Mitochondria play a key role in the regulation of insulin secretion by providing ATP and other coupling intermediates for β-cell intermediary metabolism. ATP synthesis is crucial for an increase in the ATP/ADP ratio. An increase in the ATP/ADP ratio triggers closure of ATP sensitive K⁺ channels (K_ATP channels), leading to the K⁺ influx, plasma membrane depolarization, opening of the voltage-sensitive Ca²⁺ channels and Ca²⁺ influx. Influx of Ca²⁺ is then prerequisite for insulin granule exocytosis. Thus, the rise in mitochondrial ATP is an important parameter in β-cell metabolism. Development of strategies enabling real-time monitoring of ATP production in β-cell is of critical importance to understanding metabolic modulation of secretory processes.

Progress

In the past year we created an adenovirus that allows for expression of firefly luciferase in the mitochondria of β-cells. This virus (Ad-mt-Luc), acts as a reporter for intra-mitochondrial ATP concentration. When cells infected with this virus are incubated in the presence of luciferin, the luciferin enters the mitochondria where it is oxidized by luciferase. During this reaction, photons are generated, which can be detected by the photomultiplier (PMT). The intensity of the luminescence measured is proportional to intra-mitochondrial ATP concentration, since luciferase uses ATP to oxidize luciferin.

We have confirmed the usefulness of this mt-Luc reporter in our first series of experiments in which we infected mouse β-cells with the Ad-mt-Luc and then demonstrated that exposure of β-cells to elevated concentrations of glucose results in increased mt-Luc activity. Such experiments were performed using a photomultiplier tube (PMT)-based luminometer that allows the detection of luciferase-catalyzed oxidation of luciferin in a population of cells plated onto glass coverslips. As few as 50,000 cells have been shown to be sufficient to perform an experiment under these conditions.

With these tools in hand, several in house or collaborative projects have been initiated, where measurement of mitochondrial ATP levels was critical to address questions related to the β-cell metabolism and insulin secretion. One of these projects assessed the role of cytosolic malic enzyme in insulin secretion (Heart et al., in press) The other addressed the metabolic role of GLP-1, an incretin, in the β-cell metabolism. (Glucagon Like Peptide-1 and pancreatic b-Cell Fuel and Energy Metabolism. M. Peyot, J.P. Gray, J. Lamontagne, P.J.S. Smith, G. Holz, S.R. M. Madiraju, M. Prentki and E. Heart; manuscript in preparation)

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