Reduced insulin exocytosis in human pancreatic β-cells with gene variants linked to type 2 diabetes

Abstract

The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants affected granule docking. We combined our results to create a novel genetic risk score for β-cell dysfunction that includes aberrant granule docking, decreased Ca(2+) sensitivity of exocytosis, and reduced insulin release. Individuals with a high risk score displayed an impaired response to intravenous glucose and deteriorating insulin secretion over time. Our results underscore the importance of defects in β-cell exocytosis in T2D and demonstrate the potential of cellular phenotypic characterization in the elucidation of complex genetic disorders.

FIG. 1.

Characterization of islets from diabetic and ND individuals. A: Fold-stimulation of insulin secretion by glucose in batch-incubated islets from non-T2D (n = 42) and T2D (n = 17) donors, nonobese (n = 37) and obese (n = 5) ND donors, as well as nonobese (n = 12) and obese T2D donors (n = 5). Fold-stimulation of insulin secretion in non-T2D and T2D donors separated by center (1, Lund; 2, Oxford) is also displayed. B: Total exocytosis evoked by a train of 10 depolarizations from −70 to 0 mV (ΣΔC) (n = 189 and 60 cells from 28 non-T2D and 8 T2D donors, respectively). C: Electron micrographs of human islets from non-T2D donors with low (25 kg/m²) and high (29 kg/m²) BMI. Lipid droplets (*), nucleus (N), and plasma membrane (PM, solid line) are indicated; dashed lines show 150-nm distance from PM. Scale bars, 0.5 μm. The histograms show the total granule number as volume density (Nv; granules/μm³) and the number of docked granules as surface density (Ns; granules/μm²) in β-cells from non-T2D and T2D donors (n = 75 and 42 cells from 12 and 6 donors, respectively). A granule was defined as docked when the center of the granule was located within 150 nm from the plasma membrane. Data are presented as means ± SEM. *P < 0.05 and **P < 0.01.

FIG. 2.

Genotype effects on insulin secretion and β-cell exocytosis. A: Glucose-stimulated insulin secretion in islets from different genotype carriers of rs7903146 (T is risk allele). Data from 28 CC, 17 CT, and 2 TT carriers. B: Glucose-stimulated insulin secretion in islets from donors with different genotype for rs553668 (A is risk allele). Data from 36 GG, 12 GA, and 2 AA carriers. C–F: Depolarization-evoked increase in cell capacitance (ΔC) in β-cells representative for individuals carrying risk or nonrisk alleles for rs7903146 (TCF7L2), rs553668 (ADRA2A), rs5219 (KCNJ11), and rs2237895 (KCNQ1). G and H: Exocytotic response in human β-cells treated with control siRNA or siRNA targeting KCNQ1. Histogram shows average total exocytosis (ΣΔC) from 22 and 21 cells per group, respectively. *P < 0.05.

FIG. 3.

Effects of the genetic risk score. A: Depolarization-evoked exocytosis in β-cells from donors with different scores. B: Electron micrographs of human islet sections from an individual with low (top) and high (bottom) score. Docked insulin granules (arrows), lipid droplets (*), nuclei (N), lipofusic bodies (LB), and plasma membrane (PM) are indicated. Left scale bars, 2 μm; right, 0.5 μm. C: Effects of the risk score on insulin secretion during IVGTT in 604 individuals. Data are means ± SEM. *P < 0.05.