J C E M O N L I N E

A d v a n c e s i n G e n e t i c s

Epigenetic Alterations in Human Liver From Subjects

With Type 2 Diabetes in Parallel With Reduced Folate
Levels

Emma Nilsson, Ashok Matte, Alexander Perfilyev, Vanessa D. de Mello,

Pirjo Käkelä, Jussi Pihlajamäki,* and Charlotte Ling*
Epigenetics and Diabetes Unit (E.N., A.P., C.L.), Department of Clinical Sciences, Lund University
Diabetes Centre, 205 02 Malmö, Sweden; Department of Clinical Nutrition (A.M., V.D.d.M., J.P.),
Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Department of Surgery
(P.K.), University of Eastern Finland and Kuopio University Hospital, and Clinical Nutrition and Obesity
Center (J.P.), Kuopio University Hospital, 70211 Kuopio, Finland

Objective: Epigenetic variation may contribute to the development of complex metabolic diseases
such as type 2 diabetes (T2D). Hepatic insulin resistance is a hallmark of T2D. However, it remains
unknown whether epigenetic alterations take place in the liver from diabetic subjects. Therefore,
we investigated the genome-wide DNA methylation pattern in the liver from subjects with T2D and
nondiabetic controls and related epigenetic alterations to gene expression and circulating folate
levels.

Research Design and Methods: Liver biopsies were obtained from 35 diabetic and 60 nondiabetic
subjects, which are part of the Kuopio Obesity Surgery Study. The genome-wide DNA methylation
pattern was analyzed in the liver using the HumanMethylation450 BeadChip. RNA expression was
analyzed from a subset of subjects using the HumanHT-12 Expression BeadChip.

Results: After correction for multiple testing, we identified 251 individual CpG sites that exhibit
differential DNA methylation in liver obtained from T2D compared with nondiabetic subjects (Q
⬍ .05). These include CpG sites annotated to genes that are biologically relevant to the develop-
ment of T2D such as GRB10, ABCC3, MOGAT1, and PRDM16. The vast majority of the significant CpG
sites (94%) displayed decreased DNA methylation in liver from subjects with T2D. The hypomethy-
lation found in liver from diabetic subjects may be explained by reduced folate levels. Indeed,
subjects with T2D had significantly reduced erythrocyte folate levels compared with nondiabetic
subjects. We further identified 29 genes that displayed both differential DNA methylation and
gene expression in human T2D liver including the imprinted gene H19.

Conclusions: Our study highlights the importance of epigenetic and transcriptional changes in the
liver from subjects with T2D. Reduced circulating folate levels may provide an explanation for
hypomethylation in the human diabetic liver. (J Clin Endocrinol Metab 100: E1491–E1501, 2015)

he prevalence of type 2 diabetes (T2D) is rapidly in- uncover disease-causing mechanisms in T2D. However,
T creasing worldwide, and it is well established that
combinations of genetic and nongenetic factors such as
the identified genetic variants explain only a modest pro-
portion of the estimated heritability of the disease (1).
obesity, physical inactivity, and aging increase the suscep- Additional studies, going beyond the genome, are there-
tibility of this complex metabolic disorder. Recent ge- fore needed to dissect the molecular mechanisms that con-
nome-wide association studies (GWAS) were expected to tribute to T2D. These investigations may include epige-

ISSN Print 0021-972X ISSN Online 1945-7197 * J.P. and C.L. contributed equally to this work.
Printed in USA Abbreviations: BMI, body mass index; FDR, false discovery rate; GWAS, genome-wide
Copyright © 2015 by the Endocrine Society association studies; NASH, nonalcoholic steatohepatitis; qPCR, quantitative PCR; T2D, type
Received August 19, 2015. Accepted September 24, 2015. 2 diabetes; UTR, untranslated region; VIF, variance inflation factor.
First Published Online September 29, 2015

doi: 10.1210/jc.2015-3204 J Clin Endocrinol Metab, November 2015, 100(11):E1491–E1501 press.endocrine.org/journal/jcem E1491

