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; notsignificant.

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)

Probe name / Human orthologue
(Symbol) / OF / OF+Erio / Log ratio / FDR
A_15_P106002 / XYLB / 0.35 / 1.72 / -0.55 / -0.46 / 1.24 / -3.72 / 0.61 / -3.81 / -5.36 / 0.13
A_15_P190701 / HNRNPA0 / 1.03 / -1.64 / 0.69 / 0.90 / 1.44 / -1.37 / -1.93 / -2.77 / -4.73 / 0.18
A_15_P211791 / NFKBIA / -0.39 / -0.09 / -0.62 / -0.35 / 0.08 / 1.65 / 1.79 / 2.41 / -4.10 / 0.18
A_15_P113821 / CISH / -1.04 / 0.60 / 0.76 / 1.15 / 0.94 / -1.75 / -1.47 / -2.25 / -3.08 / 0.13
A_15_P143731 / PNP / -1.09 / -0.39 / 0.00 / -0.19 / 0.00 / 2.26 / 1.16 / 1.62 / -3.01 / 0.19
A_15_P107051 / PDK2 / 0.59 / 2.49 / 1.56 / -1.03 / -3.33 / -1.14 / -3.10 / 1.63 / -1.64 / 0.01
A_15_P105956 / ERRFI1 / -1.96 / 0.40 / -0.90 / -0.71 / -0.56 / 1.88 / 1.08 / 2.62 / -1.59 / 0.15
A_15_P107432 / MAPK12 / -0.07 / -0.46 / 0.02 / -2.22 / -0.02 / 1.79 / 1.24 / 1.08 / -1.50 / 0.25
A_15_P209091 / CELA1 / 5.51 / -3.54 / -2.88 / -0.24 / 3.36 / -3.81 / 1.88 / 0.21 / -1.42 / 0.20
A_15_P101274 / HGS / -0.36 / 0.51 / 1.19 / 0.95 / -1.92 / 0.28 / -0.97 / -0.63 / -1.41 / 0.25
A_15_P112691 / GLUL / -2.19 / -0.62 / -0.25 / 0.21 / 0.67 / -0.40 / 1.65 / 1.64 / -1.25 / 0.27
A_15_P162361 / GADD45G / 0.08 / 1.39 / 0.11 / 2.30 / -0.08 / -1.47 / -2.25 / -0.98 / -1.23 / 0.01
A_15_P106051 / G6PC / -2.75 / -0.05 / 0.05 / -0.57 / -1.02 / 1.53 / 1.73 / 1.58 / -1.15 / 0.20
A_15_P120937 / ARL5C / -2.48 / -1.23 / -1.01 / -0.28 / 0.24 / 1.34 / 2.25 / 1.92 / -1.15 / 0.03
A_15_P528837 / HMOX1 / -1.05 / -2.06 / -2.08 / 2.72 / 2.07 / 0.58 / 1.01 / -0.98 / -1.09 / 0.20
A_15_P115492 / ARL4A / -1.79 / 0.13 / -0.14 / -1.21 / -0.97 / 0.65 / 1.46 / 2.04 / -1.05 / 0.28
A_15_P106261 / LPIN1 / -1.61 / -1.00 / 0.35 / -1.51 / -0.47 / 2.32 / 0.60 / 1.42 / -1.03 / 0.13
A_15_P177326 / PPP1R1B / 0.37 / 1.95 / 0.46 / 0.03 / -0.64 / -0.25 / -1.92 / -0.03 / -1.01 / 0.26
A_15_P172616 / GLS2 / -0.59 / 0.36 / 0.92 / 1.89 / 1.12 / -0.48 / -0.90 / -2.28 / -0.98 / 0.26
A_15_P167731 / MAF / -1.53 / -1.88 / -0.04 / -0.36 / 0.04 / 1.30 / 1.01 / 1.37 / -0.98 / 0.22
A_15_P150786 / ARG2 / -1.17 / -1.79 / 0.45 / -1.61 / -0.67 / 2.15 / 1.12 / 1.31 / -0.95 / 0.19
A_15_P539397 / CYYR1 / 0.06 / -0.07 / 1.61 / 1.40 / 0.83 / -0.58 / -0.60 / -2.44 / -0.93 / 0.19
