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5-IP7 is a GPCR messenger mediating neural control of synaptotagmin-dependent insulin exocytosis and glucose homeostasis

Abstract

5-diphosphoinositol pentakisphosphate (5-IP7) is a signalling metabolite linked to various cellular processes. How extracellular stimuli elicit 5-IP7 signalling remains unclear. Here we show that 5-IP7 in β cells mediates parasympathetic stimulation of synaptotagmin-7 (Syt7)-dependent insulin release. Mechanistically, vagal stimulation and activation of muscarinic acetylcholine receptors triggers Gαq–PLC–PKC−PKD-dependent signalling and activates IP6K1, the 5-IP7 synthase. Whereas both 5-IP7 and its precursor IP6 compete with PIP2 for binding to Syt7, Ca2+ selectively binds 5-IP7 with high affinity, freeing Syt7 to enable fusion of insulin-containing vesicles with the cell membrane. β-cell-specific IP6K1 deletion diminishes insulin secretion and glucose clearance elicited by muscarinic stimulation, whereas mice carrying a phosphorylation-mimicking, hyperactive IP6K1 mutant display augmented insulin release, congenital hyperinsulinaemia and obesity. These phenotypes are absent in mice lacking Syt7. Our study proposes a new conceptual framework for inositol pyrophosphate physiology in which 5-IP7 acts as a GPCR second messenger at the interface between peripheral nervous system and metabolic organs, transmitting Gq-coupled GPCR stimulation to unclamp Syt7-dependent, and perhaps other, exocytotic events.

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Fig. 1: The M3R−Gαq/11–PLC–PKC–PKD–IP6K1 signalling axis activates IP6K1 by S118/S121 phosphorylation.
Fig. 2: The phospho-mimic IP6K1DD mutant mice display insulin hypersecretion, whereas β-cell-specific IP6K1 deletion decreases insulin secretion.
Fig. 3: Neural regulation of insulin secretion requires the pancreatic M3R–PKC–IP6K1 phosphorylation axis.
Fig. 4: 5-IP7 does not affect insulin biogenesis or Ca2+ influx.
Fig. 5: 5-IP7 promotes Syt7-triggered insulin secretion.
Fig. 6: 5-IP7 clamps Syt7 in a Ca2+-releasable manner to regulate Syt7–PIP2 interaction.

Data availability

The atomic coordinates of the C2B domain of Syt-7 with IP6 were deposited to the Protein Data Bank under the accession codes PDB: 6LCY. All other data generated or analysed during this study are included in this published article (and its supplementary information files). Raw data and reagent requests should be addressed to F. Rao (raof@sustech.edu.cn). Source data are provided with this paper.

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Acknowledgements

We thank S. H. Snyder and F. Yu for generously sharing reagents and tools for this research, the Southern University of Science and Technology (SUSTech) Core Research Facilities and Peking University Laboratory Animal Center of Shenzhen Graduate School for technical assistance, and the Shanghai Synchrotron Radiation Facility beamline BL17U1 and BL19U1 for X-ray beam time. This work was supported by grants from the National Natural Science Foundation of China (31872798 and 91853129 to F.R.; 31670734 and 91953110, to C. W.), the Shenzhen Science and Technology Program (KQTD20200820113040070 to F.R.) and the Shenzhen Municipal Government (KQJSCX20180322152418316, JCYJ20170412153517422 and JCYJ20170817104311912 to F. R.), the Department of Science and Technology of Guangdong Province (2018A030313207, to F. R.), the Ministry of Science and Technology of the People’s Republic of China (2019YFA0508402, to C. W.), USTC Research Funds of Double First-Class Initiative (YD9100002006), the Fundamental Research Funds for the Central Universities (WK9100000029 and WK9100000013) and the Chinese Academy of Sciences Pioneer Hundred Talents Program (C. W.).

Author information

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Authors

Contributions

F. R., C. W., X. Z., J. Z., C. M., and C. H. W. designed research; X. Z., J. Z., Y. Z., Y. Y., Y. L., B. Z., Z.X., N. L., Xiuyan Yang, Xiaoli Yang, D. C., A. W., B. W., N. M., S. W., Z. Z., C. Y., D. Y., K.Z., B. L. and Z. K. performed research; F. R., C. W., C. H. W., C. M., Y. R. and W. Z. supervised research; N. J., Z. L., M. L., Q. W., Z. H., X. Q., G. X. and W. H. contributed new reagents and analytic tools; H. L., Y. D., Q. F., T.-N. Z. and F. R. analysed data; and F. R. wrote the draft of the paper with input from X. Z., N. L., J. Z. and C. W.

