mtdna haplogroup n9a increases the risk of type 2 diabetes ... · mtdna haplogroup hv in t2d, but...

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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 Lyu 1,3 Diabetes 2018;67:14411453 | https://doi.org/10.2337/db17-0974 Mitochondrial DNA (mtDNA) haplogroups have been as- sociated with the incidence of type 2 diabetes (T2D); how- ever, their underlying role in T2D remains poorly elucidated. Here, we report that mtDNA haplogroup N9a was associ- ated with an increased risk of T2D occurrence in Southern China (odds ratio 1.999 [95% CI 1.2293.251], P = 0.005). By using transmitochondrial technology, we demonstrated that the activity of respiratory chain complexes was lower in the case of mtDNA haplogroup N9a (N9a1 and N9a10a) than in three non-N9a haplogroups (D4j, G3a2, and Y1) and that this could lead to alterations in mitochondrial func- tion and mitochondrial redox status. Transcriptome anal- ysis revealed that OXPHOS function and metabolic regulation differed markedly between N9a and non-N9a cybrids. Furthermore, in N9a cybrids, insulin-stimulated glu- cose uptake might be inhibited at least partially through enhanced stimulation of ERK1/2 phosphorylation and sub- sequent TLR4 activation, which was found to be mediated by the elevated redox status in N9a cybrids. Although it remains unclear whether other signaling pathways (e.g., Wnt pathway) contribute to the T2D susceptibility of haplo- group N9a, our data indicate that in the case of mtDNA haplogroup N9a, T2D is affected, at least partially through ERK1/2 overstimulation and subsequent TLR4 activation. Millions of people worldwide live with diabetes, and .90% of these people have been diagnosed with type 2 diabetes (T2D) (1). Although the molecular mechanisms underly- ing T2D remain incompletely elucidated, deregulation of mitochondrial oxidative phosphorylation (OXPHOS) is widely accepted as one of the major causes of T2D and insulin resistance (2). Diminished OXPHOS function might contribute generally to insulin resistance through elevated generation of reactive oxygen species (ROS) production, a major regulatory signal in T2D-related insulin receptor signaling and inammation (3,4). The OXPHOS pathway comprises ve complexes, of which four complexes are dually regulated by nuclear DNA (nDNA) and mitochondrial DNA (mtDNA); thus, as expected, variants in both nuclear and mitochondrial genomes have been associated with T2D (5,6). An mtDNA haplogroup is a speci c mtDNA genetic background dened by variants in human mtDNA (i.e., single nucleotide polymorphisms [SNPs]) that are inherited during long-term evolution. Initial evidence indicated that mtDNA haplogroups inuence cellular respira- tion and ROS production, which implied the importance of mtDNA haplogroups in the regulation of mitochondrial func- tion (7). Subsequently, mtDNA haplogroups were shown to play a pathophysiological role in rats with T2D (8) and regulate physical performance in mice (9). Shortly thereafter, diagnostic SNPs of two human macro haplogroups, M and N, were shown to alter mitochondrial matrix pH and intracellular calcium dynamics (10), and mtDNA haplogroups have thus far been reported to regulate mtDNA replication and transcriptional efciency (11), the activity of respiratory chain complex (RCC) I, and the assembly dynamics of multiple RCCs (12,13). Re- cently, to comprehensively elucidate the mechanisms under- lying the roles of mtDNA haplogroups in diseases such as 1 Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China 2 Department of Endocrinology, The First Afliated Hospital of Wenzhou Medical University, Wenzhou Medical University, Wenzhou, Zhejiang, China 3 Zhejiang Provincial Peoples Hospital, Afliated Peoples Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, China Corresponding authors: Hezhi Fang, [email protected], and Jianxin Lyu, ljx@ wmu.edu.cn or [email protected]. Received 14 August 2017 and accepted 26 April 2018. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0974/-/DC1. H.F., N.H., Q.Z., and B.W. contributed equally to this work. © 2018 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for prot, and the work is not altered. More information is available at http://www.diabetesjournals .org/content/license. Diabetes Volume 67, July 2018 1441 GENETICS/GENOMES/PROTEOMICS/METABOLOMICS

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  • mtDNA Haplogroup N9a Increases the Risk of Type 2Diabetes by Altering Mitochondrial Function andIntracellular Mitochondrial SignalsHezhi 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

    Mitochondrial DNA (mtDNA) haplogroups have been as-sociated with the incidence of type 2 diabetes (T2D); how-ever, their underlying role in T2D remains poorly elucidated.Here, we report that mtDNA haplogroup N9a was associ-ated with an increased risk of T2D occurrence in SouthernChina (odds ratio 1.999 [95%CI 1.229–3.251], P = 0.005). Byusing transmitochondrial technology, we demonstratedthat the activity of respiratory chain complexes was lowerin the case of mtDNA haplogroup N9a (N9a1 and N9a10a)than in three non-N9ahaplogroups (D4j,G3a2, andY1) andthat this could lead to alterations in mitochondrial func-tion andmitochondrial redox status. Transcriptome anal-ysis revealed that OXPHOS function and metabolicregulation differed markedly between N9a and non-N9acybrids.Furthermore, inN9acybrids, insulin-stimulatedglu-cose uptake might be inhibited at least partially throughenhanced stimulation of ERK1/2 phosphorylation and sub-sequent TLR4 activation, which was found to be mediatedby the elevated redox status in N9a cybrids. Although itremains unclear whether other signaling pathways (e.g.,Wnt pathway) contribute to the T2D susceptibility of haplo-group N9a, our data indicate that in the case of mtDNAhaplogroup N9a, T2D is affected, at least partially throughERK1/2 overstimulation and subsequent TLR4 activation.

    Millions of people worldwide live with diabetes, and.90%of these people have been diagnosed with type 2 diabetes(T2D) (1). Although the molecular mechanisms underly-ing T2D remain incompletely elucidated, deregulation of

    mitochondrial oxidative phosphorylation (OXPHOS) iswidely accepted as one of the major causes of T2D andinsulin resistance (2). Diminished OXPHOS function mightcontribute generally to insulin resistance through elevatedgeneration of reactive oxygen species (ROS) production,a major regulatory signal in T2D-related insulin receptorsignaling and inflammation (3,4).