A_15_P139571 / BCL6 / 1.25 / -1.22 / -1.04 / -2.17 / -1.37 / 0.60 / 1.93 / 1.69 / -0.90 / 0.28
A_15_P101970 / PNPO / 1.71 / 2.62 / 1.68 / -2.06 / -1.42 / -0.92 / 0.56 / -1.70 / -0.89 / 0.05
A_15_P178061 / XPOT / 0.28 / -0.36 / 0.76 / 2.12 / 0.77 / -1.19 / -1.50 / -0.48 / -0.85 / 0.27
A_15_P102146 / SULT2B1 / -0.86 / -1.80 / 3.40 / 1.70 / 0.56 / 0.45 / -0.66 / -2.38 / -0.83 / 0.21
A_15_P177906 / SLC22A2 / -0.65 / -0.05 / -2.21 / -0.58 / 0.40 / 0.04 / 0.75 / 1.66 / -0.82 / 0.29
A_15_P148136 / IGFBP1 / -0.40 / 2.17 / 0.76 / 1.18 / -1.26 / -1.13 / -0.60 / 0.32 / -0.72 / 0.13
A_15_P192056 / DNM3 / -1.15 / -1.31 / -0.56 / -1.06 / 0.50 / 0.40 / 0.53 / 1.20 / -0.64 / 0.21
A_15_P220861 / LOC153328 / -1.66 / 0.57 / -0.65 / -1.51 / -3.55 / 1.93 / 0.45 / 3.02 / -0.57 / 0.15
A_15_P193116 / INHBA / 0.08 / -0.54 / -1.74 / -3.27 / -0.09 / 1.58 / 0.94 / 0.44 / -0.53 / 0.15
A_15_P208486 / MRPS7 / -0.85 / -4.89 / -0.48 / 1.33 / 1.16 / 1.15 / -0.25 / 0.22 / -0.47 / 0.25
A_15_P101502 / UPP2 / -1.30 / 0.15 / -0.98 / -2.27 / -0.12 / 0.11 / 0.46 / 1.52 / -0.45 / 0.26
A_15_P105218 / ARRDC3 / 1.09 / 3.34 / 0.10 / 0.39 / -0.47 / -0.30 / -0.11 / -0.81 / -0.34 / 0.21
A_15_P365290 / FKBP5 / -4.74 / -4.29 / -0.22 / -0.45 / 0.19 / 0.96 / 0.72 / 0.96 / -0.29 / 0.05
A_15_P193306 / EXOC1 / -0.57 / 2.67 / 2.54 / 2.35 / -0.70 / -0.75 / 0.21 / -0.24 / -0.21 / 0.02
A_15_P264991 / ULK2 / -2.69 / -2.39 / 0.05 / -0.30 / 0.43 / 0.50 / -0.05 / 0.23 / -0.21 / 0.26
A_15_P101781 / PRKACG / 1.95 / 2.45 / 1.92 / -0.23 / -0.21 / -0.50 / -0.21 / 0.18 / -0.12 / 0.13
A_15_P108161 / TMEM43 / 0.38 / 0.90 / -4.07 / -3.53 / -0.18 / -0.02 / 0.02 / 0.81 / -0.10 / 0.17
A_15_P290496 / LECT2 / 3.30 / -0.09 / 4.58 / -0.95 / -1.03 / -0.50 / 0.09 / 0.87 / -0.08 / 0.13
A_15_P208476 / KLF13 / -2.78 / -5.13 / 0.26 / -0.14 / 0.19 / 0.13 / 0.17 / -0.46 / 0.00 / 0.19
A_15_P111545 / CYP2R1 / -4.09 / 0.46 / -0.31 / -2.20 / 0.25 / 0.47 / -1.72 / 0.83 / 0.03 / 0.23
A_15_P149406 / FABP2 / 7.26 / 1.14 / -0.14 / -1.24 / -1.55 / -0.16 / 1.78 / 0.13 / 0.03 / 0.26
A_15_P210696 / TSR2 / -1.98 / -2.34 / -3.80 / 2.15 / 1.03 / -3.19 / 0.97 / 0.80 / 0.06 / 0.19
A_15_P108721 / VDAC1 / -4.44 / 0.77 / -0.22 / -4.83 / -4.47 / 0.65 / 0.19 / 1.29 / 0.27 / 0.18
A_15_P170116 / RPL36 / 0.78 / -5.64 / 0.18 / -0.08 / -5.95 / 0.07 / -5.21 / 1.27 / 2.06 / 0.08

