Supplementary Material
Supplementary Methods
Gene expression analysis by RT-qPCR
Control RNA from lean (BMI<25 kg/m2) and obese (BMI 32.1 kg/m2) human adipose tissue (Amsbio, UK)
was used to validate the expression of selected genes by real-time quantitative PCR system (CFX96, BioRad, UK) with TaqMan technology (Applied Biosystems, Europe). The reactions were performed following
the manufacturer’s guidelines in a final volume of 20 µL (IQ Supermix 170-8860, Bio-Rad, UK). The
optimal number of reference genes was evaluated by Genorm analysis (qBASEplus, Biogazelle,
Netherlands) and ACTB and GAPDH were chosen for normalization of the results. The results were
analyzed by qBASEplus (Biogazelle, Netherlands). The commercially available TaqMan probes (Applied
Biosystems, Europe) were used as follows: ZBTB16 (Hs00232313_m1), E2F5 (Hs00231092_m1),
EHBP1L1
(Hs00411094_m1),
MRPL23
(Hs00221699_m1),
PPARγ
(Hs01115513_m1),
ASAP2
(Hs00187341_m1), RBPMS (Hs00199302_m1), FGFRL1 (Hs00222484_m1), CHST11 (Hs00218229_m1),
TBC1D16 (Hs00292666_m1), ACTB (Hs01060665_g1), and GAPDH (Hs02758991_g1).
Immunohistochemistry
Immunohistochemistry was performed on paraffin-embedded SAT sections from a lean individual with
antibodies to PPARγ (ab19481, Abcam, UK), CD163 (MS-1103-S0, Nuppulinna, Finland), CD31 (MS-353S0, Thermo Scientific, USA), ZBTB16 (HPA001499, Sigma, USA), MRPL23 (HPA050406, Sigma, USA),
CHST11 (HPA052828, Sigma-Aldrich, Sweden), and RBPMS (HPA056999, Sigma-Aldrich, Sweden). A
poly-HRP-anti-mouse/rabbit/rat IgG kit was used for detection (Thermo Scientific, USA), and the sections
were imaged with a Zeiss AxioImager microscope with a 63 x objective. Hematoxylin-eosin staining was
performed by a standard protocol.
Supplementary Table 1. Anthropometric and metabolic measures of the 26 BMI-discordant and 11 BMI-concordant MZ twin
pairs.
BMI-discordant pairs
BMI-concordant pairs
Number of twin pairs
Age, years (range)
26
11
30 (23-36)
32 (23-36)
9/17
7/4
Sex (m/f)
Heavier
Within-Pair
(Heavy-Lean)
P-value a
Leaner
Heavier
Within-Pair
(Heavy-Lean)
P-value a
Leaner
BMI (kg/m2)
25.28±4.52
31.25±5.18
5.97±2.78
Body fat (%)
32.62±9.09
41.15±6.83
8.53±5.61
<0.001
26.30±3.72
27.66±4.21
1.36±0.82
0.0033
<0.001
30.19±10.04
31.15±9.60
0.96±2.70
0.3739
3813.77±2125.58
6358.86±2755.28
2545.093±1598.13
<0.001
3223.95±1444.35
3618.25±1607.74
394.30±494.38
0.0329
790.16±912.63
1643.67±1261.71
853.52±690.78
<0.001
1099.02±687.47
1190.69±792.60
91.66±394.50
1.0000
1.12±1.61
4.52±5.09
3.40±4.17
<0.001
2.26±3.74
4.02±7.23
1.76±3.64
0.075
81.51±13.07
95.26±14.69
13.75±8.37
<0.001
84.85±10.29
86.65±10.01
1.80±3.01
0.176
fP-glucose (mmol/l)
5.15±0.35
5.28±0.53
2.63±4.99
0.010
5.3±0.45
5.41±0.53
0.11±0.26
0.138
fP-insulin (mU/l)
4.91±2.52
8.50±6.05
3.57±5.33
<0.001
6.08±1.36
6.01±2.36
-0.07±3.13
0.625
HOMA-IR index
1.14±0.62
2.07±1.69
0.94±1.45
<0.001
1.47±1.14
1.47±0.64
0.003±0.71
0.477
Matsuda index
9.07±4.71
5.91±3.08
-3.40±4.18
<0.002
10.56±9.24
8.14±5.17
-2.42±4.81
0.286
fS-Total cholesterol (mmol/l)
4.40±0.76
4.69±0.96
0.28±1.05
0.131
4.67±0.89
4.7±0.75
0.04±0.56
0.789
fS-LDL cholesterol (mmol/l)
2.57±0.73
2.98±0.83
0.41±0.93
0.034
2.97±0.70
3.20±2.56
0.03±0.45
0.893
fS-HDL cholesterol (mmol/l)
1.61±0.37
1.33±0.42
-0.28±0.33
<0.001
1.36±0.36
1.39±0.29
0.03±0.27
0.3739
fS-Triglycerides (mmol/l)
0.94±0.45
1.32±0.81
0.38±0.68
0.016
1.05±0.78
0.94±0.66
-0.10±0.45
0.965
Subcutaneous fat (cm3)
3
