Islet-on-a-chip device reveals first phase glucose-stimulated respiration is substrate limited by glycolysis independent of Ca²⁺activity

Abstract

Pancreatic islets respond metabolically to glucose by closing KATP channels resulting in Ca2+-influx and insulin secretion. Previous work showed that 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). To tease apart the metabolism of first phase glucose-stimulated insulin secretion, 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 the 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 in the device 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 showed islet OxPhos is limited by substrate-NADH 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+. Ultimately, we validated our device and successfully used it to show islet OxPhos is determined by glycolytic flux and not amplified by Ca2+-activity.

Insulin C-peptide secretion on-a-chip to measure the dynamics of secretion and metabolism from individual islets

“…We previously designed several islet-on-a-chip devices to treat and image living pancreatic islets. 32 , 33 , 34 To measure insulin secretion from individual islets, we aimed to adapt one of our islet-on-chip devices for the FAIA of C-peptide ( Figures 2 and S5 ). Microfluidic devices are generally fabricated using PDMS due to biocompatibility, oxygen permeability, and standard fabrication that translates to other research laboratories.…”

Insulin secretion chip (InS-chip) reveals two peaks within first-phase that temporally coincide with a transition in glucose metabolism

“…We previously designed several islet-on-a-chip devices to treat and image living pancreatic islets [28]- [30]. To measure insulin secretion from individual islets, we aimed to adapt one of our islet-on-chip devices for the FAIA of C-peptide (Fig.…”