Maintenance of ß-Cell Heterogeneity Is Required for Normal Islet Function

Aim: Recent studies have shown the existence of discrete β-cell subpopulations with differing levels of maturity and function. We sought here to understand how this diversity may influence islet function and insulin secretion using a ‘loss of heterogeneity‘ model.

Material and Methods: Overexpression was achieved using an adenoviral polycistronic construct for Pdx1, MafA, Ngn3 and mCherry (Ad3-NPM). Pdx1 was silenced using short hairpin RNAs (shRNA). Gene and protein levels were detected by qPCR and immunohistochemistry (IHC), respectively. Ca²⁺ fluxes, ATP/ADP ratios and cAMP levels were analysed using dyes/biosensors and high-speed spinning disk microscopy. Glucose- (GSIS) and incretin-stimulated insulin secretion (ISIS) were measured using HTRF assays.

Results: Ad3-NPM increased Pdx1 (12-fold vs. CT; P<0.01) and MafA levels (2-fold vs. CT) without affecting Ngn3. IHC showed that Pdx1 overexpression occurred predominantly in Pdx1^low β-cells, forcing higher uniformity throughout the β-cell complement. The α/β-cell ratio did not change, indicating absence of transdifferentiation. Ca²⁺ responses to glucose were markedly blunted (ΔF=0.81 vs. 0.44 AU, CT vs. Ad3-NPM; P<0.01), and this was accompanied by a decrease in β-cell-β-cell cell connectivity and hub number (12.6 vs. 5.6 % hubs, CT vs. Ad3-NPM; P<0.05), the latter previously shown to be relatively immature cells with pacemaker features. ATP/ADP ratios and cAMP levels were unaffected. Glucose- and incretin-stimulated insulin secretion was impaired (% content secreted = 8.7 vs. 5.2 for GSIS and 56.4 vs. 29.02 for ISIS, CT vs. Ad3-NPM; both P<0.01). Similar results for Ca²⁺ dynamics, cellular connectivity and insulin secretion were obtained in islets treated with Pdx1 shRNA to increase the size of the Pdx1^low population.

Conclusion: Loss of β-cell heterogeneity, whether due to increased or decreased maturity, leads to islet failure and impaired insulin secretion. Thus, cellular diversity appears necessary for normal islet function.