Description:

Luminescent (photon emitting) reporters offer different perspectives on cell activity than the more conventional fluorescent reporters – the latter requiring an excitation frequency to react. Luminescent reporters, although far less diverse, depend upon and chemical interaction between two molecules where the association is dependent on the target to be measured. In the case being developed here we wish to record the cellular activity of ATP through the photon emitting luciferin/luciferase reaction. Luciferin is membrane permeable and as luciferase is a protein it can targeted and expressed if a suitable vector is available. Acquisition of such vectors, and their testing in our photon counting/imaging systems (see SPID 0094) are the goals of this subproject.

Progress:

In the past year we created an adenovirus that allows for expression of firefly luciferase in the mitochondria of beta-cells. This virus, designated here as mt-Luc, acts as a reporter for intra-mitochondrial ATP concentration because its expression is targeted to the inner mitochondrial matrix. When cells infected with this virus are incubated in a solution containing luciferin, the luciferin enters the mitochondria where it is oxidized by luciferase, thereby generating photons that are detected by luminometry. The intensity of the luminescence measured in this manner is proportional to intra-mitochondrial ATP concentration since luciferase uses ATP to oxidize its substrate luciferin.
We have confirmed the usefulness of this mt-Luc reporter in our first series of experiments in which we infected mouse beta-cells with the virus and then demonstrated that exposure of beta-cells to elevated concentrations of glucose results in increased mt-Luc activity. This result is expected because oxidative beta-cell glucose metabolism stimulates mitochondrial ATP production. 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. Subsequently, we have confirmed that the activity of this reporter is also stimulated by ketoisocaproate (KIC), a cell-permeant substrate for Kreb's cycle-catalyzed generation of hydrogen ions that are used to initiate mitochondrial respiratory chain electron transport that underlies mitochondrial ATP production.
With these tools and protocols in hand, we have now turned our attention to the main focus of Sub Proposal 0076. In year 2 we will assess to what extent GLP-1 or GLP-1 mimetics (e.g. Exendin-4) facilitate glucose-dependent mitochondrial ATP production. Our use of a PMT to perform such experiments is a new approach not previously reported when performing assays of this type. We are particularly interested in determining the physiological significance of prior reports in which GLP-1 was reported to stimulate mitochondrial ATP production in an insulin-secreting cell line known as MIN6 cells. These prior studies of MIN6 cells were performed using single photon imaging techniques that allowed for the measurement of ATP in individual clusters of cells. However, such an approach introduces a sample bias since only a small proportion of the cells are tested for their responsiveness to GLP-1. Thus, it is not clear to what extent most beta cells act in the manner described for MIN6 cells. For this reason, we wish to evaluate the efficacy of GLP-1 in assays of mouse or rat beta-cells in which we measure a population response derived from 10,000-50,000 cells simultaneously. Our rationale for this approach is that if the previously described action of GLP-1 measured in MIN6 is truly of physiological significance, an action of GLP-1 to stimulate mitochondrial ATP production in a large population of beta-cells should be readily demonstratable. The outcome of studies performed in Year 2 are expected to yield two possible results. Either GLP-1 will be shown not to have such an effect on mitochondrial ATP production, or alternatively, no such effect will be confirmed. Regardless of the outcome, we expect to obtain important publication quality information using this approach.