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, D4j, and Y1, are 60.34 6 12.839 years; median 60, range 17–93) with T2D referred to as non-N9a haplogroups in this study. were recruited at the The First Affiliated Hospital of Wenzhou By using the standard protocol (30), transmitochondrial Medical University (Zhejiang, China) from March 2009 to cybrids were generated through the fusion of mtDNA-lacking December 2017. T2D was diagnosed according to the China r0 human osteosarcoma 143B cells with platelets of haplo- Medical Nutrition Therapy Guideline For Diabetes (26). A total group N9a1, N9a10a, G3a2, Y1, or D4j obtained from five of 974 geographically matched and sex-matched control volunteers. Platelets containing blood was collected from participants (mean 6 SD age 53.76 6 15.919 years; median thevolunteerswhentheywere22yearsoldduringphysical 54, range 17–89) with no history of T2D were also recruited examination before their enrollment in the graduate school at the same hospital (at its physical examination center). The of Wenzhou Medical University. The transformant cybrid participants without diabetes were people in whom the clones were cultured in high-glucose DMEM (Thermo Fisher fasting plasma glucose concentration was ,6.1 mmol/L and Scientific, Waltham, MA) containing 10% Cosmic Calf Serum diabetes.diabetesjournals.org Fang and Associates 1443

(Sigma, St. Louis, MO). Pathogenic mtDNA mutations and was determined by sequentially injecting 10 mmol/L glucose, cross contamination during single-clone selection were ruled 1 mmol/L oligomycin, and 50 mmol/L 2-deoxy-D-glucose by out through Sanger sequencing of the mitochondrial ge- using the Seahorse system. nome in both the five volunteers and the cybrid cells during culture (Supplementary Table 1). ATP, Mitochondrial Membrane Potential, NAD+/NADH Ratio, and ROS Measurements mtDNA Content, mtRNA, and Inflammatory Mitochondrial membrane potential (MMP), total ATP Gene Expression content, and NAD+/NADH ratio were determined using Both mtDNA content and the mRNA levels of 13 mito- the cationic fluorescent redistribution dye tetramethyl- chondrially encoded OXPHOS subunits were determined fi (–DDCT) rhodamine, methyl ester (Thermo Fisher Scienti c),anATP using the 2 method as previously described (31). measurement kit (Thermo Fisher Scientific), and an NAD+/ fl Brie y, genomic DNA and total RNA were extracted using NADH ratio assay kit (Abcam), respectively (32). Mito- standard protocols, and the total RNA was then treated chondrial and cytoplasmic ROS production was measured with DNase and reverse-transcribed using random 6-mers using MitoSOX and carboxy-DCFDA (both from Thermo primers (Takara Biotechnology, Dalian, China). Quantita- Fisher Scientific), respectively. Briefly, cells in 12-well tive real-time PCR was performed using primers targeted plates were treated with MitoSOX (5 mmol/L) or carboxy- fl to mtDNA, mtRNA, a subset of in ammatory genes, DCFDA (40 mg/mL) for ,1 h at 37°C in the dark and then and related nuclear housekeeping genes on a StepOne washed with HBSS and analyzed immediately using a fluo- fi Real-Time PCR System (Thermo Fisher Scienti c) by using rescence microscope (Eclipse Ti-E, Nikon Eclipse Ti-S; Nikon SYBR Green qPCR Master Mix (Takara Biotechnology). All Instruments, Inc., Tokyo, Japan). At least five regions were primers used in these analyses are listed in Supplementary quantitatively analyzed for each cybrid to generate the aver- Table 2. age fluorescence intensity in one independent experiment by using ImageJ (Bethesda, MD). Mitochondrial RCC Enzymatic Activity Assay Mitochondria from cultured cells were isolated as pre- viously described (30). The enzymatic activity of four Fluorescence Microscopy for Examining Mitochondrial Morphology RCCs was measured in the mitochondria of cybrids as Cells were incubated with 500 nmol/L MitoTracker Red described (30). The RCC enzymatic activity in each case (Thermo Fisher Scientific) for 30 min and fixed for 15 min was normalized against that of citrate synthase, a mito- with 4% paraformaldehyde at room temperature. The cells chondrial matrix marker enzyme. were then permeabilized with 0.2% Triton X-100 (Sigma), Immunoblotting and Antibodies stained with DAPI (Thermo Fisher Scientific), and exam- Proteins were extracted using RIPA lysis buffer (Cell Signal- ined using an Olympus imaging system (Olympus FV1000; ing Technology, Danvers, MA) supplemented with a protease- Olympus, Melville, NY). Mitochondrial length and com- fi inhibitor cocktail (Sigma-Aldrich). Proteins separated using plexity were quanti ed by measuring the form factor and SDS-PAGE were blotted with these antibodies: anti-VDAC, aspect ratio, respectively. anti-JNK 1/2, anti–phospho-JNK 1/2, anti-p38, anti–phospho- p38 (Thr389), anti-ERK1/2, anti–phospho-ERK (Thr202/ Sample Preparation and RNA Sequencing Tyr204), anti–NF-kB, anti–phospho-NF-kB, anti-SRC, anti– Total RNA was isolated from three biological triplicates of phospho-SRC, anti-MEK1/2, anti–phospho-MEK1/2, anti- each group of cybrids by using an RNeasy Mini extraction kit AMPK, and anti–phospho-AMPK (all from Cell Signaling (Qiagen, Valencia, CA), and mRNA from 20 mgofthetotal fi – Technology;1:1,000);anti-TOMM20,anti-SDHA,anti-RXRA, RNA was puri ed using poly-T attached magnetic beads. fi anti–POLY-g,anti-TFAM,anti-NRF1,anti-AFG3L2,anti-ClpP, After fragmenting the mRNA, rst-strand cDNA was synthe- anti-ClpX, anti-HSP60, anti-PINK1, anti-DRP1, anti–phospho- sized and then sequenced using an Illumina HiSeq 2000 plat- DRP1,anti-OPA1,anti-MFN1,and anti-MFN2 (all from Abcam, form (Illumina, San Diego, CA) as described previously (32). Cambridge, MA; 1:1,000); and anti–b-actin (1:5,000), anti- SOD2 (1:1,000), anti-TFAM (1:1,000), and anti-GRP75 Analysis of Gene Expression Data (1:2,000) (all from Santa Cruz Biotechnology, Santa Cruz, CA). To obtain high-quality reads, reads containing adaptor sequences and poly-N and low-quality reads were removed Measurement of Endogenous Oxygen Consumption from the raw data. Reference-genome and gene-model Endogenous oxygen consumption in intact cells was deter- annotation files were downloaded from genome websites mined using a Seahorse XF24 Extracellular Flux Analyzer directly. The reference genome was built using STAR, and (Seahorse Bioscience, North Billerica, MA) as described in our paired-end high-quality reads were aligned to the reference previous study (2). Briefly, 4 3 104 cells were seeded into genome by using STAR (v2.5.1b). HTSeq v0.6.0 was used to 24-well Seahorse plates together with 250 mLofgrowth count the reads mapped to each gene, after which the medium 1 day before