    The OXPHOS pathway comprises five complexes, ofwhich four complexes are dually regulated by nuclear DNA(nDNA) and mitochondrial DNA (mtDNA); thus, as expected,variants in both nuclear and mitochondrial genomes havebeen associated with T2D (5,6). An mtDNA haplogroup isa specific mtDNA genetic background defined by variants inhumanmtDNA (i.e., single nucleotide polymorphisms [SNPs])that are inherited during long-term evolution. Initial evidenceindicated that mtDNA haplogroups influence cellular respira-tion and ROS production, which implied the importance ofmtDNA haplogroups in the regulation of mitochondrial func-tion (7). Subsequently, mtDNA haplogroups were shown toplay a pathophysiological role in rats with T2D (8) and regulatephysical performance inmice (9). Shortly thereafter, diagnosticSNPs of twohumanmacro haplogroups,M andN,were shownto alter mitochondrial matrix pH and intracellular calciumdynamics (10), and mtDNA haplogroups have thus far beenreported to regulate mtDNA replication and transcriptionalefficiency (11), the activity of respiratory chain complex (RCC)I, and the assembly dynamics of multiple RCCs (12,13). Re-cently, to comprehensively elucidate the mechanisms under-lying the roles of mtDNA haplogroups in diseases such as

    1Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang ProvincialKey Laboratory of Medical Genetics, College of Laboratory Medicine and LifeSciences, Wenzhou Medical University, Wenzhou, Zhejiang, China2Department of Endocrinology, The First Affiliated Hospital of Wenzhou MedicalUniversity, Wenzhou Medical University, Wenzhou, Zhejiang, China3Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital of HangzhouMedical College, Hangzhou, Zhejiang, China

    Corresponding authors: Hezhi Fang, [email protected], and Jianxin Lyu, [email protected] or [email protected].

    Received 14 August 2017 and accepted 26 April 2018.

    This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0974/-/DC1.

    H.F., N.H., Q.Z., and B.W. contributed equally to this work.

    © 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

    Diabetes Volume 67, July 2018 1441

    GENETIC

    S/G

    ENOMES/P

    ROTEOMIC

    S/M

    ETABOLOMIC

    S

    https://doi.org/10.2337/db17-0974http://crossmark.crossref.org/dialog/?doi=10.2337/db17-0974&domain=pdf&date_stamp=2018-06-02mailto:[email protected]:[email protected]:[email protected]:[email protected]://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0974/-/DC1http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0974/-/DC1http://www.diabetesjournals.org/content/licensehttp://www.diabetesjournals.org/content/license

  • T2D, mitochondrial retrograde signaling has been frequentlyanalyzed using microarray or RNA sequencing technologies(9,14). This approach has yielded clues regarding disease-causing factors such as alterations in genemethylation statusand shifts inmetabolic pathways in diseases like T2D (14,15).

    Mitochondrial haplogroups play a critical role in bothmitochondrial function and mitochondria-mediated sig-naling pathways; accordingly, mtDNA haplogroups have beensuggested to be involved in a series of metabolic diseases suchas metabolic syndrome, obesity, T2D, and T2D-associatedcomplications in distinct populations (16–19). However, cer-tain contradictory observations remain unresolved, particu-larly in studies related to T2D. As in the case of studiesreporting varying disease phenotypes associated with dis-orders such as Alzheimer disease and Parkinson disease,several pitfalls in the T2D studies might account for a few ofthe contradictions (20,21). However, differences in thenuclear genetic background might be responsible for mostof the discrepancies, which have been found in populationsfrom both Asia (16–19) and Europe (16,22–24). One po-tential underlying reason is that the nuclear genetic back-ground might contribute to the difference in the functionalperformance of mitochondria, which has been referred to ina recent study as mitochondrial–nuclear coevolution (24).Another reason is that the environment can also contrib-ute to divergent responses of the same mtDNA in humandiseases (25). Nevertheless, how mtDNA haplogroups af-fect T2D is currently unknown. For example, NDUFC2 hasbeen recognized to influence the disease susceptibility ofmtDNA haplogroup HV in T2D, but whether and howhaplogroup HV itself affects the susceptibility remainsunresolved (23,24).

    In this study, we conducted a large-scale case-controlledstudy to validate the effect of mtDNA haplogroup N9a onthe pathogenesis of T2D in the Han Chinese population.Because nuclear gene expression is unfailingly alteredwhen mitochondrial function is affected in haplogroupN9a, we analyzed the mitochondrial retrograde signalingin two N9a cybrids and three cybrids other than N9a.Last, we tested the effect of alterations in this retrogradesignaling on diabetes by using a cellular model.

    RESEARCH DESIGN AND METHODS

    Study ParticipantsIn this study, 1,295 unrelated patients (mean 6 SD age60.34 6 12.839 years; median 60, range 17–93) with T2Dwere recruited at the The First AffiliatedHospital ofWenzhouMedical University (Zhejiang, China) from March 2009 toDecember 2017. T2D was diagnosed according to the ChinaMedical Nutrition Therapy Guideline For Diabetes (26). A totalof 974 geographically matched and sex-matched controlparticipants (mean 6 SD age 53.76 6 15.919 years; median54, range 17–89) with no history of T2D were also recruitedat the same hospital (at its physical examination center). Theparticipants without diabetes were people in whom thefasting plasma glucose concentration was,6.1 mmol/L and

    blood glycosylated hemoglobin (HbA1c) level was ,6.2%(44mmol/mol).Wehavedescribed675patients and649controlsubjects used here in a previous study (27). Informed consentwas obtained from all participants under protocols approved bythe ethics committee of Wenzhou Medical University. Allexperimental methods were performed in accordance withapproved guidelines of Wenzhou Medical University.

    mtDNA Sequencing and GenotypingGenomic DNA from peripheral blood was extracted usinga standard SDS lysis protocol. Complete mtDNA sequencewas Sanger sequenced for 347 T2D patients and 383 con-trol subjects by using 24 previously reported pairs ofmtDNA primers (28). The mtDNA of 235 T2D patientsand 141 control subjects was completely sequenced pre-viously (27); for all other study participants, Sanger se-quencing was performed using two pairs of mtDNA primers(Supplementary Table 1). SNPs of each participant wereidentified by comparing the obtained sequences with therevised Cambridge Reference Sequence by using CodonCodeAligner 3.0.1 (CodonCode Corporation, Centerville, VA). Weused the HaploGrep program (http://haplogrep.uibk.ac.at/)to annotate the mtDNA haplogroup for the cases where themtDNA was completely sequenced. For all other study par-ticipants, mtDNA haplogroup was assigned by comparingthe target SNPs from the D-loop, ND3, and ND4L with thediagnostic SNPs of the most up-to-date Chinese mtDNAhaplogroup tree (29).

