Eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial
biogenesis
Masanori Hiramitsu, Yasuhito Shimada, Junya Kuroyanagi, Takashi Inoue, Takao Katagiri,
Zang Liquing, Yuhei Nishimura, Norihiro Nishimura, and Toshio Tanaka
Figure S1. Food intake during feeding experiment
Figure S2. Clustering analysis of DNA microarray data
Figure S3. qRT-PCR of genes related to lipid metabolism in lipid-loaded HepG2 cells
All values are means±SEM; n=4, *P<0.05.
Figure S4. qRT-PCR of genes related to mitochondrial functions in lipid-loaded HepG2
cells
All values are means±SEM; n=4, *P<0.05.
Figure S5. Eriocitrin effect on development of zebrafish
Zebrafish were exposed to eriocitrin from 6 hpf to 5 dpf. The number of survivors was
counted at 5 dpf. Each experiment contained 30 embryos and was conducted 3 times,
independently. ns; not significant.
Figure S6. Eriocitrin effect on locomotor activities in zebrafish.
The effects of appetite-regulating drug treatment on spontaneous swimming (15 min) were
measured in young zebrafish. Zebrafish were exposed to eriocitrin from 6 hpf to 5 dpf. n=16,
ns; no significant.
Table S1. 152 human orthologues were altered by eriocitrin in the OF group (clusters 7 and 10)
Human
Probe name
orthologue
OF
OF+Erio
(Symbol)
A_15_P106002 XYLB
0.35
1.72
-0.55
-0.46
1.24
-3.72
0.61
A_15_P190701 HNRNPA0
1.03
-1.64
0.69
0.90
1.44
-1.37
-1.93
Log
ratio
FDR
-3.81
-2.77
-5.36
-4.73
0.13
0.18
A_15_P211791
A_15_P113821
A_15_P143731
A_15_P107051
A_15_P105956
A_15_P107432
A_15_P209091
NFKBIA
CISH
PNP
PDK2
ERRFI1
MAPK12
CELA1
-0.39
-1.04
-1.09
0.59
-1.96
-0.07
5.51
-0.09
0.60
-0.39
2.49
0.40
-0.46
-3.54
-0.62
0.76
0.00
1.56
-0.90
0.02
-2.88
-0.35
1.15
-0.19
-1.03
-0.71
-2.22
-0.24
0.08
0.94
0.00
-3.33
-0.56
-0.02
3.36
1.65
-1.75
2.26
-1.14
1.88
1.79
-3.81
1.79
-1.47
1.16
-3.10
1.08
1.24
1.88
2.41
-2.25
1.62
1.63
2.62
1.08
0.21
-4.10
-3.08
-3.01
-1.64
-1.59
-1.50
-1.42
0.18
0.13
0.19
0.01
0.15
0.25
0.20
A_15_P101274
A_15_P112691
A_15_P162361
A_15_P106051
A_15_P120937
A_15_P528837
A_15_P115492
HGS
GLUL
GADD45G
G6PC
ARL5C
HMOX1
ARL4A
-0.36
-2.19
0.08
-2.75
-2.48
-1.05
-1.79
0.51
-0.62
1.39
-0.05
-1.23
-2.06
0.13
1.19
-0.25
0.11
0.05
-1.01
-2.08
-0.14
0.95
0.21
2.30
-0.57
-0.28
2.72
-1.21
-1.92
0.67
-0.08
-1.02
0.24
2.07
-0.97
0.28
-0.40
-1.47
1.53
1.34
0.58
0.65
-0.97
1.65
-2.25
1.73
2.25
1.01
1.46
-0.63
1.64
-0.98
1.58
1.92
-0.98
2.04
-1.41
-1.25
-1.23
-1.15
-1.15
-1.09
-1.05
0.25
0.27
0.01
0.20
0.03
0.20
0.28
A_15_P106261
A_15_P177326
A_15_P172616
A_15_P167731
A_15_P150786
LPIN1
PPP1R1B
GLS2
MAF
ARG2
-1.61
0.37
-0.59
-1.53
-1.17
-1.00
1.95
0.36
-1.88
-1.79
0.35
0.46
0.92
-0.04
0.45
-1.51
0.03
1.89
-0.36
-1.61
-0.47
-0.64
1.12
0.04
-0.67
2.32
-0.25
-0.48
1.30
2.15
0.60
-1.92
-0.90
1.01
1.12
1.42
-0.03
-2.28
1.37
1.31
-1.03
-1.01
-0.98
-0.98
-0.95
0.13
0.26
