Genome-wide association study and functional characterization identifies candidate genes for insulin-stimulated glucose uptake

Abstract

Distinct tissue-specific mechanisms mediate insulin action in fasting and postprandial states. Previous genetic studies have largely focused on insulin resistance in the fasting state, where hepatic insulin action dominates. Here we studied genetic variants influencing insulin levels measured 2 h after a glucose challenge in >55,000 participants from three ancestry groups. We identified ten new loci (P < 5 × 10^-8) not previously associated with postchallenge insulin resistance, eight of which were shown to share their genetic architecture with type 2 diabetes in colocalization analyses. We investigated candidate genes at a subset of associated loci in cultured cells and identified nine candidate genes newly implicated in the expression or trafficking of GLUT4, the key glucose transporter in postprandial glucose uptake in muscle and fat. By focusing on postprandial insulin resistance, we highlighted the mechanisms of action at type 2 diabetes loci that are not adequately captured by studies of fasting glycemic traits.

Extended Data Figure 1. Overview of genetic discovery for insulin fold change and Modified Stumvoll ISI, and downstream genetic and in vitro studies.

Created with BioRender.com.

Extended Data Figure 2. Observational Correlation of Insulin Fold Change and Modified Stumvoll ISI with metabolic traits in the Fenland Study.

Pairwise Spearman’s Rank correlation. Red shades denote positive correlation, blue shared denote negative correlation between trait pairs. X denotes no significant correlation (P > 0.05).

Extended Data Figure 3. Observational Correlation of insulin sensitivity and clearance related traits in the RISC study.

Pairwise Spearman’s Rank correlation. Red shades denote positive correlation, blue shared denote negative correlation between trait pairs, legend along bottom of heatmap shows colour scale relative to rho value. X denotes no significant correlation (P > 0.05). Abbreviations denote: IFC_OGTT – IFC calculated using OGTT measures, ISI_OGTT – Modified Stumvoll ISI calculated using OGTT measures. M_I – M/I index of insulin sensitivity measured by clamp. InsC0c – insulin measured at 0min during clamp. InsO0c - insulin measured at 0min during OGTT. InsC120c – insulin measured at 120min during clamp. InsO120c - insulin measured at 120min during OGTT. InsC_20c – insulin measured at 20min before clamp. EGP_B -basal glucose production, EGP_SS - glucose production during clamp, GCR_B - basal glucose clearance, ml/min/kg lean body mass, GCR_SS - steady state glucose clearance, ml/min/kg lean body mass, icl_clamp - peripheral insulin clearance (1/min/m²), icl_OGTT - endogenous "pre-hepatic" clearance during the OGTT, hie_0 - hepatic insulin extraction during clamp, OGIS - oral glucose insulin sensitivity index (ml min-1 m-2). ISR5dr - insulin secretion 5 mM glucose, beta cell dose response (pmol min-1m-2). ISR0 basal insulin secretion (pmol min-1m-2). ISRtot - total insulin secretion (nmol m-2).

Extended Data Figure 4. Meta-analysis workflow for genetic discovery analyses

Analysis workflow for the meta-analysis of study-level GWAS results for Insulin fold change and Modified Stumvoll ISI. Created with BioRender.com

Extended Data Figure 5. Two independent signals were identified at PPP1R3B for insulin fold change

Conditional analyses identify a second independent signal at PPP1R3B for insulin fold change adjusted for BMI. The regional association plot shows the primary signal in red and the secondary signal in blue for marginal summary statistics for insulin fold change adjusted for BMI. Shade of point indicates pairwise LD (R²) with indicated lead variant.

Extended Data Figure 6. Forest plot of beta estimates for the association of rs60453193 with insulin fold change in individual cohorts.

Labels on the right-hand side indicate the ancestry of the study and study name. EUR-European ancestry, HISAMR – Hispanic American ancestry, EAS – East Asian ancestry. Left-hand side values are beta estimate and 95% confidence interval. Error bars denote a 95% confidence interval. X-axis denotes the beta estimate of associations with insulin fold change in BMI adjusted analyses.

Extended Data Figure 7. Regional association plot at BICC1 (rs60453193)for insulin fold change in meta-analysis of non-European cohorts.

Unadjusted -log10 p-values are indicated on the y axis. Lead variant indicated by purple diamond.

Extended Data Figure 8. rs117643180 exhibits allelic differences in transcription factor binding.

An EMSA using 6μg per lane of nuclear extract from undifferentiated LHCN-M2 cells shows protein-DNA interactions for probes centered around each both alleles of rs117643180. The probe containing rs117643180-A shows allele-specific protein binding (Arrow A, lane 6), relative to the probe containing rs117643180-C (lane 2). A 25-fold excess unlabeled probe containing the A allele competed away A-specific bands more effectively (lane 7) than 25-fold excess unlabeled probe containing the C allele (lane 8). Arrow B shows a biotinylated free probe (200 fmol per lane). Uncropped image is available in Source Data.