    Generation of Cell Lines and Culture ConditionsTwo N9a haplogroups (N9a1 and N9a10a) were used toexclude the effect of private SNPs (such as mt.13214)in the terminal clades of the mtDNA tree (SupplementaryFig. 1). As control haplogroups, we included haplogroups G(G3 in this study) and D4 (D4j in this study), both of whichwere not positively associated with metabolic diseases inprevious work and were evenly distributed in this studyamong T2D patients (haplogroup G, 2.5%; haplogroup D4,10.9%) and control subjects (haplogroup G, 2.2%; haplo-group D4, 11.4%). Moreover, haplogroup Y (Y1 in thisstudy) (0.8% in T2D patients; 0.6% in control subjects),which forms a neighboring clade of haplogroup N9a, wasincluded as additional control haplogroup to exclude thepotential phenotypic effects produced by haplogroup N9-defining SNPs such as mt.5417 (Supplementary Fig. 1).These three control haplogroups, G3, D4j, and Y1, arereferred to as non-N9a haplogroups in this study.

    By using the standard protocol (30), transmitochondrialcybrids were generated through the fusion ofmtDNA-lackingr0 human osteosarcoma 143B cells with platelets of haplo-group N9a1, N9a10a, G3a2, Y1, or D4j obtained from fivevolunteers. Platelets containing blood was collected fromthe volunteers when they were 22 years old during physicalexamination before their enrollment in the graduate schoolof Wenzhou Medical University. The transformant cybridclones were cultured in high-glucose DMEM (Thermo FisherScientific,Waltham,MA) containing 10%Cosmic Calf Serum

    1442 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018

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  • (Sigma, St. Louis, MO). Pathogenic mtDNA mutations andcross contamination during single-clone selection were ruledout through Sanger sequencing of the mitochondrial ge-nome in both the five volunteers and the cybrid cells duringculture (Supplementary Table 1).

    mtDNA Content, mtRNA, and InflammatoryGene ExpressionBoth mtDNA content and the mRNA levels of 13 mito-chondrially encoded OXPHOS subunits were determinedusing the 2(–DDCT) method as previously described (31).Briefly, genomic DNA and total RNA were extracted usingstandard protocols, and the total RNA was then treatedwith DNase and reverse-transcribed using random 6-mersprimers (Takara Biotechnology, Dalian, China). Quantita-tive real-time PCR was performed using primers targetedto mtDNA, mtRNA, a subset of inflammatory genes,and related nuclear housekeeping genes on a StepOneReal-Time PCR System (Thermo Fisher Scientific) by usingSYBR Green qPCR Master Mix (Takara Biotechnology). Allprimers used in these analyses are listed in SupplementaryTable 2.

    Mitochondrial RCC Enzymatic Activity AssayMitochondria from cultured cells were isolated as pre-viously described (30). The enzymatic activity of fourRCCs was measured in the mitochondria of cybrids asdescribed (30). The RCC enzymatic activity in each casewas normalized against that of citrate synthase, a mito-chondrial matrix marker enzyme.

    Immunoblotting and AntibodiesProteins were extracted using RIPA lysis buffer (Cell Signal-ing Technology, Danvers,MA) supplemented with a protease-inhibitor cocktail (Sigma-Aldrich). Proteins separated usingSDS-PAGE were blotted with these antibodies: anti-VDAC,anti-JNK 1/2, anti–phospho-JNK 1/2, anti-p38, anti–phospho-p38 (Thr389), anti-ERK1/2, anti–phospho-ERK (Thr202/Tyr204), anti–NF-kB, anti–phospho-NF-kB, anti-SRC, anti–phospho-SRC, anti-MEK1/2, anti–phospho-MEK1/2, anti-AMPK, and anti–phospho-AMPK (all from Cell SignalingTechnology; 1:1,000); anti-TOMM20, anti-SDHA, anti-RXRA,anti–POLY-g, anti-TFAM, anti-NRF1, anti-AFG3L2, anti-ClpP,anti-ClpX, anti-HSP60, anti-PINK1, anti-DRP1, anti–phospho-DRP1, anti-OPA1, anti-MFN1, and anti-MFN2 (all fromAbcam,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(1:2,000) (all from Santa Cruz Biotechnology, Santa Cruz, CA).

    Measurement of Endogenous Oxygen ConsumptionEndogenous oxygen consumption in intact cells was deter-mined using a Seahorse XF24 Extracellular Flux Analyzer(Seahorse Bioscience, North Billerica, MA) as described in ourprevious study (2). Briefly, 4 3 104 cells were seeded into24-well Seahorse plates together with 250 mL of growthmedium 1 day before experiments. Oxygen consumptionrate (OCR) was determined with and without the inclusionof 1mmol/L oligomycin. Extracellular acidification rate (ECAR)

    was determined by sequentially injecting 10 mmol/L glucose,1 mmol/L oligomycin, and 50 mmol/L 2-deoxy-D-glucose byusing the Seahorse system.

    ATP, Mitochondrial Membrane Potential, NAD+/NADHRatio, and ROS MeasurementsMitochondrial membrane potential (MMP), total ATPcontent, and NAD+/NADH ratio were determined usingthe cationic fluorescent redistribution dye tetramethyl-rhodamine, methyl ester (Thermo Fisher Scientific), an ATPmeasurement kit (Thermo Fisher Scientific), and an NAD+/NADH ratio assay kit (Abcam), respectively (32). Mito-chondrial and cytoplasmic ROS production was measuredusing MitoSOX and carboxy-DCFDA (both from ThermoFisher Scientific), respectively. Briefly, cells in 12-wellplates were treated with MitoSOX (5 mmol/L) or carboxy-DCFDA (40 mg/mL) for ,1 h at 37°C in the dark and thenwashed with HBSS and analyzed immediately using a fluo-rescence microscope (Eclipse Ti-E, Nikon Eclipse Ti-S; NikonInstruments, Inc., Tokyo, Japan). At least five regions werequantitatively analyzed for each cybrid to generate the aver-age fluorescence intensity in one independent experiment byusing ImageJ (Bethesda, MD).

    Fluorescence Microscopy for Examining MitochondrialMorphologyCells were incubated with 500 nmol/L MitoTracker Red(Thermo Fisher Scientific) for 30 min and fixed for 15 minwith 4% paraformaldehyde at room temperature. The cellswere then permeabilized with 0.2% Triton X-100 (Sigma),stained with DAPI (Thermo Fisher Scientific), and exam-ined using an Olympus imaging system (Olympus FV1000;Olympus, Melville, NY). Mitochondrial length and com-plexity were quantified by measuring the form factor andaspect ratio, respectively.

