Background: A normal islet includes both mature and immature β-cells, with the former possessing higher insulin content and the latter displaying better proliferative capacity. However, it remains unknown whether immature β-cells also contribute to the regulation of insulin release, especially by commanding the activity of their mature counterparts.
Materials and methods: Pdx1, Mafa, and Ngn3 were overexpressed in β-cells by either transduction with adenovirus (Ad-M3C) or using a doxycycline-inducible mouse line (RIP7rtTA+/-; TetO/M3C+/- (Tet-MAT)). Conditional chemogenetic β-cell silencing was achieved in islets expressing the inhibitory DREADD h4MDi under the control of the Ins1Cre driver line (D-MAT). Confocal microscopy, coupled with biosensors or organic dyes, was used for measurement of Ca2+, cAMP and ATP/ADP. HTRF assay was used to measure insulin secretion. RNAseq was performed using Lexogen QuantSeq 3 mRNA-Seq. Glucose tolerance was assessed by intraperitoneal glucose tolerance test.
Results: Pdx1 and Mafa expression levels were increased, while Ngn3 showed no change. PDX1 and MAFA overexpression was largely restricted to PDX1LOW/MAFALOW β-cells (defined as immature β-cells), and confirmed using Pdx1-BFP islets reporting historic cell PDX1 levels. Loss of immature β-cells presented with impaired islet Ca2+, cAMP and ATP/ADP fluxes, as well as insulin secretion in response to glucose and/or Exendin-4. RNAseq analyses revealed dysregulation of gene pathways involved in carbohydrate and lipid analysis, and upregulation of transcripts involved in inhibitory signalling. Chemogenetic silencing in D-MAT islets showed that immature β-cells were dependent on islet Ca2+ signaling dynamics for their phenotype. Induction of immature β-cell loss in vivo by placing the Tet-MAT animals on doxycycline diet for 2 weeks resulted in glucose intolerance.
Conclusion: The current study redefines immature β-cells as a functionally competent, but islet-dependent β-cell subpopulation. Findings from single-cell screening studies or studies in dissociated cells should be interpreted carefully in light of differences arising from the islet context.