The Rab-GTPase-activating proteins TBC1D1 and TBC1D4 (AS160) were previously shown to regulate GLUT4 translocation in response to activation of AKT and AMPdependent kinase. However, knockout mice lacking either Tbc1d1 or Tbc1d4 displayed only partially impaired insulin-stimulated glucose uptake in fat and muscle tissue. The aim of this study was to determine the impact of the combined inactivation of Tbc1d1 and Tbc1d4 on glucose metabolism in double-deficient (D1/4KO) mice. D1/4KO mice displayed normal fasting glucose concentrations but had reduced tolerance to intraperitoneally administered glucose, insulin, and AICAR. D1/4KO mice showed reduced respiratory quotient, indicating increased use of lipids as fuel. These mice also consistently showed elevated fatty acid oxidation in isolated skeletal muscle, whereas insulin-stimulated glucose uptake in muscle and adipose cells was almost completely abolished. In skeletal muscle and white adipose tissue, the abundance of GLUT4 protein, but not GLUT4 mRNA, was substantially reduced. Cell surface labeling of GLUTs indicated that RabGAP deficiency impairs retention of GLUT4 in intracellular vesicles in the basal state. Our results show that TBC1D1 and TBC1D4 together play essential roles in insulin-stimulated glucose uptake and substrate preference in skeletal muscle and adipose cells.
To identify novel disease genes for type 2 diabetes (T2D) we generated two backcross populations of obese and diabetessusceptible New Zealand Obese (NZO/HI) mice with the two lean mouse strains 129P2/OlaHsd and C3HeB/FeJ. Subsequent wholegenome linkage scans revealed 30 novel quantitative trait loci (QTL) for T2D-associated traits. The strongest association with blood glucose [12 cM, logarithm of the odds (LOD) 13.3] and plasma insulin (17 cM, LOD 4.8) was detected on proximal chromosome 7 (designated Nbg7p, NZO blood glucose on proximal chromosome 7) exclusively in the NZOxC3H crossbreeding, suggesting that the causal gene is contributed by the C3H genome. Introgression of the critical C3H fragment into the genetic NZO background by generating recombinant congenic strains and metabolic phenotyping validated the phenotype. For the detection of candidate genes in the critical region (30-46 Mb), we used a combined approach of haplotype and gene expression analysis to search for C3H-specific gene variants in the pancreatic islets, which appeared to be the most likely target tissue for the QTL. Two genes, Atp4a and Pop4, fulfilled the criteria from our candidate gene approaches. The knockdown of both genes in MIN6 cells led to decreased glucosestimulated insulin secretion, indicating a regulatory role of both genes in insulin secretion, thereby possibly contributing to the phenotype linked to Nbg7p. In conclusion, our combined-and comparative-cross analysis approach has successfully led to the identification of two novel diabetes susceptibility candidate genes, and thus has been proven to be a valuable tool for the discovery of novel disease genes.
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