Corresponding authors

Correspondence to Chao Wang or Feng Rao.

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The authors declare no competing interests.

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Peer review information Nature Metabolism thanks Guy Rutter and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary handling editor: Christoph Schmitt.

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Extended data

Extended Data Fig. 1 PKC and PKD phosphorylates IP6K1 at S121 and S118, respectively.

(a) Phos-tag gel electrophoresis of immunoprecipitated IP6K1 reveals a slower migrating species that is absent under normal PAGE. (b-c) Mass-spectrum of immunoprecipitated IP6K1 reveals phosphorylation at S118/S121 (b) and S127 (c). (d) Sequence alignments of the three human IP6K isoforms. The poorly-conserved unstructured region is marked by red rectangles. (e) The disorder tendency of IP6K1 determined by IUPred (http://iupred.enzim.hu/). IDR: intrinsic disordered region. (f) Phosphorylation status of IP6K1 and its mutants examined with antibodies specifically recognizing phosphorylated PKC and PKD substrates. (g-h) In vitro phosphorylation of GST-IP6K1 purified from HEK293 cells by purified PKCβ (g) and PKD (h). GST-IP6K1 overexpressed in a 10-cm plate was pull-down, washed extensively and then incubated with commercial PKCβ and PKD.

Source data

Extended Data Fig. 2 Activation of IP6K1 by PKC/D-mediated phosphorylation and consequent conformational changes.

(a) The catalytic activity of IP6K1 with or without phosphorylation by PKC or PKD, assayed by the PAGE gel method, suggest that single kinase phosphorylation only modestly improves IP6K1 enzymatic activity. (b) The catalytic activities of the S118D/S121D (DD) phospho-mimic mutant is higher than wildtype IP6K1. (c) The catalytic activity of IP6K1 wildtype, S118D and S121D single mutants are comparable. (d) Limited tryptic proteolysis of unmodified or phosphorylated IP6K1 reveal slowed digest upon phosphorylation. (e) Circular dichroism analysis of WT (upper panel) or DD mutant (lower panel) IP6K1 with or without urea at the indicated concentrations. 3-4 M urea totally denatures WT but not DD mutant IP6K1. (f) Scheme depicting the proposed mechanism of IP6K1 activation by PKC phosphorylation.

Source data

Extended Data Fig. 3 A M3R-Gαq/11-PLC-nPKC-IP6K1 signaling axis revealed by a phospho-IP6K1 antibody specifically detecting p-S118/S121.

(a) The specificity of a rabbit monoclonal antibody (p-IP6K1) that specifically recognizes IP6K1 phosphorylated at S118/S121 is validated by using IP6K1 knockdown and knockout cells as well as male IP6K1 knockout mice. (b) Effect of in-lysate treatment with calf intestinal phosphatase (CIP, 1:100) on IP6K1-S118/S121 phosphorylation. An asterisk (*) indicates a nonspecific band. (c) The signal of the p-IP6K1 antibody is not affected by the neighboring S127A mutation. (d) Effect of in cell treatment with the PKC activator PMA (5 μM, 5 min) and the PKC inhibitor Go6983 (5 μM, 20 min) on IP6K1-S118/S121 phosphorylation. (e) Time-dependent analysis of IP6K1 S118/S121 phosphorylation upon carbachol (CCH) stimulation. (f) Effect of M3R antagonists Atropine (10 μM, 20 min) and J104129 (100 nM, 20 min) on carbachol stimulated IP6K1 phosphorylation. (g) Effect of overexpressing Gαq, Gα11, and their constitutively active Q209L mutants on carbachol stimulated IP6K1 phosphorylation. (h) Effect of Gαq and Gα11 knockdown on carbachol stimulated IP6K1 phosphorylation. An asterisk (*) indicates a nonspecific band. (i) Effect of histamine (50 μM) stimulation on IP6K1 S118/121 phosphorylation in HeLa cells. (j) Effect of Gαq/11 inhibition by YM-254890 (100 nM, 30 min), PLC inhibition by U73122 (10 μM, 30 min), PKC inhibition by Go6983 (5 μM, 30 min), and PKD inhibition CRT0066101 (2 μM, 30 min) on Histamine-stimulated (50 μM, 3 min) IP6K1 phosphorylation.