    Sample Preparation and RNA SequencingTotal RNA was isolated from three biological triplicates ofeach group of cybrids by using an RNeasy Mini extraction kit(Qiagen, Valencia, CA), and mRNA from 20 mg of the totalRNA was purified using poly-T–attached magnetic beads.After fragmenting the mRNA, first-strand cDNA was synthe-sized and then sequenced using an Illumina HiSeq 2000 plat-form (Illumina, San Diego, CA) as described previously (32).

    Analysis of Gene Expression DataTo obtain high-quality reads, reads containing adaptorsequences and poly-N and low-quality reads were removedfrom the raw data. Reference-genome and gene-modelannotation files were downloaded from genome websitesdirectly. The reference genome was built using STAR, andpaired-end high-quality reads were aligned to the referencegenome by using STAR (v2.5.1b). HTSeq v0.6.0 was used tocount the reads mapped to each gene, after which thefragments per kilobase million of each gene was calculatedbased on the length of the gene and the count of the readsmapped to the gene. Differential expression analysis under

    diabetes.diabetesjournals.org Fang and Associates 1443

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  • two conditions (three biological replicates per condition) wasperformed using the DESeq2 R package. DESeq2 providesstatistical routines for determining differential expressionin digital gene expression data by using a model based onnegative binomial distribution. The resulting P values wereadjusted using the Benjamini and Hochberg approach forcontrolling the false discovery rate (33). Genes found usingDESeq2 that feature an adjusted P , 0.05 were regarded asdifferentially expressed genes (DEGs). To identify the genesthat differed between the two N9a and three non-N9acybrids, we respectively compared N9a1 and N9a10a withnon-N9a cybrids (G3a2, Y1, and D4j), and the genes thatoverlapped in both comparisons were confirmed as the finalDEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG)pathway and gene ontology (GO) biological performanceenrichment analyses of DEGs were implemented using theclusterProfiler R package. GO and KEGG pathway termsfeaturing P values of ,0.05 were regarded as significantlyenriched among DEGs.

    Insulin-Stimulated Glucose UptakeBefore measurements, cells were serum starved for 5 h,incubated with 0.1 mmol/L insulin plus 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG)(0.1 mmol/L) in a CO2 incubator for 30 min, and thenwashed thrice with cold phosphate-buffered saline. The fluo-rescence signal was measured after 30 min with excitationand emission at 485 and 535 nm, by using a Varioskan FlashMultimode Reader (Thermo Fisher Scientific). Insulin-stimulated glucose uptake was also measured in with thepresence of N-acetyl-L-cysteine (NAC; Sigma) (15 mmol/L,24 h), nicotinamide (NAM; Sigma) (15mmol/L, 24 h), U0126(Selleckchem, Houston, TX) (50 mmol/L, 6 h), C34 (Sigma)(10 mmol/L, 30 min), or TAK-242 (MedChemExpress,Monmouth Junction, NJ) (10 mmol/L, 1 h).

    Statistical AnalysisIn this case-controlled study, haplogroups featuring a fre-quency of .5% in either the control participants or T2Dpatients were analyzed to evaluate the effect of commonmtDNA haplogroups on T2D. Haplogroups that featuredfrequencies of ,5% were regarded as “other haplogroups.”Multivariate logistic regression analysis was applied to adjustthe risks associated with age, sex, and mtDNA haplogroupsin case and control subjects. T2D was a dependent variable,whereas age, sex, and genotype of each mtDNA haplogroupwere independent variables. In the case of nine mtDNAhaplogroups, dummy coding was applied due to their numer-ical variability. Bonferroni correction indicating a P valueof ,0.006 (0.05/9) was considered statistically significantwhen analyzing these nine haplogroups (with the otherhaplogroups excluded). The significance of “other haplogroups”was not considered here because the group included multiplemtDNA haplogroups. An independent Student t test was usedto evaluate cybrid data, and a null hypothesis was rejectedwhen P , 0.05. All statistical analyses were performed usingSPSS 21.0 (IBM, Armonk, NY).

    RESULTS

    Association of mtDNA Haplogroups With T2DTo investigate the relationship between mtDNA haplogroupN9a and T2D in the Chinese population, we performeda large-cohort case-controlled study that included 1,295T2D patients and 974 geographically matched asymptomaticcontrol participants. The frequency of haplogroup N9a wasfound to be significantly higher in patients than in controlsubjects when multivariate logistic regression analysis wasperformed with adjustment for age, sex, and haplogroups(odds ratio [OR] 1.999 [95% CI 1.229–3.251], P = 0.005)(Table 1). By contrast, a significantly decreased frequency ofhaplogroupN9awas previously observed in T2Dpatients fromKorea and Japan (Table 1). This finding suggests that distinctpopulations could present divergent responses in terms of theeffect of haplogroup N9a on T2D occurrence. Furthermore,we found that the frequency of haplogroup N9, which com-prised haplogroups Y and N9a, was also significantly higherin T2D patients than in control participants (OR 1.967 [95%CI 1.238–3.124], P = 0.004), but the statistical significancewas not retained in the case of ethnic Chinese people fromTaiwan (Table 1). The distribution of haplogroup Ywas similarbetween T2D patients (0.8%) and control subjects (0.6%).

    N9a Cybrids Exhibited Lower RCC Activity ThanNon-N9a CybridsTo analyze the effect of mtDNA haplogroups on the regu-lation of mitochondrial function, we determined the mtDNAcontent and the RNA level of mtDNA-encoded OXPHOSsubunits in two N9a and three non-N9a cybrids. The mtDNAcontent in non-N9a cybrids was ;30% higher than that inN9a cybrids (Fig. 1A). The lower mtDNA content measuredin N9a cybrids was not because of the presence of fewermitochondria, and the mitochondrial mass was roughly thesame in the N9a and non-N9a cybrids (Fig. 1B and E). Next,examination of the RNA level of mtDNA-encoded OXPHOSsubunits revealed that the RNA levels of ATP8, ND1, ND5,and CO1 were higher in all non-N9a cybrids than in N9acybrids (Fig. 1C). This result suggested superior mitochon-drial function in non-N9a cybrids than in N9a cybrids (34),and to test this possibility, we examined the activity of threeRCCs containing mtDNA-encoded subunits. After normali-zation of the RCC activities relative to citrate synthaseactivity, we found that the activities of complexes I and IVwere significantly higher in non-N9a cybrids than in N9acybrids, whereas the activity of complex III did not differ(Fig. 1D and E). Although the activity of certain RCCs wasdiminished inN9a cybrids, transcriptomic analysis performedusing RNA sequencing technology revealed that N9a cybridsexhibited increased mRNA levels of most nDNA-encodedOXPHOS subunits as compared with non-N9a cybrids (Fig.1F), and, notably, the mRNA levels of complex II subunitswere not significantly affected, particularly those of thesubunits SDHA and SDHB (Fig. 1F). The observed patternof mtDNA-encoded OXPHOS subunits was not reliablein the experiment performed here using RNA sequencing,as the length of poly-A tails varied among distinct

