Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

Aims/hypothesis ATP links changes in glucose metabolism to electrical activity, Ca²⁺ signalling and insulin secretion in pancreatic beta cells. There is evidence that beta cell metabolism oscillates, but little is known about ATP dynamics at the plasma membrane, where regulation of ion channels and exocytosis occur. Methods The sub-plasma-membrane ATP concentration ([ATP]_pm) was recorded in beta cells in intact mouse and human islets using total internal reflection microscopy and the fluorescent reporter Perceval. Results Glucose dose-dependently increased [ATP]_pm with half-maximal and maximal effects at 5.2 and 9 mmol/l, respectively. Additional elevations of glucose to 11 to 20 mmol/l promoted pronounced [ATP]_pm oscillations that were synchronised between neighbouring beta cells. [ATP]_pm increased further and the oscillations disappeared when voltage-dependent Ca²⁺ influx was prevented. In contrast, K⁺-depolarisation induced prompt lowering of [ATP]_pm. Simultaneous recordings of [ATP]_pm and the sub-plasma-membrane Ca²⁺ concentration ([Ca²⁺]_pm) during the early glucose-induced response revealed that the initial [ATP]_pm elevation preceded, and was temporarily interrupted by the rise of [Ca²⁺]_pm. During subsequent glucose-induced oscillations, the increases of [Ca²⁺]_pm correlated with lowering of [ATP]_pm. Conclusions/interpretation In beta cells, glucose promotes pronounced oscillations of [ATP]_pm, which depend on negative feedback from Ca²⁺ _. The bidirectional interplay between these messengers in the sub-membrane space generates the metabolic and ionic oscillations that underlie pulsatile insulin secretion.

OBJECTIVE cAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown. RESEARCH DESIGN AND METHODS The subplasma-membrane cAMP concentration ([cAMP]_pm) was recorded in α- and β-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and β-cells. RESULTS In islets exposed to 3 mmol/L glucose, [cAMP]_pm was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP]_pm, often with oscillations synchronized among β-cells. Whereas glucagon also induced [cAMP]_pm oscillations in most α-cells, <20% of the α-cells responded to GLP-1. Elevation of the glucose concentration to 11–30 mmol/L in the absence of hormones induced slow [cAMP]_pm oscillations in both α- and β-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in the β-cells but not caused by the changes in [Ca²⁺]_i. The transmembrane adenylyl cyclase (AC) inhibitor 2′5′-dideoxyadenosine suppressed the glucose- and hormone-induced [cAMP]_pm elevations, whereas the preferential inhibitors of soluble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-β-triol perturbed cell metabolism and lacked effect, respectively. CONCLUSIONS Oscillatory [cAMP]_pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]_pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.

Insulin secretion from pancreatic β-cells is tightly regulated by glucose and other nutrients, hormones, and neural factors. The exocytosis of insulin granules is triggered by an elevation of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and is further amplified by cyclic AMP (cAMP). Cyclic AMP is formed primarily in response to glucoincretin hormones and other G(s)-coupled receptor agonists, but generation of the nucleotide is critical also for an optimal insulin secretory response to glucose. Nutrient and receptor stimuli trigger oscillations of the cAMP concentration in β-cells. The oscillations arise from variations in adenylyl cyclase-mediated cAMP production and phosphodiesterase-mediated degradation, processes controlled by factors like cell metabolism and [Ca(2+)](i). Protein kinase A and the guanine nucleotide exchange factor Epac2 mediate the actions of cAMP in β-cells and operate at multiple levels to promote exocytosis and pulsatile insulin secretion. The cAMP signaling system contains important targets for pharmacological improvement of insulin secretion in type 2 diabetes.