Abstract
Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic β-cells causes overt diabetes in mice; thus, therapies that sensitize β-cells to insulin may protect patients with diabetes against β-cell failure1,2,3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse β-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir−/−) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir−/− mice showed an increase in the activation of INSR–IGF1R in Iir−/− pancreatic tissue, resulting in an increase in the proliferation and mass of β-cells. Similarly, inducible β-cell-specific Iir−/− knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR–IGF1R and increased proliferation of β-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR–IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR–IGF1R in β-cells. Together, our findings show that inceptor shields insulin-producing β-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR–IGF1R sensitization and diabetes therapy.
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Data availability
The microarray data have been deposited at the GEO with the accession code GSE144519. Source data are provided with this paper.
Change history
12 March 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41586-021-03347-z
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Acknowledgements
We thank M. Bakhti, E. Schlüssel, A. Böttcher and M. Catani for comments and discussions; R. Fimmen, J. Schultheiß, J. Beckenbauer and L. Appel for their technical support; J. Miyazaki for the MIN6 K8 and K20 clones; and R. Scharfman for the EndoC-βH1 cells. This work was supported by funds from the Helmholtz Future Topic ‘Aging and Metabolic programming’ (AMPro), the Helmholtz Society, Helmholtz Portfolio Theme ‘Metabolic Dysfunction and Common Disease’, the German Research Foundation and the German Center for Diabetes Research (DZD e.V.). F.F.F. was supported by a PhD fellowship from the Hans-Seidl-Stiftung e.V.
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A., C.J., F.F.F., K.W., S.H., F.G.v.H., A.R., S.S., S.B., J.S., J.W., L.O., A.M, A.B.-P., G.C., A.F. and M.G. performed experiments and analysed the data. M.S., M.I. and J.B. performed and analysed microarray experiments. C.A. and O.P. generated monovalent labelled insulin. Ü.C. provided all polyclonal antibodies against the complete inceptor ectodomain. R.F. generated all monoclonal anti-inceptor peptide antibodies. R.F. and Ü.C. generated monoclonal antibodies against the complete inceptor ectodomain and M.G. performed biochemical validation experiments thereof. T.D.M., M.M., Ü.C., M.H.T. and H.L. supervised the study and A., C.J., S.H. and H.L. prepared the manuscript. H.L. conceived and designed the study, supervised the work and secured funding.
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The Helmholtz Center Munich GmbH has filed a patent application “Novel IGFR-like receptor and uses thereof” (inventor: H.L.), which is pending (WO2017042242), covering the targeting of inceptor for diabetes therapy. Another Helmholtz Center Munich GmbH priority application is under preparation. No application number is yet available, but the title of the patent application will be “Novel IGFR-like 1 monoclonal antibodies and uses thereof”, covering the targeting of inceptor for various medical applications (inventors will be H.L., Ü.C. and M.G.).
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Peer review information Nature thanks Anil Bhushan, Eunhee Choi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Inceptor is expressed in all pancreatic lineages and inceptor knockout embryos show an increase in the proliferation of endocrine cells.
a, In situ hybridization showing mRNA expression of Iir at embryonic stage E14.5 (genepaint.org). b, Body weight at postnatal day P0 (data are mean ± s.e.m.; P = 0.62; Iir+/+, n = 23; Iir−/−, n = 11). Significance was calculated using a two-tailed, unpaired t-test. c, Serum glucagon levels after 2–5 h starvation at E19.5 (data are mean ± s.e.m.; P = 0.20, Iir+/+, n = 8; Iir−/−, n = 8). Significance was calculated using a two-tailed, unpaired t-test. d, α-cell area at E19.5 (data are mean ± s.e.m.; P = 0.7906, Iir+/+, n = 5; Iir−/−, n = 5 mice). Significance was calculated using a two-tailed, unpaired t-test. e, Confocal images of the expression of inceptor (green) in endocrine (insulin/glucagon, red/blue), exocrine (amylase, red) and ductal (SOX9, red) cells in the embryonic pancreas at E14.5, E16.5 and E18.5. Scale bars, 50 μm. f, Confocal images of proliferative (EdU+, green) endocrine (ChgA+, magenta) cells in Iir+/+ and Iir−/− pancreases at E16.5 (n = 3 mice). White arrowheads indicate EdU and ChgA co-positive cells. For quantification, see Fig. 1i. Scale bar, 50 μm. g, Kaplan–Meier curve for Iir+/+ (n = 6) and Iir−/− (n = 12) P0 pups after glucose administration. Log-rank (Mantel–Cox) test with Welch’s t-test was performed to compare survival curves (***P ≤ 0.001) h, Blood glucose levels during glucose tolerance test in P0 pups (Iir+/+, n = 6–12 each time point; Iir−/−, n = 4–12 each time point). Data are mean ± s.e.m.; *P ≤ 0.05. Significance was calculated by two-way ANOVA followed by Bonferroni’s multiple comparisons test. i, qPCR of selected genes for microarray validation. Significance was calculated from Iir+/+ and Iir−/− groups (n = 4 pups) using multiple t-test. Data are mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.01.