experiments. Oxygen consumption fragments per kilobase million of each gene was calculated rate (OCR) was determined with and without the inclusion based on the length of the gene and the count of the reads of 1 mmol/L oligomycin. Extracellular acidification rate (ECAR) mapped to the gene. Differential expression analysis under 1444 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018 two conditions (three biological replicates per condition) was RESULTS performedusingtheDESeq2Rpackage.DESeq2provides Association of mtDNA Haplogroups With T2D statistical routines for determining differential expression To investigate the relationship between mtDNA haplogroup in digital gene expression data by using a model based on N9a and T2D in the Chinese population, we performed P negative binomial distribution. The resulting values were a large-cohort case-controlled study that included 1,295 adjusted using the Benjamini and Hochberg approach for T2D patients and 974 geographically matched asymptomatic controlling the false discovery rate (33). Genes found using control participants. The frequency of haplogroup N9a was P , DESeq2 that feature an adjusted 0.05 were regarded as found to be significantly higher in patients than in control differentially expressed genes (DEGs). To identify the genes subjects when multivariate logistic regression analysis was that differed between the two N9a and three non-N9a performed with adjustment for age, sex, and haplogroups cybrids, we respectively compared N9a1 and N9a10a with (odds ratio [OR] 1.999 [95% CI 1.229–3.251], P = 0.005) non-N9a cybrids (G3a2, Y1, and D4j), and the genes that (Table 1). By contrast, a significantly decreased frequency of fi fi overlapped in both comparisons were con rmed as the nal haplogroup N9a was previously observed in T2D patients from DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) Korea and Japan (Table 1). This finding suggests that distinct pathway and gene ontology (GO) biological performance populations could present divergent responses in terms of the enrichment analyses of DEGs were implemented using the effect of haplogroup N9a on T2D occurrence. Furthermore, fi clusterPro ler R package. GO and KEGG pathway terms we found that the frequency of haplogroup N9, which com- P , fi featuring values of 0.05 were regarded as signi cantly prised haplogroups Y and N9a, was also significantly higher enriched among DEGs. in T2D patients than in control participants (OR 1.967 [95% CI 1.238–3.124], P = 0.004), but the statistical significance Insulin-Stimulated Glucose Uptake was not retained in the case of ethnic Chinese people from Before measurements, cells were serum starved for 5 h, Taiwan (Table 1). The distribution of haplogroup Y was similar incubated with 0.1 mmol/L insulin plus 2-deoxy-2-[(7- between T2D patients (0.8%) and control subjects (0.6%). nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) (0.1 mmol/L) in a CO2 incubator for 30 min, and then N9a Cybrids Exhibited Lower RCC Activity Than washed thrice with cold phosphate-buffered saline. The fluo- Non-N9a Cybrids rescence signal was measured after 30 min with excitation To analyze the effect of mtDNA haplogroups on the regu- and emission at 485 and 535 nm, by using a Varioskan Flash lation of mitochondrial function, we determined the mtDNA Multimode Reader (Thermo Fisher Scientific). Insulin- content and the RNA level of mtDNA-encoded OXPHOS stimulated glucose uptake was also measured in with the subunits in two N9a and three non-N9a cybrids. The mtDNA presence of N-acetyl-L-cysteine (NAC; Sigma) (15 mmol/L, content in non-N9a cybrids was ;30% higher than that in 24 h), nicotinamide (NAM; Sigma) (15 mmol/L, 24 h), U0126 N9a cybrids (Fig. 1A). The lower mtDNA content measured (Selleckchem, Houston, TX) (50 mmol/L, 6 h), C34 (Sigma) in N9a cybrids was not because of the presence of fewer (10 mmol/L, 30 min), or TAK-242 (MedChemExpress, mitochondria, and the mitochondrial mass was roughly the Monmouth Junction, NJ) (10 mmol/L, 1 h). same in the N9a and non-N9a cybrids (Fig. 1B and E). Next, examination of the RNA level of mtDNA-encoded OXPHOS Statistical Analysis subunits revealed that the RNA levels of ATP8, ND1, ND5, In this case-controlled study, haplogroups featuring a fre- and CO1 were higher in all non-N9a cybrids than in N9a quency of .5% in either the control participants or T2D cybrids (Fig. 1C). This result suggested superior mitochon- patients were analyzed to evaluate the effect of common drial function in non-N9a cybrids than in N9a cybrids (34), mtDNA haplogroups on T2D. Haplogroups that featured and to test this possibility, we examined the activity of three frequencies of ,5% were regarded as “other haplogroups.” RCCs containing mtDNA-encoded subunits. After normali- Multivariate logistic regression analysis was applied to adjust zation of the RCC activities relative to citrate synthase the risks associated with age, sex, and mtDNA haplogroups activity, we found that the activities of complexes I and IV in case and control subjects. T2D was a dependent variable, were significantly higher in non-N9a cybrids than in N9a whereas age, sex, and genotype of each mtDNA haplogroup cybrids, whereas the activity of complex III did not differ were independent variables. In the case of nine mtDNA (Fig. 1D and E). Although the activity of certain RCCs was haplogroups, dummy coding was applied due to their numer- diminished in N9a cybrids, transcriptomic analysis performed ical variability. Bonferroni correction indicating a P value using RNA sequencing technology revealed that N9a cybrids of ,0.006 (0.05/9) was considered statistically significant exhibited increased mRNA levels of most nDNA-encoded when analyzing these nine haplogroups (with the other OXPHOS subunits as compared with non-N9a cybrids (Fig. haplogroups excluded). The significance of “other haplogroups” 1F), and, notably, the mRNA levels of complex II subunits was not considered here because the group included multiple were not significantly affected, particularly those of the mtDNA haplogroups. An independent Student t test was used subunits SDHA and SDHB (Fig. 1F). The observed pattern to evaluate cybrid data, and a null hypothesis was rejected of mtDNA-encoded OXPHOS subunits was not reliable when P , 0.05. All statistical analyses were performed using in the experiment performed here using RNA sequencing, SPSS 21.0 (IBM, Armonk, NY). as the length of poly-A tails varied among distinct diabetes.diabetesjournals.org Fang and Associates 1445

