Glucose generates sub-plasma membrane ATP microdomains in single islet beta-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+ (KATP) 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 MIN6 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 ( approximately 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 KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration

Targeting of reporter molecules to mitochondria to measure calcium, ATP, and pH.

The study of isolated mitochondria, dating back to the 60ties, has provided a wealth of information on the biochemical routes allowing these organelles, deriving from the adaptation of primordial symbionts, to couple oxidation of substrates to the production of ATP. In this work, concepts were acquired (such as, to name a few, the chemiosmotic mechanism of energy conservation, the import into mitochondria of most organelle proteins, the existence of a resident genome with a different genetic code encoding the remaining 13 polypeptides) that are now established dogmas of modern biology. At the same time, the availability of highly efficient probes and imaging systems has allowed cell biologists to obtain a deep insight into how extracellular signals are conveyed and translated in living cells. An example of this insight has been the demonstration that a variety of extracellular stimuli cause a rise in intracellular Ca2+ concentration of high spatio-temporal complexity, that in turn is specifically decoded by intracellular effectors. Among these effectors are mitochondria themselves, that, endowed with low-affinity transport system for Ca2+, can however be recruited by microdomains generated in their proximity by the opening of Ca2+ channels