Table S2. GSEA of DIO-zebrafish with or without eriocitrin

Gene set / Detail / FDR q-value
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 / 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 / Downregulated in five primary endothelial cell types (lung, aortic, iliac, dermal, and colon) by IFNG. / 0.187
VEGF_MMMEC_3HRS_UP / Upregulated at 3 h follwing VEGF treatment of human myometrial microvascular endothelial cells. / 0.197
NING_COPD_DN / Downregulated genes in lung tissue of smokers with chronic obstructive pulmonary disease (COPD) vs smokers without disease. / 0.199

FDR,false discovery rate

Table S3.Primer sequences for qRT-PCR

ZF symbol / ZF gene name / ZF locus / forward primer / reverse primer
tfam / Transcription factor A, mitochondrial / NM_001077389.1 / TGTGGAGGCAAAGGGAACTACT / CGTCCTCTGCCAGCTGTATGT
nrf1 / Nuclear respiratory factor 1 / NM_131680.1 / CTCTGAACTTCCTCCGCTGACT / CCCTCGGCCTGTGGAATACT
pparab / Peroxisome proliferator activated receptorαb / NM_001102567.1 / GTTTGTCGCTGCCATCATCTG / GCAGATGGAGAACGTTAACAATGC
acox1 / Acyl-coenzyme A oxidase 1, palmitoyl / NM_001005933.1 / ATGCTTTTGACTACCGTGATGAGAT / TGGGACTCATGGACCTCAGTATG
acadm / Acyl-coenzyme A dehydrogenase, C-4 to C-12 straight chain / NM_213089.1 / AGGAGAACGTTTTGATTGGAGAGG / AGCATACTTTGTGGCCTCTTCTAG
cox4i1 / Cytochrome c oxidase subunit IV isoform 1 / NM_214701.1 / CTCTCAAGGAGAAAGAGAAGGGATC / CAACCACAGATTTCCACTCTCCTG
atp5j / ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 / NM_213307.1 / GCTGGTCCGGTGTATCAGAAGAA / GTGAAGCTGAACTGAGGGAACT
ppargc1al / Peroxisome proliferator-activated receptor γ, coactivator 1α like / XM_002667531.2 / CCTCAGTCCCAGTCGCATTC / TTCTCATAGTCGCGTCGGTACTC
b2m / β2-Microglobulin / NM_001159768.1 / GGGAAAGTCTCCACTCCGAAA / CCGACATAACCTGTCCGTTCTT
HS symbol / HS gene name / HS LOCUS / forward primer / reverse primer
TFAM / Transcription factor A, mitochondrial / NM_003201.1 / GCTCAGAACCCAGATGCAAAA / GCCACTCCGCCCTATAAGC
NRF1 / Nuclear respiratory factor 1 / NM_001040110.1 / CAGACGACGCAAGCATCAGA / CGCTGTTAAGCGCCATAGTG
PPARA / Peroxisome proliferator activated receptor α / NM_001001928.2 / GGGATGCTGGTAGCGTATGG / AACTTCATGGCAAAATCAAACTTG
ACOX1 / Acyl-CoA oxidase 1, palmitoyl / NM_001185039.1 / CGCTATGATGGGAATGTGTATGA / GCTTGGACTGCAGTGACTTCAG
ACADM / Acyl-CoA dehydrogenase, C-4 to C-12 straight chain / NM_000016.4 / CCAAGTATGCCCTGGAAAGG / ACCAGAATCAACCTCCCAAGCT
COX4I1 / Cytochrome c oxidase subunit IV isoform 1 / NM_001861.3 / GAGATGAACAGGGGCTCGAAC / TGCTTCTGCCACATGATAACGAG
ATP5J / ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 / NM_001003696.1 / GAGGAACATTGGTGTTACAGCAGT / TCCTCCAGATGTCTGTCGCTTAG
CYTB / Cytochrome B / NC_012920.1 / CAAGGCCCAGAGAAGTATGATG / GTGGCAGGGAAGGTCTAGGTAG
PK / Pyruvate kinase / NM_000298.5 / ATGAAACTTCGGCTCACTCCT / TGTATGGGATGGCGGATAGTA
B2M / β2-Microglobulin / NM_004048.2 / TTTCCTGAATTGCTATGTGTCTGGGTT / CTGAATGCTCCACTTTTTCAATTCTCT