Intra-abdominal fat (cm )
Liver fat (%)
Adipocyte diameter (μm)
Hs-CRP
2.53±3.31
4.02±5.59
1.46±4.53
0.075
1.45±2.30
1.23±0.76
-0.21±2.54
0.306
fP-Adiponectin (ng/ml)
1490±1614
1178±1270
-312±535
0.0002
1284±1323
1267±1293
-17±208
0.790
fP-Leptin (pg/ml)
18790±18448
33099±20977
14309±13980
<0.001
13649±15442
15122±17910
1472±4533
0.328
Systolic blood pressure (mmHg)
120.65±11.87
123.55±12.93
2.90±10.88
0.183
122.91±8.89
119.36±8.45
-3.55±9.49
0.211
Diastolic blood pressure (mmHg)
76.70±7.97
80.10±7.37
3.40±5.51
0.024
78.82±7.49
79.36±5.08
0.55±5.48
0.651
Heart rate (beats/min)
Alcohol intake dose/wk
68.15±15.15
70.90±14.71
7.45±8.78
2.75±10.33
0.303
65±15.92
0.449
1.51±10.23
0.154
6.56±8.40
60.45±11.40
8.97±7.22
-4.55±13.69
5.94±10.5
2.42±3.59
0.154
8.87±1.23
8.29±1.13
-0.58±1.40
0.203
8.59±1.90
2141±433
8.73±1.65
0.14±1.11
86±865
1.0000
1
2
Baecke Index
Energy intake (kcal)
One twin smokesb
2094±543
2166±515
3
3
72±687
0.932
Non-smokers
16 pairs
6 pairs
Both smokers
4 pairs
2 pairs
2228±580
0.594
Data are mean ± SD.
fP, fasting plasma; fS, fasting serum; hs-CRP, high-sensitivity C-reactive protein;
a
Comparisons of the clinical parameters within twin pairs were made by matched-pairs Wilcoxon’s signed rank tests.
b
The number in the cell reports which twin is the smoker.
Supplementary Table 2. Within-pair gene expression differences in adipocytes of the 14 BMIdiscordant MZ twin pairs.
Entrez ID
8853
11030
7704
53834
50515
125058
6150
1875
254102
Gene
ASAP2
RBPMS
ZBTB16
FGFRL1
CHST11
TBC1D16
MRPL23
E2F5
EHBP1L1
Log Fold Change in heavy
vs. lean co-twins
0.03
-0.05
-0.03
-0.08
0.1
0.01
-0.01
-0.02
0.03
P-value
0.003
0.003
0.048
0.051
0.176
0.245
0.522
0.49
0.599
Supplementary Table 3. Expression of genes in differentially methylated pathways of the 24
BMI-discordant MZ twin pairs.
Entity
Pathway
ChREBP activates metabolic
gene expression
Regulation of cholesterol
biosynthesis by SREBP
(SREBF)
Lipid metabolism
Fatty acyl-CoA biosynthesis
Regulation of signaling by
NODAL
Acyl chain remodeling of
DAG and TAG
Activation of gene
expression by SREBF
(SREBP)
Triglyceride biosynthesis
Inflammation
Linoleic acid (LA)
metabolism
ABC transporters in lipid
homeostasis
Downregulation of TGF-beta
receptor signaling
PTM: gamma carboxylation,
hypusine formation, and
arylsulfatase activation
TGF-beta receptor signaling
activates SMADs
Genes upregulated
Genes downregulated
ACACA, ACACB,
–
ACLY, FASN, MLX,
MLXIPL
ACACA, ACACB,
GGPS1, MBTPS1, NFYA, ELOVL6, FASN, FDFT1,
NFYB, NFYC, SEC24C,
HMGCS1, KPNB1, LSS,
SEC24D, SAR1B, SQLE
SC5D, SEC24B, SP1,
TM7SF2
ACACA, ACLY, FASN,
ACSL3, ELOVL1,
SLC25A1, TECR,
ELOVL4, ELOVL7
HSD17B12, ELOVL5,
ELOVL6
ACVR1B, ACVR2B,
–
ACVR1C
–
GGPS1, NFYA, NFYB,
NFYC, SQLE
ACSL3, ELOVL1,
ELOVL4, ELOVL7,
AGPAT3, AGPAT4,
AGPAT5, AGPAT6,
LPCAT1, LCLAT1
DGAT1
ACACA, ACACB,
ELOVL6, FASN, FDFT1,
HMGCS1, LSS, SC5D,
SP1, TM7SF2
ACACA, ACLY, FASN,
SLC25A1, DGAT1,
TECR, LPIN2, GPD1L,
LPIN1, HSD17B12,
ELOVL5, ELOVL6,
AGPAT9
FADS3, ELOVL5
ACSL1
ABCA2
PEX19
BAMBI, PPP1CB,
PPP1CA, TGFB1,
TGFBR2, PMEPA1,
SMAD3, UCHL5
SMAD2, NEDD4L,
UBA52, ZFYVE9,
USP15, SMURF1
ARSB, GGCX, PROS1,
SUMF1
DHPS
BAMBI, FKBP1A,
PPP1CB, PPP1CA,
TGFB1, TGFBR2,
PMEPA1, SMAD3,
UCHL5
NEDD4L, SMAD2,
SMAD4, SMURF1,
UBA52, USP15, ZFYVE9
Signaling
Vitamin
metabolism
Remodeling of the adipose
tissue ECM
Deactivation of the betacatenin transactivating
complex
FasL/ CD95L signaling
Lysosome vesicle biogenesis
