Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic β-cells

Abstract

The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine α-amidating monooxygenase (PAM) are associated with both T2D risk and insulinogenic index. Here, we demonstrate that the T2D risk alleles impact negatively on overall PAM activity via defects in expression and catalytic function. PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in a human β-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in β-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.

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Fig. 1: Analysis of wild-type and variant PAM function and expression.
Fig. 2: PAM expression in human pancreatic islets and EndoC-βH1 cells.
Fig. 3: Effects of endogenous PAM on β-cell function.
Fig. 4: Analysis of β-cell ultrastructural features following PAM silencing.
Fig. 5: Effects of rs35658696 genotype status on insulin secretion measures from intact human islets.
Fig. 6: Effects of rs35658696 genotype status on exocytosis measurements in dispersed human islets.
Fig. 7: Effects of endogenous CgA on β-cell function.

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Acknowledgements

We acknowledge sharing of data from the GoT2D and T2D-GENES consortia before publication. We thank J. Galvanovskis (University of Oxford) for microscopy assistance, J. Lyon (Alberta Diabetes Institute IsletCore) for his work on human islet isolations, and J. Buteau and Y. Wang (both Alberta Diabetes Institute) for their assistance with imaging human pancreatic sections. We also thank the Human Organ Procurement and Exchange Program (Edmonton) and the Trillium Gift of Life Network (Toronto) and other organ procurement agencies for their efforts in obtaining human pancreata for research. A.L.G. is a Wellcome Trust Senior Fellow in Basic Biomedical Science. P.E.M. holds a 2016–2017 Killam Annual Professorship. M.I.M. is a Wellcome Senior Investigator. S.K.T. is a Radcliffe Department of Medicine Scholar. S.S. is funded by the Medical Research council (MC_ST_15019 [2015 DTG/DTA]). J.C. is funded by the Oxford – Medical Research Council Doctoral Training Partnership and the Nuffield Department of Clinical Medicine. This work was funded by the Wellcome Trust (095101 (A.L.G.), 200837 (A.L.G.), 098381 (M.I.M.), 106130 (A.L.G., M.I.M.), 203141 (M.I.M.), 090531 (P.R.)), Medical Research Council (MR/L020149/1) (M.I.M., A.L.G., P.R.), European Union Horizon 2020 Programme (T2D Systems) (A.L.G.), and National Institutes of Health (U01-DK105535; U01-DK085545) (M.I.M., A.L.G.). Human islet isolation and phenotyping was supported by funding from the Alberta Diabetes Foundation (P.E.M.F.). The research was funded by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (A.L.G., M.I.M., P.R.). The views expressed are those of the author(s) and not necessarily those of the NHS, NIHR or the Department of Health.

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S.K.T., A.R., B.H., and A.L.G. conceived the study. A.R., B.H., S.S., M.M.U., A. Barrett, C.J.G., N.L.B., P.R., and A.L.G. performed kinetic and cellular characterization of T2D-associated alleles. S.K.T., A.R., B.H., S.S., A.C., P.R., and A.L.G. performed the characterization of PAM knockdown in β-cells. S.K.T., X.-Q.D., A. Bautista, A.F.S., J.E.M.F., P.E.M., and A.L.G. performed characterization of primary human islets. A.J.P., M.I.M., and A.L.G. performed islet transcriptomics. J.C., M.I.M., and A.M. performed plasma proteomics. A.R., S.K.T., and A.L.G. wrote the manuscript. All authors approved the final draft of the manuscript.

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Correspondence to Anna L. Gloyn.

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Competing interests

S.K.T. is now an employee of Vertex Pharmaceuticals, and N.L.B. is now an employee of Novo Nordisk, although all experimental work was carried out under employment at the University of Oxford. A.L.G. has received research funding and honoraria from Novo Nordisk. M.I.M. serves on advisory panels for Pfizer, Novo Nordisk and Zoe Global; has received honoraria from Pfizer, Novo Nordisk and Eli Lilly; has stock options in Zoe Global; and has received research funding from AbbVie, Astra Zeneca, Boehringer Ingelheim, Eli Lilly, Janssen, Merck, Novo Nordisk, Pfizer, Roche, Sanofi Aventis, Servier and Takeda.

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Supplementary Figure 1 Analysis of wild-type and variant PAM function.

a, Amidating activity of wild-type (WT)-PAM (circles), p.Asp563Gly-PAM (squares), and p.Ser539Trp-PAM (crosses) (n = 1). b, Amidating activity of WT-PAM (circles), p.Asp563Gly-PAM (squares), and empty vector (EV) (triangles) over a substrate concentration range (n = 5 independent experiments). Data are shown as mean ± s.e.m.