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E1492 Nilsson et al Epigenetic Alterations in Human T2D Liver J Clin Endocrinol Metab, November 2015, 100(11):E1491–E1501

netic mechanisms such as DNA methylation and histone Table 1. Clinical Characteristics of Subjects Included
modifications. Indeed, recent studies from our group and in the Study
others have identified altered DNA methylation patterns
in some of the primary tissues for T2D including pancre- Type 2 P
Nondiabetics Diabeticsa Valueb
atic islets, adipose tissue, and skeletal muscle from diabetic
n (men/women) 60 (17/43) 35 (17/18)
vs nondiabetic subjects (2– 6). However, whether the Age, y 48.9 ⫾ 7.9 50.5 ⫾ 7.3 ns
DNA methylation pattern is altered in the liver from sub- BMI, kg/m2 43.6 ⫾ 5.5 42.2 ⫾ 6.0 ns
jects with T2D remains unknown. fP-glucose, mmol/L 5.8 ⫾ 0.8 7.7 ⫾ 2.9 4.1 ⫻ 10⫺6
fS-insulin, mU/L 16.4 ⫾ 8.7 57.8 ⫾ 113.0 .052
The liver plays an important role in maintaining glu- (n ⫽ 42) (n ⫽ 30)
cose homeostasis during both the fed state, when it syn-
Abbreviation: fP, fasting plasma; fS, fasting serum; ns, not significant.
thesizes and stores glycogen in response to insulin stimu- Data are shown as mean ⫾ SD.
lation, and the fasted state, when gluconeogenesis and the a
In type 2 diabetic subjects, 22 (63%) received oral agents (metformin,
release of glucose take place in response to glucagon stim- sitagliptin, glimepiride, or rosiglitazone), 10 (29%) received both
ulation. This fine-tuned balance is lost in subjects with insulin and oral agents, and one (3%) received insulin treatment.
b
T2D, in whom hepatic insulin resistance contributes to Refers to Mann-Whitney statistics.
hyperglycemia. Epigenetic changes may potentially alter Diabetes was defined according to the World Health Or-
gene expression in the liver from subjects with T2D and ganization’s criteria (15). Plasma glucose and serum insu-
thereby contribute to an impaired metabolism and hyper- lin were analyzed as described before (12–14). Overall
glycemia. Such epigenetic changes could potentially be in- histological assessment of liver biopsy samples was per-
duced by a methyl donor supply-consumption imbalance. formed by one pathologist according to the standard cri-
Dietary components, including folate, serve to modulate teria (16, 17). Histological diagnosis was divided into the
the availability of methyl donors for methylation reactions following three categories: 1) normal liver without any
in vivo. Folate can thereby influence the levels of DNA steatosis, inflammation, ballooning, or fibrosis, 2) simple
methylation and consequently affect gene expression and steatosis (steatosis ⬎ 5%) without evidence of hepatocel-
cell functions (7). lular ballooning, inflammation, or fibrosis, and 3) nonal-
Although epigenetic studies in livers from subjects with coholic steatohepatitis (NASH). Steatosis was graded into
T2D are lacking, DNA methylation changes have been four categories (⬍5%, 5%–33%, 33%– 66%, and ⬎
identified in the liver from subjects with obesity and non- 66%). Of the 60 nondiabetic subjects, 27 (45%) had a
alcoholic fatty liver disease (8, 9). Interestingly, obesity normal liver phenotype, 21 (35%) had simple steatosis,
has been shown to accelerate epigenetic ageing of human and 12 (20%) had NASH. Of the 35 T2D subjects, eight
liver, which may partly explain why obese subjects often (23%) had a normal liver phenotype, 13 (37%) had simple
suffer from an early onset of age-related diseases such as steatosis, and 14 (40%) had NASH (P␹2 ⬍ .05 comparing
liver phenotype distribution between T2D and nondia-
T2D (8). Also, animal studies support a role for epigenetic
betic subjects). The level of lobular inflammation was as-
modifications in hepatic insulin resistance (10, 11).
sessed by histology as previously described (12). Erythro-
To identify epigenetic alterations in liver from subjects
cyte folate levels were measured in 20 of the subjects in the
with T2D, we used a genome-wide approach in which
fasted state using an electrochemiluminescense immuno-
DNA methylation of approximately 455 000 sites was an-
assay (Roche Diagnostics). One measurement was in a
alyzed in the liver from 35 diabetic and 60 nondiabetic
normal probability plot defined as an outlier (2.5 SD from
subjects. We further tested whether epigenetic changes in
the mean) and discarded from the analysis. Vitamin B12
the liver were associated with a differential gene expres-
levels were measured in 16 of the subjects using an elec-
sion and altered folate levels in humans.
trochemiluminescense immunoassay.
The study was performed in accordance with the Dec-
laration of Helsinki. Written informed consent was ob-
Research Design and Methods tained from all participants, and the study protocol was
approved by the Ethics Committee of the Northern Savo
Study participants and analyses of clinical and
Hospital District (numbers 54/2005, 104/2008, and
metabolic parameters
27/2010).
Liver biopsies obtained from 35 subjects with T2D and
60 nondiabetic controls as a wedge biopsy during a Roux- Genome-wide analysis of DNA methylation in
en-Y gastric bypass operation were included in this study. human liver
The subjects are part of the Kuopio Obesity Surgery Study DNA was extracted from human liver biopsies by
(12–14), and their characteristics are described in Table 1. DNeasy blood and tissue kit (QIAGEN) according to the