    1444 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018

  • mitochondrial genes during RNA capture. However, the ratioof nDNA-encoded subunits to mtDNA-encoded subunitswas found to be higher in N9a cybrids than in non-N9acybrids when we compared the nDNA-encoded gene expres-sion determined from the transcriptome analysis with themtDNA-encoded gene expression measured using quantita-tive real-time PCR analysis (Fig. 1C and F). Furthermore, thelevel of NRF1, an essential transcription factor for nucleargenes required for respiration, was higher in N9a cybridsthan in non-N9a cybrids (Fig. 1G), which suggests thata retrograde signaling machinery might be involved incompensatory protection of mitochondrial function inN9a cybrids (35). Together with this mitonuclear imbal-ance of OXPHOS subunits, the detection of a higher levelof the mitochondrial quality-control protein ClpP in N9acybrids than in non-N9a cybrids supported the notion thatN9a cybrids exhibit increased mitochondrial unfolded pro-tein response (mtUPR) as compared with non-N9a cybrids(Fig. 1H). However, we found that other mtUPR proteins didnot differ between the N9a and non-N9a cybrids (Fig. 1H),and thus the mtUPR level in N9a cybrids might be limited.

    Mitochondrial Function Is Lower in N9a Cybrids ThanNon-N9a CybridsNext, we measured mitochondrial respiratory profilesby using a Seahorse XF24 Extracellular Flux Analyzer.A respiration assay of the cybrids revealed that both in-tracellular respiration and proton leakage were significantlyhigher in non-N9a cells than in N9a cells (Fig. 2A). Themeasured ratio of coupled-to-uncoupled respiration indi-cated that the coupling efficiency did not differ significantlybetween N9a and non-N9a cells (Fig. 2B), and an increasedECAR andOCR/ECAR ratio confirmed that glycolytic function

    was lower but mitochondrial function was higher in non-N9a cells than in N9a cells (Fig. 2C and D), which suggesteddistinct mitochondrial retrograde signaling pathways be-tween N9a and non-N9a cells. Accordingly, the mean valuesofMMP and total ATP content in non-N9a cells were;50%higher than those measured for N9a cells (Fig. 2E and F).Notably, total ATP content and mtDNA content in periph-eral blood mononuclear cells (PBMCs) from non-N9a haplo-group study participants were significantly higher thanthose in PBMCs from N9a haplogroup participants (Fig. 2Gand H), which suggested that mitochondrial function wasdistinctly affected by N9a and non-N9a haplogroups. Fur-thermore, we analyzed mitochondrial fragmentation bymeasuring the form factor (an index ofmitochondrial branch-ing) and the aspect ratio (an index of mitochondrial branchlength) of single mitochondrion in the case of these haplo-groups (Fig. 2I); a low degree of mitochondrial fragmentationis indicated by high values of aspect ratio and form factor,parameters that represent increased mitochondrial length/width and branching, respectively. Our results revealed a lowerpercentage of mitochondrial fragmentation in non-N9a cellsthan in N9a cells (Fig. 2J and K). Last, we examined mito-chondrial fission/fusion proteins and found that the level ofthe long form of OPA1, which is considered to promotemitochondrial fusion, was higher in non-N9a cells than inN9a cells (Fig. 2L). Collectively, our results demonstrated thatN9a cybrids exhibit diminished mitochondrial function rela-tive to non-N9a cybrids.

    N9a and Non-N9a Cybrids Feature Distinct Profiles ofMitochondrial Signaling Mediators and TranscriptomeFine-tuning of mitochondrial function can activate diverseretrograde signaling pathways in the nucleus by affecting

    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. *Haplogroupswith 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 Bonferronicorrection while 16 haplogroups were studied (17). §P , 0.005 (0.05/10), adjusted P value with Bonferroni correction while10 haplogroups were studied (18).

    diabetes.diabetesjournals.org Fang and Associates 1445

  • the levels of mitochondrial signaling mediators (33). Be-cause redox signaling pathways play a major role in cellularphysiology, we measured the mitochondrial redox signal,the ROS level, and the NAD+/NADH ratio in N9a and non-N9a cybrids. N9a cybrids generated more ROS than non-N9a cybrids did, as determined using either the cytosolic ROSprobe carboxy-DCFDA (Fig. 3A and B) or the mitochondrial

    ROS probe MitoSOX (Fig. 3C and D), but mitochondrialantioxidant activity did not differ between the cybrids, asrevealed by measurement of the antioxidant protein SOD2(Fig. 1B). Furthermore, the NAD+/NADH ratio was lower inN9a cybrids than in non-N9a cybrids (Fig. 3E). These resultssuggested that N9a and non-N9a cybrids feature distinctmitochondrial retrograde signaling profiles.

    Figure 1—N9a cybrids (N9a1 and N9a10a) exhibit lower RCC activity than non-N9a cybrids (D4j, G3a2, and Y1). A: Relative mtDNA contentin N9a and non-N9a cybrids. mtDNA content in non-N9a cybrids was normalized relative to that in N9a cells (n = 4). B: RepresentativeWestern blot of mitochondrial marker proteins. VDAC, TOMM20, SOD2, and SDHAwere stained in whole-cell lysates fromN9a and non-N9acybrids. 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-N9acybrids (n = 4) (D), and mitochondrial complex enzyme activity was normalized with citrate synthase activity (E ). F: Heat map showingtranscriptional 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 fromupregulation to downregulation.G: Representative Western blot of RXRA, POLY-g, TFAM, and NRF1 levels in whole-cell extracts of N9a andnon-N9a cybrids from 143B cells. Actin was used as a total protein loading control. Protein levels in all cybridswere normalized relative to thatin N9a1 cybrids. H: Immunoblotting analysis of the levels of AFG3L2, ClpX, PINK1, GRP75, HSP60, and ClpP in whole-cell extracts of N9aand 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 threeindependent tests per experiment. *P # 0.05, **P # 0.01, ***P # 0.001. OD, optical density.