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Extended Data Fig. 4 The phosphor-mimic IP6K1DD mutant mice display insulin hypersecretion, whereas β cell-specific IP6K1 deletion decrease insulin secretion.

(a) levels of 5-IP7 and IP6 levels in the various organs of male WT and IP6K1DD (DD) mouse littermates. (b) Levels of IP6K1 protein in the pancreas of male WT and DD mutant mice. (c) Lelves of 5-IP7 and IP6 in male WT and DD mouse embryonic fibroblasts (MEF) (*p = 0.02). (d) Weight of the various adipose tissues from male WT and DD mice at 4-months old (*p = 0.01, **p < 0.01). Right panel: Representative photographs of the white adipose tissues (WAT). EWAT: epididymal WAT; RWAT: retroperitoneal WAT; IWAT: inguinal WAT. (e-h) O2 consumption (e), CO2 production (f), respiratory exchange ratio (g), energy expenditure (h) in male WT and DD mutant mice, measured using CLAMS cages for 48 h (n = 6 mice per group). (i-j) Levels of serum glycerol (i) and non-esterified fatty acids (j) in male WT and DD mice before and after fasting (*p < 0.05, **p = 0.006). (k) Glucose-induced insulin secretion with glucose applied by oral gavage. (2 g/Kg body weight) tolerance test (GTT) (*p < 0.05, **p = 0.007). (l-n) Body weight (l), levels of feeding and fasting glucose (m), and levels of feeding and fasting insulin (n) in female WT vs DD mice (*p < 0.05, **p = 0.009, ***p = 0.0006). (o) Intraperitoneal glucose-induced insulin secretion in female WT and DD mice (*p < 0.05). (p) Bodyweight and serum insulin levels of male WT and DD mice prior to and six weeks after streptozocin treatment (STZ, 50 mg/kg, 4 days) (**p = 0.004). (q) Bodyweight and serum insulin levels of male WT and DD mice prior to and six weeks after streptozocin treatment (STZ, 50 mg/kg, 4 days) (*p = 0.02, **p = 0.007). (r) Insulin secretion from pancreatic islets isolated from male WT and mice (*p = 0.009). (s) Western-blot demonstrating successful depletion of IP6K1 protein in male IP6K1fl/fl:MIP-CreERT mice upon tamoxifen induction (*p = 9.5 x 10-11). (t-u) Levels of feeding and fasting glucose (t) and feeding and fasting insulin (u) in male IP6K1fl/fl and IP6K1fl/fl:MIP-CreERT mice after tamoxifen-induced IP6K1 deletion. n.s.: not significant. For Extended Data Fig. 4b-d and i-u, data represent means ± SEM; n represents number of mice (each data point = 1 mouse). Two-tailed t-tests were used for statistical analysis.

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Extended Data Fig. 5 Glucose does not stimulate IP6K1 phosphorylation.

(a) Effect of glucose on IP6K1 phosphorylation in 293 and INS1 cells. An asterisk (*) indicates a nonspecific band. (b) Effect of glucose, applied via oral gavage or i.p. injection, on IP6K1 phosphorylation in pancreas. (c) Effect of IP6K1 knockdown on insulin secretion from INS1 cells at 2.8 mM and 16.8 mM glucose concentrations. For Extended Data Fig. 5c, data represent means ± SEM; n represents number of mice (each data point = 1 mouse). Two-tailed t-tests were used for statistical analysis.

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Extended Data Fig. 6 Cholinergic, but not adrenergic, regulation of insulin secretion requires the pancreatic M3R-PKC-IP6K1 phosphorylation axis.

(a) Time-dependent stimulation of IP6K1 S118/S121 phosphorylation by the muscarinic agonist Oxotremorine M (Oxo-M, 2.5 μM) in isolated mouse islets. (b) Carbachol-induced IP6K1 phosphorylation is reversed by pretreatment with the Gαq inhibitor YM254890 (100 nM, 15 min). (c) Isoprenaline (i.p., 750 μg/kg, 10 min)-induced insulin secretion is similar between tamoxifen-treated male IP6K1fl/fl and IP6K1fl/fl:MIP-CreERT mice. (d) Intracisternal injection of thyrotropin releasing hormone (TRH) (0.5 μg in 100 nL) application elicits IP6K1 phosphorylation in male mouse pancreas. For Extended Data Fig. 6c, data represent means ± SEM; n = 3 independent biological experiments. Two-tailed t-tests were used for statistical analysis.