Extended Data Fig. 2 Iir gene structure and inceptor protein domains.
a–d, Schematic representation of the 5330417C22Rik (Iir) gene (a), predicted alternative, protein coding splice variants of the 5330417C22Rik (Iir) gene (b), protein domains predicted for the three transcripts of the 5330417C22Rik (Iir) gene (c) and various motifs found in the transmembrane domain and cytoplasmic tail of inceptor (d). Images were modified from ensemble.org.
Extended Data Fig. 3 Domain structure similarities of inceptor with INSR, IGF1R, CD-M6PR and CI-M6PR (IGF2R).
a, b, Amino acid alignment of the first (a) and second (b) predicted cysteine-rich domain (CRD) of inceptor (amino acids (aa) 272–400 and 574–660, respectively) with the CRD of INSR (aa 180–336) and IGF1R (aa 169–328). Cysteine residues of INSR and IGF1R conserved in inceptor are indicated in red boxes and non-conserved residues in blue boxes. c, Amino acid alignment of the M6PR binding domain predicted for inceptor (aa 654–857) with the CD-M6PR (aa 22–278). d, Amino acid alignment of the M6PR binding domain predicted for inceptor (aa 654–857) with the 15 repeats of the CI-M6PR (IGF2R). Red box indicates 13 aa with the highest similarities (uniprot.org). Amino acids 1897–1929 (repeat 13) are not shown. Amino acids are coloured according to their side chain’s chemical properties at pH 7.4: A, F, I, L, M, V, W: hydrophobic (cyan); N, Q, S, T: polar, uncharged (green); R, K: basic (red); C, D, E: acidic (magenta); G (orange), H, Y (blue), P (yellow). * indicates single, fully conserved residues. “:” and “.” indicate conservation of strong or weak groups according to the Gonnet PAM250 matrix (score >0.5 or ≤0.5, respectively).
Extended Data Fig. 4 Generation of Iir knockout MIN6 cell line, and production and validation of inceptor-specific mono- and polyclonal antibodies.
a, Affinity-purified human inceptor ectodomain from human embryonic kidney cells (HEK293F) showing inceptor protein (purified by size-exclusion chromatography) and its validation on SDS–PAGE. b, Schematic representation of the CRISPR–Cas9 targeting strategy for the generation of MIN6 Iir−/− cells. MIN6 cells were transfected followed by fluorescence-activated cell sorting of Venus-positive cells and colonies were picked for genotyping. Two sgRNAs were used to delete the start codon from exon 1. c, Schematic representation of inceptor domains and an indication of antibodies generated against either the extracellular domain or the cytoplasmic domain. d, Immunostaining in MIN6 Iir+/+ and Iir−/− cells using the mouse (31A11, 36D7), rat (14F1, 16F6) and rabbit (1374, 1692) anti-inceptor (green) antibodies. Scale bar, 10 μm. e, Immunostaining in pancreases from E19.5 Iir+/+ and Iir−/− embryos using rat (19A6) and rabbit (1374) anti-inceptor (green) antibodies. Scale bar, 50 μm f, Immunostaining using rat (16F6) and rabbit (1374) anti-inceptor (green) antibodies in adult mouse pancreas (6 months old). Scale bar, 50 μm. g, Validation of mouse, rat and rabbit anti-inceptor antibodies in MIN6 Iir+/+ and Iir−/− cells by western blot analysis.
