Functional analysis of the novel mitochondrial tRNA and tRNA variants associated with type 2 diabetes mellitus.

BACKGROUND

Mutations in mitochondrial tRNA () genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus (T2DM). We pre-viously reported a large Chinese pedigree with maternally inherited T2DM that harbors novel and variants, however, the effects of these variants on T2DM progression are largely unknown.

AIM

To assess the potential pathogenicity of T2DM-associated and variants at genetic, molecular, and biochemical levels.

METHODS

Cytoplasmic hybrid (cybrid) cells carrying both and variants, and healthy control cells without these mitochondrial DNA (mtDNA) variants were generated using trans-mitochondrial technology. Mitochondrial features, including steady-state level, levels of adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mtDNA copy number, nicotinamide adenine dinucleotide (NAD)/NADH ratio, enzymatic activities of respiratory chain complexes (RCCs), 8-hydroxy-deo-xyguanine (8-OhdG), malondialdehyde (MDA), and superoxide dismutase (SOD) were examined in cell lines with and without these variants.

RESULTS

Compared with control cells, the variant caused an approximately 35% reduction in the steady-state level of ( < 0.0001); however, the variant did not affect the steady-state level ( = 0.5849). Biochemical analysis revealed that cells with both and variants exhibited more severe mitochondrial dysfunctions and elevated oxidative stress than control cells: ATP, MMP, NAD/NADH ratio, enzyme activities of RCCs and SOD levels were markedly decreased in mutant cells ( < 0.05 for all measures). By contrast, the levels of ROS, 8-OhdG and MDA were significantly increased ( < 0.05 for all measures), but mtDNA copy number was not affected by and variants ( = 0.5942).

CONCLUSION

The variant impaired metabolism, which subsequently caused mitochondrial dysfunction. The variant did not alter the steady-state level of , indicating that it may be a modifier of the variant. The variant may exacerbate the pathogenesis and progression of T2DM in this Chinese pedigree.