Insulin secretion from beta-cells is affected by deletion of nicotinamide nucleotide transhydrogenase.
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.
Shimomura
Kenju
K
Henry Wellcome Center for Gene Function, Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, United Kingdom.
Galvanovskis
Juris
J
Goldsworthy
Michelle
M
Hugill
Alison
A
Kaizak
Stephan
S
Lee
Angela
A
Meadows
Nicholas
N
Quwailid
Mohamed Mohideen
MM
Rydström
Jan
J
Teboul
Lydia
L
Ashcroft
Fran
F
Cox
Roger D
RD
D000818:Animals; D002118:Calcium / Q000032:analysis; D002460:Cell Line; D020868:Gene Silencing; D006861:Hydrogen Peroxide / Q000032:analysis; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000201:enzymology / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D009250:NADP Transhydrogenases / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation
D007328:Insulin; D006861:Hydrogen Peroxide; D009250:NADP Transhydrogenases; D002118:Calcium