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doi: 10.1210/jc.2015-3204 press.endocrine.org/journal/jcem E1493

manufacturer’s protocol. Nucleic acid concentration and Validation of expression data with real-time
purity were determined using a Nano Drop 1000 spectro- quantitative PCR (qPCR)
photometer (NanoDrop Technologies). DNA methyl- Total RNA was reverse transcribed with a high-capacity
ation was analyzed in liver from 35 subjects with T2D and cDNA reverse transcription kit (Applied Biosystems). The
60 nondiabetic controls using the Infinium HumanMethy- qPCR was performed with the 7500 Fast real-time PCR sys-
lation450 BeadChip (Illumina) (18). Five hundred nano- tem using SYBR Green (Applied Biosystems). The following
grams of genomic DNA were bisulfite treated using the EZ primer sequences were used for the quantification of
DNA methylation kit (Zymo Research) and subsequently RIPK4 mRNA (receptor-interacting serine-threonine kinase
used for analysis of DNA methylation following the In- 4): forward primer, 5⬘-GTTAGGCCCACCTTCCAAGA-
finium HD assay methylation protocol (Illumina). The 3⬘, reverse primer, 5⬘-GGGGCTTTTCACGTCCAGAT-3⬘.
BeadChips’ images were captured using the Illumina Gene expression values were normalized to the endogenous
iScan. All included samples showed a high-quality bisul- expression of RPLP0 mRNA (ribosomal phosphoprotein
fite conversion efficiency (intensity signal ⬎ 4000) and large P0): forward primer, 5⬘-GGCGACCTGGAAGTC-
passed all GenomeStudio quality control steps based on CAACT-3⬘, reverse primer, 5⬘-CCATCAGCACCACAGC-
built-in control probes for staining, hybridization, exten- CTTC-3⬘ and analyzed based on the comparative cycle
sion, and specificity. We filtered away 65 rs-probes, threshold method (2-␦␦Ct). RPLP0 was selected as an endog-
14 548 cross-reactive probes based on annotation from enous control based on its previous use as a control gene in
Chen et al (19), and 10 817 X chromosome and 50 Y liver studies and because its expression is stable between sub-
chromosome probes as well as 2973 non-CpG probes. jects with T2D and nondiabetic controls in our microarray
Additionally, 1598 individual probes were filtered away expression data from human liver (P ⫽ .8 for probe identi-
based on mean detection values P ⬎ .01. The raw meth- fication 1470349) (25).
ylation data were exported from the GenomeStudio and
analyzed using Bioconductor and the lumi/methylumi Statistical analyses
package, and ␤-values were converted to M-values (M ⫽ To identify the differences in DNA methylation and
log2[␤/(1-␤)]) (20 –22). Data were background corrected mRNA expression in the liver from diabetic vs nondiabetic
and normalized using quantile normalization and ␤-mix- subjects, a linear regression model was used including
ture quantile normalization to correct for probe design T2D, gender, body mass index (BMI), age, NASH diag-
bias (23). The DNA methylation array data were corrected nosis, and degree of steatosis as covariates and DNA meth-
for batch effects using COMBAT (24). To easier interpret ylation or mRNA expression as the dependent variable.
the results, the M-values were reconverted to ␤-values, Variance inflation factors (VIFs), which provides infor-
which was used when describing the data and creating the mation about potential multicollinearity of studied phe-
figures. notypes, were calculated (26). To account for multiple
testing in the genome-wide analysis of DNA methylation,
Genome-wide analysis of gene expression in we applied false discovery rate (FDR) analysis and Q ⬍ .05
human liver (FDR ⬍ 5%) was considered significant. Spearman cor-
Total RNA was extracted from human liver tissue using relations were used to relate folate levels and fasting glu-
the miRNeasy minikit (QIAGEN). Nucleic acid concen- cose levels, folate levels and average degree of methyl-
tration and purity were determined using the Nano Drop ation, and RIPK4 mRNA expression and DNA
1000 spectrophotometer (NanoDrop Technologies). The methylation.
RNA quality was determined using the Agilent 2100 bio-
analyzer (Agilent Technologies). RNA expression was an-
alyzed in the liver from a subset of subjects (19 T2D and Results
23 nondiabetic subjects), due to the limited size of human
liver biopsies, available amounts of liver RNA, and re- Altered DNA methylation in the liver from subjects
sources. The clinical characteristics of these subjects are pre- with T2D
sented in Supplemental Table 1 and shows that the subcohort To dissect the epigenetic basis of T2D, we analyzed
is a representative sample of the full cohort, despite that there DNA methylation of 455 526 CpG sites in the liver from
was no significant difference in fasting insulin between cases 35 diabetic and 60 nondiabetic subjects included in the
and controls in the subcohort. RNA expression was analyzed Kuopio Obesity Surgery Study. The clinical characteristics
using the HumanHT-12 Expression BeadChip (Illumina), of these subjects are shown in Table 1. Although there
which covers 28 688 coding transcripts, according to the were no significant differences in age or BMI, subjects with
manufacturer’s recommendations. diabetes had elevated fasting plasma glucose levels (P ⫽

