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Full Text (PDF) Diabetes Volume 67, July 2018 1441 mtDNA Haplogroup N9a Increases the Risk of Type 2 Diabetes by Altering Mitochondrial Function and Intracellular Mitochondrial Signals Hezhi Fang,1 Nianqi Hu,1 Qiongya Zhao,1 Bingqian Wang,1 Huaibin Zhou,1 Qingzi Fu,1 Lijun Shen,1 Xiong Chen,2 Feixia Shen,2 and Jianxin Lyu1,3 Diabetes 2018;67:1441–1453 | https://doi.org/10.2337/db17-0974 GENETICS/GENOMES/PROTEOMICS/METABOLOMICS Mitochondrial DNA (mtDNA) haplogroups have been as- mitochondrial oxidative phosphorylation (OXPHOS) is sociated with the incidence of type 2 diabetes (T2D); how- widely accepted as one of the major causes of T2D and ever, their underlying role in T2D remains poorly elucidated. insulin resistance (2). Diminished OXPHOS function might Here, we report that mtDNA haplogroup N9a was associ- contribute generally to insulin resistance through elevated ated with an increased risk of T2D occurrence in Southern generation of reactive oxygen species (ROS) production, China (odds ratio 1.999 [95% CI 1.229–3.251], P = 0.005). By a major regulatory signal in T2D-related insulin receptor using transmitochondrial technology, we demonstrated signaling and inflammation (3,4). that the activity of respiratory chain complexes was lower The OXPHOS pathway comprises five complexes, of in the case of mtDNA haplogroup N9a (N9a1 and N9a10a) which four complexes are dually regulated by nuclear DNA than in three non-N9a haplogroups (D4j, G3a2, and Y1) and (nDNA) and mitochondrial DNA (mtDNA); thus, as expected, that this could lead to alterations in mitochondrial func- variants in both nuclear and mitochondrial genomes have tion and mitochondrial redox status. Transcriptome anal- been associated with T2D (5,6). An mtDNA haplogroup is ysis revealed that OXPHOS function and metabolic fi fi regulation differed markedly between N9a and non-N9a aspeci cmtDNAgeneticbackgroundde ned by variants in cybrids. Furthermore, in N9a cybrids, insulin-stimulated glu- human mtDNA (i.e., single nucleotide polymorphisms [SNPs]) cose uptake might be inhibited at least partially through that are inherited during long-term evolution. Initial evidence fl enhanced stimulation of ERK1/2 phosphorylation and sub- indicated that mtDNA haplogroups in uence cellular respira- sequent TLR4 activation, which was found to be mediated tion and ROS production, which implied the importance of by the elevated redox status in N9a cybrids. Although it mtDNA haplogroups in the regulation of mitochondrial func- remains unclear whether other signaling pathways (e.g., tion (7). Subsequently, mtDNA haplogroups were shown to Wnt pathway) contribute to the T2D susceptibility of haplo- play a pathophysiological role in rats with T2D (8) and regulate group N9a, our data indicate that in the case of mtDNA physical performance in mice (9). Shortly thereafter, diagnostic haplogroup N9a, T2D is affected, at least partially through SNPs of two human macro haplogroups, M and N, were shown ERK1/2 overstimulation and subsequent TLR4 activation. to alter mitochondrial matrix pH and intracellular calcium dynamics (10), and mtDNA haplogroups have thus far been reported to regulate mtDNA replication and transcriptional Millions of people worldwide live with diabetes, and .90% efficiency (11), the activity of respiratory chain complex (RCC) of these people have been diagnosed with type 2 diabetes I, and the assembly dynamics of multiple RCCs (12,13). Re- (T2D) (1). Although the molecular mechanisms underly- cently, to comprehensively elucidate the mechanisms under- ing T2D remain incompletely elucidated, deregulation of lying the roles of mtDNA haplogroups in diseases such as 1Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Received 14 August 2017 and accepted 26 April 2018. Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life This article contains Supplementary Data online at http://diabetes Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0974/-/DC1. 2Department of Endocrinology, The First Affiliated Hospital of Wenzhou Medical H.F., N.H., Q.Z., and B.W. contributed equally to this work. University, Wenzhou Medical University, Wenzhou, Zhejiang, China 3Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital of Hangzhou © 2018 by the American Diabetes Association. Readers may use this article as Medical College, Hangzhou, Zhejiang, China long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals Corresponding authors: Hezhi Fang, [email protected], and Jianxin Lyu, ljx@ .org/content/license. wmu.edu.cn or [email protected]. 