Significance and molecular mechanisms of uncoupling protein 2 during insulin secretion in pancreatic ß-cells
Final Report Abstract
Mitochondrial function and dysfunction play major role for many metabolic diseases but the underlying molecular mechanisms considering mitochondrial energy transduction and ATP output remain unknown in most cases. Quantitative plate-based respirometry in pancreatic beta cell culture used in this project illustrates that energy transduction can be dissected in different modules (ATP synthesis, proton leak, maximal substrate oxidation) and that UCP2 reduces coupling efficiency (energy used for ATP synthesis). We identified a non-canonical regulatory pathway of insulin secretion, involving modulation of mitochondrial reactive oxygen species by UCP2. However, as the absence of UCP2 decreases both absolute values of proton leak as well as substrate oxidation, it was not clear whether UCP2 affects causatively proton leak or substrate oxidation. To resolve this ambiguity, we developed the measurement of absolute mitochondrial membrane potential in adherent cell culture during this project. This task was accomplished towards the end of the project by measuring the kinetics of potentiometric fluorescent dyes microscopically, corrected for various parameters such as mitochondrial-to-cytosol volume fractions. Pairing respiration (mitochondrial flux) and mitochondrial membrane potential (steady state of the proton motive force), and titrating those over a range of membrane potentials will precisely resolve the behavior of mitochondrial oxidative phosphorylation upon manipulation. The established methods will have a major impact in the field of mitochondrial biology as any manipulation of energy metabolism (e.g. genetically, drugs, etc.) can be monitored in the living cell, disease phenotypes can be identified, and the impact on direct mitochondrial output (e.g. ATP/ROS) or indirectly downstream output (e.g. insulin secretion) can be determined. Also important for a broad use was the transfer of the originally microscopic method to a high-throughput plate reader format allowing large-scale screening approaches. We are still in progress to resolve the precise function of UCP2 in beta cells, but more importantly, novel mitochondrial targets that affect energy transduction can now be identified in the cellular system of interest.
Publications
- The regulation and turnover of mitochondrial uncoupling proteins. Biochim Biophys Acta. 2010 Jun-Jul;1797(6-7):785-91
Azzu V, Jastroch M, Divakaruni AS, Brand MD
- Uncoupling protein-2 attenuates glucose-stimulated insulin secretion in INS-1E insulinoma cells by lowering mitochondrial reactive oxygen species. Free Radic Biol Med. 2011;50(5):609-16
Affourtit C, Jastroch M, Brand MD
- Expression of uncoupling proteins in a mammalian cell culture system (HEK293) and assessment of their protein function. Methods Mol Biol. 2012;810:153-64
Jastroch M
- Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria. J Physiol. 2012 [Epub ahead of print]
Gerencser AA, Chinopoulos C, Birket MJ, Jastroch M, Vitelli C, Nicholls DG, Brand MD