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E1494 Nilsson et al Epigenetic Alterations in Human T2D Liver J Clin Endocrinol Metab, November 2015, 100(11):E1491–E1501

4.1 ⫻ 10⫺6) and fasting insulin levels (borderline signifi- Here 18 genes previously associated with diabetes were
cance, P ⫽ .052) compared with nondiabetic subjects (Ta- found among the genes that showed differential DNA
ble 1). methylation in the T2D liver (Figure 1F) (27– 43). These
A linear regression model was then used to identify include GRB10, BCAT1, ANK1, and ZMIZ1 identified
differences in DNA methylation in the liver from diabetic through GWAS (32, 35, 38).
vs nondiabetic subjects. However, to estimate the multi- Although we adjusted our statistical analysis for
collinearity among the covariates included in the linear NASH, we cannot fully exclude that some of the 251 CpG
regression model, we first calculated the VIF for all cova- sites showing altered DNA methylation in the liver from
riates (T2D, gender, BMI, age, NASH diagnosis, and de- subjects with T2D compared with nondiabetic controls
gree of steatosis). All calculated VIFs were close to 1 reflect NASH rather than T2D. We therefore looked for an
(1.06 –1.41), demonstrating that the problem with multi- overlap between our 251 CpG sites significantly associ-
collinearity among these variables is very limited. Based on ated with T2D in liver and CpG sites significantly associ-
the linear regression model and after correction for mul- ated with NASH in human liver in a previous study by
tiple testing using FDR analysis, we identified 251 CpG Ahrens et al (44). Only 4 of our 251 significant CpG sites
sites, representing 162 unique genes, with significant dif- were found to also be significant in liver from subjects with
ferences in DNA methylation in the liver from T2D vs NASH compared with controls in this study (marked in
nondiabetic subjects (Q ⬍ .05, Supplemental Table 2). Supplemental Table 2). We also looked for an overlap
The vast majority of the significant CpG sites (236 sites; between our 251 CpG sites significantly associated with
94%) displayed decreased DNA methylation in subjects T2D and CpG sites significantly associated with NASH in
with T2D (Figure 1A). Because hypomethylation may be the liver samples included in our study. Here 41 of our 251
explained by a methyl donor supply-consumption imbal- significant sites were found to be significantly associated
ance, we analyzed subjects’ vitamin B12 and folate levels. with NASH (marked in Supplemental Table 2). Addition-
There was no significant difference in vitamin B12 levels ally, we performed the analysis restricted to those without
between T2D (n ⫽ 6) and nondiabetic subjects (n ⫽ 10) NASH (21 T2D and 48 nondiabetic subjects) and re-
(400.0 ⫾ 191.3 pmol/L vs 372.6 ⫾ 89.9 pmol/L, P ⫽ .8). stricted to those without NASH or simple steatosis (eight
Interestingly, subjects with T2D (n ⫽ 8) had significantly T2D and 27 nondiabetic subjects). Importantly, although
reduced erythrocyte folate levels compared with nondia- reducing the statistical power, all of the 251 CpG sites
betic subjects (n ⫽ 11) (Figure 1B). Importantly, folate were differentially methylated in the T2D compared with
levels correlated negatively with fasting glucose levels al- the nondiabetic subjects when excluding those with
ready in nondiabetic subjects (␳ ⫽ ⫺0.67, P ⫽ .026). Ad- NASH, and 151 of the CpG sites were still significant
ditionally, although folate levels correlated significantly when excluding those with NASH and simple steatosis (Q
with fasting glucose levels when combining nondiabetic ⬍ .05, Supplemental Table 2). These analyses suggest that
and diabetic subjects (␳ ⫽ ⫺0.59, P ⫽ .008), the correla- most our significant CpG sites are specific for T2D and not
tion was not significant when including only subjects with linked to NASH. Additionally, because an altered cell
T2D (␳ ⫽ ⫺0.41, P ⫽ .32). Although there were no sig- composition potentially could affect DNA methylation,
nificant correlation between the average degree of meth- we further tested whether the level of lobular inflamma-
ylation of all analyzed sites and folate levels (␳ ⫽ ⫺0.25, tion based on histology affects DNA methylation of our
P ⫽ .3), we observed a nominal significant positive cor- 251 CpG sites significantly associated with T2D in the
relation between the average degree of methylation of the liver. However, none of these CpG sites were differentially
236 significant CpG sites with decreased methylation in methylated based on the level of lobular inflammation in
T2D subjects and folate levels (␳ ⫽ 0.45, P ⫽ .051). the human liver.
The distribution of the absolute differences in DNA We proceeded to examine the average DNA methyl-
methylation between T2D vs nondiabetic subjects is ation levels of different genomic regions in human liver
shown in Figure 1C. Among the significant sites annotated from T2D and nondiabetic subjects, either based on their
to a protein coding gene, the largest difference in methyl- relation to the nearest gene and functional genome distri-
ation between T2D and nondiabetic subjects was ob- bution (Figure 1G) or in relation to the CpG content (Fig-
served for cg02772880 in the 5⬘-untranslated region ure 1H). There were no significant differences in average
(UTR) of HYDIN (encoding hydrocephalus-inducing DNA methylation for these regions between diabetic and
protein, Figure 1D) and cg24867790 in the TSS1500 of nondiabetic subjects. Although the average methylation
NPHS2 (encoding nephrosis 2, Figure 1E). We next per- level was high within the gene body, 3⬘UTR, and inter-
formed a literature search, in which diabetes and each gene genic regions, it was low in the proximal promoter regions,
name of the 162 identified genes were used as search terms. defined as regions 200 or 1500 bp upstream of the tran-

