Loss of ZnT8 function protects against diabetes by enhanced insulin secretion

Abstract

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived β-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human β cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced K_ATP channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D.

Extended Data Fig. 1. Families of SLC30A8-p.Arg138* carriers involved in genotype-based recall for human in vivo study.

Families (≥ 2 members per family) of SLC30A8-p.Arg138* carriers participated in genotype-based recall (test meal) study. To protect anonymity of the carriers, the gender of the offspring is not revealed and some pedigrees have been split to smaller nuclear families. The carrier status of p.Arg138* is shown by yellow (p.Arg138Arg) or magenta (p.Arg138*), that of p.Trp325Arg by vertical (p.Arg325Arg), horizontal (p.Trp325Trp) or diagonal (p.Trp325Arg) lines. The white color (with no lines) represents individuals with unknown status for genotype and phenotype.

Extended Data Fig. 2. Association of SLC30A8 p.Arg138* and p.Trp325Arg with free fatty acids, hormones, and insulin clearance during test meal.

Association of SLC30A8 p.Arg138* and p.Trp35Arg variant with a, serum (S)-C-peptide b, S-proinsulin c, plasma (P)-Glucagon d, Total S-GLP-1 e, S-free fatty acid (FFA) concentrations f, Insulin-C-peptide ratio and g, model-based insulin clearance index during test meal. Left panel: Carriers (red, n=50-54) vs. non-carriers (black, n=37-47) of p.Arg138*. Middle panel: Carriers of p.Arg138* (red, n=50-54) vs. p.Arg138Arg having the common risk variant p.Arg325 (blue, n=25-31). Right panel: Carriers of p.Trp325Trp (grey, n=12-16) vs. p.Arg325 (blue, n=25-31). Exact numbers used for genetic association analysis are available in Source Data files. Data are Mean ±SEM; A star (*p < 0.05, ** p < 0.01) indicates significance in family based association (using QTDT35) after 100,000 permutations, adjusted for age, sex and BMI for left panel and age, sex, BMI and genotype of p.Trp325Arg for middle panel. A hash sign (# p < 0.05) indicates significance in QFAM (as implemented in PLINK) test using 100,000 permutations (see Methods).

Extended Data Fig. 3. Effect of p.Trp325Arg genotype on insulin secretion during intravenous glucose tolerance test (IVGTT) and β-cell sensitivity to glucose during OGTT

a-b, (a) Serum (S-) insulin concentrations, p.Trp325Trp (grey, n=116) and p.Arg325 (blue, n=733), and (b) S-insulin -plasma glucose ratio, p.Trp325Trp (grey, n=86) and p.Arg325 (blue, n=458) during IVGTT. Data are Mean ± SEM. Analysis was performed using mixed model adjusting for age, sex, BMI and genetic relatedness. * p < 0.05 c, β-cell sensitivity to glucose is presented as insulin secretion rate in response to plasma glucose during oral glucose tolerance test (OGTT) in people with newly diagnosed type 2 diabetes. Data are Mean ± SEM. Analysis was performed using a generalized linear model (log-transformed data) for repeated measures, adjusting for age, sex and BMI.

Extended Data Fig. 4. Generation of SLC30A8-p.Lys34Serfs50* and SLC30A8-p.Arg138* hiPSC lines.

a, CRISPR-Cas9 strategy to generate SLC30A8-p.Lys34Serfs50* (Exon 2) and SLC30A8-p.Arg138* (Exon 3) hiPSC lines. Orange font highlights the nucleotide changes: c.101-107del; p.Lys34Serfs50* and c.412C>T; p.Arg138*. The gRNA (blue font) and PAM sequences (red font) are indicated on the partial genomic sequence of SLC30A8. b-c, FACS data from undifferentiated b, SLC30A8-p.Arg138* and c, SLC30A8-p.Lys34Serfs50* iPSCs and relevant isotype controls using antibodies against: OCT3/4, SSEA, SOX2, and NANOG. d, Expression of INSULIN in hiPSC-derived beta-like cells. Black bars represent p.Arg138Arg control cells, red bars represent p.Arg138*, and yellow bars represent p.Lys34Serfs50*. (n=6-8 wells from three differentiations) e-g, RNAscope analysis of the number of e, INSULIN- and f, SLC30A8-transcript positive cells in hiPSC-derived beta-like cells. 7-21 image fields were quantified and presented as % of DAPI+ cells. Representative images used for quantification shown in g (scale bar = 50 μm). Data are presented as Mean±SEM. Statistical analysis was performed using the one-way ANOVA and Tukey’s multiple comparison test (n = 5-9 wells from three differentiations, ****p<0.0001).

