads to inflammation affecting β cell proliferation and function in obesity. Cell Metab. 29, 457–474 (2019).

    Article  CAS  PubMed  Google Scholar 

  17. Minhas, P. S. et al. Restoring metabolism of myeloid cells reverses cognitive decline in ageing. Nature 590, 122–128 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lin, P. & Ye, R. D. The lysophospholipid receptor G2A activates a specific combination of G proteins and promotes apoptosis. J. Biol. Chem. 278, 14379–14386 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Obinata, H., Hattori, T., Nakane, S., Tatei, K. & Izumi, T. Identification of 9-hydroxyoctadecadienoic acid and other oxidized free fatty acids as ligands of the G protein-coupled receptor G2A. J. Biol. Chem. 280, 40676–40683 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Hua, T. et al. Crystal structures of agonist-bound human cannabinoid receptor CB1. Nature 547, 468–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Morimoto, K. et al. Crystal structure of the endogenous agonist-bound prostanoid receptor EP3. Nat. Chem. Biol. 15, 8–10 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Yang, F. et al. Structural basis of GPBAR activation and bile acid recognition. Nature 587, 499–504 (2020).

    Article  CAS  PubMed  Google Scholar 

  23. Latorraca, N. R., Venkatakrishnan, A. J. & Dror, R. O. GPCR dynamics: structures in motion. Chem. Rev. 117, 139–155 (2017).

    Article  CAS  PubMed  Google Scholar 

  24. Bokoch, M. P. et al. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463, 108–112 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ping, Y. Q. et al. Structures of the glucocorticoid-bound adhesion receptor GPR97–Go complex. Nature 589, 620–626 (2021).

    Article  CAS  PubMed  Google Scholar 

  26. Xiao, P. et al. Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4. Nature 604, 771–778 (2022).

    Article  CAS  PubMed  Google Scholar 

  27. Yang, F. et al. Structure, function and pharmacology of human itch receptor complexes. Nature 600, 164–169 (2021).

    Article  CAS  PubMed  Google Scholar 

  28. Ichimura, A. et al. Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human. Nature 483, 350–354 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Chavakis, T., Alexaki, V. I. & Ferrante, A. W. Macrophage function in adipose tissue homeostasis and metabolic inflammation. Nat. Immunol. 24, 757–766 (2023).

    Article  CAS  PubMed  Google Scholar 

  30. Hammerich, L. & Tacke, F. Hepatic inflammatory responses in liver fibrosis. Nat. Rev. Gastroenterol. Hepatol. https://doi.org/10.1038/s41575-023-00807-x (2023).

    Article  PubMed  Google Scholar 

  31. Llovet, J. M. et al. Nonalcoholic steatohepatitis-related hepatocellular carcinoma: pathogenesis and treatment. Nat. Rev. Gastroenterol. Hepatol. 20, 487–503 (2023).

    Article  CAS  PubMed  Google Scholar 

  32. Lim, G. B. Diabetes: No benefit of canakinumab in diabetes prevention. Nat. Rev. Cardiol. 15, 256 (2018).

    PubMed  Google Scholar 

  33. Moran, A. et al. Interleukin-1 antagonism in type 1 diabetes of recent onset: two multicentre, randomised, double-blind, placebo-controlled trials. Lancet 381, 1905–1915 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Donath, M. Y. & Shoelson, S. E. Type 2 diabetes as an inflammatory disease. Nat. Rev. Immunol. 11, 98–107 (2011).

    Article  CAS  PubMed  Google Scholar 

  35. Diana, J. & Lehuen, A. Macrophages and β-cells are responsible for CXCR2-mediated neutrophil infiltration of the pancreas during autoimmune diabetes. EMBO Mol. Med. 6, 1090–1104 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Donath, M. Y. Targeting inflammation in the treatment of type 2 diabetes: time to start. Nat. Rev. Drug Discov. 13, 465–476 (2014).

    Article  CAS  PubMed  Google Scholar 

  37. Hanefeld, M. et al. Orally-administered chemokine receptor CCR2 antagonist CCX140-B in type 2 diabetes: a pilot double-blind, randomized clinical trial. J. Diabetes Metab. 3, 1–8 (2012).

  38. Giraldez, M. D., Carneros, D., Garbers, C., Rose-John, S. & Bustos, M. New insights into IL-6 family cytokines in metabolism, hepatology and gastroenterology. Nat. Rev. Gastroenterol. Hepatol. 18, 787–803 (2021).

    Article  CAS  PubMed  Google Scholar 

  39. Brown, A. J. H. et al. From structure to clinic: design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer’s disease. Cell 184, 5886–5901 (2021).

    Article  CAS  PubMed  Google Scholar 

  40. Manglik, A. et al. Structure-based discovery of opioid analgesics with reduced side effects. Nature 537, 185–190 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lyu, J. et al. Ultra-large library docking for discovering new chemotypes. Nature 566, 224–229 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Stein, R. M. et al. Virtual discovery of melatonin receptor ligands to modulate circadian rhythms. Nature 579, 609–614 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wang, S. et al. D4 dopamine receptor high-resolution structures enable the discovery of selective agonists. Science 358, 381–386 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Cao, D. et al. Structure-based discovery of nonhallucinogenic psychedelic analogs. Science 375, 403–411 (2022).

    Article  CAS  PubMed  Google Scholar 

  45. Du, Y. Q. et al. Endogenous lipid-GPR120 signaling modulates pancreatic islet homeostasis to different extents. Diabetes 71, 1454–1471 (2022).

    Article  CAS  PubMed  Google Scholar 

  46. Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).

    Article  CAS  PubMed  Google Scholar 

  47. Li, Q. et al. A cullin 4B-RING E3 ligase complex fine-tunes pancreatic delta cell paracrine interactions. J. Clin. Invest. 127, 2631–2646 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Zhang, D. L. et al. Gq activity- and beta-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility. Elife https://doi.org/10.7554/eLife.33432 (2018).

  49. Li, Q. et al. A cullin 4B-RING E3 ligase complex fine-tunes pancreatic δ cell paracrine interactions. J. Clin. Invest. 127, 2631–2646 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Olsen, R. H. J. et al. TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome. Nat. Chem. Biol. 16, 841–849 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Liu, C. H. et al. Arrestin-biased AT1R agonism induces acute catecholamine secretion through TRPC3 coupling. Nat. Commun. 8, 14335 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Ping, Y.-Q. et al. Structures of the glucocorticoid-bound adhesion receptor GPR97–Go complex. Nature 589, 620–626 (2021).

    Article  CAS  PubMed  Google Scholar 

  53. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  PubMed  Google Scholar 

  55. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    Article  CAS  PubMed  Google Scholar 

  56. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  57. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  58. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Halgren, T. A. Identifying and characterizing binding sites and assessing druggability. J. Chem. Inf. Model. 49, 377–389 (2009).

    Article  CAS  PubMed  Google Scholar 

  60. Shelley, J. C. et al. Epik: a software program for pKa prediction and protonation state generation for drug-like molecules. J. Comput. Aided Mol. Des. 21, 681–691 (2007).

    Article  CAS  PubMed  Google Scholar 

  61. Harder, E. et al. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 12, 281–296 (2016).

    Article  CAS  PubMed  Google Scholar 

  62. Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).

    Article  CAS  PubMed  Google Scholar 

  63. Le Grand, S., Götz, A. W. & Walker, R. C. SPFP: Speed without compromise—a mixed precision model for GPU accelerated molecular dynamics simulations. Comput. Phys. Commun. 184, 374–380 (2013).

    Article  Google Scholar 

  64. Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Dickson, C. J. et al. Lipid14: the amber lipid force field. J. Chem. Theory Comput. 10, 865–879 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174 (2004).

    Article  CAS  PubMed  Google Scholar 

  67. Roe, D. R. & Cheatham, T. E. 3rd PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 9, 3084–3095 (2013).

    Article  CAS  PubMed  Google Scholar 

  68. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 27–38 (1996).

