Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion

Abstract

The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-βH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.

Figure 1

Impaired insulin secretion in Sox4mt islets. A-B: Insulin secretion (A) and content (B) measured in intact islets in static incubations. Islets were exposed to increasing glucose concentrations as indicated. Data are from 3 animals per genotype (with 3 technical replicates of 5 islets per animal) experiments. *P<0.05. C: Dynamic measurements (perifusion) in Sox4mt (red) and Sox4wt (black) islets. Data are from 6 animals per genotype with 50 islets per animal and normalized to insulin content *P<0.05 using a one-way Anova, repeated measures.

Figure 2

No impairment of [Ca²⁺]_i signaling, exocytosis (measured via whole cell capacitance) and Ca²⁺ currents. A: Glucose- and tolbutamide-induced increases in cytosolic Ca²⁺ in Sox4wt (black) or Sox4mt (red) islets. The glucose concentration was varied as indicated above the traces. Tolbutamide was added at a concentration of 0.2 mM. Traces are mean ± S.E.M. of 9 experiments for each genotype (3 islets per animal and 3 animals per genotype) and have been baseline corrected to correct for fading of the fluorophore. B: Increase in membrane capacitance (ΔC) during a train of ten 50 ms depolarizations from -70 mV to 0 mV (V) in Sox4wt (black) and Sox4mt (red) β-cells. Histogram (below) compares the total increase in membrane capacitance (ΣΔC) evoked by the train of depolarizations in Sox4wt (black) and Sox4mt (red) β-cells (n=24-28 β-cells from 4 mice per genotype). C: Peak current (I)-voltage (V) relationship for voltage-gated Ca²⁺ currents recorded from single Sox4wt (black) and Sox4mt (red) β-cells during depolarizations from -70 mV to membrane potentials between -60 and +80 mV (n=13-16 β-cells from 4 mice per genotype). The horizontal dotted line indicates zero current level.

Figure 3

Subtle effects on cellular ultrastructure in Sox4mt β-cells. A-B: Representative electron micrographs from Sox4wt (A) andSox4mt (B) β-cells. Scale bars: 1μm. C-E: Granule density (C) and box plots, depicting groups of data according to their quartiles, of the dense core area (D) and granule diameter (E) in Sox4wt (black) and Sox4mt β-cells (grey). At least 6 β-cells from 2 animals per genotype were analyzed. Data in (D) are mean ± S.E.M. **P<0.01. Distributions of core areas and granule diameters were estimated by counting >382 granules per genotype. **P<0.005; ***P<0.0005 (by Mann-Whitney U-test).

Figure 4

Delayed granule emptying in Sox4mt β-cells. A: Release of ATP monitored in individual Sox4wt β-cells expressing ionotropic P2X₂ receptors infused with 2 µM free [Ca²⁺]_i. B: As in (A) but using from Sox4mt β-cells. Arrows indicate slow ('kiss-and-run') events. C: Rapid event recorded in a Sox4wt β-cell (black) and slow 'kiss-and-run' events recorded in a Sox4mt (red) β-cell. D: Cumulative histogram for the rise times in Sox4wt (black) and Sox4mt (red) β-cells. (P<0.005 by independent-samples Kolmogorov-Smirnov test). E: As in (D) for half-widths. The arrow indicates that ~20% of release events are of longer duration (P<0.005 by independent-samples Kolmogorov-Smirnov test). F: As in (D-E) for total charge (Q). Data shown in (D-F) based on >368 events obtained from 10 (Sox4mt) and 14 (Sox4wt) β-cells from 2 mice of each genotype. See also Supplemental Figure 1.

Figure 5

Impaired fusion pore expansion in Sox4mt β-cells. A: On-cell measurements of membrane capacitance (C) and conductance (G) showing two examples of full fusion (FF) from a Sox4wt β-cell (left) where the first event is associated with a prominent but short-lived increase in conductance (arrow) and an example of transient exocytosis (kiss-and-run; abbreviated KR) recorded from a Sox4mt β-cell (right) where C returns to baseline and the capacitance step is associated with a persistent increase in G. B: Pie charts summarizing the fraction of full fusion or kiss-and-run exocytosis in Sox4wt (n=119 events in 23 cells from 5 mice) and Sox4mt (120 events in 30 cells from 5 mice) β-cells (P=8.10^-7 by χ² test). C: Cumulative histogram of the distribution of estimated fusion pore diameters in Sox4mt β-cells. The dashed vertical line indicates a fusion pore diameter of 1.1 nm (enough to allow the exit of ATP). Also note that 10% of the events have an estimated pore diameter >6nm. See also Supplemental Table 1.