Table 1—Multivariate logistic regression analysis of mitochondrial haplogroups associated with T2D with adjustment for age, sex, and haplogroup Haplogroups Patients (n = 1,295) Control subjects (n = 974) OR (95% CI) P value A 70 (5.4) 57 (5.9) 1.215 (0.777–1.901) 0.394 B4 115 (8.9) 117 (12.0) 1.0 B5 80 (6.2) 61 (6.3) 1.374 (0.892–2.117) 0.150 CZ 74 (5.7) 73 (7.5) 1.014 (0.662–1.553) 0.950 D4 141 (10.9) 111 (11.4) 1.256 (0.869–1.816) 0.225 D5 93 (7.2) 69 (7.1) 1.278 (0.843–1.939) 0.248 F1 107 (8.3) 90 (9.2) 1.203 (0.813–1.781) 0.355 M7 111 (8.6) 83 (8.5) 1.320 (0.888–1.960) 0.169 N9a 73 (5.6) 37 (3.8) 1.999 (1.229–3.251) 0.005† Others* 431 (33.3) 276 (28.3) N9 (Taiwan) 25 (2.9% of 859) 39 (3.4% of 1,151) 0.77 (0.44–1.30) 0.305‡ N9a (Korea) 19 (2.6% of 732) 40 (6.3% of 633) 0.43 (0.24–0.77) 0.005‡ N9a (Japan) 41 (3.2% of 1,289) 79 (4.9% of 1,617) 0.43 (0.24–0.74) 0.004§ Data are n (%) unless otherwise indicated. *Haplogroups with frequencies ,5% in both control subjects and patients. †P , 0.006 (0.05/9), adjusted P value with Bonferroni correction while 9 haplogroups were studied. ‡P , 0.0031 (0.05/10), adjusted P value with Bonferroni correction while 16 haplogroups were studied (17). §P , 0.005 (0.05/10), adjusted P value with Bonferroni correction while 10 haplogroups were studied (18). mitochondrial genes during RNA capture. However, the ratio was lower but mitochondrial function was higher in non- of nDNA-encoded subunits to mtDNA-encoded subunits N9a cells than in N9a cells (Fig. 2C and D), which suggested was found to be higher in N9a cybrids than in non-N9a distinct mitochondrial retrograde signaling pathways be- cybrids when we compared the nDNA-encoded gene expres- tween N9a and non-N9a cells. Accordingly, the mean values sion determined from the transcriptome analysis with the of MMP and total ATP content in non-N9a cells were ;50% mtDNA-encoded gene expression measured using quantita- higher than those measured for N9a cells (Fig. 2E and F). tive real-time PCR analysis (Fig. 1C and F). Furthermore, the Notably, total ATP content and mtDNA content in periph- level of NRF1, an essential transcription factor for nuclear eral blood mononuclear cells (PBMCs) from non-N9a haplo- genes required for respiration, was higher in N9a cybrids group study participants were significantly higher than than in non-N9a cybrids (Fig. 1G), which suggests that those in PBMCs from N9a haplogroup participants (Fig. 2G a retrograde signaling machinery might be involved in and H), which suggested that mitochondrial function was compensatory protection of mitochondrial function in distinctly affected by N9a and non-N9a haplogroups. Fur- N9a cybrids (35). Together with this mitonuclear imbal- thermore, we analyzed mitochondrial fragmentation by ance of OXPHOS subunits, the detection of a higher level measuring the form factor (an index of mitochondrial branch- of the mitochondrial quality-control protein ClpP in N9a ing) and the aspect ratio (an index of mitochondrial branch cybrids than in non-N9a cybrids supported the notion that length) of single mitochondrion in the case of these haplo- N9a cybrids exhibit increased mitochondrial unfolded pro- groups (Fig. 2I); a low degree of mitochondrial fragmentation tein response (mtUPR) as compared with non-N9a cybrids is indicated by high values of aspect ratio and form factor, (Fig. 1H).However,wefoundthatothermtUPRproteinsdid parameters that represent increased mitochondrial length/ not differ between the N9a and non-N9a cybrids (Fig. 1H), width and branching, respectively. Our results revealed a lower and thus the mtUPR level in N9a cybrids might be limited. percentage of mitochondrial fragmentation in non-N9a cells than in N9a cells (Fig. 2J and K). Last, we examined mito- fi Mitochondrial Function Is Lower in N9a Cybrids Than chondrial ssion/fusion proteins and found that the level of Non-N9a Cybrids the long form of OPA1, which is considered to promote Next, we measured mitochondrial respiratory profiles mitochondrial fusion, was higher in non-N9a cells than in L by using a Seahorse XF24 Extracellular Flux Analyzer. N9a cells (Fig. 2 ). Collectively, our results demonstrated that A respiration assay of the cybrids revealed that both in- N9a cybrids exhibit diminished mitochondrial function rela- tracellular respiration and proton leakage were significantly tive to non-N9a cybrids. higher in non-N9a cells than in N9a cells (Fig. 2A). The measured ratio of coupled-to-uncoupled respiration indi- N9a and Non-N9a Cybrids Feature Distinct Profiles of cated that the coupling efficiency did not differ significantly Mitochondrial Signaling Mediators and Transcriptome between N9a and non-N9a cells (Fig. 2B), and an increased Fine-tuning of mitochondrial function can activate diverse ECAR and OCR/ECAR ratio confirmed that glycolytic function retrograde signaling pathways in the nucleus by affecting 1446 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018

Figure 1—N9a cybrids (N9a1 and N9a10a) exhibit lower RCC activity than non-N9a cybrids (D4j, G3a2, and Y1). A: Relative mtDNA content in N9a and non-N9a cybrids. mtDNA content in non-N9a cybrids was normalized relative to that in N9a cells (n = 4). B: Representative Western blot of mitochondrial marker proteins. VDAC, TOMM20, SOD2, and SDHA were stained in whole-cell lysates from N9a and non-N9a cybrids. Actin was used as a total protein loading control. Protein levels in all cybrids were normalized relative to that in N9a1 cybrids. C: mtRNA levels in N9a and non-N9a cybrids (n $3). Relative mtRNA levels in the non-N9a cybrids were normalized to N9a cells. D and E: Enzyme activity levels of mitochondrial complexes I (CI), III (CIII), and IV (CIV) were measured in mitochondria isolated from N9a and non-N9a cybrids (n =4)(D), and mitochondrial complex enzyme activity was normalized with citrate synthase activity (E). F: Heat map showing transcriptional changes of nuclear-encoded OXPHOS subunits in the N9a and non-N9a cybrids (n = 3). Data were obtained by high- throughput RNA sequencing of N9a and non-N9a cybrids. The gradual color change from red to blue represents the changing process from upregulation to downregulation. G: Representative Western blot of RXRA, POLY-g, TFAM, and NRF1 levels in whole-cell extracts of N9a and non-N9a cybrids from 143B cells. Actin was used as a total protein loading control. Protein levels in all cybrids were normalized relative to that in N9a1 cybrids. H: Immunoblotting analysis of the levels of AFG3L2, ClpX, PINK1, GRP75, HSP60, and ClpP in whole-cell extracts of N9a and non-N9a cybrids from 143B cells (n = 3). TOMM20 was used as a loading control. Data are presented as means 6 SD of at least three independent tests per experiment. *P # 0.05, **P # 0.01, ***P # 0.001. OD, optical density.