0.26
0.22
0.19
A_15_P539397
A_15_P139571
A_15_P101970
A_15_P178061
A_15_P102146
A_15_P177906
CYYR1
BCL6
PNPO
XPOT
SULT2B1
SLC22A2
0.06
1.25
1.71
0.28
-0.86
-0.65
-0.07
-1.22
2.62
-0.36
-1.80
-0.05
1.61
-1.04
1.68
0.76
3.40
-2.21
1.40
-2.17
-2.06
2.12
1.70
-0.58
0.83
-1.37
-1.42
0.77
0.56
0.40
-0.58
0.60
-0.92
-1.19
0.45
0.04
-0.60
1.93
0.56
-1.50
-0.66
0.75
-2.44
1.69
-1.70
-0.48
-2.38
1.66
-0.93
-0.90
-0.89
-0.85
-0.83
-0.82
0.19
0.28
0.05
0.27
0.21
0.29
A_15_P148136
A_15_P192056
A_15_P220861
A_15_P193116
A_15_P208486
A_15_P101502
A_15_P105218
IGFBP1
DNM3
LOC153328
INHBA
MRPS7
UPP2
ARRDC3
-0.40
-1.15
-1.66
0.08
-0.85
-1.30
1.09
2.17
-1.31
0.57
-0.54
-4.89
0.15
3.34
0.76
-0.56
-0.65
-1.74
-0.48
-0.98
0.10
1.18
-1.06
-1.51
-3.27
1.33
-2.27
0.39
-1.26
0.50
-3.55
-0.09
1.16
-0.12
-0.47
-1.13
0.40
1.93
1.58
1.15
0.11
-0.30
-0.60
0.53
0.45
0.94
-0.25
0.46
-0.11
0.32
1.20
3.02
0.44
0.22
1.52
-0.81
-0.72
-0.64
-0.57
-0.53
-0.47
-0.45
-0.34
0.13
0.21
0.15
0.15
0.25
0.26
0.21
A_15_P365290
A_15_P193306
A_15_P264991
A_15_P101781
A_15_P108161
A_15_P290496
A_15_P208476
FKBP5
EXOC1
ULK2
PRKACG
TMEM43
LECT2
KLF13
-4.74
-0.57
-2.69
1.95
0.38
3.30
-2.78
-4.29
2.67
-2.39
2.45
0.90
-0.09
-5.13
-0.22
2.54
0.05
1.92
-4.07
4.58
0.26
-0.45
2.35
-0.30
-0.23
-3.53
-0.95
-0.14
0.19
-0.70
0.43
-0.21
-0.18
-1.03
0.19
0.96
-0.75
0.50
-0.50
-0.02
-0.50
0.13
0.72
0.21
-0.05
-0.21
0.02
0.09
0.17
0.96
-0.24
0.23
0.18
0.81
0.87
-0.46
-0.29
-0.21
-0.21
-0.12
-0.10
-0.08
0.00
0.05
0.02
0.26
0.13
0.17
0.13
0.19
A_15_P111545
A_15_P149406
A_15_P210696
A_15_P108721
A_15_P170116
CYP2R1
FABP2
TSR2
VDAC1
RPL36
-4.09
7.26
-1.98
-4.44
0.78
0.46
1.14
-2.34
0.77
-5.64
-0.31
-0.14
-3.80
-0.22
0.18
-2.20
-1.24
2.15
-4.83
-0.08
0.25
-1.55
1.03
-4.47
-5.95
0.47
-0.16
-3.19
0.65
0.07
-1.72
1.78
0.97
0.19
-5.21
0.83
0.13
0.80
1.29
1.27
0.03
0.03
0.06
0.27
2.06
0.23
0.26
0.19
0.18
0.08
Table S2. GSEA of DIO-zebrafish with or without eriocitrin
Gene set
Detail
HCC_SURVIVAL_GOOD_VS_POOR_DN
Highly expressed in hepatocellular carcinoma with poor survival.
0.072
INOS_ALL_DN
Downregulated following iNOS induction in hepatocytes.
0.080
NEMETH_TNF_UP
Induced after 3 h in LPS stimulated RAW 2647 macrophages.
0.082
FLOTHO_CASP8AP2_MRD_DIFF
Associated with minimal residual disease in ALL. Genes significantly associated with MRD
on day 46
FDR q-value
0.092
HIPPOCAMPUS_DEVELOPMENT_PRENATAL Highly expressed in prenatal hippocampus.
0.109
HUMAN_MITODB_6_2002
Mitochondrial genes.
0.110
ELECTRON_TRANSPORT_CHAIN
Electron transport chain.
0.119
UVB_NHEK1_UP
Upregulated in NHEK cells (normal keratinocytes) by UV-B irradiation.
0.122
AGED_RHESUS_DN
Downregulated in the vastus lateralis muscle of aged vs young adult rhesus monkeys
0.125
LEE_MYC_UP
Upregulated in hepatocellular carcinoma (HCC) induced by overexpression of MYC.