Extended Data Fig. 5. Confirmation of the ddPCR probe specificity and target SLC30A8 mRNA sequencing.

a, R138 (pGEM_CT) and X138 (pGEM_TT) sequences were inserted in pGEM vector and used as template for digital droplet PCR. Original probe configuration confirmed specificity as R138 droplets were only detected by FAM (CT-FAM; channel 1) and X138 droplets were only detected by VIC (TT-VIC; channel 2). In the swapped probe configuration, FAM and VIC probes were swapped and ddPCR was performed using pGEM_CT or pGEM_TT as template. b, Detection of R138 allele (Channel 1) and X138 allele (Channel 2) using cDNA from the heterozygous hiPSC-derived beta-like cells (B1 clone) as a template. In the swapped probe configuration, FAM and VIC probes were swapped and ddPCR was performed using cDNA from hiPSC-derived beta-like cells as template. c, Depicting the unique sequencing reads coverage at p.Arg138* and p.Ala139Ala obtained by SLC30A8 target mRNAs sequencing in edited clones (B1 and A3) and unedited cells (wildtype).

Extended Data Fig. 6. Silencing of ZnT8 tends to lower granule Zn²⁺ content.

EndoC-βH1 cells were transfected with siRNA control (siCtrl) or targeted against SLC30A8 (siZnT8) for 72 hours prior to imaging. a, ZnT8 knock-down was confirmed at the protein level by Western-blot. b, Control cells were incubated for 20 min. with the membrane-targeted zinc probe ZIMIR to monitor zinc secretion after cell stimulation with 20 mM KCl using total internal reflection of fluorescence (TIRF) microscopy (see accompanying movies, Supplementary video 1-4). c, Fluorescence intensity at the membrane was monitored upon time and traces obtained were averaged for cells transfected with siCtrl (14 cells) or with siZnT8 (14 cells). d, Fluorescence intensity increase due to zinc secretion after stimulation with KCl was determined for each cell. An outlier data point in the siZnT8 condition, likely to reflect release from a non-silenced cell, was excluded by Grubb’s test and statistical significance determined by Student’s t-test (Graph Pad Prism 7.0). Scale bar in b, 5 μm. Blots have been cropped and corresponding full blots are available in Source Data files.

Extended Data Fig. 7. RNA (mRNAs) sequencing of SLC30A8 knock down and control EndoC-βH1 cells.

Effect of SLC30A8 knock down (KD) on expression of genes involved in a, proinsulin processing, b, insulin production and β-cell development, c, β-cell excitability and insulin exocytosis (Supplementary Data Set 1). d, Over-representation analysis of differentially expressed genes (red dots) and depiction of most enriched WNT pathway genes (Supplementary Data Set 1) along with a gene set enrichment analysis (GSEA) considering all expressed genes. *p<0.05, ***p<0.001; Differential expression analysis was done using sequencing reads count based data using method similar to Fisher's Exact test as implemented in edgR software package and further corrected for multiple testing using Bonferroni correction (see Supplementary Note).

Extended Data Fig. 8. Expression and localization of p.Arg138* and impact on cytosolic free Zn²⁺ concentrations in cultured INS1 β-cells.

a-d, Rat INS1e cells were transiently transfected with p.Arg138*-mCherry fusion construct followed by fluorescence microscopy imaging and immunodetection. a, Fusion protein localized to distinct subcellular compartments in INS1e cells at 48 h and 96 h after transfection. b, Expression of mCherry in control INS1e cells indicated cytoplasmic localization. c, Control experiments with immunostaining of p.Arg138* with HA or Myc-His (both are significantly smaller additions than mCherry) confirmed localization of fusion proteins to distinct subcellular compartments in the INS1e cells. d, Immunological detection (anti-mCherry) of the fusion protein at indicated time points after transfection confirms protein expression and indicate protein stability. Tubulin is used as control. e-f, INS1(832/13) cells were transfected constructs expressing p.Arg138*-Myc-His or eCALWY-4, or co-transfected with both, followed by (e) immunostaining or (f) immunofluorescence imaging at 24 h post-transfection using anti-c-Myc antibody. g, Cytosolic free Zn²⁺ concentrations in INS-1 (832/13) cells. Data are combined from three fully independent experiments. Scale bars are 50 μm (a, b), 10 μm (c) and 25 μm (f). Blots have been cropped and corresponding full blots are available in Source Data files.

Fig. 1. A flow-chart describing the study design.

