Basic Study
Copyright ©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Diabetes. Aug 15, 2024; 15(8): 1753-1763
Published online Aug 15, 2024. doi: 10.4239/wjd.v15.i8.1753
Functional analysis of the novel mitochondrial tRNATrp and tRNASer(AGY) variants associated with type 2 diabetes mellitus
Yu Ding, Xue-Jiao Yu, Qin-Xian Guo, Jian-Hang Leng
Yu Ding, Qin-Xian Guo, Jian-Hang Leng, Central Laboratory, Hangzhou First People’s Hospital, Hangzhou 310006, Zhejiang Province, China
Xue-Jiao Yu, Clinical Laboratory, Quzhou People’s Hospital, Quzhou 324000, Zhejiang Province, China
Co-first authors: Yu Ding and Xue-Jiao Yu.
Author contributions: Ding Y contributed to conceptualization; Ding Y and Yu XJ contributed to formal analysis; Ding Y, Guo QX, and Leng JH contributed to investigation; Ding Y and Yu XJ contributed to writing-original draft preparation; Ding Y contributed to writing-review and editing; and all authors have read and agreed to the published version of the manuscript.
Supported by the Hangzhou Joint Fund of the Zhejiang Provincial Natural Science Foundation of China, No. LHZY24H020002; Hangzhou Municipal Health Commission, No. ZD20220010; and Quzhou Bureau of Science and Technology, No. 2022K51.
Institutional review board statement: The study was reviewed and approved by the Ethics Committee of Hangzhou First People’s Hospital (Approval No. KY-20240327-0100-01).
Conflict-of-interest statement: The authors declare that they have no conflict of interest to disclose.
Data sharing statement: The datasets for this study will be available from the corresponding authors upon reasonable request.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Yu Ding, MD, Associate Professor, Central Laboratory, Hangzhou First People’s Hospital, No. 261 Huansha Road, Hangzhou 310006, Zhejiang Province, China. dingyu_zj@126.com
Received: February 15, 2024
Revised: May 9, 2024
Accepted: June 18, 2024
Published online: August 15, 2024
Processing time: 162 Days and 0 Hours
Abstract
BACKGROUND

Mutations in mitochondrial tRNA (mt-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 mt-tRNATrpA5514G and tRNASer(AGY)C12237T variants, however, the effects of these mt-tRNA variants on T2DM progression are largely unknown.

AIM

To assess the potential pathogenicity of T2DM-associated m.A5514G and m.C12237T variants at genetic, molecular, and biochemical levels.

METHODS

Cytoplasmic hybrid (cybrid) cells carrying both m.A5514G and m.C12237T variants, and healthy control cells without these mitochondrial DNA (mtDNA) variants were generated using trans-mitochondrial technology. Mitochondrial features, including mt-tRNA 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 mt-tRNA variants.

RESULTS

Compared with control cells, the m.A5514G variant caused an approximately 35% reduction in the steady-state level of mt-tRNATrp (P < 0.0001); however, the m.C12237T variant did not affect the mt-tRNASer(AGY) steady-state level (P = 0.5849). Biochemical analysis revealed that cells with both m.A5514G and m.C12237T 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 (P < 0.05 for all measures). By contrast, the levels of ROS, 8-OhdG and MDA were significantly increased (P < 0.05 for all measures), but mtDNA copy number was not affected by m.A5514G and m.C12237T variants (P = 0.5942).

CONCLUSION

The m.A5514G variant impaired mt-tRNATrp metabolism, which subsequently caused mitochondrial dysfunction. The m.C12237T variant did not alter the steady-state level of mt-tRNASer(AGY), indicating that it may be a modifier of the m.A5514G variant. The m.A5514G variant may exacerbate the pathogenesis and progression of T2DM in this Chinese pedigree.

Keywords: Type 2 diabetes mellitus; Mitochondrial tRNA genes; Novel variants; Oxidative stress; Mitochondrial dysfunctions

Core Tip: We established cytoplasmic hybrid (cybrid) cells with m.A5514G and m.C12237T variants, and control cells without these variants. The m.A5514G variant decreased mt-tRNATrp stability, whereas the m.C12237T variant did not alter the stability of mt-tRNASer(AGY). More severe mitochondrial dysfunction was observed in mutant cybrids than in control cells, indicating that the m.A5514G variant impaired mt-tRNATrp metabolism and mitochondrial functions and increased cellular oxidative stress, which play central roles in type 2 diabetes mellitus (T2DM) progression. By contrast, the m.C12237T variant acted as a modifier of the m.A5514G variant. Our study provides novel insight into the pathophysiology of maternally transmitted T2DM caused by novel mt-tRNA variants.