The Role Of Mitochondrial TRNA Mutations Associated With Maternally Transmitted Diabetes

Type 2 diabetes mellitus (T2DM [MIM125853]) is a group of metabolic diseases characterized by high blood glucose. It is often accompanied by the disorder of carbohydrate, fat and protein metabolism. Long-standing hyperglycemia can induce chronic damage and dysfunction of many tissues and organs including eyes, kidneys, heart, vessels, and nerves. Genetic and enviromental factors are involved in T2DM. The maternal transmission of diabetes in several studies further supported that mitochondrial DNA (mtDNA) mutation may be one of the molecular basis of the pathogenesis of diabetes. As part of the genetic screening program for T2DM in the Chinese population, we performed a clinical, genetic, molecular and biochemical characterization of two Han Chinese families with materally transmitted diabetes. Mutational analysis of their mitochondrial genomes identified the glycine transfer RNA (tRNAGly) T10003C mutation belonging to the Eastern Asian haplogroup Mllb and the glutamic acid transfer RNA (tRNAGlu) A14692G mutation belonging to the Eastern Asian haplogroup B5.The primary and secondary structures of human mitochondrial transfer RNAs (tRNAs) differ significantly from those of canonical bacterial and cytoplasmic tRNAs, and tRNAs in human mitochondria are less thermodynamically stable as they generally contain higher numbers of mismatched and A/U base pairs. Meanwhile, human mitochondrial tRNAs are highly susceptible to point mutations, which are primary causes of mitochondrial dysfunctions associated with a wide range of pathologies.The T10003C mutation is localized at a highly conserved nucleotide (U13) of tRNAGly, which is important for the stability and identity of tRNA. Tha anticipated formation of a base-pairing (13C-22G) at the D-stem by the T10003C mutation might affect secondary structure to decrease the steady-state level and affect function of tRNAGly. Functional significance of this mutation is further supported by an ～70% reduction in the steady-state level of tRNAGly observed in cells carrying the T10003C mutation, relative to that in control cell lines.The decrease of the tRNAGly caused an ampairment of mitochondrial protein synthesis. An in vivo mitochondrial protein labeling analysis showed -32.5% reduction in the rate of mitochondrial translation in muatant cells. Then the altered mitochondrial protein synthesis led to the decreasing activities of the enzyme complexes of the respiratory chain, thus causing defects in overall respiratory capacity. The basal oxygen consumption rate (OCR), adenosine triphosphate (ATP)-linked OCR, proton leak OCR, maximal OCR, reserve OCR and non-mitochondrial OCR in mutant cell lines were ～53%,45%,84%,40%,39% and 32%, relative to the mean value measured in the control cell lines respectively. Abnormal mitochondrial respiration resulted in increasing reactive oxygen species (ROS) production, uncoupling of the oxidative pathways for ATP synthesis, and subsequent failure of cellular energetic process, contributed to the pathogenesis of T2DM.The m.A14692G mutation located the highly conserved uridine at position 55 (U55) in the TΨC loop of tRNAGlu, in the elbow domain of the L-shaped structure that is critical for tRNA folding and structure stability. In particular, the long-distance base pair (U55:A18) is formed by the pseudouridine (Ψ) modified from U55, with adenine at position 18 (A18) of tRNAGlu. Thus, it is anticipated that the primary defect by the m.A14692G mutation destabilizes the base-pairing (18A-Ψ55), and subsequently alter the structure and function of tRNAGlu. In PAGE analysis under nature conditions, wild type (U55) tRNAGlu transcript slightly migrated faster than mutant (C55) tRNAGlu transcript. The perturbed tertiary structure then makes the mutant tRNAGluto be unstable and more subject to degradation. The stability of tRNA transcripts was examined by the measurement of the melting temperatures (Tm) by calculating the derivatives of the absorbance against a temperature curve. The results showed that the Tms for wild-type (U55) and mutant (C55) tRNAGlu transcripts were 50℃ and 46℃ respectively. This suggested that the global folding of mutant tRNAGlu was less stable at high temperature than that of wild-type tRNAGlu. Furthermore, -60% reductions in the steady-state level of tRNAGlu were observed in the cell lines carrying the m.A14692G mutation, relative to that in the wild type cell lines. The failure of tRNAGlu metabolism resulted in variable reductions in mtDNA-encoded polypeptides in mutant cells, ranging from 20% to 66%, with the average of ～29% reduction, as compared with those of control cells. The basal OCR, ATP-linked OCR, proton leak OCR, maximal OCR, reserve OCR and non-mitochondrial OCR in mutant cell lines were 59.5%,60%,56%,64%,67%,71.5%, relative to those of control cells. In present study,39% drop was found in mitochondrial ATP production in mutant cells, which may be caused by the defective activity of complex I and IV. Moreover, the deficient activities of respiratory chain complexes led to 35% reduction of mitochondrial membrane potentials, as well as 33% increase the ROS production.Thus, our finding suggested that m.T10003C and m.A14692G mutations are an inherited risk factor necessary for the development of diabetes. The variable phenotypes, late onset and incomplet penetrance of diabetes observed in two Chinese families indicated that these mutations may by themselves be insufficient to lead to a clinical phenotype and the modifiers should contribute to the phenotypic manifestation, especially genes required for the mitochodrial tRNA metabolism. Moreover, epigenetic and enviromental factors may also be involved in the development of diabetes.