OGTT; oral glucose tolerance test, IVGTT; intravenous glucose tolerance test, GTT; glucose tolerance test a, The study design including various model systems (left panels), methods (middle panels) and the purpose of these experiments (right panels). b, Detailed description of the human in vivo studies, including a genotype-based recall study for p.Arg138* carriers and their relatives for metabolic studies.

Fig. 2. SLC30A8-p.Arg138* enhances insulin secretion and proinsulin processing during test meal.

Association of SLC30A8 p.Arg138* and p.Trp325Arg variants with a, plasma glucose b, serum insulin c, insulin/glucose ratio d, proinsulin/C-peptide ratio and e, proinsulin/insulin ratio during test meal. Left panel: Carriers (red, n=53-54) vs. non-carriers (black, n=37-47) of p.Arg138*. Middle panel: Carriers of p.Arg138* (red, n=53-54) vs Arg138Arg having the common risk variant p.Arg325 (blue, n=25-31). Right panel: Carriers of p.Trp325Trp (grey, n=12-16) vs. p.Arg325 (blue, n=31).). Exact numbers used for genetic association analysis are available in the Source Data files. Data are Mean ± SEM. A star (*p < 0.05, ** p < 0.01) indicates significance in family based association (using QTDT) after 100,000 permutations, adjusted for age, sex and BMI for left panel and age, sex, BMI and genotype of p.Trp325Arg for middle panel. A hash sign (# p < 0.05) indicates significance in family-based QFAM (as implemented in PLINK) test using 100,000 permutations (see Methods).

Fig. 3. SLC30A8 p.Arg138* and p.Trp325 enhance insulin secretion during OGTT.

Association of SLC30A8 p.Arg138* and p.Trp325Arg with a, plasma glucose b, serum insulin c, insulin/glucose ratio during an oral glucose tolerance test (OGTT). Left panel: Carriers (red, n=34-35) vs. non-carriers (black, n=7,954-8,141) of p.Arg138*. Middle panel: Carriers of p.Arg138* (red, n=34-35) vs. p.Arg138Arg having the common risk variant p.Arg325 (blue, n=6,728-6,893). Right panel: Carriers of p.Trp325Trp (grey n=1,226-1,248) vs. p.Arg325 (blue, n=6,728-6,893). Exact numbers used for genetic association analysis are available in Supplementary Table 5. Data are shown as Mean ±SEM. A star (*) indicates significance (*p < 0.05, **p< 0.01) for additive effects, calculated using mixed model adjusting for genetic relationship, age, sex, BMI and additionally genotype of p.Trp325Arg only for middle panel as implemented in GCTA (see Methods).

Fig. 4. Beta like cells derived from SLC30A8-p.Arg138* iPSCs display haploinsufficiency of SLC30A8.

a-c, Characterization of SLC30A8 expression at the (a) mRNA and (b-c) protein level in cells heterozygous for SLC30A8-p.Arg138* or homozygous for SLC30A8-p.Lys34Serfs50*. Gene expression data normalized to TBP and expressed as fold change relative to p.Arg138Arg control (n=7-13 wells from three differentiations). d-e, Allele-specific expression (ASE) of p.Arg138Arg (black bar) and p.Arg138* (red bar) in clone A3 or clone B1 derived cells using (d) Digital Droplet PCR based probes also validated by (e) target SLC30A8 mRNA sequencing of p.Arg138* clones (n=number of unique sequencing reads for each allele). f-g, Allele-specific expression of p.Arg138Arg (black bar) and p.Arg138* (red bar) in f, clone A3 and g, clone B1 derived cells treated with DMSO (Dimethyl sulfoxide) or cycloheximide (CHX) for four hours. ASE data (Mean±SEM) determined by Digital Droplet PCR were presented as fold change relative to p.Arg138Arg transcript (d, n=9 wells from three differentiations) or to DMSO control (f-g, n=4-7 wells from two differentiation). Blots have been cropped and corresponding full blots are available in the Source Data files. *p<0.05, **p<0.01, ****p<0.0001 one-way ANOVA Holm-Sidak’s multiple comparison test (a) or one-sample t-test (d, f and g) or binominal test (one-tailed) considering 0.5 as expected probability (e).

Fig. 5. SLC30A8 knockdown (KD) leads to enhanced insulin secretion, proinsulin processing and cell viability in the human pancreatic EndoC-βh1 cells.

