Design of an islet-on-a-chip device reveals glucose-stimulated respiration is substrate limited by glycolytic flux through PKM2

Abstract

Pancreatic islets respond metabolically to glucose by closing KATP channels resulting in Ca2+-influx and insulin secretion. Previous work has revealed the importance of glycolytic flux in triggering insulin secretion. However, it is unclear whether the triggered ( first phase) secretion is further amplified by Ca2+-stimulation of mitochondrial NADH production and/or oxidative phosphorylation (OxPhos). Although commercially available tools have been developed to explore islet metabolism, these methods often overlook islet variability and have poor spatiotemporal resolution. To tease apart first phase glucose-stimulated respiration, we designed an islet-on-a-chip microfluidic device to simultaneously measure O2-consumption rate (OCR) and Ca2+-activity of individual islets with high temporal resolution. We used finite element analysis to optimize placement of sensor in optically clear microwells on a thin glass coverslip. The microfluidic channels were subsequently fabricated using O2-impermeable plastic to limit outside-in diffusion and push islets against the microsensor. We validated our device using living mouse islets and well-established modulators of respiration. By inhibiting glycolysis and mitochondrial pyruvate transport, we show that islet OxPhos is limited by NADH-substrate rather than ADP in low and high glucose. We subsequently imaged glucose-stimulated OCR and Ca2+-influx simultaneously to reveal a biphasic respiratory response that is determined by glycolytic flux through pyruvate kinase (PKM2) and independent of Ca2+. These data demonstrate the unique utility of our modular and optically clear O2-sensor to simultaneously measure glucose-stimulated OCR and Ca2+ activity of multiple individual islets.