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doi: 10.1210/jc.2015-3204 press.endocrine.org/journal/jcem E1495

Figure 1. The human methylome in the liver from 35 T2D and 60 nondiabetic subjects. A, Pie chart describing the number of sites that exhibit
increased or decreased DNA methylation in liver from T2D compared with nondiabetic subjects (Q ⬍ .05). B, Subjects with T2D (n ⫽ 8) have
significantly reduced fasting erythrocyte folate levels compared with nondiabetic subjects (n ⫽ 11). C, The absolute difference in DNA methylation
in liver from T2D compared with nondiabetic subjects (Q ⬍ .05). The largest difference in methylation between T2D and nondiabetic subjects was
observed for cg02772880 in the 5⬘UTR of HYDIN (D) and cg24867790 in the TSS1500 of NPHS2 (E). F, Genes previously associated with diabetes
that exhibit differential DNA methylation in T2D compared with nondiabetic subjects. Global DNA methylation in human liver from T2D and
nondiabetic subjects is shown for each gene region (G) and CpG island regions (H). Global DNA methylation is calculated as the average DNA
methylation based on all CpG sites in each annotated region on the Infinium HumanMethylation450 BeadChip. Folate levels and methylation data
are presented as mean ⫾ SD. fE, fasting erythrocyte; N, northern; S, southern; Shelf, regions flanking island shores (2000 – 4000 bp from the CpG
island); Shore, flanking region of CpG islands (0 –2000 bp); TSS, proximal promoter, defined as 200 or 1500 bp upstream of the transcription start
site. #, Q ⬍ .05; *, P ⬍ .05.

