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ENU mutagenesis is a powerful tool for the identification of novel models of human disease. Haploinsufficiency of the insulin receptor (IR) and associated mild insulin resistance has been utilised to sensitise an ENU screen to identify novel mutations resulting in impaired glucose tolerance and diabetes. An intraperitoneal glucose tolerance test (IPGTT) was utilised in a phenotype driven screen to identify F1 individuals with impaired glucose tolerance. Animals deviating more than 2 SDs from an unmutagenised control population entered inheritance testing. Here we describe two dominant mutations in the highly conserved HMG box of the transcription factor Sox4, one identified in the phenotype driven screen and the other subsequently obtained from an embryonic screen. Sox4 has previously been associated with pancreas development and, as expected, homozygotes for either mutation or compound heterozygotes were embryonic lethal, suggesting both mutations affect gene function and are nulls. Heterozygous Sox4 adult mice had mildly impaired glucose tolerance, and insulin secretion assays in both whole animals and in isolated islets indicated a defect in insulin secretion. In association with IR+/- induced insulin resistance, this insulin secretory defect causes a more severe impaired glucose tolerance and represents a novel polygenic model of type 2 diabetes. Knockdown of Sox4 in INS1 cells with two different siRNAs, targeted to separate regions of the gene, abolished glucose-stimulated insulin release. Interestingly, we also observed a significant decrease in insulin release in response to tolbutamide in Sox4 siRNA treated cells. Tolbutamide stimulates insulin secretion in pancreatic beta cells by closing KATP channels in the plasma membrane, leading to membrane depolarisation and activation of voltage-gated calcium channels. This results in calcium influx and a rise in intracellular calcium ([Ca2+]i) that triggers insulin release. Changes in [Ca2+]i evoked by glucose in Sox4-siRNA treated cells, however, were normal, indicating that KATP channels close normally. Taken together with the impaired secretory response to tolbutamide, this suggests the insulin secretory defect produced by Sox4 knockdown lies downstream of calcium signalling, perhaps at the level of exocytosis. [ABSTRACT FROM AUTHOR]