Interleukin-1 processing
Trafficking and processing
of endosomal TLR
Hyaluronan uptake and
degradation
Other semaphorin
interactions
Cell-extracellular matrix
interactions
Regulation of cytoskeletal
remodeling and cell
spreading by IPP complex
components
Vitamin B5 (pantothenate)
metabolism
FMO oxidizes nucleophiles
Vitamin B6 activation to
pyridoxal phosphate
SOS-mediated signaling
Signal attenuation
CTNNB1, CTBP1, APC,
TLE4, YWHAZ, MEN1,
TLE3, TLE2
CASP10, FADD
CLTC, DNM2, DNASE2,
AP1B1, AP1G1, GNS,
VAMP8
CASP1, IL18
CNPY3, CTSB, CTSS,
LGMN
CD44
ITGB1, PLXNA1, PTPRC,
TYROBP, PLXNC1,
PLXNA4, PLXND1
ACTB, ACTG1, ACTN1,
ITGB1, FBLIM1, PARVA,
VASP
AKT2, TLE1, TCF7L1,
TCF7L2, ASH2L, UBA52
CASP8
ARRB1
–
CHP1
SEMA6D, SEMA3E
FERMT2
ACTN1, PARVA
–
SLC25A16, PPCDC,
PANK3, PPCS
AASDHPPT, FASN,
PANK2
–
FMO2
AOX1, PNPO
–
GRB2, HRAS, NRAS,
MAPK1, MAP2K1, SOS1,
YWHAB
CRK, GRB2, MAPK1,
SHC1, SOS1
IRS2, IRS1
GRB10, INSR, IRS1,
IRS2, SHC3
Supplementary Figures
Supplementary Figure 1. Similarity of methylation between unrelated individuals and cotwins. Methylation differences between unrelated twin individuals (blue) and co-twins (red) were
plotted as an empirical cumulative distribution function. The plot shows that MZ twin pairs are
more similar in their methylation patterns than unrelated individuals matched by age, sex and BMI.
Euclidean distance (ED) was used as a measure for methylation differences between the samples.
The mean ED was 19.6 for twin pairs and 28.1 for unrelated twin individuals. Eight MZ twin pairs
and 16 same-sex unrelated twin individuals were used in plotting the graph. All subjects were nonsmokers.
Supplementary Figure 2. Relative methylation of the differentially methylated CpG sites in heavy
compared with lean monozygotic co-twins discordant for body mass index (n=24 pairs), and their
locations in relation to genes and CpG density. Half of the 22 differentially methylated CpG sites
were hypermethylated in the heavy co-twins. Most of the hypermethylated CpG sites were located
in gene bodies and associated with CpG islands, whereas most of the hypomethylated CpGs were
found in intergenic regions as isolated CpG sites (open sea).
Supplementary Figure 3. Validation of differential gene expression. A) RT-qPCR analysis of
the nine differentially methylated and differentially expressed genes by using commercial adipose
tissue RNA validated the upregulation of CHST11, TBC1D16, ASAP2, EHBP1L1 and MRPL23 and
the downregulation of ZBTB16, FGFRL1, RBPMS, and E2F5. B) The master upstream regulator
PPARγ was found to be downregulated as expected. The bars show the mean ratio of gene
expression in obese individuals to that in lean individuals.
Supplementary Figure 4. Expression of the differentially methylated genes during adipocyte
differentiation. Analysis of gene expression during adipogenesis published by Mikkelsen et al
(2010) showed increased expression of CHST11, ZBTB16, FGFRL1, and RBPMS during induction
of adipogenesis. The expression patterns of ZBTB16 and RBPMS closely follow that of PPARG, the
master regulator of adipogenesis. Numbers on the X-axis denote days in culture. Day 0 consists of
preadipocytes, day 3 immature adipocytes, and day 9 mature adipocytes. The Y-axis shows the
level of gene expression.
Mikkelsen TS, Xu Z, Zhang X, Wang L, Gimble JM, Lander ES, et al. Comparative epigenomic
analysis of murine and human adipogenesis. Cell 2010; 143: 156-169.