    1446 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018

  • Figure 2—Non-N9a cybrids (D4j, G3a2, and Y1) present higher mitochondrial function and superior mitochondrial morphology than N9acybrids (N9a1 and N9a10a). A: Mitochondrial OCR was determined in N9a and non-N9a cybrids by using a Seahorse XF24 Extracellular FluxAnalyzer. 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 andnon-N9a cybrids was determined using the Seahorse XF24 Extracellular Flux Analyzer by sequentially injecting 10mmol/L glucose, 1 mmol/Loligomycin, 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 weremeasured in N9a and non-N9a cybrids treatedwith 30 nmol/L tetramethylrhodamine for 30min. RelativeMMP levels in non-N9a cybrids werenormalized to that in N9a cells (n = 4). MMP values were normalized relative to protein concentration. F and G: Relative ATP content wasmeasured in approximately 13 106 cells each of N9a and non-N9a cybrids (n = 4) (F ) and in approximately 13 106 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 valueswere 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-N9acybrids in which mitochondria were stained with MitoTracker Red (n = 3). Images are shown at 6003magnification. The upper and lower tworows 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 mitochondriallength/width and branching, respectively. J and K: Quantification of aspect ratio (J) and form factor (K) in N9a and non-N9a cybrids (n = 3).

    diabetes.diabetesjournals.org Fang and Associates 1447

  • To uncover the differences between the transcriptomeof N9a and non-N9a cybrids, we used high-throughputRNA sequencing for transcriptomic profiling of the twoN9a cybrids and three non-N9a cybrids. Our analysisrevealed 826 statistically significant DEGs between thetwo N9a cybrids and three non-N9a cybrids, of which604 and 222 genes were upregulated and downregulated,respectively, in both N9a cybrids as compared with thelevels in three non-N9a cybrids; moreover, among theseDEGs, 52 genes encoded transcription factors and wererelated to signaling pathways such as the ERK1/2 pathway(Fig. 4A and Supplementary Data Set 1) (full access to thedata set is available upon request to the author). We nextperformed both GO and KEGG pathway enrichment anal-yses to determine the contribution of theseDEGs in biologicalperformance. As shown in Fig. 4B and Supplementary DataSet 2 (full access to the data set is available upon request tothe author), multiple mitochondrial OXPHOS-related path-ways and three signal transduction pathways (Wnt, ERK1/2,and p38-MAPK) were presented in all 79 GO biologicalprocesses that showed significantly different enrichmentbetween the two N9a and three non-N9a cybrids. KEGGenrichment analysis further revealed that the two N9a andthree non-N9a cybrids exhibit distinct performance in termsof metabolic regulation and pathways related to metabolicdiseases such as nonalcoholic fatty liver disease (Fig. 4C). Thestatistical significance of most pathways was retained whenwe excluded the DEGs encoding OXPHOS subunits (Fig. 4Dand E and Supplementary Data Set 2), which indicated thatmitochondrial retrograde signaling contributed substantiallyto the difference in biological performance between the twoN9a and three non-N9a cybrids. Notably, although only90 DEGs remained after we applied the criterion of fold-change.2 (Fig. 4F), the difference in biological performancebetween N9a and non-N9a cybrids was retained in severalaspects (Fig. 4G and H).

    Mitochondrial Redox Signal–Mediated ERK1/2Phosphorylation Contributes to Cellular GlucoseUptakeWe sought to functionally assess how and through whichpathway mitochondrial retrograde signaling influencesT2D susceptibility in N9a and non-N9a cybrids. Includingthe candidate ERK1/2 pathway, we tested seven pathwaysthat are commonly associated with mitochondrial retro-grade signaling pathway (33). As expected, ERK1/2 phos-phorylation levels differed between the two N9a and threenon-N9a cybrids (Fig. 5A), and p38 phosphorylation waslower in non-N9a cybrids than in N9a cybrids (Fig. 5A). To

    examine how redox signals affect p38 and ERK1/2, wetreated N9a cells, which exhibited higher ROS generationand a lower NAD+/NADH ratio than non-N9a cells, withNAC and NAM to reduce ROS generation and increase theNAD+/NADH ratio, respectively. Our results showed thatonly ERK1/2 phosphorylation was affected by the mito-chondrial redox signals in our cybrids (Fig. 5B and C).ERK1/2 activation has been associated with the expressionof inflammation (36) and with inflammation-induced in-sulin resistance (37). Therefore, we measured the mRNAlevels of 38 inflammation-related genes, which revealedthat mRNA levels of four genes, IL13, TLR4, CSF3, andCCL3, were lower in non-N9a cybrids than in N9a cybrids(Fig. 5D). Because TLR4 expression is closely associatedwith ERK1/2 activation (36), we tested whether TLR4is downregulated upon ERK1/2 inhibition. Treatment ofcells with either U0126, a specific ERK1/2 inhibitor, or NAM,an effective antioxidant, caused a significant decrease in themRNA level of TLR4 (Fig. 5E and F). Physiologically, non-N9a cells exhibited higher insulin-stimulated glucose uptakethan N9a cells (Fig. 5G), whereas administration of thetwo antioxidants, NAC and NAM, upregulated insulin-stimulated glucose uptake in N9a cells (Fig. 5H). Further-more, the upregulation of insulin-stimulated glucose uptakein N9a cells was mimicked when ERK1/2 phosphorylationwas inhibited in N9a cells through U0126 treatment (Fig.5H). Last, blockage of TLR4 signaling by using two TLR4inhibitors increased the insulin-stimulated glucose uptake(Fig. 5I). Our results support the proposal that regardlessof the other signaling pathways that might regulate insulinsensitivity and cellular glucose uptake, mitochondrial re-dox signal–mediated ERK1/2 phosphorylation contributesto the insulin-stimulated glucose uptake, at least partiallythrough TLR4 activation.

    DISCUSSION

    Previously, a study showed that haplogroup N9a is asso-ciated with diminished T2D occurrence (i.e., N9a actsa “protective factor”) in both Japanese and Koreanpatients (18). However, this reported effect of N9a inthe case of Japanese T2D patients has been challenged(19). In Taiwan, haplogroup B4, but not haplogroup N9,was found to be associated with T2D (17). Recently, wefound that mtDNA haplogroup N9a was marginally asso-ciated with an increased occurrence of T2D and signifi-cantly associated with diabetic nephropathy incidence (27).Here, to evaluate the causal role of haplogroup N9a in T2D,we conducted another large-scale case-controlled study, whichconfirmed 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 mitochondrialfission and fusion proteins; p-DRP1, total DRP1, OPA1, and MFN1/2 were stained in whole-cell lysates prepared from N9a and non-N9acybrids. 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.