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E1496 Nilsson et al Epigenetic Alterations in Human T2D Liver J Clin Endocrinol Metab, November 2015, 100(11):E1491–E1501

scription start site (TSS200 and TSS1500, respectively), methylated between T2D and nondiabetic subjects (tran-
5⬘UTR, and the first exon (Figure 1G). Moreover, the av- scripts with significant CpG sites within the cis distance
erage methylation level was low within the CpG islands 500 kb upstream and 100 kb downstream of the gene). We
and intermediate within shores (flanking regions of CpG found that 26 CpG sites with significant association be-
islands [0 –2000 bp]), whereas shelves (regions flanking tween T2D and DNA methylation (Q ⬍ .05) had one or
island shores [2000 – 4000 bp from the CpG islands]) and more nearby gene(s) in which the liver mRNA expression
open sea showed the highest average methylation levels differed between T2D and nondiabetics (in total 29 genes,
(Figure 1H). P ⬍ .05, Table 2). Among these CpG sites, 52% had meth-
ylation and expression differences going in the opposite
Altered mRNA expression of genes with
direction, whereas 48% had differences going in the same
differential DNA methylation in the liver from T2D
subjects direction.
Epigenetic modifications have been associated with Based on a literature search in which diabetes and each
transcriptional regulation, eg, whereas methylation close name of the 29 identified genes were used as search terms,
to transcription sites often correlates negatively with ex- we further examined whether genes of potential impor-
pression, methylation in the gene body may enhance gene tance in diabetes were among the differentially expressed
elongation and be positively associated with gene expres- transcripts. It should be noted that this second literature
sion (45). Therefore, we investigated expression of nearby search includes some different genes compared with the
genes of the 251 CpG sites significantly differentially first literature search because a cis distance was applied in

Table 2. Altered mRNA Expression (P ⬍ .05) of Genes With Differential DNA Methylation (Q ⬍ .05) in the Liver
From Subjects With T2D Compared With Nondiabetic Subjects
Absolute
Difference in
Difference DNA Methylation
Probe Non-T2D, Mean T2D, Mean in Expression T2D P T2D vs non-T2D, Q
Gene Symbol Identification Expression ⴞ SD Expression ⴞ SD vs non-T2D, % Value CpG Site % Points Value
KCTD12 5560500 38.2 ⫾ 14.4 51.3 ⫾ 24.6 34.3 .005 cg01665118 ⫺4.1 .025
NUDT15 3450370 27.3 ⫾ 6.3 23.2 ⫾ 8.1 ⫺14.9 .006 cg18026416 ⫺2.9 .018
RNF144 3450136 32.0 ⫾ 12.3 43.9 ⫾ 17.3 37.1 .009 cg23335736 ⫺2.4 .023
AP3M2 3420128 28.8 ⫾ 6.2 35.1 ⫾ 8.6 21.8 .013 cg05885688 ⫺3.1 .018
MYH10 1770685 251.5 ⫾ 63.9 275.2 ⫾ 68.1 9.4 .014 cg24655262 ⫺4.3 .018
PPP1R1A 2750563 470.5 ⫾ 164.2 362.6 ⫾ 120.8 ⫺22.9 .015 cg16784745 ⫺4.3 .028
PLA2G4C 1990672 63.9 ⫾ 11.9 86.5 ⫾ 35.2 35.4 .018 cg26873392 ⫺1.8 .044
TMCO7 6330437 25.5 ⫾ 8.0 22.4 ⫾ 7.8 ⫺12.2 .021 cg07735969 ⫺4.9 .018
(TANGO6)
GOLPH4 5870221 81.4 ⫾ 37.2 59.6 ⫾ 39.1 ⫺26.8 .022 cg18142906 ⫺5.2 .046
(GOLIM4)
UPF2 5390603 187.6 ⫾ 53.6 221.1 ⫾ 66.8 17.9 .023 cg23421114 ⫺6.2 .031
TICAM1 5570730 61.0 ⫾ 17.8 74.5 ⫾ 19.2 22.2 .024 cg04410777 ⫺0.5 .018
H19 5340017 1053.5 ⫾ 742.0 1267.8 ⫾ 1237.9 20.3 .025 cg09575189 ⫺5.5 .042
ARFGEF1 3710681 26.0 ⫾ 6.9 20.9 ⫾ 7.0 ⫺19.5 .026 cg21462844 ⫺5.1 .018
SUMO1P3 4050358 204.8 ⫾ 39.2 172.5 ⫾ 42.6 ⫺15.8 .028 cg19308497 ⫺2.3 .048
IL23Ap19 6280750 26.7 ⫾ 9.3 38.4 ⫾ 23.4 43.9 .029 cg14940636 2.2 .020
ZFP90 5900286 60.0 ⫾ 10.0 69.0 ⫾ 16.0 15.0 .031 cg07735969 ⫺4.9 .018
SLAMF6 7200743 35.0 ⫾ 15.8 46.4 ⫾ 24.0 32.6 .032 cg19308497 ⫺2.3 .048
PRCC 6520156 30.5 ⫾ 9.6 27.0 ⫾ 7.0 ⫺11.6 .034 cg05954120 ⫺3.3 .023
ELOF1 630709 37.6 ⫾ 8.7 33.5 ⫾ 8.5 ⫺11.1 .035 cg14296903 ⫺2.0 .023
CYB561D1 1990092 59.9 ⫾ 12.0 69.5 ⫾ 14.1 16.0 .035 cg19244300 ⫺6.5 .033
RAD50 1400253 30.3 ⫾ 8.8 25.1 ⫾ 8.3 ⫺17.0 .037 cg03046705 ⫺1.1 .018
ZNF23 4260221 31.6 ⫾ 9.5 26.0 ⫾ 6.6 ⫺17.7 .038 cg02772880 ⫺16.8 .043
FAM173B 7320368 29.6 ⫾ 6.5 26.6 ⫾ 5.4 ⫺10.2 .038 cg07429192 ⫺5.0 .043
RIPK4 5860138 37.2 ⫾ 12.7 51.9 ⫾ 33.6 39.5 .040 cg01303480 ⫺8.3 .048
cg13520715 ⫺9.8 .018
CCNY 1340291 73.6 ⫾ 15.8 66.9 ⫾ 13.1 ⫺9.1 .040 cg16832648 ⫺1.7 .044
LOC148413 2060541 168.0 ⫾ 43.5 193.4 ⫾ 31.0 15.1 .043 cg22627753 ⫺3.7 .018
C4orf19 670475 78.7 ⫾ 19.3 67.9 ⫾ 23.1 ⫺13.7 .045 cg23862925 ⫺4.7 .018
ZNF295 6060326 28.6 ⫾ 7.7 32.8 ⫾ 8.0 15.0 .048 cg01303480 ⫺8.3 .048
cg13520715 ⫺9.8 .018
VWA1 50682 36.4 ⫾ 8.1 30.9 ⫾ 7.9 ⫺15.1 .048 cg22627753 ⫺3.7 .018