    Article  Google Scholar 

  69. Hou, T., Wang, J., Li, Y. & Wang, W. Assessing the performance of the MM/PBSA and MM/GBSA methods. 1. the accuracy of binding free energy calculations based on molecular dynamics simulations. J. Chem. Inf. Model. 51, 69–82 (2011).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Key R&D Program of China (2019YFA0904200 to J.-P.S.), a National Natural Science Foundation of China grant (82225011 to X.Y., 92057121 to X.Y., 32130055 to J.-P.S., 81825022 to J.-P.S., 91939301 to J.-P.S., 82071445 to Y.H., 22131002 to N.J., 22161142019 to N.J., 22293014 to N.J. and 81821004 to N.J.), the Major Fundamental Research Program of Natural Science Foundation of Shandong Province, China (ZR2021ZD18 to X.Y.), the Key Research Project of the Natural Science Foundation of Beijing, China (Z2000129 to J.-P.S.), the Cutting Edge Development Fund of Advanced Medical Research Institute (to J.-P.S. and J.C.), the Changping Laboratory and the New Cornerstone Science Foundation through the XPLORER PRIZE (to N.J.).

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Author notes

  1. These authors contributed equally: Jia-Le Wang, Xiao-Dong Dou, Jie Cheng, Ming-Xin Gao, Guo-Feng Xu, Wei Ding, Jin-Hui Ding.

Authors and Affiliations

  1. State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing, China

    Jia-Le Wang, Jin-Hui Ding, Yu Li, Xue-Ying Sha & Jin-Peng Sun

  2. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China

    Xiao-Dong Dou, Guo-Feng Xu, Xin-Yi Zhao, Tong-Yu Huo, Cai-Fang Zhang, Ya-Meng Liu & Ning Jiao

  3. Advanced Medical Research Institute, Cheeloo College of Medicine, Shandong University, Jinan, China

    Jie Cheng & Jin-Peng Sun

  4. Key Laboratory Experimental Teratology of the Ministry of Education and Department of Physiology, School of Basic Medical Sciences, Shandong University, Jinan, China

    Jie Cheng, Ming-Xin Gao, Si-Han Wang, Zhao-Wei Ji, Jia-Rui Gao, Wen-Hui Zhang & Xiao Yu

  5. Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China

    Wei Ding

  6. Changping Laboratory, Yard 28, Science Park Road, Chanaping District,, Beijing, China

    Ya-Meng Liu & Ning Jiao

  7. Department of Neurology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yong Hao

  8. The Key Laboratory of Cardiovascular Remodelling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital, Shandong University, Jinan, China

    Cheng Zhang

  9. NHC Key Laboratory of Otorhinolaryngology, Qilu hospital and advanced Medical Research Institute, Meili Lake Translational Research Park, Cheeloo College of Medicine, Shandong University, Jinan, China

    Jin-Peng Sun

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  1. Jia-Le Wang
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Contributions

X.Y., J.-P.S. and N.J. designed and supervised the overall project. X.Y. conceived the method to identify important signalling circuits within pancreatic islets contributing to macrophage reprogramming during diabetes and designed all functional assays. N.J. conceived all chemical design and development of ligands targeting GPR132. J.P.S. designed all pharmacology, cell signalling and structural studies. X.Y., J.-P.S., N.J., J.C., J.-L.W., M.-X.G., G.-F.X. and X.-D.D. participated in data analysis and interpretation. J.C. and M.-X.G. performed metabolic and histological studies of mice. J.C., M.-X.G., J.-R.G. and W.-H.Z. isolated mice islets, performed ex vivo experiments and performed a lipidomic analysis. J.C., M.-X.G. and S.-H.W. performed macrophage isolation, cytokine studies and RNA sequencing. J.C., M.-X.G. and Z.-W.J. performed IF and mice experiments. J.-L.W. generated the GPR132 insect cell expression construct, established the GPR132–Gi1–scFv16, 9(S)-HODE–GPR132–Gi1–scFv16, NPGLY–GPR132–Gi1–scFV16 and NOX-6-7–GPR132–Gi1–scFV16 complex purification protocol, and prepared samples for the cryo-EM with assistance from J.-H.D. and X.-Y.S. J.-L.W., Y.L. and J.-H.D. prepared the cryo-EM grids. J.-L.W. and Y.L. collected the cryo-EM images. W.D. and J.-L.W. performed the cryo-EM map calculation, model building and refinement. J.-P.S. and X.Y. designed the FlAsH-BRET assays and provided insightful ideas and experimental designs. M.-X.G., J.-H.D., J.-L.W. and J.C. performed the G-protein dissociation assay and FlAsH-BRET assay. N.J. and X.-D.D. provided the rational design ideas for GPR132 agonists and antagonists. X.-D.D. performed the binding pockets calculation, MD simulation, molecular docking and virtual screening. N.J., X.-D.D. and G.-F.X. designed the target compounds. G.-F.X., X.-Y.Z., T.-Y.H., C.-F.Z. and X.-D.D. synthesized all target compounds. N.J., G.-F.X., X.-Y.Z., T.-Y.H., C.-F.Z., X.-D.D. and Y.-M.L. performed analytical chemical analysis and wrote the lead compounds discovery parts. J.C., J.-L.W., M.-X.G., G.-F.X., X.-D.D. and J.-H.D. prepared the figures. J.-P.S. and X.Y. wrote the manuscript. N.J., Y.H. and C.Z. revised the manuscript. All the authors commented on the manuscript.

Corresponding authors

Correspondence to Jin-Peng Sun, Ning Jiao or Xiao Yu.

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Nature Metabolism thanks Ivet Bahar and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Identification of activation of 9(S)-HODE-GPR132 signaling during the reprogramming of islet-resident macrophages.