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  • against T2D incidence in China (Table 1). Such conflictingreports on mtDNA population variants in common diseaseshave been highlighted by previously (38) and are also knownto be common in the case of other diseases such as Leberhereditary optic neuropathy (39). Several major factors mightcontribute to the distinct reported effects of mtDNA lineageon human diseases, including inappropriate research designand statistical performance (20,21), divergent nuclear geneticbackgrounds (24), and different environment factors (25). Inthe investigation of haplogroup N9a/N9 and T2D, distinctinclusion criteria used for T2D patients might also contributeto the conflicting conclusions reached regarding the relation-ship between haplogroup N9a/N9 and T2D (17,18). Here, wedid not set a cutoff value for the age of the T2D patients, butthe patients included in other studies were aged.40 or.30years old (17,18). We do not believe that the use of age as aninclusion criterion affected the results here because only 17 ofthe 1,295 patients were,30 years old and none of themweregenotyped as N9a; by comparison, 94 control participantswere ,30 years old, with 8 genotyped as N9a. The T2D riskpresented by haplogroup N9a would be even higher than thatwe have reported if these patients and control subjects wereexcluded. Moreover, although a limited amount of lifestyleinformation and clinical data are available for the study par-ticipants, it is unclear whether other factors such as smokingcontributed to the distinct effects of haplogroup N9a on T2Doccurrence. The only recognized difference that could affectthe contribution of these factors might be the inclusion

    criterion “HbA1c” for the control participants (HbA1c ,6.2%[44 mmol/mol] in this study, ,5.6% [38 mmol/mol] inJapan/Korea, ,6.0% [2 mmol/mol] in Taiwan). Overall, wespeculate that environment factors might contribute to thedivergent responses of the same mtDNA in healthy humansbecause Chinese populations were studied both by us and byLiou et al. (17).

    Cytoplasmic hybrid technology is widely used for in-vestigating the effects of distinct mtDNA haplogroups oncellular physiological conditions, including insulin sensi-tivity (32,40,41). The mtDNA-lacking r0 human osteo-sarcoma 143B cells represent the most accepted cellularmodel for studying how mtDNA haplogroups influencecellular functions. Although the use of a disease-related cellmodel as the nuclear donor is the optimalmethod to uncoverthe pathogenic role of specific mtDNA haplogroups, 143Bcells are widely used in the study of Parkinson disease (42),T2D (40), Alzheimer disease (41), and Leber hereditary opticneuropathy (43). Thus, as in other studies (3,40), we used143B cells to evaluate how mtDNA haplogroup N9a affectsinsulin sensitivity. Furthermore, 143B cells express GLUT4,which can translocate to the plasmamembrane upon insulinstimulation, and by performing glucose uptake experiments,we obtained data supporting the view that relative to N9acells, the three non-N9a cybrids are more sensitive to insulinand exhibit higher levels of insulin-stimulated glucose up-take (Fig. 5G–I). Our analysis of mitochondrial function byusing PBMCs obtained from N9a and non-N9a control

    Figure 3—Differential mitochondrial redox status between N9a (N9a1 and N9a10a) and non-N9a (D4j, G3a2, and Y1) cybrids. A: Relativecytoplasmic ROS levels in N9a and non-N9a cybrids. Cells were stained with the probe carboxy-DCFDA. Representative images are shownat 2003 magnification. B: Quantification of cytoplasmic ROS levels in N9a and non-N9a cybrids. The ROS level in non-N9a cells wasnormalized relative to that in N9a cells (n = 3). C: Mitochondrial ROS levels were determined by staining cells with MitoSOX. Representativeimages are shown at 2003magnification. 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+ andNADH levelsin cell extracts were quantified based on fluorescence intensity (n = 3). Data are presented as means6 SD of at least three independent testsper experiment. **P # 0.01, ***P # 0.001.

    diabetes.diabetesjournals.org Fang and Associates 1449

  • Figure 4—N9a (N9a1 and N9a10a) and non-N9a (G3a2, Y1, and D4j) cybrids feature distinct mitochondrial signaling mediators andtranscriptome profiles. A: Venn diagrams showing the numbers of DEGs that were shared by or specific to N9a and non-N9a cybrids andmetthe 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: Venndiagrams 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 andabsolute 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. Thehorizontal axis (B, C, D, E, G, and H) represents the number of genes in each category. Down, downregulated; Up, upregulated.

    1450 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018

  • participants further indicated that haplogroup N9a couldaffectmitochondrial function in disparate global populations(Fig. 2G and H). Moreover, distinct nuclear genetic back-grounds, such as the presence of NDUFC2 polymorphisms,might act as secondary genetic modifiers that enhance orreduce the effect of mtDNA haplogroup N9a in T2D (24).

    Conflicting reports have been published on the effect ofmtDNA haplogroup on T2D (16–19,22–24,44), and N9a isa haplogroup regarding which incongruent findings havebeen reported in Asian populations (17–19); therefore, it isnecessary to comprehensively elucidate the biological roleof mtDNA haplogroup N9a in the development of T2D.

    Figure 5—Mitochondrial redox signal–mediated ERK1/2 phosphorylation contributes to cellular glucose uptake. A: Representative Westernblotting 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 andC: 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 ofphosphorylated proteins in N9a1 and N9a10 cybrids treated with NAC or NAM were normalized relative to the levels of phosphorylatedproteins in the same cybrids in the absence of NAC or NAM treatment. D: Heat map showing inflammation-related genes that weredifferentially 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 changefrom upregulation to downregulation. Black arrows, genes upregulated in N9a cybrids. E: mRNA level of TLR4 was measured in N9a cellstreated with or without U0126 (50 mmol/L, 6 h) (n = 3). The TLR4 value obtained for U0126-treated N9a cells was normalized relative to thatmeasured for untreated cells. F: TLR4mRNA level was determined in N9a cells treated with or without NAM (5mmol/L, 24 h) (n = 3). The TLR4value obtained for NAM-treated N9a cells was normalized relative to thatmeasured for untreated cells.G–I: Insulin-stimulated glucose uptakein 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 thatmeasured for N9a cells that were not treated with any chemical (n = 3–4). Data are presented as means 6 SD of at least threeindependent tests per experiment. **P # 0.01, ***P # 0.001.