the levels of mitochondrial signaling mediators (33). Be- ROS probe MitoSOX (Fig. 3C and D), but mitochondrial cause redox signaling pathways play a major role in cellular antioxidant activity did not differ between the cybrids, as physiology, we measured the mitochondrial redox signal, revealed by measurement of the antioxidant protein SOD2 the ROS level, and the NAD+/NADH ratio in N9a and non- (Fig. 1B). Furthermore, the NAD+/NADH ratio was lower in N9a cybrids. N9a cybrids generated more ROS than non- N9a cybrids than in non-N9a cybrids (Fig. 3E). These results N9a cybrids did, as determined using either the cytosolic ROS suggested that N9a and non-N9a cybrids feature distinct probe carboxy-DCFDA (Fig. 3A and B)orthemitochondrial mitochondrial retrograde signaling profiles. diabetes.diabetesjournals.org Fang and Associates 1447

Figure 2—Non-N9a cybrids (D4j, G3a2, and Y1) present higher mitochondrial function and superior mitochondrial morphology than N9a cybrids (N9a1 and N9a10a). A: Mitochondrial OCR was determined in N9a and non-N9a cybrids by using a Seahorse XF24 Extracellular Flux Analyzer. Basal, basal mitochondrial respiration; Oligo, uncoupled mitochondrial respiration, measured in the presence of oligomycin (1 mmol/L) (n = 4). B: Ratios of oligomycin-sensitive to oligomycin-resistant respiration rates calculated from A (n = 4). C: ECAR in N9a and non-N9a cybrids was determined using the Seahorse XF24 Extracellular Flux Analyzer by sequentially injecting 10 mmol/L glucose, 1 mmol/L oligomycin, and 50 mmol/L 2-deoxy-D-glucose (2-DG) (n = 4). D: OCR/ECAR ratios calculated from C (n = 4). E: Relative MMP levels were measured in N9a and non-N9a cybrids treated with 30 nmol/L tetramethylrhodamine for 30 min. Relative MMP levels in non-N9a cybrids were normalized to that in N9a cells (n = 4). MMP values were normalized relative to protein concentration. F and G: Relative ATP content was measured in approximately 1 3 106 cells each of N9a and non-N9a cybrids (n =4)(F) and in approximately 1 3 106 PBMCs from N9a (n = 13) and non-N9a haplogroup (n = 15) participants (G). Relative ATP content in non-N9a cells was normalized to that in N9a cells. MMP values were normalized relative to protein concentration. H: Relative mtDNA content in PBMCs from N9a and non-N9a haplogroup participants (n = 16 each). mtDNA content in non-N9a PBMCs was normalized relative to that in N9a PBMCs. I: Confocal micrographs of N9a and non-N9a cybrids in which mitochondria were stained with MitoTracker Red (n = 3). Images are shown at 6003 magnification. The upper and lower two rows show cybrid cells featuring macro haplogroups N9a and non-N9a haplogroups, respectively; mtDNA haplogroups are shown in yellow. Mitochondrial fragmentation was evaluated by measuring the aspect ratio and form factor; higher values represent increased mitochondrial length/width and branching, respectively. J and K: Quantification of aspect ratio (J) and form factor (K) in N9a and non-N9a cybrids (n = 3). 1448 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018

To uncover the differences between the transcriptome examine how redox signals affect p38 and ERK1/2, we of N9a and non-N9a cybrids, we used high-throughput treated N9a cells, which exhibited higher ROS generation RNA sequencing for transcriptomic profiling of the two and a lower NAD+/NADH ratio than non-N9a cells, with N9a cybrids and three non-N9a cybrids. Our analysis NAC and NAM to reduce ROS generation and increase the revealed 826 statistically significant DEGs between the NAD+/NADH ratio, respectively. Our results showed that two N9a cybrids and three non-N9a cybrids, of which only ERK1/2 phosphorylation was affected by the mito- 604 and 222 genes were upregulated and downregulated, chondrial redox signals in our cybrids (Fig. 5B and C). respectively, in both N9a cybrids as compared with the ERK1/2 activation has been associated with the expression levels in three non-N9a cybrids; moreover, among these of inflammation (36) and with inflammation-induced in- DEGs, 52 genes encoded transcription factors and were sulin resistance (37). Therefore, we measured the mRNA related to signaling pathways such as the ERK1/2 pathway levels of 38 inflammation-related genes, which revealed (Fig. 4A and Supplementary Data Set 1) (full access to the that mRNA levels of four genes, IL13, TLR4, CSF3, and data set is available upon request to the author). We next CCL3, were lower in non-N9a cybrids than in N9a cybrids performed both GO and KEGG pathway enrichment anal- (Fig. 5D). Because TLR4 expression is closely associated yses to determine the contribution of these DEGs in biological with ERK1/2 activation (36), we tested whether TLR4 performance.AsshowninFig.4B and Supplementary Data is downregulated upon ERK1/2 inhibition. Treatment of Set 2 (full access to the data set is available upon request to cells with either U0126, a specific ERK1/2 inhibitor, or NAM, the author), multiple mitochondrial OXPHOS-related path- an effective antioxidant, caused a significant decrease in the ways and three signal transduction pathways (Wnt, ERK1/2, mRNA level of TLR4 (Fig. 5E and F). Physiologically, non- and p38-MAPK) were presented in all 79 GO biological N9a cells exhibited higher insulin-stimulated glucose uptake processes that showed significantly different enrichment than N9a cells (Fig. 5G), whereas administration of the between the two N9a and three non-N9a cybrids. KEGG two antioxidants, NAC and NAM, upregulated insulin- enrichment analysis further revealed that the two N9a and stimulated glucose uptake in N9a cells (Fig. 5H). Further- three non-N9a cybrids exhibit distinct performance in terms more, the upregulation of insulin-stimulated glucose uptake of metabolic regulation and pathways related to metabolic in N9a cells was mimicked when ERK1/2 phosphorylation diseases such as nonalcoholic fatty liver disease (Fig. 4C). The was inhibited in N9a cells through U0126 treatment (Fig. statistical significance of most pathways was retained when 5H). Last, blockage of TLR4 signaling by using two TLR4 we excluded the DEGs encoding OXPHOS subunits (Fig. 4D inhibitors increased the insulin-stimulated glucose uptake and E and Supplementary