(a) Kyoto Encyclopedia of Genes and Genomes annotation of signaling pathways changes by whole transcriptome RNA-seq analysis of CD11c+ labeled islet cells in pancreatic islet isolated from HFD mice compared with NCD mice. Genes involved in signaling networks composed of cluster of ‘differentiation molecules’, ‘cytokine‒cytokine receptor interactions’ and ‘viral protein interaction with cytokine’ and ‘cytokine receptor’ were highly upregulated in CD11c+ labeled islet cells derived from islets of HFD-fed mice compared with macrophages of NCD-fed mice. (b) Gating strategy to identify CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from NCD-fed mice, according to indicated markers. We used BD Horizon™ Fixable Viability Stain 450 (FVS450) in the flow cytometry experiment to ensure the viability of isolated pancreatic islet cells. The expression of marker genes in isolated macrophages, including Mertk and CD64, was assessed by qRT‒PCR. Flow cytometric analysis of the viable pancreatic islet cells from NCD mice (left), and the expression of CD11c and Ly6C in the viable pancreatic islet cells isolated from the age-matched NCD-fed mice (middle), and the fractionations of islet resident macrophages based on their expression of CD11c+Ly6C− and F4/80 and analyzed by flow cytometry (right). Shown are the percentages of each subset in islet single cells isolated from the age-matched NCD mice. (c) mRNA level of Mertk and CD64 in CD11c+Ly6C−F4/80low cells by flow cytometry in NCD mice. Approximately 5*104 CD11c+Ly6C−F4/80low cells isolated from 15–20 NCD-fed mice were grouped (5*104 islet resident macrophages/group), Data are from three independent experiments (n = 3). (from left to right, P < 0.0001, P < 0.0001). (d) Gating strategy to identify CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from HFD-fed mice, according to indicated markers. Flow cytometric analysis of the viable pancreatic islet cells from HFD-fed (with HFD for 16 weeks) mice (left), and the expression of CD11c and Ly6C in the viable pancreatic islet cells isolated from the age-matched HFD-fed (with HFD for 16 weeks) mice (middle), and the fractionations of islet resident macrophages based on their expression of CD11c+Ly6C− and F4/80 and analyzed by flow cytometry (right). Shown are the percentages of each subset in islet single cells isolated from the age-matched HFD (with HFD for 16 weeks) mice. (e) Relative mRNA level of Mertk and CD64 in CD11C+Ly6c−F4/80low cells by flow cytometry in HFD (with HFD for 16 weeks) mice. Approximately 5*104 CD11C+Ly6c−F4/80low cells isolated from 8–10 HFD-fed mice were grouped (5*104 islet single cells/group), Data are from three independent experiments (n = 3). (from left to right, P < 0.0001, P < 0.0001). (f) Secretion levels of the cytokines IL-1β, TNF-α, CCL2 and CXCL1 into the culture supernatant of pancreatic CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from HFD-fed or NCD-fed mice. IL-1β has been proposed to be an important marker of inflamed pancreatic islets and has been shown to induce β-cell apoptosis and inhibit glucose-stimulated insulin secretion (GSIS). Whereas activation of NF-κB-inducing kinase (NIK) by TNF-α causes β cell failure in diet-induced obesity and T2DM, Ccl2 promotes macrophage proliferation and inflammation within pancreatic islet tissue. Moreover, CXCL1 recruits CXCR2-expressing neutrophils from the blood to pancreatic islets, a process that has been suggested to be important in autoimmune type 1 diabetes mellitus (T1DM) development. These three markers might play important roles in the reduction of diabetes progression. A total of 3*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 4–6 HFD- or 12–15 NCD-fed mice were grouped into each well of a 96-well plate, incubated with 100 μl of DMEM for 12 hours, and subjected to IL-1β, TNF-α, CCL2 and CXCL1 abundance measurement for an independent experiment. Data are from three independent experiments (n = 3). (from left to right, P = 0.0001, P = 0.0005, P < 0.0001, P = 0.0033). (g) Relative mRNA level of Il-1β, Tnf-α, Ccl2 and Cxcl1 in mouse CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from HFD mice or NCD mice. 1*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 4–6 NCD-fed mice or 2-3 HFD-fed mice were grouped (1*104 islet-resident macrophages/group), Data are from three independent experiments (n = 3). (from left to right, P < 0.0001, P < 0.0001, P = 0.0002, P = 0.0025). (h) Importantly, many lipids with significant differences in the supernatant/lysates derived from HFD-fed mice and NCD-fed mice are known agonists of lipid-sensing GPCRs, such as free fatty acid receptor (FFAR) 1–4 and GPR132. We therefore examined the mRNA levels of seven FA-related GPCRs in pancreatic islet-resident macrophages of HFD-fed and NCD mice. Relative mRNA levels of fatty acids sensing GPCRs in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages or islet isolated from WT mice, using mRNA levels of these GPCRs in whole islet as a control. 1*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 4–6 WT mice were grouped (1*104 islet-resident macrophages/group), Data are from three independent experiments. (n = 3). (from left to right, P = 0.0309, P < 0.0001, P = 0.0340, P = 0.0104, P = 0.9825, P = 0.5623, P < 0.0001). (i) Relative mRNA levels of Il-1β, Tnf-α, Ccl2 and Cxcl1 in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice in response to stimulations with 1 μM 9(S)-HODE, 11-HETE, ARA DHA or vehicle for 24h. Fold changes represented the fold of mRNA level of indicated genes which were normalized to CD11c+Ly6C−F4/80low-labeled islet-resident macrophages treated with vehicle. 5*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 15–20 WT mice were grouped (1*104 islet-resident macrophages/group), Data are from three independent experiments. (n = 3). (j, k) For a detailed analysis, we examined the proportion of GPR132+ islet macrophages in HFD-fed mice at different ages during obese development (4 weeks, 10 weeks and 16 weeks mice) by immunofluorescence (IF). We used BD Horizon™ Fixable Viability Stain 450 (FVS450) in the flow cytometry experiment to ensure the viability of isolated pancreatic islet cells. The immunofluorescence (A) and proportion (B) of GPR132+ cells (red) to CD11c+ labeled islet cells (green) were analyzed by immunofluorescence staining in NCD or 4 weeks, 10 weeks and 16 weeks HFD. Proportion of GPR132+ cells to CD11c+ labeled islet cells of mice fed a NCD or 4 weeks, 10 weeks and 16 weeks HFD. The percentage was normalized to CD11c+ labeled islet cells. The results indicated that the proportion of GPR132+ islet cells normalized to the number of CD11c+-labeled islet cells increased during obesity development, ranging from 15.3 ± 0.2% to 27.5 ± 0.5%. Scale bar: 50 μm. n = 6 mice; each mouse was used for slicing and counted as an independent experiment; 3–5 random areas were selected from each islet section, and 6–8 sections were randomly selected from each mouse. (from left to right, K, P < 0.0001, P < 0.0001, P < 0.0001). (l) The expression of Gpr132 in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages in NCD-fed mice or HFD-fed mice (4 weeks, 10 weeks, 16 weeks) were examined by single cell RT-PCR. Approximately 1*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 4–6 NCD-fed mice or 2-3 HFD-fed mice were grouped (1*104 CD11c+Ly6C−F4/80low macrophages/group). 12 individual cells were randomly selected from each group and then conducted with single cell RT-PCR. Agrose electrophoresis gel of RT-PCR product showing correct molecular weight. (m) The proportion of Gpr132+ cells in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages were analyzed by single cell RT-PCR of mice fed with NCD or different week’s HFD (4 weeks, 10 weeks, 16 weeks). The single-cell PCR results were similar to the IF staining results, which showed percentages of CD11c+Ly6C−F4/80lowGPR132+ islet macrophages vs. CD11c+Ly6C−F4/80low islet macrophages were 11.1 ± 1.8% (NCD), 15.3 ± 1.4% (4-week HFD), 22.2 ± 2.8% (10-week HFD) and 29.2 ± 1.9% (16-week HFD), respectively. Data are from six independent experiments. (n = 6). (from left to right, P = 0.0924, P = 0.0070, P < 0.0001). (C, E) *** < 0.001, CD11c+Ly6C−F4/80low cells compared with CD11c− cells in NCD (B) and HFD (D) mice. (F-G) **P < 0.01, ***P < 0.001; CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from HFD-fed mice compared with those isolated from NCD-fed mice. (H) *P < 0.05; ***P < 0.001, ns, no significant difference; CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice compared with pancreatic islets. (K) ***P < 0.001; Percentage of CD11c+GPR132+-labeled islet single cells in HFD-fed mice (4 weeks, 10 weeks, 16 weeks) compared with these in NCD-fed mice. (M) **P < 0.01; ***P < 0.001; ns, no significant difference; CD11c+Ly6C−F4/80low Gpr132+-labeled islet-resident macrophages in HFD-fed mice (4 weeks, 10 weeks, 16 weeks) compared with these in NCD-fed mice. The bars indicate the mean ± SEM values. Data were statistically analyzed using one-way ANOVA with Dunnett’s post hoc test (K, M) or two-way ANOVA with Dunnett’s post hoc test (C, E-H).

Extended Data Fig. 2 Strategy for Gpr132 deficiency in pancreatic islet macrophages.

