Studies implicating sodium–glucose cotransporter 2 (SGLT2) inhibitors in glucagon secretion by pancreatic α-cells reported controversial results. We hypothesized that interindividual heterogeneity in SGLT2 expression and regulation may affect glucagon secretion by human α-cells in response to SGLT2 inhibitors. An unbiased RNA-sequencing analysis of 207 donors revealed an unprecedented level of heterogeneity of SLC5A2 expression. To determine heterogeneity of SGLT2 expression at the protein level, the anti-SGLT2 antibody was first rigorously evaluated for specificity, followed by Western blot and immunofluorescence analysis on islets from 10 and 12 donors, respectively. The results revealed a high interdonor variability of SGLT2 protein expression. Quantitative analysis of 665 human islets showed a significant SGLT2 protein colocalization with glucagon but not with insulin or somatostatin. Moreover, glucagon secretion by islets from 31 donors at low glucose (1 mmol/L) was also heterogeneous and correlated with dapagliflozin-induced glucagon secretion at 6 mmol/L glucose. Intriguingly, islets from three donors did not secrete glucagon in response to either 1 mmol/L glucose or dapagliflozin, indicating a functional impairment of the islets of these donors to glucose sensing and SGLT2 inhibition. Collectively, these data suggest that heterogeneous expression of SGLT2 protein and variability in glucagon secretory responses contribute to interindividual differences in response to SGLT2 inhibitors.
Genome-wide association studies have shown that the rs340874 single nucleotide polymorphism (SNP) in PROX1 is a genetic susceptibility factor for type 2 diabetes. We conducted genetic and molecular studies to better understand the role of PROX1 in type 2 diabetes. We assessed the impact of the whole common genetic variability of PROX1 (80 SNPs) on type 2 diabetes–related biochemical traits in the HELENA (Healthy Lifestyle in Europe by Nutrition in Adolescence) study (n = 1,155). Three SNPs (rs340838, rs340837, and rs340836) were significantly associated with fasting plasma insulin levels (P ≤ 0.00295). We evaluated the impact of nine PROX1 SNPs (the three insulin-associated SNPs plus six SNPs in strong linkage disequilibrium) on luciferase reporter gene expression. The insulin-lowering alleles of rs340874, rs340873, and rs340835 were associated with lower luciferase activity in MIN6 and HepG2 cells (except for rs340874, which was in HepG2 cells only). Electrophoretic mobility shift assays indicated that specific nuclear protein bindings occur at the three SNPs in HepG2 cells, with allele-binding differences for rs340874. We also showed that the knockdown of Prox1 expression by small interfering RNAs in INS-1E cells resulted in a 1.7-fold reduction in glucose-stimulated insulin secretion. All together, we propose that reduced expression of PROX1 by cis-regulatory variants results in altered β-cell insulin secretion and thereby confers susceptibility to type 2 diabetes.
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