Data Set 2), which indicated that (Fig. 5I). Our results support the proposal that regardless mitochondrial retrograde signaling contributed substantially of the other signaling pathways that might regulate insulin to the difference in biological performance between the two sensitivity and cellular glucose uptake, mitochondrial re- N9a and three non-N9a cybrids. Notably, although only dox signal–mediated ERK1/2 phosphorylation contributes 90 DEGs remained after we applied the criterion of fold- to the insulin-stimulated glucose uptake, at least partially change .2 (Fig. 4F), the difference in biological performance through TLR4 activation. between N9a and non-N9a cybrids was retained in several aspects (Fig. 4G and H). DISCUSSION Previously, a study showed that haplogroup N9a is asso- Mitochondrial Redox Signal–Mediated ERK1/2 ciated with diminished T2D occurrence (i.e., N9a acts Phosphorylation Contributes to Cellular Glucose a “protective factor”) in both Japanese and Korean Uptake patients (18). However, this reported effect of N9a in We sought to functionally assess how and through which the case of Japanese T2D patients has been challenged pathway mitochondrial retrograde signaling influences (19). In Taiwan, haplogroup B4, but not haplogroup N9, T2D susceptibility in N9a and non-N9a cybrids. Including was found to be associated with T2D (17). Recently, we the candidate ERK1/2 pathway, we tested seven pathways found that mtDNA haplogroup N9a was marginally asso- that are commonly associated with mitochondrial retro- ciated with an increased occurrence of T2D and signifi- grade signaling pathway (33). As expected, ERK1/2 phos- cantly associated with diabetic nephropathy incidence (27). phorylation levels differed between the two N9a and three Here, to evaluate the causal role of haplogroup N9a in T2D, non-N9a cybrids (Fig. 5A), and p38 phosphorylation was we conducted another large-scale case-controlled study, which lower in non-N9a cybrids than in N9a cybrids (Fig. 5A). To confirmed that haplogroup N9a could serve as a risk factor

Aspect ratio and form factor in non-N9a cells were normalized relative to those in N9a cells. L: Representative Western blot of mitochondrial fission and fusion proteins; p-DRP1, total DRP1, OPA1, and MFN1/2 were stained in whole-cell lysates prepared from N9a and non-N9a cybrids. TOMM20 was used as a total protein loading control. Protein levels in all cybrids were normalized relative to that in N9a1 cybrids. Data are presented as means 6 SD of at least three independent tests per experiment. *P # 0.05, **P # 0.01, ***P # 0.001. diabetes.diabetesjournals.org Fang and Associates 1449

Figure 3—Differential mitochondrial redox status between N9a (N9a1 and N9a10a) and non-N9a (D4j, G3a2, and Y1) cybrids. A: Relative cytoplasmic ROS levels in N9a and non-N9a cybrids. Cells were stained with the probe carboxy-DCFDA. Representative images are shown at 2003 magnification. B: Quantification of cytoplasmic ROS levels in N9a and non-N9a cybrids. The ROS level in non-N9a cells was normalized relative to that in N9a cells (n = 3). C: Mitochondrial ROS levels were determined by staining cells with MitoSOX. Representative images are shown at 2003 magnification. D: Quantification of mitochondrial ROS levels in N9a and non-N9a cybrids. The ROS level in non- N9a cells was normalized relative to that in N9a cells (n = 3). E: Cellular NAD+/NADH ratio in N9a and non-N9a cybrids. NAD+ and NADH levels in cell extracts were quantified based on fluorescence intensity (n = 3). Data are presented as means 6 SD of at least three independent tests per experiment. **P # 0.01, ***P # 0.001.

against T2D incidence in China (Table 1). Such conflicting criterion “HbA1c” for the control participants (HbA1c ,6.2% reportsonmtDNApopulationvariantsincommondiseases [44 mmol/mol] in this study, ,5.6% [38 mmol/mol] in havebeenhighlightedbypreviously(38)andarealsoknown Japan/Korea, ,6.0% [2 mmol/mol] in Taiwan). Overall, we to be common in the case of other diseases such as Leber speculate that environment factors might contribute to the hereditary optic neuropathy (39). Several major factors might divergent responses of the same mtDNA in healthy humans contribute to the distinct reported effects of mtDNA lineage because Chinese populations were studied both by us and by on human diseases, including inappropriate research design Liou et al. (17). and statistical performance (20,21), divergent nuclear genetic Cytoplasmic hybrid technology is widely used for in- backgrounds (24), and different environment factors (25). In vestigating the effects of distinct mtDNA haplogroups on the investigation of haplogroup N9a/N9 and T2D, distinct cellular physiological conditions, including insulin sensi- inclusion criteria used for T2D patients might also contribute tivity (32,40,41). The mtDNA-lacking r0 human osteo- to the conflicting conclusions reached regarding the relation- sarcoma 143B cells represent the most accepted cellular ship between haplogroup N9a/N9 and T2D (17,18). Here, we model for studying how mtDNA haplogroups influence did not set a cutoff value for the age of the T2D patients, but cellular functions. Although the use of a disease-related cell the patients included in other studies were aged .40 or .30 model as the nuclear donor is the optimal method to uncover years old (17,18). We do not believe that the use of age as an the pathogenic role of specific mtDNA haplogroups, 143B inclusion criterion affected the results here because only 17 of cells are widely used in the study of Parkinson disease (42), the 1,295 patients were ,30 years old and none of them were T2D (40), Alzheimer disease (41), and Leber hereditary optic genotyped as N9a; by comparison, 94 control participants neuropathy (43). Thus, as in other studies (3,40), we used were ,30 years old, with 8 genotyped as N9a. The T2D risk 143B cells to evaluate how mtDNA haplogroup N9a affects presented by haplogroup N9a would be even higher than that insulin sensitivity. Furthermore, 143B cells express GLUT4, we have reported if these patients and control subjects were which can translocate to the plasma membrane upon insulin excluded. Moreover, although a limited amount of lifestyle stimulation, and by performing glucose uptake experiments, information and clinical data are available for the study par- we obtained data supporting the view that relative to N9a ticipants, it is unclear whether other factors such as smoking cells, the three non-N9a cybrids are more sensitive to insulin contributed to the distinct effects of haplogroup N9a on T2D and exhibit higher levels of insulin-stimulated glucose up- occurrence. The only recognized difference that could affect take (Fig. 5G–I). Our analysis of mitochondrial function by the contribution of these factors might be the inclusion using PBMCs obtained from N9a and non-N9a control 1450 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018