(a) Representative blots (left) and quantification (right) showing the expression of 12-LOX in islet from NCD mice or HFD mice. Consistent with the increase in the level of total 9(S)-HODE, the level of 12-LOX, an enzyme that converts linoleic acid (LA) to 9(S)-HODE, was also significantly increased in pancreatic islet cells under HFD conditions compared with NCD conditions. 600 islets from 8–12 NCD-fed mice or 4–6 HFD-fed mice were grouped (200 islets/group). Data from three independent experiments (n = 3). (P = 0.0010). (b) Generation of conditional knockout mice of GPR132 and specifically ablated GPR132 in macrophage using Cre-LoxP recombination system. The single exon of GPR132 is deleted using Lyz2-Cre-mediated recombination. (c) Schematic description of the PCR strategy for the genotyping of the conditional knockout mice targeting to Lyz2-cre and Gpr132fl/fl, respectively. (d) Relative mRNA levels of Il-1β, Tnf-α, Ccl2 and Cxcl1 in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from GPR132fl/fl mice or Lyz2-cre+/−Gpr132fl/fl mice in response to stimulations with the SN or lysate from HFD mice islets. The supernatants were collected from HFD mice islets after stimulation with 20 mM glucose for 1h in mKRBB solutions. Approximately 6*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 18–24 NCD-fed mice (1*104 CD11c+Ly6C−F4/80low cells/group), Data are from three independent experiments (n = 3). (from left to right, P = 0.0025, P = 0.0047, P = 0.0002, P = 0.0003, P = 0.0105, P = 0.0252, P = 0.0002, P = 0.0013, P = 0.0003, P = 0.0028, P = 0.0002, P = 0.0022, P = 0.0018, P = 0.0040, P < 0.0001, P < 0.0001). (e, f) Plasma glucose (E) and insulin (F) levels in Gpr132fl/fl or Lyz2-cre+/− Gpr132fl/fl mice fed with NCD or HFD for 12 weeks fasted for 16 hours. (n = 6). (from left to right, E, P = 0.9091, P < 0.0001; F, P = 0.5582, P < 0.0001). (g, h) Plasma glucose (G) and insulin levels (H) of the Gpr132fl/fl or Lyz2-cre+/− Gpr132fl/fl mice fed with NCD or HFD for 12 weeks (4h after refeeding) (n = 6). (from left to right, G, P = 0.6290, P = 0.0003; H, P = 0.8824, P = 0.0054). (i) The expression of Gpr132 and Cx3cr1 in CD11c+-labeled islet cells was examined by single-cell RT‒PCR. The results indicated that all GPR132+ and CD11c+ cells expressed Cx3cr1. Approximately 1*104 CD11c+-labeled islet cells isolated from 4–6 NCD-fed mice were grouped (1*104 CD11c+-labeled islet cells /group). Twelve individual cells were randomly selected from each group and then subjected to single-cell RT‒PCR. Agarose electrophoresis gel of the RT‒PCR product showing the correct molecular weights of Gpr132 (217 bp), Cd11c (185 bp) and Cx3cr1 (125 bp). (j) We constructed a Cx3cr1-cre adeno-associated virus (AAV-Cx3cr1-cre) to specifically knock down the expression of GPR132 in Cx3cr1+ macrophages by infecting the Gpr132fl/fl mice with AAV-Cx3cr1-cre. Representative immunostaining of GFP (green) cells and CX3CR1 (red) cells in pancreatic sections from Gpr132fl/fl mice infected with pAAV-Cx3cr1 promoter-Cre-P2A-eGFP (AAV-Cx3cr1Cre). Scale bar: 50 μm. n = 6 mice; each mouse was used for slicing and counted as an independent experiment; 4–7 random areas were selected from each section, and 10 sections were randomly selected from each mouse. (k, l) Representative western blots (K) and Quantification (L) showing the protein levels of GPR132 in pancreatic islets isolated from Gpr132fl/fl mice infected with pAAV-Cx3cr1 promoter-Cre-P2A-eGFP (AAV-Cx3cr1Cre) or pAAV-Cx3cr1 promoter-eGFP (AAV-Cx3cr1Con). Infection of pancreatic islets derived from Gpr132fl/fl mice by AAV-Cx3cr1-cre showed a more than 80% reduction in Gpr132 expression in islets. Approximately 600 islets isolated from 8–12 Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre were grouped (200 islets/group). Data are from three independent experiments (n = 3). (L, P = 0.0018). (m) Relative mRNA levels of Il-1β, Tnf-α, Ccl2 and Cxcl1 in Cx3cr1+-labeled islet cells isolated from Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre treated with 1 μM 9(S)-HODE or Vehicle for 24 h. The increased mRNA levels of Il-1, Tnf-α, Ccl-2 and Cxcl1 induced by 9(S)-HODE were significantly reduced in GPR132 downregulated Cx3cr1+ islet macrophages in AAV-Cx3cr1-cre Gpr132fl/fl mice. 2*104 Cx3cr1+-labeled islet cells isolated from 4–6 Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre were grouped (1* Cx3cr1+-labeled islet cells / group) stimulated with 1 μM 9(S)-HODE or vehicle, and subjected for RNA extraction for an independent experiment. The data are from three independent experiments (n = 3). (from left to right, P = 0.8032, P < 0.0001, P = 0.0004, P = 0.4189, P = 0.0006, P = 0.0007, P = 0.7717, P = 0.0002, P = 0.0003). (n, o) The epididymal fat mass (N) and the liver mass (O) of Gpr132fl/fl or Lyz2-cre+/−Gpr132fl/fl mice fed with NCD or HFD for 12 weeks (n = 6). (from left to right, N, P = 0.8564, P < 0.0001; O, P = 0.9214, P = 0.0001). (p) Hepatic triglyceride content of Gpr132fl/fl or Lyz2-cre+/− Gpr132fl/fl mice fed with NCD or HFD for 12 weeks. The data are from six independent experiments (n = 6). (from left to right, P = 0.6916, P < 0.0003). (q) Food intake of wild-type mice, Lyz2-cre+/− mice, GPR132fl/fl mice and Lyz2-cre+/−GPR132fl/fl mice fed a NCD or HFD (n = 6). (from left to right, P = 0.9201, P = 0.5767, P = 0.6661, P = 0.7913, P = 0.7328, P = 0.5660). (B) **P < 0.01, protein level of 12-LOX of islets isolated from HFD mice compared with those isolated from NCD mice; (D) *P < 0.05; **P < 0.01; ***P < 0.001, ns, no significant difference; CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Gpr132fl/fl mice treated with islet lysate or SN from HFD mice islets compared with vehicle; #P < 0.05; ##P < 0.01; ###P < 0.001, ns, no significant difference; CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-cre+/−Gpr132fl/fl mice compared with those from Gpr132fl/fl mice. (E-H) ns, no significant difference; **P < 0.01; ***P < 0.001. Lyz2-cre+/− Gpr132fl/fl mice were compared with Gpr132fl/fl mice fed on the same diet. (L) **P < 0.01; the protein levels of GPR132 in pancreatic islets from Gpr132fl/fl mice infected with AAV-Cx3cr1Con compared with AAV-Cx3cr1Cre. (M) *** P < 0.001; Gpr132fl/fl mice injected with AAV-Cx3cr1Con treated with 1 μM 9(S)-HODE compared with vehicle. Ns, no significant; ###P < 0.001; Gpr132fl/fl mice infected with AAV-Cx3cr1Con compared with Gpr132fl/fl mice infected with AAV-Cx3cr1Cre. (N-P) ns, no significant difference; ***P < 0.001. Gpr132fl/fl mice and were compared with Lyz2-cre+/− Gpr132fl/fl mice fed on the same diet. (Q) Ns, no significant difference, for Lyz2-cre+/− mice, GPR132fl/fl mice and Lyz2-cre+/−GPR132fl/fl mice compared with wild-type mice. The bars indicate the mean ± SEM values. Data were statistically analyzed using one-way ANOVA with Dunnett’s post hoc test (A, L) or two-way ANOVA with Dunnett’s post hoc test (H and M-Q).

Extended Data Fig. 3 Reversed islet inflammation and improved glucose homeostasis in Cx3cr1-cre induced Gpr132 deficiency in Gpr132fl/fl mice under HFD condition.