Extended Data Fig. 5 Generation of inceptor full-body knockout and β-cell-specific conditional knockout mice.
a, Schematic representation of the targeted Iir allele for the generation of full-body knockout (Iir−/−) and CKO (MIP-CreERT+;Iirfl/FD) mice. b, c, Genotyping of full-body knockout (Iir+/+, Iir+/−, Iir−/−) (b), control and CKO mice (Iirflox/+ and Iirflox/FD) (c) in combination with MIP-CreERT+ and MIP-CreERT−). d, Mating scheme for the generation of CKO and control mice. To rule out the effects of the MIP-CreERT allele and tamoxifen, we used these two indicated F1 genotypes. e, f, Body weight (e) and α-cell mass (f) in control (n = 12 and 3, respectively) and CKO (n = 14 and 4, respectively) male mice, 4 weeks after tamoxifen injection. Data are mean ± s.e.m. No significant changes were observed. g, Experimental paradigm showing in vitro tamoxifen-induced gene deletion in isolated islets. Islets were isolated from 14-week old male CKO mice and induced with tamoxifen (1 μM) or vehicle (ethanol) for 24 h, followed by a 72-h wash period. EdU (10 μM) was added to the culture medium during the wash period to label replicating cells. Islets were then fixed and immunostained for insulin and EdU co-positive cells. For signalling assays, islets were induced with 100 nM insulin for 15 min. Immunostaining (left) showing in vitro deletion efficiency of Iir (green) in β-cells of male CKO mice. EtOH injection served as control. Islet area is indicated in white dashed outline. Scale bar, 50 μm. Immunostaining (right) showing the proliferation (EdU, green) in β-cells (insulin, magenta) of islets from CKO mice. Islets from 14-week-old male MIP-CreERT;Iirfl/FD mice were induced with either tamoxifen (1 μM) or ethanol to rule out the effect of MIP-CreERT on proliferation. Scale bar, 50 μm. h, Inceptor immunoreactivity in the hypothalamus at the level of the arcuate nucleus (left) and paraventricular nucleus (right) in wild-type and CKO mice. ARH, arcuate nucleus; VMH, ventromedial nucleus; DMH, dorsomedial nucleus; PVH, paraventricular nucleus; 3V = third ventricle. Scale bars, 200 μm. i, Immunostaining for maturation markers UCN3 and MAFA in pancreases from control and CKO mice. Scale bars, 50 μm (UCN3); 20 μm (MAFA).
Extended Data Fig. 6 Inceptor is located in the secretory pathway and is quickly internalized to the Golgi–ER–lysosome compartment.
a, Representative confocal images demonstrating colocalization of inceptor (green) with giantin, CM1, EEA1, GM130, ERGIC53 or LAMP1 (magenta) and quantified by Pearson correlation coefficient (n = 3; total numbers of cells: giantin, 300; CM1, 296; EEA1, 202; GM130, 350; ERGIC53, 257; LAMP1, 273). Scale bars, 10 μm. b–e, Experimental design (b) for the endocytosis assay of inceptor. Representative confocal images (c) and quantification by Pearson correlation coefficient (d) of the internalization of inceptor (green) from the plasma membrane within COP-vesicles (CM1, magenta) or to lysosomes (LAMP2, magenta) at different time points. Within 10–30 min, inceptor was also found to a higher extent in lysosomes and COP vesicles. (CM1, n = 4, 1,225 cells in total; LAMP2, n = 3, 766 cells in total). No antibody and pre-immune serum (e) served as control. Scale bars, 10 μm. f, Representative confocal images of the colocalization of inceptor–Venus (green) with endogenous inceptor, GM130, giantin, EEA1 or clathrin (magenta) quantified as Pearson’s correlation coefficient (n = 3; Gm130, 111 cells per n, 383 cells in total; giantin, 100 cells per n, 329 cells in total; EEA1, 107 cells per n, 332 cells in total; clathrin, 80 cells per n, 495 cells in total). Scale bars, 10 μm. g, Representative confocal images of the colocalization of inceptor(AP2*)–Venus (green) with GM130 or clathrin (magenta), quantified as Pearson’s correlation coefficient (n = 3; GM130, 63 cells per n, 220 cells in total; clathrin, 136 cells per n, 432 cells in total). Scale bars, 10 μm.