Figure 4—N9a (N9a1 and N9a10a) and non-N9a (G3a2, Y1, and D4j) cybrids feature distinct mitochondrial signaling mediators and transcriptome profiles. A: Venn diagrams showing the numbers of DEGs that were shared by or specific to N9a and non-N9a cybrids and met the threshold of P , 0.05. B and C: Enriched GO biological performance (BP) (B) and KEGG pathways (C) for DEGs with P , 0.05. D and E: Enriched GO BP (D) and KEGG pathways (E) for DEGs with P , 0.05, with the DEGs of OXPHOS pathway subunits being excluded. F: Venn diagrams showing the numbers of DEGs that were shared by or specific to N9a and non-N9a cybrids and met the threshold of P , 0.05 and absolute fold-change .2. G and H: Enriched GO BP (G) and KEGG pathways (H) for DEGs with P , 0.05 and absolute fold-change .2. The horizontal axis (B, C, D, E, G, and H) represents the number of genes in each category. Down, downregulated; Up, upregulated. diabetes.diabetesjournals.org Fang and Associates 1451

Figure 5—Mitochondrial redox signal–mediated ERK1/2 phosphorylation contributes to cellular glucose uptake. A: Representative Western blotting analysis of the relative phosphorylation of NF-kB, ERK1/2, JNK, p38, MEK, SRC, and AMPK in N9a (N9a1 and N9a10a) and non-N9a (D4j, G3a2, and Y1) cybrids. The levels of phosphorylated proteins in all cybrids were normalized relative to the levels in N9a1 cybrids. B and C: Relative phosphorylation of ERK1/2 and p38 in N9a cells in the presence of 5 mmol/L NAC (B) or 5 mmol/L NAM (C). The levels of phosphorylated proteins in N9a1 and N9a10 cybrids treated with NAC or NAM were normalized relative to the levels of phosphorylated proteins in the same cybrids in the absence of NAC or NAM treatment. D: Heat map showing inflammation-related genes that were differentially expressed between N9a and non-N9a cybrids. Data were obtained through quantitative real-time PCR analysis of five cybrids. Relative RNA level was obtained by normalizing to the level in N9a1 cybrids. The gradual color change from red to blue represents the change from upregulation to downregulation. Black arrows, genes upregulated in N9a cybrids. E: mRNA level of TLR4 was measured in N9a cells treated with or without U0126 (50 mmol/L, 6 h) (n = 3). The TLR4 value obtained for U0126-treated N9a cells was normalized relative to that measured for untreated cells. F: TLR4 mRNA level was determined in N9a cells treated with or without NAM (5 mmol/L, 24 h) (n = 3). The TLR4 value obtained for NAM-treated N9a cells was normalized relative to that measured for untreated cells. G–I: Insulin-stimulated glucose uptake in N9a and non-N9a cybrids without any treatment (G) and after treatment with NAC (15 mmol/L, 24 h) (H), NAM (15 mmol/L, 24 h) (H), U0126 (50 mmol/L, 6 h) (H), C34 (10 mmol/L, 30 min) (I), or TAK-242 (10 mmol/L, 1 h) (I). The glucose uptake values were normalized relative to that measured for N9a cells that were not treated with any chemical (n =3–4). Data are presented as means 6 SD of at least three independent tests per experiment. **P # 0.01, ***P # 0.001. participants further indicated that haplogroup N9a could Conflicting reports have been published on the effect of affect mitochondrial function in disparate global populations mtDNA haplogroup on T2D (16–19,22–24,44), and N9a is (Fig. 2G and H). Moreover, distinct nuclear genetic back- a haplogroup regarding which incongruent findings have grounds, such as the presence of NDUFC2 polymorphisms, been reported in Asian populations (17–19); therefore, it is might act as secondary genetic modifiers that enhance or necessary to comprehensively elucidate the biological role reduce the effect of mtDNA haplogroup N9a in T2D (24). of mtDNA haplogroup N9a in the development of T2D. 1452 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018

Previously, we showed that impaired mitochondrial func- overactivation, TLR4 activation was increased and insulin- tion and increased ROS levels played a critical role in T2D stimulated glucose uptake was decreased in N9a cells. (2). Accordingly, we detected lower mitochondrial function However, several questions remain unanswered, such in N9a cybrids than in non-N9a cybrids (D4, G3, and Y1 as whether and how other signaling pathways (e.g., Wnt cybrids) and confirmed that the mitochondrial redox status pathway) are regulated by mitochondrial function, and differed significantly between N9a and non-N9a cybrids by thus further investigation required to completely reveal measuring mitochondrial ROS levels and the NAD+/NADH the underlying role of N9a in T2D. ratio (45–47). Notably, we found that in N9a cells, the In summary, we have presented the most comprehen- mtDNA content was higher than that in non-N9a cells (Figs. sive analysis to date of mitochondrial function, mitochon- 1A and 3H). N9a cells generated higher levels of ROS than drial retrograde signaling, and insulin-stimulated glucose non-N9a cells did, but ROS scavenging did not lead to uptake in the study of mtDNA haplogroups in relation to upregulation of the mtDNA content in N9a cybrids (Sup- T2D. Our findings support a positive association between plementary Fig. 2), and the levels mtDNA replication related the mtDNA haplogroup N9a and T2D and further dem- proteins did not differ between N9a and non-N9a cybrids; onstrate that N9a cells exhibit an altered redox status, these findings suggest that specificSNPsinhaplogroupN9a which might contribute to an increased risk of T2D might affect the mtDNA replication process as previously