0.143
RIBOSOMAL_PROTEINS
Ribosomal protein.
0.151
MOOTHA_VOXPHOS
Oxidative phosphorylation.
0.161
SANA_IFNG_ENDOTHELIAL_DN
VEGF_MMMEC_3HRS_UP
NING_COPD_DN
FDR, false discovery rate
Downregulated in five primary endothelial cell types (lung, aortic, iliac, dermal, and colon)
by IFNG.
Upregulated at 3 h follwing VEGF treatment of human myometrial microvascular
endothelial cells.
Downregulated genes in lung tissue of smokers with chronic obstructive pulmonary disease
(COPD) vs smokers without disease.
0.187
0.197
0.199
Table S3. Primer sequences for qRT-PCR
ZF
symbol
ZF gene name
ZF locus
forward primer
reverse primer
NM_001077
TGTGGAGGCAAAGGGAA
CGTCCTCTGCCAGCTGTA
389.1
CTACT
TGT
NM_131680
CTCTGAACTTCCTCCGCT
CCCTCGGCCTGTGGAATA
.1
GACT
CT
proliferator
NM_001102
GTTTGTCGCTGCCATCAT
GCAGATGGAGAACGTTA
activated receptor
567.1
CTG
ACAATGC
NM_001005
ATGCTTTTGACTACCGTG
TGGGACTCATGGACCTCA
933.1
ATGAGAT
GTATG
dehydrogenase,
NM_213089
AGGAGAACGTTTTGATTG
AGCATACTTTGTGGCCTC
C-4 to C-12
.1
GAGAGG
TTCTAG
NM_214701
CTCTCAAGGAGAAAGAG
CAACCACAGATTTCCACT
.1
AAGGGATC
CTCCTG
NM_213307
GCTGGTCCGGTGTATCAG
GTGAAGCTGAACTGAGG
.1
AAGAA
GAACT
XM_002667
CCTCAGTCCCAGTCGCAT
TTCTCATAGTCGCGTCGG
531.2
TC
TACTC
NM_001159
GGGAAAGTCTCCACTCCG
CCGACATAACCTGTCCGT
768.1
AAA
TCTT
HS
LOCUS
forward primer
reverse primer
Transcription
tfam
factor A,
mitochondrial
Nuclear
nrf1
respiratory factor
1
Peroxisome
pparab
αb
Acyl-coenzyme A
acox1
oxidase 1,
palmitoyl
Acyl-coenzyme A
acadm
straight chain
Cytochrome c
cox4i1
oxidase subunit
IV isoform 1
ATP synthase, H+
transporting,
atp5j
mitochondrial F0
complex, subunit
F6
Peroxisome
ppargc
1al
proliferator-activa
ted receptor γ,
coactivator 1α
like
b2m
β2-Microglobulin
HS
symbol
HS gene name
Transcription
TFAM
NM_003201
GCTCAGAACCCAGATGCA
GCCACTCCGCCCTATAAG
.1
AAA
C
NM_001040
CAGACGACGCAAGCATCA
CGCTGTTAAGCGCCATAG
110.1
GA
TG
proliferator
NM_001001
GGGATGCTGGTAGCGTAT
AACTTCATGGCAAAATCA
activated receptor
928.2
GG
AACTTG
NM_001185
CGCTATGATGGGAATGTG
GCTTGGACTGCAGTGACT
039.1
TATGA
TCAG
dehydrogenase,
NM_000016
CCAAGTATGCCCTGGAAA
ACCAGAATCAACCTCCCA
C-4 to C-12
.4
GG
AGCT
NM_001861
GAGATGAACAGGGGCTC
TGCTTCTGCCACATGATA
.3
GAAC
ACGAG
NM_001003
GAGGAACATTGGTGTTAC
TCCTCCAGATGTCTGTCG
696.1
AGCAGT
CTTAG
NC_012920.
CAAGGCCCAGAGAAGTAT
GTGGCAGGGAAGGTCTA
1
GATG
GGTAG
NM_000298
ATGAAACTTCGGCTCACT
TGTATGGGATGGCGGATA
.5
CCT
GTA
NM_004048
TTTCCTGAATTGCTATGTG
CTGAATGCTCCACTTTTT
.2
TCTGGGTT
CAATTCTCT
factor A,
mitochondrial
Nuclear
NRF1
respiratory factor
1
Peroxisome
PPAR
A
α
ACOX
1
Acyl-CoA
oxidase 1,
palmitoyl
Acyl-CoA
ACAD
M
straight chain
COX4I
1
Cytochrome c
oxidase subunit
IV isoform 1
ATP synthase, H+
transporting,
ATP5J
mitochondrial F0
complex, subunit
F6
CYTB
Cytochrome B
PK
Pyruvate kinase
B2M
β2-Microglobulin