Extended Data Fig. 7 Inceptor directly interacts with pAP2M1 and regulates AP2-mediated endocytosis of INSR–IGF1R.
a, b, Immunostaining showing the effect of an inhibitor of clathrin-mediated endocytosis (Dynasore) on inceptor endocytosis. Inceptor–Venus-expressing cells were treated with 80 μM Dynasore in serum-free DMEM for 2 h before labelling with CellMask Deep Red and fixation. Analysis was performed by quantifying the ratio of inceptor–Venus (green) in the membrane (red) versus the intracellular region (n = 3 biologically independent experiments; data are mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Significance was calculated using an unpaired t-test. Scale bar, 10 μm. c–e, Interaction of endogenous inceptor and inceptor–Venus with pAP2M1 in MIN6 cells when co-immunoprecipitated using an anti-inceptor ectodomain antibody under different metabolic conditions. Mutation in AP2-binding motif in inceptor(AP2*)–Venus fails to interact with pAP2M1 subunit. Beads only and IgGs served as immunoprecipitation controls (n = 3 biologically independent experiments; data are mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Significance was calculated using two-way ANOVA followed by Bonferroni’s multiple comparisons test. f–i, Western blot analysis (f) and quantification (g–i) from MIN6 cells expressing endogenous inceptor, inceptor–Venus (wild-type) and inceptor(AP2*)–Venus under different metabolic conditions. (n = 3; biologically independent experiment; data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Significance was calculated using two-way ANOVA followed by Bonferroni’s multiple comparisons test.
Extended Data Fig. 8 Interaction of inceptor with INSR–IGF1R and reduced uptake of labelled insulin in Iir-knockout MIN6 cells and mouse islets.
a, b, Interaction of endogenous inceptor with INSR in wild-type mouse islets (5-month-old mice) when co-immunoprecipitated using an anti-INSR antibody under different metabolic conditions. Beads only and IgG served as immunoprecipitation controls. Box plots (box plot elements as in Fig. 4b) showing relative density (fold change) of proteins (n = 3 biologically independent samples; data are mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Significance was calculated using one-way ANOVA followed by Bonferroni’s multiple comparisons test. c, Proximity ligation assay for endogenous inceptor alone as well as together with INSR, IGF1R and pIGF1R in MIN6 Iir−/− cells. Scale bar, 50 μm. d, Immunostaining showing the uptake of insulin-546 by INSR–IGF1R at different time points in MIN6 Iir+/+ and Iir−/− cells. For quantification, see Fig. 4 e. Scale bar, 10 μm. e, f, Immunostaining showing the uptake of insulin-546 by INSR–IGF1R at different time points in dispersed islets from control and CKO mice treated with tamoxifen (1 μM for 24 h). Live-cell imaging was performed at different time points. Scale bar, 100 μm. Around 200 cells were quantified (data are mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Significance was calculated using two-way ANOVA followed by Bonferroni’s multiple comparisons test. g, Western blot images of the input for the surface biotinylation assay as shown in Fig. 4 h, i.
Extended Data Fig. 9 Effect of inceptor monoclonal antibody on human EndoC-βH1 cell line.
a, b, Western blot analysis and quantification from EndoC-βH1 cells under growth conditions treated with rat anti-inceptor monoclonal antibody and IgG2b (control) for 15 min at three different concentrations (n = 3 biologically independent experiments; data are mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Significance was calculated using a two-way ANOVA followed by Bonferroni’s multiple comparisons test. c, d, Surface biotinylation assay showing surface pools of inceptor, pINSR–IGF1R, INSR and IGF1R in EndoC-βH1 cells treated with rat anti-inceptor (1 μg ml−1) at different time points compared to IgG2b control. Quantification of the surface pools at different time points (n = 3 biologically independent experiments; data are mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Significance was calculated using a two-way ANOVA followed by Bonferroni’s multiple comparisons test.
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Ansarullah, Jain, C., Far, F.F. et al. Inceptor counteracts insulin signalling in β-cells to control glycaemia. Nature 590, 326–331 (2021). https://doi.org/10.1038/s41586-021-03225-8
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DOI: https://doi.org/10.1038/s41586-021-03225-8
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