(a) Body weight change curves of Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre at 6 weeks and fed with NCD or HFD from 8 weeks for 12–14 weeks (n = 6). (from left to righ, P = 0.8898, P = 0.2489, P = 0.0266, P = 0.0333, P = 0.0198, P = 0.0147, P = 0.0217, P = 0.0048, P = 0.0019, P = 0.0009, P = 0.0008, P = 0.0004, 0.0003; P = 0.9517, P = 0.9804, P = 0.9686, P = 0.9720, P = 0.9242, P = 0.9984, P = 0.9683, P = 0.9260, P = 0.9881, P = 0.8191,P = 0.9686, P = 0.8715, P = 0.8799). (b, c) Plasma glucose levels of Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre fed with a NCD or HFD for 12–14 weeks before a glucose tolerance test (B) and insulin tolerance test (C) (n = 6). AAV-Cx3cr1-cre Gpr132fl/fl mice phenocopied Lyz2-cre+/−GPR132fl/fl mice in terms of decreased body weight gain, improved glucose tolerance and insulin sensitivity under HFD stress compared. (from left to right, B, P = 0.0015, P = 0.0014, P = 0.0043, P = 0.0024, P = 0.0365; P = 0.8700, P = 0.0941, P = 0.6328, P = 0.2349, P = 0.7916; C, P = 0.0272, P = 0.3660, P = 0.0236, P = 0.0067, P = 0.0495; P = 0.2456, P = 0.1676, P = 0.3613, P = 0.7053, P = 0.8903). (d, e) Plasma glucose (D) and insulin (E) levels in Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre fed with a NCD or HFD for 12–14 weeks fasted for 16 hours. (n = 6). The fasting glucose and insulin levels were also reduced in HFD-fed AAV-Cx3cr1-cre Gpr132fl/fl mice. (from left to right, D, P = 0.7730, P < 0.0001; E, P = 0.8918, P < 0.0001). (f, g) Plasma glucose (F) and insulin levels (G) of the Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre fed with a NCD or HFD for 12–14 weeks (4h after refeeding) (n = 6). The plasma glucose level was decreased and the insulin level was increased after refeeding in AAV-Cx3cr1-cre Gpr132fl/fl mice compared with AAV- Cx3cr1-control-GFP Gpr132fl/fl mice. (from left to right, F, P = 0.8276, P = 0.0053; E, P = 0.4247, P < 0.0001). (h, i) Immunostaining (H) and quantification (I) of CD11c+ labeled islet cells (red) in pancreatic sections of Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre fed with a NCD or HFD for 12–14 weeks. Representative images are shown. The number of CD11c+ labeled islet cells was normalized to total the number of islet cells. Scale bar: 50 μm. n = 6 mice; each mouse was used for slicing and counted as an independent experiment; 4–7 random areas were selected from each section, and 10 sections were randomly selected from each mouse. (from left to right, I, P = 0.7829, P = 0.0008). (j) Heatmap showing inflammatory factors, including Il-1β, Ccl1, Ccl4, Ccl5, Cxcl2, Cxcl9 and Cxcl10, in pancreatic islets isolated from Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre fed with a NCD or HFD for 12–14 weeks. 100 islets isolated from 2-3 Gpr132fl/fl mice injected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre were grouped. The IF staining indicated that the number of CD11c+ labeled islet cells and islet inflammatory response were significantly reduced in Gpr132fl/fl mice infected with the AAV-Cx3cr1-cre compared with those infected with AAV-Cx3cr1-control-GFP. Fold changes represent the differences in the mRNA levels of the indicated genes, which were normalized to the expression after vehicle treatment. Data are from three independent experiments (n = 3). (k) Phagocytosis of Cx3cr1+-labeled islet cells isolated from Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre fed with a NCD or HFD for 12–14 weeks. 1*104 Cx3cr1+-labeled islet cells isolated from 4–6 Gpr132fl/fl mice fed with NCD or 2–4 Gpr132fl/fl mice fed with HFD infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre were subjected for the assay of phagocytosis for an independent experiment. Data are from six independent experiments (n = 6). (from left to right, P = 0.7380, P = 0.0038, P = 0.0173). (l) Relative mRNA levels of Il-1β, Tnf-α, Ccl2 and Cxcl1 in Cx3cr1+-labeled islet cells isolated from Gpr132fl/fl mice infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre fed with a NCD or HFD for 12–14 weeks. The increased phagocytic capacity and mRNA levels of reprogramming biomarkers including Il-1β, Tnf-α, Ccl-2 and Cxcl1 under HFD stress were decreased in Cx3cr1+ islet macrophages in Gpr132fl/fl mice infected with AAV-Cx3cr1-cre.1*104 Cx3cr1+-labeled islet cells isolated from 4–6 Gpr132fl/fl mice fed with NCD or Gpr132fl/fl mice fed with HFD were infected with AAV-Cx3cr1Con or AAV-Cx3cr1Cre subjected for RNA extraction for an independent experiment. Data are from three independent experiments (n = 3). (from left to right, P = 0.4307, P < 0.0001, P = 0.0003, P = 0.6833, P = 0.0004, P = 0.0040, P = 0.7082, P = 0.0001, P = 0.0022, P = 0.5653, P = 0.0003, P = 0.0004). (A-I) ns, no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001. Gpr132fl/fl mice infected with AAV-Cx3cr1Con compared with Gpr132fl/fl mice infected with AAV-Cx3cr1Cre. (K-L) **P < 0.01; *** P < 0.001; Gpr132fl/fl mice fed with NCD compared with fed with HFD. ns, no significant; #P < 0.05; ##P < 0.01; ### P < 0.001; Gpr132fl/fl mice infected with AAV-Cx3cr1Cre compared with Gpr132fl/fl mice infected with AAV-Cx3cr1Con. The bars indicate the mean ± SEM values. All data were statistically analyzed using two-way ANOVA with Dunnett’s post hoc test.

Extended Data Fig. 4 GPR132-Gi signaling contributed to the reprogramming of islet-resident macrophages and the GPR132 complexes samples preparation.

(a) Heatmap showing inflammatory factors, including Il-1β, Ccl1, Ccl4, Ccl5, Cxcl2, Cxcl9 and Cxcl10, in pancreatic islets isolated from WT mice and pretreated with or without 100 ng/ml PTX after simulation with 1 μM 9(S)-HODE or vehicle for 24 hours. Four hundred islets from 6–8 WT mice were grouped (100 islets/group) for an independent experiment. Fold changes represent the differences in the mRNA levels of the indicated genes, which were normalized to the expression after vehicle treatment. Data are from three independent experiments (n = 3). (b) Relative mRNA levels of Il-1β, Tnf-α, Ccl2 and Cxcl1 in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-Cre+/− mice infected with adenovirus containing the desired genes (PTXfl/fl or control vector) in response to stimulation with 1 μM 9(S)-HODE or vehicle for 24 hours. CV, control vector. 4*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 16–20 Lyz2-cre+/− mice were grouped (1*104 islet-resident macrophages/group), and subjected for RNA extraction for an independent experiment. Data are from three independent experiments (n = 3). (from left to right, P = 0.5648, P < 0.0001, P = 0.0001, P = 0.8490, P = 0.0004, P = 0.0085, P = 0.8298, P < 0.0001, P = 0.0001, P = 0.9610, P = 0.0002, P = 0.0009). (c) Phagocytosis of CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-Cre+/− mice infected with adenovirus containing the desired genes (PTXfl/fl or control vector) in response to stimulation with 1 μM 9(S)-HODE or vehicle for 24 hours. CV, control vector. 4*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 16–20 Lyz2-cre+/− mice were grouped (1*104 islet-resident macrophages/group), and subjected for the assay of phagocytosis for an independent experiment. Data are from six independent experiments (n = 6). (from left to right, P = 0.4485, P < 0.0001, P < 0.0001). (d) Representative western blots showing the expression of PTX in pancreatic islets isolated from Lyz2-cre+/− mice infected with adenovirus containing desired genes (PTXfl/fl or control vector). CV, control vector. 400 islets from 6–8 Lyz2-cre+/− mice infected with PTXfl/fl or CV were grouped (200 islets/group). Data are from three independent experiments (n = 3). (e) Relative mRNA levels of Il-1β, Tnf-α, Il-6 and Ccl2 in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice pretreated with or without 100 ng/ml PTX in response to stimulations with 1 μM 9(S)-HODE or vehicle for 24h. Approximately 4*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 12–16 Lyz2-cre+/− mice (1*104 CD11c+ Ly6C−F4/80low cells/group), Data are from three independent experiments (n = 3). (from left to right, P = 0.0004, P = 0.0004, P = 0.0017, P = 0.0140, P = 0.0012, P = 0.0057, P = 0.0001, P = 0.0004). (f) Phagocytosis of CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice pretreated with or without 100 ng/ml PTX in response to stimulations with 1 μM 9(S)-HODE or vehicle for 24h. Approximately 4*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 12–16 Lyz2-cre+/− mice (1*104 CD11c+ Ly6C−F4/80low cells/group), Data are from three independent experiments (n = 6). (from left to right, P < 0.0001, P < 0.0001). (g) Relative mRNA levels of Il-1β, Tnf-α, Ccl2 and Cxcl1 in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-Cre+/− mice and Lyz2-cre+/− GPR132fl/fl mice infected with adenovirus containing the desired genes (PTXfl/fl or control vector) in response to stimulation with 1 μM 9(S)-HODE or vehicle for 24 hours. CV, control vector. Approximately 4*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 12–16 NCD-fed of Lyz2-Cre+/− mice and Lyz2-cre+/− GPR132fl/fl mice (1*104 CD11c+ Ly6C−F4/80low cells/group), The data are from three independent experiments (n = 3). The inflammatory response of islet-resident macrophages treated with 9(S)-HODE was not significantly different between the group of Lyz2-Cre+/−GPR132fl/fl mice infected with control adenovirus and those infected with PTXfl/fl adenovirus. This result suggested that Gi/Go inhibition had no significant additional effect on CD11c+Ly6C−F4/80low-labeled islet-resident macrophages derived from mouse islets in response to 9(S)-HODE stimulation with GPR132 deficiency. Therefore, Gi/Go signaling downstream of 9(S)-HODE stimulation in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages was mainly dependent on GPR132 but not other Gi/Go-coupled receptors. We also noted that the mRNA levels of Il-1β, Ccl2 and Cxcl1 in macrophages derived from Lyz2-Cre+/−GPR132fl/fl mouse islets were significantly decreased compared to those in macrophages derived from Lyz2-Cre+/− PTXfl/fl mouse islets (PTX was transduced by adenovirus) in response to 9(S)-HODE stimulation, suggesting that signals other than Gi/Go signaling may contribute to 9(S)-HODE-induced inflammatory responses via GPR132. (from left to right, P = 0.0060, P = 0.0391, P = 0.0075, P = 0.1621, P = 0.0023, P = 0.0025, P = 0.0023, P = 0.9964, P = 0.0042, P = 0.0075, P = 0.0041, P = 0.8655, P = 0.0002, P = 0.0004, P = 0.0002, P = 0.5992). (h) Notably, the endogenous agonists of GPR132 can be classified into at least two types: oxidized lipids (9(S)-HODE, 11-HETE, etc.) and lipoylglycine compounds (such as N‐linoleoylglycine (NLGLY)). Dose response curves in HEK293 cells overexpressing GPR132 in response to stimulations with 9(S)-HODE, 11-HETE, NPGLY or NLGLY by Gαi-Gγ dissociation assay. Among the endogenous ligands tested, 9(S)-HODE showed the highest activity, and its potency was 5-fold greater than that of 11-HETE. Among the aliphatic glycine compounds tested, NPGLY, which had the highest activity, functioned as a partial agonist compared with 9(S)-HODE (Supplementary Table 2). Data are from three independent experiments (n = 3). (i) GPR132 was constitutively coupled with Gi1. Constitutive activities of GPR132, LYN and 5-HT1A measured by BRET change ratio at similar protein expression levels. The LYN is a negative control and the 5-HT1A is a positive control. Data are from four independent experiments (n = 4). (j, k) We co-expressed BRIL-GPR132 with the Gαi1/Gβ1/Gγ2 trimer and scFv16 in Spodoptera frugiperda insect cells (SF9 cells), which we then incubated with 9(S)-HODE or NPGLY, two typical endogenous agonists. Representative size-exclusion chromatography profile (left panel) and SDS-PAGE Coomassie blue staining of the peak fraction for purification (right panel) of the NPGLY-GPR132-Gi1 complex (J), or 9(S)-HODE-GPR132-Gi1 complex (K). (B-C) ***P < 0.001; CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-Cre+/− mice and treated with 1 μM 9(S)-HODE compared with those treated with vehicle. ##P < 0.01, ###P < 0.001; CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-Cre+/− mice that had been infected with adenovirus containing desired genes PTXfl/fl compared with control vector. (E-F) **P < 0.01, ***P < 0.001, CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice treated with 9(S)-HODE compared with those treated with vehicle. #P < 0.05; ##P < 0.01; ###P < 0.001, CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice pretreated with 100 ng/ml PTX compared with those treated with vehicle. (G) **P < 0.01, ***P < 0.001; CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-Cre+/− mice and treated with 1 μM 9(S)-HODE compared with those treated with vehicle. #P < 0.05; ##P < 0.01; ###P < 0.001; CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-Cre+/− mice or Lyz2-cre+/− GPR132 fl/fl mice that had been infected with adenovirus containing desired genes PTXfl/fl and CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-cre+/− GPR132 fl/fl mice compared with CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-Cre+/− mice. Ns, $$P < 0.01; CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from Lyz2-cre+/− GPR132 fl/fl mice compared with those infected with adenovirus containing desired genes PTXfl/fl. The bars indicate the mean ± SEM values. Data were statistically analyzed using two-way ANOVA with Dunnett’s post hoc test.

