Insulin release from pancreatic β-cells is regulated by glucose metabolism. When plasma glucose levels rise, β-cell metabolism is stimulated causing intracellular ATP levels to increase, and thereby ATP-sensitive potassium (K_ATP) channels to close. This results in membrane depolarisation, activation of voltage-gated Ca²⁺ channels, influx of Ca²⁺ and exocytosis of insulin-containing vesicles. K_ATP channels in the β-cells of the inbred mouse strain, C57BL/6J, show impaired closure in response to glucose metabolism (1). However, they retain normal ATP sensitivity indicative of a defect further upstream perhaps at the level of β-cell metabolism (1). The glucose intolerance and reduced insulin secretion in these mice result in a phenotype reminiscent of human type-2 diabetes. Through quantitative trait loci (QTL) mapping we identified nicotinamide nucleotide transhydrogenase (Nnt) as a strong candidate gene for the glucose intolerant phenotype in C57BL/6J mice. C57BL/6J mice have a multi-exon deletion in Nnt and >7-fold lower gene expression compared with controls. Nnt is a nuclear encoded mitochondrial gene catalysing the reversible reduction of NADP⁺ by NADH. To investigate the role of Nnt more closely, we used siRNA to knock down the expression of Nnt in the insulin-secreting cell line MIN6. Intracellular calcium and insulin secretion were measured in response to increasing extracellular glucose. Insulin secretion in response to external glucose (10mM) is substantially reduced (12.5±1.8ng/ml, Nnt siRNA (n=6) compared with 34.7±0.7ng/ml, nonsense siRNA (n=6)). Likewise, the glucose-dependent increase in [Ca²⁺]_i was dramatically decreased (87.9±13.6nM, Nnt siRNA (n=21) compared with 180.4±11.6nM, nonsense siRNA (n=18)). We also identified two ENU-induced point mutations in the gene. These mutants were recovered as live mice by IVF, and the progeny were intercrossed to produce mice that are homozygous for the mutations. Intraperitoneal glucose tolerance tests (IPGTT), under local anaesthetic, at 12 and 16 weeks of age showed that both homozygous and heterozygous Nnt mutant mice are significantly glucose intolerant, in both males and females. They also have reduced insulin secretion in IPGTT. In conclusion, a functional linkage between Nnt and both glucose intolerance and reduced insulin secretion was established. We hypothesise that Nnt knockdown impairs β-cell mitochondrial metabolism leading to less ATP production, and thereby lowered K_ATP channel activity. Consequently, glucose-dependent β-cell electrical activity and insulin secretion are impaired.