Supplementary Figure 2 Analysis of wild-type and variant PAM expression.

a, PAM expression was investigated in (i) human tissues (n = 1), (ii) mouse tissues (n = 1), and (iii) FACS human beta cells and non-beta cells (n = 11 and n = 5 donors, respectively). RPKM, reads per kilobase of transcript per million mapped reads. Box plots display the interquartile range (IQR) with medians, whiskers indicating 1.5× the IQR, and individual data points. b, EndoC-βH1 cells were transfected with (i, iii) integral membrane or (ii) luminal variant PAM expression vectors, then labeled for PAM (green), the trans-Golgi network (TGN) (red in i), insulin (red in ii) or calnexin (ER) (red in iii). DAPI (blue) was used as a nuclear marker. Scale bar, 2 μm. Results are representative of at least two independent experiments.

Supplementary Figure 3 Allelic expression bias for rs35658696 in primary human islets.

The proportion of reference (A) to risk (G) allele expression in WASP-filtered RNA-seq data from intact islets isolated from 11 cadaveric donors heterozygous at rs35658696. The box plot displays the interquartile range (IQR) with medians, whiskers indicating 1.5× the IQR, and outliers.

Supplementary Figure 4 Gene silencing of PAM, CHGA, and IAPP and effects on beta cell function.

af, EndoC-βH1 cells were either transfected with scrambled sequence or gene-specific siRNAs, and then measured for efficiency of gene knockdown after 72 h (n = 5 biologically independent samples for a, n = 6 for b, and n = 4 for c) normalized to two housekeeping genes (PPIA and TBP). The effects of IAPP knockdown on insulin secretion (d; n = 8 biologically independent samples), insulin content (e; n = 16 for Scr and n = 8 for siIAPP), and cell numbers (f; n = 16 for Scr and n = 8 for siIAPP) are shown here, while those of PAM and CHGA knockdown are displayed in Figs. 2 and 6, respectively. *P < 0.05, *P < 0.01, ***P < 0.001 for two-tailed Student’s t-tests where a single independent variable was being tested (ac,e,f) or for two-way ANCOVA followed by Tukey’s HSD post-hoc test where two experimental variables were being tested (d). All box plots display the interquartile range (IQR) with medians, whiskers indicating 1.5× the IQR, and individual data points.

Supplementary Figure 5 Insulin secretion measures following PAM and CHGA knockdown.

ah, The data presented in Figs. 3 and 7 have been processed using alternative methods to compare the direct impact of gene silencing on insulin secretion relative to effects on insulin content and cell numbers. a and e (n = 8 biologically independent samples) show un-normalized insulin secretion (pg/hr), while b and f (n = 8) show secretion measures normalized to content values in c and g (n = 8), respectively. For reference, cell counts are shown in d and h (n = 16). *P < 0.05, **P < 0.01, ***P < 0.001 for two-tailed Student’s t-tests where a single independent variable was being tested (c,d,g,h) and by two-way ANCOVA followed by Tukey’s HSD post-hoc test where two experimental variables were being tested (a,b,e,f). All box plots display the interquartile range (IQR) with medians, whiskers indicating 1.5× the IQR, and individual data points.

Supplementary Figure 6 Calcium sensitivity of exocytosis following PAM silencing in EndoC-βH1 cells.

a, Content-normalized insulin secretion triggered by depolarizing stimuli (KCl, tolbutamide, forskolin) in 1 mM glucose (n = 3 independent experiments). b, Exocytosis and calcium current amplitude relationship (n = 15 cells from three independent experiments). The increment in exocytosis measured at the first pulse (fF) was normalized to the amplitude of the calcium current (pA) using data extracted from the capacitance measurements shown in Fig. 3. The calcium current density (normalized to the size of the cell) was measured at the first pulse, 5 ms after depolarization, to avoid interference with the sodium current component. Box plots display the interquartile range (IQR) with medians and whiskers indicating 1.5 × the IQR.

Supplementary Figure 7 Effects of rs35658696 genotype status on content-normalized insulin secretion measures from intact human islets.

The insulin secretion data shown in Fig. 5b were normalized to insulin content (Fig. 5a) on a per-donor basis. Data are shown for n = 16 donors in each group for basal (1 mM) and high (16.7 mM) glucose and n = 9 donors for medium (10 mM) glucose. All box plots display the interquartile range (IQR) with medians, whiskers indicating 1.5× the IQR, and individual data points.

Supplementary Figure 8 Western blot analysis of CgA-Gly and total CgA levels in EndoC-βH1 cells following perturbation of PAM.

a,b, The full, unedited blots corresponding to the sections shown in Fig. 6 are shown for PAM inhibition using 4P3BA (a) and PAM knockdown by siRNA (b).

Supplementary Figure 9 CgA amidation status in primary human islets from risk allele carriers and matched controls.

a, Representative co-staining of insulin (green) and total CgA (red; top) or CgA-Gly (red; bottom) in primary human islets indicative of results across all donors. b, Quantification of CgA amidation status (% of total CgA) in individuals heterozygous for the risk allele at rs35658696 (“PAM”) and matched controls (n = 6 donors per group). The graph shows means for n = 6; error bars, s.e.m.

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Thomsen, S.K., Raimondo, A., Hastoy, B. et al. Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic β-cells. Nat Genet 50, 1122–1131 (2018). https://doi.org/10.1038/s41588-018-0173-1

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