Extended Data Fig. 5 Cryo-EM data processing and Cryo-EM density map of GPR132-Gi1 complexes.

(a–d) The GPR132-Gi1 complexes were purified and then imaged using a Titan Krios microscope. The cryo-EM images and data processing of NPGLY-GPR132-Gi1 complex. Intriguingly, two distinct conformations of GPR132-Gi1-NPGLY were identified in the samples after 3D classification; one form was bound with NPGLY, and the other form was not NPGLY-bound. One of the observed conformations may be the structural state of the apo-GPR132-Gi1 complex. Cryo-EM micrograph (Scale bar: 50 nm) and reference-free two-dimensional class averages (Scale bar: 10 nm) of the NPGLY-GPR132-Gi1 complex (A). Workflow chart of cryo-EM data processing for the NPGLY-GPR132-Gi1 complex (B). After 2D and 3D classification, two distinct conformations of the GPR132-Gi1-NPGLY sample were identified after 3D classification, one (444,266 particles) with and one (363,528 particles) without NPGLY EM density (apo-GPR132-Gi1 complex). The Gold-standard Fourier shell correlation (FSC) curves showing an overall resolution at 2.95 Å and 2.97 Å for the NPGLY-GPR132-Gi1 complex (C) and Apo-GPR132-Gi1 (D). Cryo-EM map colored based on local resolution for NPGLY-GPR132-Gi1 complex (top right panel) and apo-GPR132-Gi1 complex (bottom right panel), respectively. (e–g) Cryo-EM density map of the Apo-GPR132-Gi1 complex (E), NPGLY-GPR132-Gi1 complex (F) and 9(S)-HODE-GPR132-Gi1 complex (G). NPGLY is shown in green, 9(S)-HODE in orange, GPR132 in purple (E) or blue (F-G), Gαi1 in yellow, Gβ in cyan, Gγ in pink and scFv16 in grey. (h–j) The ECL2 assumed different conformations in the apo state or between different endogenous agonist-bound states of GPR132, suggesting that the flexibility of ECL2 may be a key feature to accommodate diverse GPR132 ligands and may participate in receptor activation to different degrees. Top view of the cryo-EM density map of the apo-GPR132-Gi1 complex (H), NPGLY-GPR132-Gi1 complex (I) and 9(S)-HODE-GPR132-Gi1 complex (J). NPGLY is green, 9(S)-HODE is orange, GPR132 is purple (H) or blue (I-J), and extracellular loop 2 (ECL2) is red. The yellow rectangle indicates the orthosteric sites binding with endogenous ligands NPGLY and 9(S)-HODE.

Extended Data Fig. 6 GPR132 activation induced by ECL2.

(a, b) The entrance of the ligand pocket of the apo-GPR132-Gi1 (A) and the NPGLY-GPR132-Gi1 (B). The entrance of pocket is outlined, and the surrounding residues are indicated and shown as sticks. GPR132 in NPGLY-GPR132-Gi1 and apo-GPR132-Gi1 structure are shown in blue and grey, respectively. NPGLY in pale red, conserved residues in yellow and non-conserved residues in red. (c) GPR132 in NPGLY/9(S)-HODE-GPR132-Gi1 and Apo-GPR132-Gi1 structure are shown in blue and grey, respectively, NPGLY in pale red, 9(S)-HODE in orange. The GPR132 residues Q191ECL2 is shown as sticks. The red arrows indicate the movements of the secondary structures. (d) Comparison of the Gpr132 mRNA level in CD11c+ labeled islet cells isolated from NCD-fed or HFD-fed mice with those in CD11c+ labeled islet cells from WT mice infected with varying amounts of adenovirus encoding Gpr132 for 48 hours. CD11c+ labeled islet cells infected with 15 μl adenovirus showed similar expression levels of Gpr132 as that isolated from HFD-fed mice. The 15 μl adenovirus encoding GPR132 gene was thus selected for the following analysis of Gpr132 overexpression in CD11c+ labeled islet cells to mimic HFD conditions. ns, no significant difference. CD11c+ labeled islet cells from NCD-fed mice infected with 15 μl adenovirus were compared with CD11c+ labeled islet cells from HFD-fed mice. The bars represent mean ± SEM. Data statistics were analyzed using one-way ANOVA with Dunnett’s post hoc test. Data are from 3 independent experiments and were related to Fig. 5a. (n = 3). (e) Representative western images showing the expression of Flag-tagged GPR132 in CD11c+ labeled islet cells from mice infected with 15 μl adenovirus encoding control vector or GPR132 for 48 hours, and the GPR132 protein level in CD11c+ labeled islet cells isolated from NCD-fed or HFD-fed mice. (P = 0.8542). (f) Predicted ligand binding pockets of apo-GPR132-Gi1 complexes using SiteMap algorithm. The predicted binding pockets were filled with yellow balls. (g) Interactions between ECL2 and the propagating path of the 7TM bundle of receptor in apo-GPR132-Gi1 complex. The ECL2 is shown in cyan and the core of the 7TM bundle of receptor and key residues of 7TMs are shown in gray and the F2556.48 is shown in blue. The hydrogen bond is shown as blue dashed lines. (h) Effects of mutations in the extracellular domain of GPR132 on its basal activities. Relative differences between WT and its mutants at a relative expression level of 3 for GPR132 (refer to ELISA data on receptor’s cell surface expression level in Supplementary Fig. 8c) are shown as the mean ± SEM of three experiments (n = 3) performed in triplicate. (i) Effects of mutations in the propagating path of GPR132 on its basal activities. Mutations in the key sites involved in ECL2 interactions, such as H106ECL1, W108ECL1, C1153.25, and Y2877.32, or the hydrophobic packing chain residues connecting inserted ECL2 to the toggle switch residue F2556.48, including Y2005.39, L2626.55, and Y2686.51, significantly decreased the constitutive activity of GPR132. Relative differences between WT and its mutants at a relative expression level of 3 for GPR132 (refer to ELISA data on receptor’s cell surface expression level in Supplementary Fig. 8e) are shown as the mean ± SEM of three experiments (n = 3) performed in triplicate.

