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.
Henry Wellcome Center for Gene Function, Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, United Kingdom.
Kenju
K
Shimomura
Juris
J
Galvanovskis
Michelle
M
Goldsworthy
Alison
A
Hugill
Stephan
S
Kaizak
Angela
A
Lee
Nicholas
N
Meadows
Mohamed Mohideen
MM
Quwailid
Jan
J
Rydström
Lydia
L
Teboul
Fran
F
Ashcroft
Roger D
RD
Cox
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