Black bar graph; control cells (siScramble); Red bar graph: SLC30A8 knock down cells (siSLC30A8). a-b, Effect of siRNA mediated knock down (KD) on SLC30A8 mRNAs (n=5) and protein. Intensities of the signal normalized to control condition are reported below the blot. c, Measurement of intracellular zinc using zinc-specific fluorescent dye Zinpyr-1 (n=6, scale bars=50 μm). d-j, Effect of KD on (d) insulin secretion (normalized to total insulin content and average siScramble basal secretion) stimulated by glucose and K_ATP channel regulators (as labelled, and n=12), (e) glucose stimulation index induced by 20 mmol.L^-1 glucose stimulation (n=12), and (f) insulin content (n=48). g, Effect of siSLC30A8 KD on K_ATP channel conductance (Gm, siScramble n=10 cells, siSLC30A8 n=9 cells), (h) cell size (n=5), (i) expression of K_ATP channel subunits (n=5), and (j) on insulin secretion stimulated by KCL and high glucose (n=10). k-l, Effect of KD on proinsulin processing estimated by (k) proinsulin/insulin ratio (n=7) and (l) proinsulin concentration (n=7). m-q, Effect of KD on protein expression of proinsulin processing enzymes PC1/3, CPE (immunoblot- m and densitometry- n, o; n, n=5 and o, n=4) and PC2 (densitometry- p, n=4 and immunoblot- q). r-t, Effect of KD on basal (5.5 mM glucose) AKT phosphorylation (densitometry- r, n=5 and immunoblot- s; phospho-AKT-Ser473, total AKT) and cell viability under ER stress (t, MTT assay, n=5). Blots have been cropped and corresponding full blots are available in the Source Data files. Data are Mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 using Mann-Whitney test; #p<0.05 one-sample t-test; d (Bonferroni multiple correction for 4 conditions).

Fig. 6. Male Slc30a8 p.Arg138* mice on high-fat diet show enhanced insulin secretion and proinsulin processing.

Black boxes; wild type mice (WT), Red boxes; Slc30a8 p.Arg138* mice. a, Body weight (n=10/17 WT/p.Arg138*), circulating b, glucose (n=10/17 WT/p.Arg138*) c, insulin (n=9/17 WT/p.Arg138*) d, proinsulin (*p=0.011, n=10/17 WT/p.Arg138*) e, C-peptide (n=10/16 WT/p.Arg138*) f, proinsulin/insulin ratio (n=9/17 WT/p.Arg138*) g, proinsulin/C-peptide ratio (n=10/16 WT/p.Arg138*) and h, insulin/C-peptide ratio (n=9/16 WT/p.Arg138*) in fasted WT and p.Arg138* mice after 20 weeks on HFD. i, Insulin response to oral glucose (2g/kg) exposure (n=5/9 WT/p.Arg138*) after 30 weeks on HFD. Blood glucose levels over time after j, oral glucose (2g/kg) exposure (n=5/11 WT/p.Arg138*) after 29 weeks on HFD and k, interperitoneal injection of insulin (1.75 U/kg) after 28 weeks on HFD (n=11/13 WT/p.Arg138*). Boxplot depicts the interquartile range, median and minimum/maximum values. *p<0.05, ***p<0.001, ****p<0.0001 using Mann Whitney test; ^#p<0.05, ^##p<0.01 using two-way Anova (repeated measurements) and Sidak’s multiple comparison test.

Fig. 7. SLC30A8- p.Trp325 leads to enhanced insulin secretion in human islets.

Experiments have been performed in two different centers: LUDC (a, b, g, h, i and j) and Oxford (c, d, e and f). a, Effect of p.Trp325Arg genotype on static insulin secretion in presence of low and high glucose stimulatory conditions. b, Effect of p.Trp325Arg genotype on static insulin secretion in presence of low or high glucose and KCL. c-d, Effect of p.Trp325Arg genotype on static insulin secretion in (c) sub-maximal stimulatory conditions (6mM glucose) and their (d) insulin contents. e, Static glucagon response to glucose and f, glucagon content at basal glucose. g-h, Correlation of SLC30A8 expression with candidate genes of INS, GCG, proinsulin processing genes and K_ATP channel subunits genes among (g) p.Arg325Arg individuals and (h) p.Trp325 carriers and effect of p.Trp325Arg genotype (p.Arg325Arg=65, p.Trp325Arg=63 and p.Trp325Trp=11) on expression (cpm=log2 of counts per million) of (i) SLC30A8 and (j) other candidate genes. Data are Mean ±SEM, Glu; glucose, N; number of experiments. *p<0.05, ***p<0.0001 using Mann-Whitney test (b, c, d, e and f) or Spearman correlation coefficient (r) with two-tailed p values (g and h). Three genotype comparison (a, i and j) by linear regression using additive effect adjusting for age, sex and islet purity as implemented in PLINK (see Methods).