through mitochondrial retrograde signaling pathways such described (11). In this scenario, the distinct mtDNA repli- as those involving ERK1/2 activation. cation capacities of N9a and non-N9a cybrids might con- tribute to the difference in mitochondrial function (48). Here, we did not detect any TFAM-binding diagnostic SNPs Acknowledgments. The authors thank the members of J.L.’s laboratory in N9a cells, but currently we cannot exclude the possibility for valuable discussions on this work. that the binding abilities of mtDNA replication–related Funding. This work was supported by grants from the Chinese National proteins differ between N9a and other haplogroups. Science Foundation (31671486 and 31501156), Zhejiang Provincial Natural The association of mtDNA haplogroup with degenera- Science Foundation of China (LY15H060007), and Specialized Research Fund tive disease such as Parkinson disease could be due to not for the Doctoral Program of Higher Education (20133321110001). fl only a decline in RCC activity (41), but also the subsequent Duality of Interest. No potential con icts of interests relevant to this article difference in nuclear signaling pathways caused by the were reported. Author Contributions. H.F. and J.L. designed the study. H.F., N.H., Q.Z., disparity in mitochondrial function (33,41). In a previous – B.W., H.Z., Q.F., L.S., X.C., and F.S. produced the data. H.F., N.H., Q.Z., B.W., and study, mtDNA haplogroup responsive retrograde signal- J.L. analyzed the data. H.F. and J.L. wrote the manuscript. H.F. and J.L. are the ing pathways were linked to the insulin pathway (3). Here, guarantors of this work and, as such, had full access to all the data in the study we noted that the insulin pathway was less active, as and take responsibility for the integrity of the data and the accuracy of the indicated by phospho-IRS1 (Y896) levels, and the fold data analysis. increase in plasma membrane GLUT4 was considerably lower in N9a cybrids than in D4 cybrids (3). Therefore, it References appears highly likely that haplogroup N9a represents a risk 1. Absetz P, Valve R, Oldenburg B, et al. Type 2 diabetes prevention in the “real factor for T2D. In this study, we adopted a widely used world”: one-year results of the GOAL Implementation Trial. Diabetes Care 2007; quantitative RNA sequencing technology to identify mi- 30:2465–2470 tochondrial retrograde signaling pathways, which could 2. Chen H, Bai J, Dong F, et al. Hepatic DsbA-L protects mice from diet-induced – uncover the mechanism underlying the effect of N9a in hepatosteatosis and insulin resistance. FASEB J 2017;31:2314 2326 3. Weng SW, Kuo HM, Chuang JH, et al. Study of insulin resistance in cybrid T2D. By performing transcriptome analysis, we deter- cells harboring diabetes-susceptible and diabetes-protective mitochondrial haplo- mined that nuclear-encoded OXPHOS gene expression groups. Mitochondrion 2013;13:888–897 was higher in N9a cybrids than in non-N9a cybrids, which 4. West AP, Shadel GS. Mitochondrial DNA in innate immune responses likely arises as a compensatory effect for mitochondrial and inflammatory pathology. Nat Rev Immunol 2017;17:363–375 redox stress as a result of the reduction in mtRNA levels in 5. Picard M, Zhang J, Hancock S, et al. Progressive increase in mtDNA 3243A.G N9a cybrids (49). We then obtained further evidence in- heteroplasmy causes abrupt transcriptional reprogramming. Proc Natl Acad Sci dicating that N9a might be involved in T2D: most iden- U S A 2014;111:E4033–E4042 tified changes in biological performance were related to 6. Olsson AH, Rönn T, Ladenvall C, et al. Two common genetic variants near metabolic regulation (Fig. 4C). In another study, micro- nuclear-encoded OXPHOS genes are associated with insulin secretion in vivo. Eur array analysis used for gene expression profiling yielded J Endocrinol 2011;164:765–771 data indicating that N9a probably does not represent a pro- 7. Moreno-Loshuertos R, Acín-Pérez R, Fernández-Silva P, et al. Differences in reactive oxygen species production explain the phenotypes associated with tective factor in the case of T2D (14). Here, by targeting common mouse mitochondrial DNA variants. Nat Genet 2006;38:1261–1268 candidate signaling pathways, we demonstrated that mito- – 8. Pravenec M, Hyakukoku M, Houstek J, et al. Direct linkage of mitochondrial chondrial redox signal mediated ERK1/2 phosphorylation/ genome variation to risk factors for type 2 diabetes in conplastic strains. Genome activation, a pathway that has been frequently related to in- Res 2007;17:1319–1326 sulin sensitivity (50), contributes to the differences in insulin- 9. Latorre-Pellicer A, Moreno-Loshuertos R, Lechuga-Vieco AV, et al. Mito- stimulated glucose uptake between N9a and non-N9a chondrial and nuclear DNA matching shapes metabolism and healthy ageing. cells. Furthermore, we found that in response to ERK1/2 Nature 2016;535:561–565 diabetes.diabetesjournals.org Fang and Associates 1453

10. Kazuno AA, Munakata K, Nagai T, et al. Identification of mitochondrial DNA 30. Fang H, Zhang F, Li F, et al. Mitochondrial DNA haplogroups modify the risk polymorphisms that alter mitochondrial matrix pH and intracellular calcium dy- of osteoarthritis by altering mitochondrial function and intracellular mitochondrial namics. PLoS Genet 2006;2:e128 signals. Biochim Biophys Acta 2016;1862:829–836 11. Suissa S, Wang Z, Poole J, et al. Ancient mtDNA genetic variants modulate 31. Fragouli E, Spath K, Alfarawati S, et al. Altered levels of mitochondrial DNA mtDNA transcription and replication. PLoS Genet 2009;5:e1000474 are associated with female age, aneuploidy, and provide an independent measure 12. Ji F, Sharpley MS, Derbeneva O, et al. Mitochondrial DNA variant associated of embryonic implantation potential. PLoS Genet 2015;11:e1005241 with Leber hereditary optic neuropathy and high-altitude Tibetans. Proc Natl Acad 32. Fang H, Zhang F, Li F, et al. Mitochondrial DNA haplogroups modify the risk Sci U S A 2012;109:7391–7396 of osteoarthritis by altering mitochondrial function and intracellular mitochon- 13. Pello R, Martín MA, Carelli V, et al. Mitochondrial DNA background modulates drial signals. Biochim Biophys Acta 2016;1862:829–836 the assembly kinetics