1442 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018 T2D, mitochondrial retrograde signaling has been frequently blood glycosylated hemoglobin (HbA1c)levelwas,6.2% analyzed using microarray or RNA sequencing technologies (44 mmol/mol). We have described 675 patients and 649 control (9,14). This approach has yielded clues regarding disease- subjects used here in a previous study (27). Informed consent causing factors such as alterations in gene methylation status was obtained from all participants under protocols approved by and shifts in metabolic pathways in diseases like T2D (14,15). the ethics committee of Wenzhou Medical University. All Mitochondrial haplogroups play a critical role in both experimental methods were performed in accordance with mitochondrial function and mitochondria-mediated sig- approved guidelines of Wenzhou Medical University. naling pathways; accordingly, mtDNA haplogroups have been suggestedtobeinvolvedinaseriesofmetabolicdiseasessuch mtDNA Sequencing and Genotyping as metabolic syndrome, obesity, T2D, and T2D-associated Genomic DNA from peripheral blood was extracted using complications in distinct populations (16–19). However, cer- a standard SDS lysis protocol. Complete mtDNA sequence tain contradictory observations remain unresolved, particu- was Sanger sequenced for 347 T2D patients and 383 con- larly in studies related to T2D. As in the case of studies trol subjects by using 24 previously reported pairs of reporting varying disease phenotypes associated with dis- mtDNA primers (28). The mtDNA of 235 T2D patients orders such as Alzheimer disease and Parkinson disease, and 141 control subjects was completely sequenced pre- several pitfalls in the T2D studies might account for a few of viously (27); for all other study participants, Sanger se- the contradictions (20,21). However, differences in the quencing was performed using two pairs of mtDNA primers nuclear genetic background might be responsible for most (Supplementary Table 1). SNPs of each participant were of the discrepancies, which have been found in populations identified by comparing the obtained sequences with the from both Asia (16–19) and Europe (16,22–24). One po- revised Cambridge Reference Sequence by using CodonCode tential underlying reason is that the nuclear genetic back- Aligner 3.0.1 (CodonCode Corporation, Centerville, VA). We ground might contribute to the difference in the functional used the HaploGrep program (http://haplogrep.uibk.ac.at/) performance of mitochondria, which has been referred to in to annotate the mtDNA haplogroup for the cases where the a recent study as mitochondrial–nuclear coevolution (24). mtDNA was completely sequenced. For all other study par- Another reason is that the environment can also contrib- ticipants, mtDNA haplogroup was assigned by comparing ute to divergent responses of the same mtDNA in human the target SNPs from the D-loop, ND3, and ND4L with the diseases (25). Nevertheless, how mtDNA haplogroups af- diagnostic SNPs of the most up-to-date Chinese mtDNA fect T2D is currently unknown. For example, NDUFC2 has haplogroup tree (29). been recognized to influence the disease susceptibility of mtDNA haplogroup HV in T2D, but whether and how Generation of Cell Lines and Culture Conditions haplogroup HV itself affects the susceptibility remains Two N9a haplogroups (N9a1 and N9a10a) were used to unresolved (23,24). exclude the effect of private SNPs (such as mt.13214) In this study, we conducted a large-scale case-controlled in the terminal clades of the mtDNA tree (Supplementary study to validate the effect of mtDNA haplogroup N9a on Fig. 1). As control haplogroups, we included haplogroups G the pathogenesis of T2D in the Han Chinese population. (G3 in this study) and D4 (D4j in this study), both of which Because nuclear gene expression is unfailingly altered were not positively associated with metabolic diseases in when mitochondrial function is affected in haplogroup previous work and were evenly distributed in this study N9a, we analyzed the mitochondrial retrograde signaling among T2D patients (haplogroup G, 2.5%; haplogroup D4, in two N9a cybrids and three cybrids other than N9a. 10.9%) and control subjects (haplogroup G, 2.2%; haplo- Last, we tested the effect of alterations in this retrograde group D4, 11.4%). Moreover, haplogroup Y (Y1 in this signaling on diabetes by using a cellular model. study) (0.8% in T2D patients; 0.6% in control subjects), which forms a neighboring clade of haplogroup N9a, was included as additional control haplogroup to exclude the RESEARCH DESIGN AND METHODS potential phenotypic effects produced by haplogroup N9- Study Participants defining SNPs such as mt.5417 (Supplementary Fig. 1). In this study, 1,295 unrelated patients (mean 6 SD age These three control haplogroups, G3,
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