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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
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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
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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
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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
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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
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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
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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
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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.
1448 mtDNA Haplogroup N9a and T2D Diabetes Volume 67, July
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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
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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
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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
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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.
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