of OXPHOS complexes in a cellular model of mitochondrial 33. Chae S, Ahn BY, Byun K, et al. A systems approach for decoding mito- disease. Hum Mol Genet 2008;17:4001–4011 chondrial retrograde signaling pathways. Sci Signal 2013;6:rs4 14. Hwang S, Kwak SH, Bhak J, et al. Gene expression pattern in trans- 34. Taylor RW, Turnbull DM. Mitochondrial DNA transcription: regulating the mitochondrial cytoplasmic hybrid cells harboring type 2 diabetes-associated power supply. Cell 2007;130:211–213 mitochondrial DNA haplogroups. PLoS One 2011;6:e22116 35. Piao Y, Kim HG, Oh MS, Pak YK. Overexpression of TFAM, NRF-1 and myr- 15. Atilano SR, Malik D, Chwa M, et al. Mitochondrial DNA variants can mediate AKT protects the MPP(+)-induced mitochondrial dysfunctions in neuronal cells. methylation status of inflammation, angiogenesis and signaling genes. Hum Mol Biochim Biophys Acta 2012;1820:577–585 Genet 2015;24:4491–4503 36. An H, Yu Y, Zhang M, et al. Involvement of ERK, p38 and NF-kappaB signal 16. Saxena R, de Bakker PI, Singer K, et al. Comprehensive association testing of transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced common mitochondrial DNA variation in metabolic disease. Am J Hum Genet by lipopolysaccharide in mouse dendritic cells. Immunology 2002;106:38–45 2006;79:54–61 37. Reading CL, Stickney DR, Flores-Riveros J, et al. A synthetic anti- 17. Liou CW, Chen JB, Tiao MM, et al. Mitochondrial DNA coding and control inflammatory sterol improves insulin sensitivity in insulin-resistant obese region variants as genetic risk factors for type 2 diabetes. Diabetes 2012;61: impaired glucose tolerance subjects. Obesity (Silver Spring) 2013;21:E343–E349 2642–2651 38. van der Westhuizen FH, Sinxadi PZ, Dandara C, et al. Understanding 18. Fuku N, Park KS, Yamada Y, et al. Mitochondrial haplogroup N9a confers the implications of mitochondrial DNA variation in the health of black Southern resistance against type 2 diabetes in Asians. Am J Hum Genet 2007;80:407–415 African populations: the 2014 workshop. Hum Mutat 2015;36:569–571 19. Takasaki S. Mitochondrial haplogroups associated with Japanese cen- 39. Tong Y, Sun YH, Zhou X, et al. Very low penetrance of Leber’s hereditary tenarians, Alzheimer’s patients, Parkinson’s patients, type 2 diabetic patients and optic neuropathy in five Han Chinese families carrying the ND1 G3460A mutation. healthy non-obese young males. J Genet Genomics 2009;36:425–434 Mol Genet Metab 2010;99:417–424 20. Salas A, Elson JL. Mitochondrial DNA as a risk factor for false positives in 40. Kuo HM, Weng SW, Chang AY, et al. Altered mitochondrial dynamics case-control association studies. J Genet Genomics 2015;42:169–172 and response to insulin in cybrid cells harboring a diabetes-susceptible mito- 21. Fachal L, Mosquera-Miguel A, Pastor P, et al. No evidence of association chondrial DNA haplogroup. Free Radic Biol Med 2016;96:116–129 between common European mitochondrial DNA variants in Alzheimer, Parkinson, 41. Wilkins HM, Carl SM, Swerdlow RH. Cytoplasmic hybrid (cybrid) cell lines as and migraine in the Spanish population. Am J Med Genet B Neuropsychiatr Genet a practical model for mitochondriopathies. Redox Biol 2014;2:619–631 2015;168B:54–65 42. Liou CW, Chuang JH, Chen JB, et al. Mitochondrial DNA variants as genetic 22. Chinnery PF, Mowbray C, Patel SK, et al. Mitochondrial DNA haplogroups and risk factors for Parkinson disease. Eur J Neurol 2016;23:1289–1300 type 2 diabetes: a study of 897 cases and 1010 controls. J Med Genet 2007;44:e80 43. Ghelli A, Porcelli AM, Zanna C, Martinuzzi A, Carelli V, Rugolo M. Protection 23. Feder J, Ovadia O, Blech I, et al. Parental diabetes status reveals association against oxidant-induced apoptosis by exogenous glutathione in Leber hereditary of mitochondrial DNA haplogroup J1 with type 2 diabetes. BMC Med Genet 2009; optic neuropathy cybrids. Invest Ophthalmol Vis Sci 2008;49:671–676 10:60 44. Li S, Besenbacher S, Li Y, et al. Variation and association to diabetes 24. Gershoni M, Levin L, Ovadia O, et al. Disrupting mitochondrial-nuclear in 2000 full mtDNA sequences mined from an exome study in a Danish population. coevolution affects OXPHOS complex I integrity and impacts human health. Eur J Hum Genet 2014;22:1040–1045 Genome Biol Evol 2014;6:2665–2680 45. de Graef MR, Alexeeva S, Snoep JL, Teixeira de Mattos MJ. The steady-state 25. Wallace DC. A mitochondrial paradigm of metabolic and degenerative internal redox state (NADH/NAD) reflects the external redox state and is correlated diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet with catabolic adaptation in Escherichia coli. J Bacteriol 1999;181:2351–2357 2005;39:359–407 46. Blacker TS, Duchen MR. Investigating mitochondrial redox state using NADH 26. Chen W, Jiang H, Tao YX, Shu XL. Development and interpretation of China and NADPH autofluorescence. Free Radic Biol Med 2016;100:53–65 Medical Nutrition Therapy Guideline For Diabetes (2010). Zhongguo Yi Xue Ke Xue 47. Willems PH, Rossignol R, Dieteren CE, Murphy MP, Koopman WJ. Redox Yuan Xue Bao 2011;33:253–256 homeostasis and mitochondrial dynamics. Cell Metab 2015;22:207–218 27. Niu Q, Zhang W, Wang H, Guan X, Lu J, Li W. Effects of mitochondrial 48. Jeng JY, Yeh TS, Lee JW, Lin SH, Fong TH, Hsieh RH. Maintenance haplogroup N9a on type 2 diabetes mellitus and its associated complications. Exp of mitochondrial DNA copy number and expression are essential for preservation Ther Med 2015;10:1918–1924 of mitochondrial function and cell growth. J Cell Biochem 2008;103:347–357 28. Rieder MJ, Taylor SL, Tobe VO, Nickerson DA. Automating the identification of 49. Reinecke F, Smeitink JA, van der Westhuizen FH. OXPHOS gene expression DNA variations using quality-based fluorescence re-sequencing: analysis of the and control in mitochondrial disorders. Biochim Biophys Acta 2009;1792:1113–1121 human mitochondrial genome. Nucleic Acids Res 1998;26:967–973 50. Fujishiro M, Gotoh Y, Katagiri H, et al. Three mitogen-activated protein 29. Yao YG, Kong QP, Bandelt HJ, Kivisild T, Zhang YP. Phylogeographic dif- kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes. ferentiation of mitochondrial DNA in Han Chinese. Am J Hum Genet 2002;70:635–651 Mol Endocrinol 2003;17:487–497