Extended Data Figure 9. Confirmation of knockdown of positive control genes in wildtype 3T3-L1 adipocytes by Western Blot.

Representative blot from N=2. Marker indicates protein size in kDa is outlined on the right-hand side of the blot. siGenome and OT+ represent siRNA pools with their corresponding targets indicated below (see methods) and NT denotes non-targeting control. Antibodies are outlined on the left-hand side of the blot with Tubulin and B-actin used as loading controls. Uncropped blots are provided in Source Data.

Figure 1. Loci associated with post-challenge insulin resistance overlap with associations with other glycaemic traits and T2D

Manhattan plot of results for IFC, ISI and relative association with key glycaemic traits of interest. Y-axis denotes the -log10(p-value) of association for each trait. X-axis outlines the chromosomal position, with alternate chromosomes represented in light and dark grey. The dashed line defines genome-wide significance threshold (P = 5x10^-8), and the dotted line denotes P = 1x10^-5. Points highlighted in blue on each plot represent those that meet genome-wide significance for either IFC and ISI (P < 5x10^-8). Labels for indicate the nearest gene at a locus defined at genome-wide significance for either IFC and ISI, and their relative association with other traits. Published GWAS were used for fasting insulin and 2 h glucose,, and T2D. For T2D y axis is cut at 1x10^-80 for clarity, with the minimum p value truncated to P = 1x10^-300. Uncorrected p-values are shown. All GWAS are in cohorts of European ancestry and adjusted for BMI, except T2D which is unadjusted for BMI.

Figure 2. rs117643180 at SLC2A4 affects GLUT4 expression and transcription regulation.

A) Regional association plot showing ± 500kb around rs117643180 (SLC2A4) : insulin fold change (IFC) - blue, Modified Stumvoll (ISI) – red, and 2 h glucose – green, fasting insulin – purple, type 2 diabetes – pink. Y-axis denotes the unadjusted log10(p-value) of association. The dashed lines and dotted line indicate genome-wide (P = 5x10^-8) and suggestive (P = 1x10^-5) significance threshold, respectively. All association statistics are from BMI adjusted analyses in studies of European ancestry, except T2D which is unadjusted for BMI. Labels indicate the lead variant for each trait, with location indicating position. B) Association of rs117643180 with key metabolic traits of interest. Plot shows beta estimate or odds ratio of association for rs117643180 with traits outlined in (A) and fasting glucose and waist-to-hip ratio in grey. Point represents effect size and error bars indicate 95% CI. C) rs117643180 overlaps a transcriptionally active region in muscle tissues. rs117643180 is located ~730 bp downstream of the main SLC2A4 transcription start site (TSS) and ~330 bp downstream of an alternate SLC2A4 TSS. A 229-bp region flanking the variant, as shown by the orange bar, overlaps with accessible chromatin in LHCN-M2 (DNAase), chromatin marks of regulatory activity in fetal muscle leg tissue , a region conserved across species (vertebrate conservation), and a skeletal muscle ChromHMM (red =ActiveTSS). Created using http://genome.ucsc.edu D-E) rs117643180 exhibits allelic differences in transcriptional activity. Values represent fold-change of firefly luciferase/Renilla activity normalized to empty pGL4.23 vector in C2C12 cells, for the A (effect) and C (non-effect) alleles clones in forward and reverse orientation (See Methods) (D) and LHCN-M2 myotubes (E). Error bars represent the SEM of 4 to 5 independent clones tested in duplicate wells; points represent individual biological replicates. P-values are calculated from two-sided t-tests.

Figure 3. 15 genes at loci associated with insulin fold change were identified to significantly impact GLUT4 trafficking in adipocytes

a) Effect of gene knockdown on GLUT4 trafficking and glucose transport at 0.5 nM insulin stimulation in WT 3T3-L1 adipocytes (N = 3 biologically independent replicates, 2 technical replicates per N). Genes are clustered using hierarchical clustering. All results are normalised to non-targeting (NT) control. Grey represents genes for which glucose transport data was not collected as their role is established in the literature. Scale represents relative effect of gene knockdown compared to NT control for given measures – red positive, and blue negative, with the maximal scale representing the maximum absolute response relative to NT (0) across all measures, expressed as a percentage, to allow direct comparison across measures.-. B-D) Representative fluorescence microscopy images of example siRNA knockdown in WT 3T3-L1 adipocytes (N = 3 biologically independent replicates, 2 technical replicates per N) at 0.5 and 100nM insulin stimulation. Green represents the surface GLUT4 signal, orange the total GLUT4 signal and blue indicates individual nuclei. Scale bars indicate 100 μm. Quantification of Slc36a4 and Pdzk1ip1 compared to NT control and images at 0 nM are shown in Supplementary Figure 25. b) shows non-targeting control, c) Slc36a4 and d) Pdzk1ip1.