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Extended Data Fig. 7 Muscarinic stimulation of human islets elicits IP6K1 phosphorylation and insulin secretion.

(a) Oxo-M treatment (2.5 μM, 10 min) of isolated human islets stimulates IP6K1 S118/121 phosphorylation, which can be blocked by Atropine pretreatment (10 μM, 15 min). (b) Effect of IP6K1 inhibition on Oxo-M-induced insulin secretion under low and high glucose conditions (***p = 0.0004, ****p = 1.7 x 10-6). (c) Effect of IP6K1 knockdown on Oxo-M-induced insulin secretion under low and high glucose conditions (**p = 0.001, ***p = 0.0005, ****p = 2 x 10-5). For Extended Data Fig. 7b-c, data represent means ± SEM; n = 4 independent biological experiments. Two-tailed t-tests were used for statistical analysis.

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Extended Data Fig. 8 IP6K1 does not affect insulin biogenesis.

(a-b) H&E staining-based morphometric analysis of pancreatic islets from tamoxifen-treated male IP6K1fl/fl and IP6K1fl/fl:MIP-CreERT mice at 3 months old. Number of islets per pancreas (a) and average islet area (b) were measured by examining 20 slides with every five slides in between, covering the whole pancreas (n = 4 mice). (c) Total insulin content of pancreatic islet from male IP6K1fl/fl or IP6K1fl/fl:MIP-CreERT mice measured using ELISA assay. (*p = 0.03, n = 5 randomly selected islets). (d) Average cytoplasmic Ca2+ response to depolarization induced by 5 μM Oxo-M, assayed with Fura2-AM (5 μM, 30 mins) on islets from tamoxifen-treated male IP6K1fl/fl and IP6K1fl/fl:MIP-CreERT mice. Images were acquired at 2 seconds intervals. n = 4 (IP6K1fl/fl) or 3 independent (IP6K1fl/fl:MIP-CreERT) biological experiments from mouse islets. For Extended Data Fig. 8a-d, data represent means ± SEM; n represents number of mice (each data point = 1 mouse). Two-tailed t-tests were used for statistical analysis. n.s.: not significant.

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Extended Data Fig. 9 5-IP7 does not affect calcium influx, but cooperates with Ca2+ to promote Syt7-dependent insulin secretion.

(a) Effect of IP6K1 knockdown on insulin secretion from INS1 cells at 5mM and 30 mM KCl concentrations (***p = 0.0006, ****p = 9 x 10-5). (b) Extracellular calcium (Ca2+o) removal greatly abolishes insulin secretion from male wildtype (WT) and IP6K1DD (DD) islets (**p = 0.001, *p = 0.048). (c) Deleting the juxtamembrane segment of Syt7 (aa 38-104) abolishes Flag-IP6K1 co-immunoprecipitation with HA-Syt7.

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Extended Data Fig. 10 5-IP7 clamps Syt7 in a Ca2+-releasable manner to regulate Syt7-PIP2 interaction.

(a) FRET assay measuring the binding of NBD-labelled Syt7 to Rhodamine-PE labelled liposome, in the presence of 0.5 mM MgCl2 and increasing concentrations of IP6 (middle panel) or 5-IP7 (right panel). In this experiment, 1 µM IANBD-Syt7, 100 µM (total lipids) liposome, 25 µM CaCl2, 0.5 mM MgCl2, and indicated concentrations of IP6/5-IP7 were mixed and measured in 1-cm cuvette at 25 °C. Data points are presented as means ± s.d. (n = 3, technical replicates). (b) Concentration-dependent effect of 5-IP7 and IP6 on the interaction between Ca2+ (2 μM) and Fura2 dye (2 μM).

Supplementary information

Supplementary Information

Supplementary Figure 1, Supplementary Tables 1 and 2 and Supplementary Protocol for 5-IP7 synthesis.

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Zhang, X., Li, N., Zhang, J. et al. 5-IP7 is a GPCR messenger mediating neural control of synaptotagmin-dependent insulin exocytosis and glucose homeostasis. Nat Metab 3, 1400–1414 (2021). https://doi.org/10.1038/s42255-021-00468-7

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