(DCP014) KNOCK-IN OF MCARDINAL AT THE MOUSE GCK LOCUS AS A TOOL TO EXPLORE PANCREATIC BETA CELL HETEROGENEITY

Background: Glucokinase is a critical beta cell glucose sensor, catalysing the flux-generating step of glycolysis. Immunocytochemical studies more than 30 years ago (Jetton & Magnusson, J Clin Invest, 1982) demonstrated that GCK is expressed heterogeneously in rat pancreatic β cells. More recently, our own findings (Johnston et al, Cell Metab, 2016) indicated that a highly-connected “hub” cell population, representing ~5 % of all beta cells and required for sustained Ca2+ oscillations across the islet, is characterised by an elevated GCK: insulin ratio. By using published scRNASeq data sets it was subsequently possible (Salem et al, Nat Metab, 2019) to impute a discrete transcriptome for hub vs follower cells, revealing an enrichment for genes involved in glycolytic and mitochondrial metabolism in the former.

METHODS AND RESULTS: As a step to towards the purification and study of living hub cells, or other populations based on the level of GCK-expression, we have developed a mouse model in which the far red-shifted fluorescent protein mCardinal is knocked in at the endogenous Gck locus. cDNA corresponding to exons 4-11 of the mouse Gck gene was introduced into the mouse genome immediately downstream of exon 3 by homologous recombination. An internal ribosome entry site then allowed independent expression of mCardinal. Suggesting faithful reporting by the probe of endogenous Gck expression, confocal microscopy revealed highly heterogenous red fluorescence across the beta cell population in isolated islets.

Conclusion: We anticipate that after crossing to mice carrying Ins1-H2B-mCherry transgene this model will provide a useful tool to purify living hub (and follower) beta cells at scale using fluorescence-activated cell sorting based on relative mCardinal: mCherry fluorescence (high in hub cells). Reaggregation of “hub” or “follower” -enriched islets will then permit the molecular (transcriptomic, proteomic etc.) and functional (glucose-stimulated insulin secretion, Ca2+ dynamics, glucose metabolism etc.) characterisation of these sub-populations.