Glucose generates sub-plasma membrane ATP microdomains in single islet β-cells -: Potential role for strategically located mitochondria

Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP sensitive K+ (K-ATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MING and primary beta-cells. Resting [ATP] in the cytosol ([ATP](c)), in the mitochondrial matrix ([ATP](m)), and beneath the plasma membrane ([ATP](pm)) were similar (similar to 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP](m) and [ATP](pm) were observed, but only a transient increase in [ATP](c). However, detectable increases in [ATP](c) and [ATP](pm), but not [ATP](m), required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP](c) and [ATP](m) increases but augmented the [ATP](pm) increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP](m) both through enhanced substrate supply and by progressive Ca2+ dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP](pm), which may be essential for the sustained closure of K-ATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.