Metformin is the first-line antidiabetic drug with over 100 million users worldwide, yet its mechanism of action remains unclear1. Here the Metformin Genetics (MetGen) Consortium reports a three-stage genome-wide association study (GWAS), consisting of 13,123 participants of different ancestries. The C allele of rs8192675 in the intron of SLC2A2, which encodes the facilitated glucose transporter GLUT2, was associated with a 0.17% (P = 6.6 × 10−14) greater metformin-induced reduction in hemoglobin A1c (HbA1c) in 10,577 participants of European ancestry. rs8192675 was the top cis expression quantitative trait locus (cis-eQTL) for SLC2A2 in 1,226 human liver samples, suggesting a key role for hepatic GLUT2 in regulation of metformin action. Among obese individuals, C-allele homozygotes at rs8192675 had a 0.33% (3.6 mmol/mol) greater absolute HbA1c reduction than T-allele homozygotes. This was about half the effect seen with the addition of a DPP-4 inhibitor, and equated to a dose difference of 550 mg of metformin, suggesting rs8192675 as a potential biomarker for stratified medicine.
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We acknowledge G.I. Bell (University of Chicago) for providing the expression vector for SLC2A2 (pSP64T-SLC2A2), and D.L. Minor and F. Findeisen for their guidance in performing oocyte injection and preparing cRNA. For full acknowledgments, see the Supplementary Note.
The authors declare no competing financial interests.
Integrated supplementary information
All 44 independent associations with P < 5.0 × 10−4 (blue line) and 23 independent signals from the next tier (P < 0.001 above the red line) with plausible biological candidacy were followed up in stage 1 replication.
The C allele was used as the effect allele in association tests. HbA1c was measured as a percentage.
(a,b) Luciferase activity in two liver cell lines transfected with reporter construct of the genomic region that include SLC2A2 intron variant rs8192675 (chr3:170724883). The construct (chr3:170724251–170727543) (195 ng) along with Renilla constructs (5 ng) was transiently transfected into Huh-7 and HepaRG liver cell lines for analysis of luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity. The methods for cloning and luciferase assays have been previously described by our group (PLoS Genet. 10, e1004648, 2014, and Clin. Pharmacol. Ther. 89, 571–578, 2011). Pooled genomic DNA were used to clone the genomic region using the In-Fusion HD Cloning kit (Clontech) with forward and reverse primers using the following primer sequences: forward (+ strand) GCTCGCTAGCCTCGAGGCAACCAGATAGAATAATAC; reverse (+ strand) CGCCGAGGCCAGATCTGGTTCTCGTCCATGGCAATG. The genomic region was cloned into XhoI- and BglII-digested pGL4.23 using the Infusion HD cloning system (Clontech). The underlined region is the digestion site for XhoI and BglII. The reference allele of rs8192675 (T allele) showed significantly greater luciferase activity than the alternate allele (C allele) (P < 0.05). Data are reported as the relative fold increase compared with the pGL4.23 vector (white bar) containing the SNP rs8192675 (black and light gray bar). Each bar represents the mean ± s.e.m. from three or four replicates from one experiment. The experiments were repeated three times with similar significance and trend. The APOE basal promoter was used as a positive control.
Supplementary Figure 5 Uptake and inhibition studies in Xenopus laevis oocytes expressing human GLUT2 (SLC2A2).
(a–c) Uptake of model substrate (14C-2-deoxyglucose (2-DG)) (a) and metformin (Metf.) (b,c) in Xenopus laevis oocytes expressing GLUT2. (a) At 30 min, uptake of the model substrate is significantly higher than in oocytes injected with saline. In the presence of GLUT2 inhibitor, phloretin (200 μM), GLUT2-mediated uptake of 14C-2-deoxyglucose is inhibited. (b,c) However, uptake of 14C-metformin (at 30 and 60 min) is not significantly different between oocytes injected with saline or GLUT2 and also in the presence of GLUT2 inhibitor, phloretin. (d) Inhibition of GLUT2-mediated uptake of 14C-2-deoxyglucose by phloretin (200 μM) and metformin (30 and 50 mM). Phloretin significantly inhibit GLUT2-mediated uptake of 14C-2-deoxyglucose but not metformin. Xenopus laevis oocytes were purchased from Ecocytes. Capped cRNA was synthesized in vitro from human GLUT2 expression vector (pSP64T) (from G.I. Bell, University of Chicago) linearized using the mMessage mMachine SP6 kit (Ambion). 50 ng of the synthesized cRNA was injected into each oocyte. Modified Barth solution was used as the uptake buffer. DPM, disintegrations per minute, measure of the activity of the source of 14C-2-deoxyglucose radioactivity.
Supplementary Figures 1–5, Supplementary Tables 1–8 and Supplementary Note. (PDF 2014 kb)
The first-stage replication was performed with three genotyping assays of CardioMetabochip (M), Sequenom (S) and TaqMan (T). Each P value for association was a geometric mean of two P values from the linear regression of HbA1c and the logistic regression of achieving a treatment target of HbA1c (XLSX 19 kb)
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Zhou, K., Yee, S., Seiser, E. et al. Variation in the glucose transporter gene SLC2A2 is associated with glycemic response to metformin. Nat Genet 48, 1055–1059 (2016). https://doi.org/10.1038/ng.3632
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