Figure 6

Mutant Sox4 mediates its effects on insulin secretion via Stxbp6. A: qRT PCR analysis of Stxbp6 expression in INS-1 832/13 cells transfected with scrambled siRNA + DsRed + hGH (dashed horizontal line; control), scrambled siRNA + Sox4wt + hGH (labeled '+Sox4') or scrambled siRNA + Sox4mt + hGH ('+Sox4mt'). †P<0.05 wt vs Sox4. B: hGH release (as a proxy for insulin release in transfected cells) from INS-1 832/13 cells transfected with scrambled siRNA + DsRed + hGH (labeled 'control'), si-Stxbp6 + DsRed + hGH ('-Stxbp6') and scrambled siRNA + Stxbp6 + hGH ('+Stxbp6'), scrambled siRNA + Sox4wt + hGH ('+Sox4'), si-Stxbp6 + Sox4wt + hGH ('+Sox4-Stxbp6'), scrambled siRNA + Sox4mt + hGH ('+Sox4mt') and si-Stxbp6 +Sox4mt + hGH ('+Sox4mt-Stxbp6') incubated with 1 or 20mM glucose (as indicated). Data are presented as mean ± S.E.M. of 4 experiments, each with 3 replicates. *P<0.05; **P<0.01; and ***P<0.001 for comparison vs control (1 or 20 mM glucose alone) and ††P<0.01 vs Sox4wt and ‡‡‡P<0.01 vs Sox4mt (by ANOVA and Tukey's tests). See also Supplemental Figure 2.

Figure 7

Effects of SOX4 and STXBP6 on human β-cell function. A: Relationship between SOX4 expression and HbA1c levels. R=0.305; P=0.036; n=52. B: Relationship between SOX4 expression and STXBP6 expression R=0.321; P=0.021; n=63. C: Relationship between SOX4 expression and GIIS. R=-0.292; P=0.043; n=48. For display, data in (A-C) have been grouped in quintiles. Best-fit black lines represent Pearson correlation analyses to the individual data points. D: qRT PCR analysis of SOX4 and STXBP6 expression in human cells transfected with scrambled siRNA + DsRed + hGH (dashed horizontal line; labelled 'Control'), scrambled siRNA + SOX4wt + hGH ('+SOX4'), si-STXBP6 + SOX4wt + hGH ('+SOX4-STXBP6') and si-SOX4 + DsRed + hGH ('-SOX4'). Data are presented as mean ± S.E.M. of 3 experiments, each with 3 replicates; **P<0.01 vs control (dashed horizontal line); ††P<0.01 vs '+SOX4' (by ANOVA and Tukey's tests). E-F: As in (D) but hGH release (as a proxy for insulin release in transfected cells) was measured in EndoC-βH2 cells exposed to 20mM glucose + 70mM [K⁺]o. **P<0.01 vs 'control'; ^††P<0.01 vs '+SOX4' (by ANOVA and Tukey's tests). Mean ± S.E.M. of 4 experiments, each with 3 replicates. Note that (E) and (F) were carried out independently, and for (F) data are presented as mean ± S.E.M. of 3 experiments, each with 3 replicates. See also Supplemental Figure 3A-D. G: Effects of overexpressing STXBP6 on ATP release in the human insulin-secreting cell line EndoC-βH2. Traces are labeled ‘Control’ (cells expressing DsRed alone) and ‘+STXBP6’ (cells overexpressing STXBP6). The top trace shows recordings when the cells were infused with Ca²⁺-free medium (several sweeps have been superimposed). H: Cumulative histogram for the charge (Q) of the events in ‘Control’ (black) and ‘+STXBP6’ cells (red) (P<0.005 by independent-samples Kolmogorov-Smirnov test). Data based on 1100-1400 events in 9-10 cells. I: Product of mean charge and frequency of events in ‘Control’ and ‘+STXBP6’ cells. P<0.05 vs. ‘Control’. See also Supplemental Fig. 4.