    diabetes.diabetesjournals.org Fang and Associates 1451

  • Previously, we showed that impaired mitochondrial func-tion and increased ROS levels played a critical role in T2D(2). Accordingly, we detected lower mitochondrial functionin N9a cybrids than in non-N9a cybrids (D4, G3, and Y1cybrids) and confirmed that the mitochondrial redox statusdiffered significantly between N9a and non-N9a cybrids bymeasuring mitochondrial ROS levels and the NAD+/NADHratio (45–47). Notably, we found that in N9a cells, themtDNA content was higher than that in non-N9a cells (Figs.1A and 3H). N9a cells generated higher levels of ROS thannon-N9a cells did, but ROS scavenging did not lead toupregulation of the mtDNA content in N9a cybrids (Sup-plementary Fig. 2), and the levelsmtDNA replication relatedproteins did not differ between N9a and non-N9a cybrids;these findings suggest that specific SNPs in haplogroup N9amight affect the mtDNA replication process as previouslydescribed (11). In this scenario, the distinct mtDNA repli-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 SNPsin N9a cells, but currently we cannot exclude the possibilitythat the binding abilities of mtDNA replication–relatedproteins differ between N9a and other haplogroups.

    The association of mtDNA haplogroup with degenera-tive disease such as Parkinson disease could be due to notonly a decline in RCC activity (41), but also the subsequentdifference in nuclear signaling pathways caused by thedisparity in mitochondrial function (33,41). In a previousstudy, mtDNA haplogroup–responsive retrograde signal-ing pathways were linked to the insulin pathway (3). Here,we noted that the insulin pathway was less active, asindicated by phospho-IRS1 (Y896) levels, and the foldincrease in plasma membrane GLUT4 was considerablylower in N9a cybrids than in D4 cybrids (3). Therefore, itappears highly likely that haplogroup N9a represents a riskfactor for T2D. In this study, we adopted a widely usedquantitative RNA sequencing technology to identify mi-tochondrial retrograde signaling pathways, which coulduncover the mechanism underlying the effect of N9a inT2D. By performing transcriptome analysis, we deter-mined that nuclear-encoded OXPHOS gene expressionwas higher in N9a cybrids than in non-N9a cybrids, whichlikely arises as a compensatory effect for mitochondrialredox stress as a result of the reduction in mtRNA levels inN9a cybrids (49). We then obtained further evidence in-dicating that N9a might be involved in T2D: most iden-tified changes in biological performance were related tometabolic regulation (Fig. 4C). In another study, micro-array analysis used for gene expression profiling yieldeddata indicating that N9a probably does not represent a pro-tective factor in the case of T2D (14). Here, by targetingcandidate signaling pathways, we demonstrated that mito-chondrial redox signal–mediated ERK1/2 phosphorylation/activation, a pathway that has been frequently related to in-sulin sensitivity (50), contributes to the differences in insulin-stimulated glucose uptake between N9a and non-N9acells. Furthermore, we found that in response to ERK1/2

    overactivation, TLR4 activation was increased and insulin-stimulated glucose uptake was decreased in N9a cells.However, several questions remain unanswered, suchas whether and how other signaling pathways (e.g., Wntpathway) are regulated by mitochondrial function, andthus further investigation required to completely revealthe underlying role of N9a in T2D.

    In summary, we have presented the most comprehen-sive analysis to date of mitochondrial function, mitochon-drial retrograde signaling, and insulin-stimulated glucoseuptake in the study of mtDNA haplogroups in relation toT2D. Our findings support a positive association betweenthe mtDNA haplogroup N9a and T2D and further dem-onstrate that N9a cells exhibit an altered redox status,which might contribute to an increased risk of T2Dthrough mitochondrial retrograde signaling pathways suchas those involving ERK1/2 activation.

    Acknowledgments. The authors thank the members of J.L.’s laboratoryfor valuable discussions on this work.Funding. This work was supported by grants from the Chinese NationalScience Foundation (31671486 and 31501156), Zhejiang Provincial NaturalScience Foundation of China (LY15H060007), and Specialized Research Fundfor the Doctoral Program of Higher Education (20133321110001).Duality of Interest. No potential conflicts of interests relevant to this articlewere reported.Author Contributions. H.F. and J.L. designed the study. H.F., N.H., Q.Z.,B.W., H.Z., Q.F., L.S., X.C., and F.S. produced the data. H.F., N.H., Q.Z., B.W., andJ.L. analyzed the data. H.F. and J.L. wrote the manuscript. H.F. and J.L. are theguarantors of this work and, as such, had full access to all the data in the studyand take responsibility for the integrity of the data and the accuracy of thedata analysis.

    References1. Absetz P, Valve R, Oldenburg B, et al. Type 2 diabetes prevention in the “realworld”: one-year results of the GOAL Implementation Trial. Diabetes Care 2007;30:2465–24702. Chen H, Bai J, Dong F, et al. Hepatic DsbA-L protects mice from diet-inducedhepatosteatosis and insulin resistance. FASEB J 2017;31:2314–23263. Weng SW, Kuo HM, Chuang JH, et al. Study of insulin resistance in cybridcells harboring diabetes-susceptible and diabetes-protective mitochondrial haplo-groups. Mitochondrion 2013;13:888–8974. West AP, Shadel GS. Mitochondrial DNA in innate immune responsesand inflammatory pathology. Nat Rev Immunol 2017;17:363–3755. Picard M, Zhang J, Hancock S, et al. Progressive increase in mtDNA 3243A.Gheteroplasmy causes abrupt transcriptional reprogramming. Proc Natl Acad SciU S A 2014;111:E4033–E40426. Olsson AH, Rönn T, Ladenvall C, et al. Two common genetic variants nearnuclear-encoded OXPHOS genes are associated with insulin secretion in vivo. EurJ Endocrinol 2011;164:765–7717. Moreno-Loshuertos R, Acín-Pérez R, Fernández-Silva P, et al. Differences inreactive oxygen species production explain the phenotypes associated withcommon mouse mitochondrial DNA variants. Nat Genet 2006;38:1261–12688. Pravenec M, Hyakukoku M, Houstek J, et al. Direct linkage of mitochondrialgenome variation to risk factors for type 2 diabetes in conplastic strains. GenomeRes 2007;17:1319–13269. Latorre-Pellicer A, Moreno-Loshuertos R, Lechuga-Vieco AV, et al. Mito-chondrial and nuclear DNA matching shapes metabolism and healthy ageing.Nature 2016;535:561–565

    1452 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July 2018

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