Nicotinamide nucleotide transhydrogenase (NNT) is an inner mitochondrial membrane transmembrane protein involved in regenerating NADPH, coupled with proton translocation across the inner membrane. We have shown that a defect in Nnt function in the mouse, and specifically within the beta-cell, leads to a reduction in insulin secretion. This chapter describes methods for examining Nnt function in the mouse. This includes generating in vivo models with point mutations and expression of Nnt by transgenesis, and making in vitro models, by silencing of gene expression. In addition, techniques are described to measure insulin secretion, calcium and hydrogen peroxide concentrations, membrane potential, and NNT activity. These approaches and techniques can also be applied to other genes of interest.
Journal article
2009-01-01T00:00:00+00:00
457
451 - 480
29
Animals, Calcium, Cell Line, Gene Silencing, Hydrogen Peroxide, Insulin, Insulin Secretion, Insulin-Secreting Cells, Membrane Potential, Mitochondrial, Mice, Mice, Inbred C3H, Mice, Inbred C57BL, Mice, Transgenic, Microscopy, Confocal, Mitochondria, NADP Transhydrogenases, Point Mutation