Extended Data Fig. 7 Comparison of activation mechanism among three GPR132-Gi1 complexes and design of Gi-biased GPR132 agonist.

(a) Chemical structures of NPGLY and 9(S)-HODE. The C3 of NPGLY and the C9, C10, C12 of 9(S)-HODE is colored red. Whereas NPGLY had an amide-carbonyl bond close to the carboxylic end, 9(S)-HODE contained a hydroxyl substitution at the C9 position and two unsaturated double bonds at the C10 and C12 positions. (b) The functional importance of the interacting NPGLY residues and residues along the propagating path connecting the NPGLY binding site to the toggle switch residue F2556.48 was validated by mutagenesis studies. Effects of mutations of key residues in the ligand-binding pocket and key residues in the propagation path along the ligand pocket to the cytoplasmic side of GPR132 after NPGLY stimulation. The color of the heatmap is based on the ΔpEC50 and Emax values (ΔpEC50 = pEC50 of mutant-pEC50 of wild type; pEC50, that is, -logEC50); nd, signal not detectable. (c) Comparison of TM6-TM7 between NPGLY-GPR132-Gi1 and the apo-GPR132-Gi1 structure. The binding of NPGLY induced inward movements of the extracellular ends of TM6 and TM7. The hydrophobic packing between NPGLY and Y2877.32 and F2696.62 contributed to structural changes in TM6 and TM7. GPR132 in the NPGLY-GPR132-Gi1 and apo-GPR132-Gi1 structures is shown in blue and gray, respectively, and NPGLY is shown in red. The GPR132 residues L2626.55, K2656.58, F2696.62 and Y2877.32 are shown as sticks. The red dashed arrows indicate the movements of the corresponding secondary structures. (d) Binding of 9(S)-HODE to the receptor promoted the outward movement of TM7 of GPR132, in contrast to the effects of NPGLY binding the GPR132-Gi complex. Comparison of TM6-7 between the apo-GPR132-Gi1 with the 9(S)-HODE-GPR132-Gi1 structure. GPR132 in 9(S)-HODE-GPR132-Gi1 and apo-GPR132-Gi1 structure are shown in blue and grey respectively, 9(S)-HODE in orange. The GPR132 residues Y2877.32, F2696.62 K2656.58are shown as sticks. The red arrows indicate the movements of the secondary structures. (e, f) Superimposition of 9(S)-HODE-GPR132-Gi1 (E) or NPGLY-GPR132-Gi1 (F) with apo-GPR132-Gi1 aligned at the ‘toggle switch’ and ‘PIF’ motif. GPR132 in 9(S)-HODE-GPR132-Gi1, NPGLY-GPR132-Gi1 and apo-GPR132-Gi1 complexes are shown in blue, cyan and gray respectively; the residues are shown as sticks. (g) 500ns MD simulation of NOX-6-1-GPR132-Gi complexes. RMSDs of the binding pocket residues (upper curve) and the compound NOX-6-1 (bottom curve) from triplicate 200 ns MD simulation. Values were calculated based on the initial complex state after equilibration (0 ns). The data are from three independent replica (n = 3). (h) Structural representation of interaction of NOX-6-1 with GPR132 according to MD simulation. The MD simulations was performed for 500ns.NOX-6-1 are represented by sticks, shown in magenta; the residues H106ECL1, T185ECL2, and Q191ECL2 are shown in yellow; GPR132 and the other residues that interact with NOX-6-1 are shown in blue; (i) The time evolution of interactions between the compound NOX-6-1 and key residues (H-bonds and water-mediated polar network) during three independent 500ns molecular dynamics simulation of NOX-6-1-GPR132 complex. Shaded regions (blue) show which residues interact with the ligand in each trajectory frame. (j) Synthetic route of target compounds NOX-6-1 to NOX-6-14.

Extended Data Fig. 8 Cryo-EM data processing of NOX-6-7-GPR132-Gi1 complex and effects of residues mutations of GPR132 in response to NOX-6-7 stimulation.

(a) BRET ratio changes curves in HEK293 cells overexpressing GPR132 in response to stimulations with NOX-6-7, NOX-6-1, dxd-190904-1, xgf-1-10 or NOX-6-13. Data are from three independent experiment (n = 3). (b) Relative mRNA levels of Il-1β, Tnf-α, Il-6 and Ccl2 in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice pretreated with or without 100 ng/ml PTX and in response to stimulations with 1 μM 9(S)-HODE, NOX-6-7 or vehicle for 24h. Approximately 6*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 18–24 WT mice (1*104 CD11c+ Ly6C−F4/80low cells/group), Data are from six independent experiments (n = 6). (from left to right, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001). (c) Phagocytosis of CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice pretreated with or without 100ng/ml PTX in response to stimulations with 1 μM 9(S)-HODE, NOX-6-7 or vehicle for 24h. Approximately 6*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 18–24 WT mice (1*104 CD11c+ Ly6C−F4/80low cells/group), Data are from six independent experiments (n = 6). (from left to right, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001). (d) Representative size-exclusion chromatography profile (left panel) and SDS-PAGE Coomassie blue staining of the peak fraction for purification (right panel) of the NOX-6-7-GPR132-Gi1 complex. (e, g) The cryo-EM images and data processing of NOX-6-7-GPR132-Gi1 complex. In total, 520250 particles were selected to construct EM density maps of the NOX-6-7-GPR132-Gi1 complex at an overall resolution of 3.0 Å. Cryo-EM micrograph (Scale bar: 50 nm) and reference-free two-dimensional class averages (Scale bar: 10 nm) of the NOX-6-7-GPR132-Gi1 complex (E). Workflow chart of cryo-EM data processing for the NOX-6-7-GPR132-Gi1 complex (F). The Gold-standard Fourier shell correlation (FSC) curves showing an overall resolution at 3.04Å for the NOX-6-7-GPR132-Gi1 complex (G). Cryo-EM map colored based on local resolution (in Extended Data Fig. 10f) for NOX-6-7-GPR132-Gi1 complex (right panel) (F). (h) Cryo-EM density map of the NOX-6-7-GPR132-Gi1 complex. NOX-6-7 is shown in blue, GPR132 in green, Gαi1 in yellow, Gβ in cyan, Gγ in pink and scFv16 in grey. (i) Effects of alanine scanning mutations of key residues in the ligand binding pocket of GPR132 in response to NOX-6-7 stimulation. The heatmap is colored according to the values of ΔpEC50 and Emax (ΔpEC50 = pEC50 of mutant-pEC50 of the wild type); nd, signal not detectable. (j) Effects of key residues allelic mutations that effect the ligand-binding pockets of GPR132 on other lipid-binding GPCRs in response to NOX-6-7 stimulation. The heatmap is colored according to the value of ΔpEC50 and Emax (ΔpEC50 = pEC50 of mutant-pEC50 of wild type); nd, signal not detectable. (B-C) ***P < 0.001, CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice treated with NOX-6-7 or 9(S)-HODE compared with those treated with vehicle. ###P < 0.001, CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice pretreated with 100 ng/ml PTX compared with those treated with vehicle in response to the stimulation with NOX-6-7. $$$P < 0.001, CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice pretreated with 100 ng/ml PTX compared with those treated with vehicle in response to the stimulation with 9(S)-HODE. The bars indicate the mean ± SEM values. Data were statistically analyzed using two-way ANOVA with Dunnett’s post hoc test.

Extended Data Fig. 9 FlAsH insertion site screening and FlAsH-BRET experiments.

(a) Schematic representation of the FlAsH-BRET assay design. The RLUC was inserted at the N-terminal of GPR132, and the FlAsH motif were inserted in the designated positions in the figure. In response to binding of agonists (9(S)-HODE and NOX-6-7) or antagonists (NOX-6-14 and NOX-6-18), the ECL1-S1/2 and the ECL2-S3 moved close to or away from the N-terminus of GPR132, respectively. (b) Detailed description of the FlAsH motif incorporation site at the extracellular loops of GPR132. FlAsH motifs are labeled in red. (c) Elisa experiments to determine the expression levels of the wild-type GPR132 and five FLAsH motif incorporated FLAsH-BRET sensors. Data from three independent experiment (n = 3). (d) The curve responses (left) and the maximal response (right) of five GPR132 FlAsH-BRET sensors in response to 9(S)-HODE stimulation Data are from three independent experiments (n = 3). (e) The dose response curves of five GPR132 FlAsH-BRET sensors in response to NOX-6-7 stimulation. Data are from three independent experiments (n = 3). (f) The NOX-6-14 induced dose-dependent antagonism of the 1 μM 9-HODE induced GPR132 activation via Gi dissociation assay in HEK293 cells overexpressing GPR132. Data are from three independent experiments (n = 3). (g) The dose response curves of five GPR132 FlAsH-BRET sensor in response to NOX-6-14 stimulation. Data are from three independent experiments (n = 3). (h) The dose response curves (left) and the maximal response (right) of five GPR132 FlAsH-BRET sensors in response to NOX-6-18 stimulation. Data are from three independent experiments (n = 3). (i) The representative conformations of NOX-6-14-GPR132 structures in 1 μs MD simulations. NOX-6-14 are represented by sticks; the start conformation colored by yellow; 1, 5, 50, 200, 800, 1000 ns conformations colored by blue. (j) Structural representation of interaction of NOX-6-14 with GPR132 according to MD simulation. The MD simulations was performed for 1μs. NOX-6-14 are represented by sticks, shown in blue; GPR132 and key residues that engaged charge-charge interaction and H-bond with NOX-6-14 are shown in magenta; the other residues are shown in gray. (k) Elisa experiments to determine the expression levels of the wild-type S2 and some S2 quick mutation. Data from three independent experiment (n = 3). (l) Effects of Allelic mutations to other lipid GPCRs of the key residues of GPR132 potentially contacting with NOX-6-18 according to MD stimulation results. ***P < 0.001; **P < 0.01; *P < 0.05; ND, no detectable signal; ns, no significant difference (comparison between the S2-GPR132 and its mutant). Values are shown as the mean±s.e.m. from three independent experiments performed in triplicate. And data were statistically analyzed using one-way ANOVA with Dunnett’s post hoc test.

Extended Data Fig. 10 Gpr132 antagonist improved glucose metabolism and islet homeostasis in HFD-fed male mice.

(a, b) Fasting plasma glucose (A) and insulin (B) levels in NCD-fed or HFD-fed Lyz2-cre+/−Gpr132fl/fl mice or Gpr132fl/fl mice treated with control vehicle or NOX-6-18 every other day (25ng/g, i.p.) for 12 weeks fasted for 16 hours. (n = 10). (from left to right, A, P = 0.1591, P = 0.3306, P < 0.0001, P = 0.4141; B, P = 0.8188, P = 0.6830, P < 0.0001, P = 0.9653). (c, d) Energy expenditure (C) and respiratory quotient (D) values in NCD mice or HFD mice treated with vehicle control or NOX-6-18 every other day (25ng/g, i.p.) for 12 weeks as measured by indirect calorimetry. (n = 6). (from left to right, C, P = 0.0384, P = 0.0107, D, P = 0.0647, P = 0.0050). (e) Heatmap showing inflammatory factors, including Il-1β, Ccl1, Ccl4, Ccl5, Cxcl2, Cxcl9 and Cxcl10 in islets isolated from NCD-fed or HFD-fed mice and pretreated with vehicle control or NOX-6-18 every other day (25 ng/g, i.p.) for four weeks. One hundred islets from 2-4 NCD-fed or 1–3 HFD-fed mice treated with vehicle control or NOX-6-18 were grouped (100 islets/group). Fold changes represent the changes in the mRNA levels of the indicated genes, which were normalized to the expression in the vehicle group. The data are from three independent experiments (n = 3). (f) Heatmap showing inflammatory factors, including Il-1β, Ccl1, Ccl4, Ccl5, Cxcl2, Cxcl9 and Cxcl10, in islets isolated from wild-type (WT) mice and pretreated with or without 1 μM NOX-6-18 in response to simulations with 1 μM 9(S)-HODE or vehicle for 24 hours. The inflammatory response of the islet cells to 9(S)-HODE treatment was significantly decreased in islet cells treated with the antagonist NOX-6-18. Four hundred islets from 6-8 WT mice were grouped (100 islets/group) for an independent experiment. Fold changes represent the changes in the mRNA levels of the indicated genes, which were normalized to the expression in the vehicle group. The data are from three independent experiments (n = 3). (g) Relative mRNA levels of Il-1β, Tnf-α, Ccl2 and Cxcl1 in CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice and pretreated with or without 1 μM NOX-6-18 in response to stimulation with 1 μM 9(S)-HODE or vehicle for 24 hours. 4*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 12–16 WT mice were grouped (1*104 CD11c+Ly6C−F4/80low macrophages /group), pretreated with or without 1 μM NOX-6-18 in response to stimulation with 1 μM 9(S)-HODE or vehicle for 24 hours, and subjected for RNA extraction for an independent experiment. The data are from three independent experiments (n = 3). (from left to right, P = 0.0005, P = 0.0002, P = 0.0001, P < 0.0001). (h) Phagocytosis of CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from WT mice and pretreated with or without 1 μM NOX-6-18 in response to stimulation with 1 μM 9(S)-HODE or vehicle for 24 hours. The extent of 9(S)-HODE-induced reprogramming of CD11c+Ly6C−F4/80low-labeled islet-resident macrophages and increased macrophage phagocytosis of isolated islet cells were significantly reduced in response to NOX-6-18 treatment4*104 CD11c+Ly6C−F4/80low-labeled islet-resident macrophages isolated from 12–16 WT mice were grouped (1*104 CD11c+Ly6C−F4/80low macrophages /group), pretreated with or without 1 μM NOX-6-18 in response to stimulation with 1 μM 9(S)-HODE or vehicle for 24 hours, and subjected for the assay of phagocytosis for an independent experiment. The data are from six independent experiments (n = 6). (from left to right, P < 0.0001, P < 0.0001). (a-b) ns, no significant difference; *P < 0.05; ***P < 0.001. NCD-fed or HFD-fed Lyz2-cre+/−Gpr132fl/fl mice or Gpr132fl/fl mice treated with NOX-6-18 compared with those treated with vehicle. (C-D, G) *P < 0.05; **P < 0.01; ***P < 0.001, HFD-fed mice or NCD-fed mice treated with NOX-6-18 (25 ng/g) for 12 weeks compared with those treated with vehicle. (H) ***P < 0.001, CD11c+Ly6C−F4/80low-labeled islet-resident macrophages of WT mice treated with 1 μM 9(S)-HODE compared with those treated with vehicle; ###P < 0.001, CD11c+Ly6C−F4/80low-labeled islet-resident macrophages pretreated with 1 μM NOX-6-18 compared with those treated with vehicle. The bars indicate the mean ± SEM values. The data were statistically analyzed using one-way ANOVA with Dunnett’s post hoc test.

Supplementary information

Supplementary Information

Supplementary Figures 1–23 and Supplementary Tables 1–14

Reporting Summary

Supplementary Data 1

NMR of compounds.

Supplementary Data 2

Synthesis of compounds.

Supplementary Data 3

Unprocessed gels and western blots for Supplementary Figs. 1–23.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8

Statistical source data.

Source Data Extended Data Fig./Table 1

Unprocessed gels.

Source Data Extended Data Fig./Table 2

Unprocessed gels and western blots.

Source Data Extended Data Fig./Table 4

Unprocessed western blots.

Source Data Extended Data Fig./Table 6

Unprocessed western blots.

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Wang, JL., Dou, XD., Cheng, J. et al. Functional screening and rational design of compounds targeting GPR132 to treat diabetes. Nat Metab 5, 1726–1746 (2023). https://doi.org/10.1038/s42255-023-00899-4

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