This analysis was restricted to transcripts with significantly differentially methylated CpG sites within the cis distance 500 kb upstream and 100 kb
downstream of the gene.

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doi: 10.1210/jc.2015-3204 press.endocrine.org/journal/jcem E1497

the combined analysis of methylation and expression data, kb downstream of the gene). We identified 57 (32 negative
whereas Illumina’s annotations were used for the analysis and 25 positive) correlations between methylation and
of DNA methylation only. We identified six diabetes-re- expression with P ⬍ .05 (Supplemental Table 3).
lated genes including RIPK4, H19, TICAM1, MYH10, To assess the accuracy and reproducibility of the
PPP1R1A, and RAD50 (39, 46 –50) (Table 2). Among microarray experiments, we performed technical repli-
these, RIPK4 displayed the largest difference in DNA cates. The correlation coefficient for pairwise compari-
methylation and mRNA expression, respectively, in liver sons of data from two liver RNA samples from the same
from the T2D compared with nondiabetic subjects (Figure individual analyzed on two different occasions showed a
2, A and B). There were nonsignificant negative correla- high reproducibility of Illumina’s HumanHT-12 expres-
tions between the RIPK4 mRNA expression and DNA sion array data (r ⫽ 0.99, P ⬍ 2.2 ⫻ 10⫺16, Supplemental
methylation (␳ ⫽ ⫺0.22, P ⫽ .16, and ␳ ⫽ ⫺0.21, P ⫽ .18, Figure 1).
for cg01303480 and cg13520715, respectively).
To validate our expression microarray data, we further
analyzed the expression of RIPK4 with qPCR in the full Discussion
cohort. The RIPK4 expression could be validated in the
full cohort and showed significant associations with T2D This study describes the methylome in human liver from a
in the same direction as the array data (Figure 2C). large number of obese T2D and nondiabetic subjects. Our
We then performed correlations between DNA meth- data support a role for epigenetic alteration in human liver
ylation and expression for the transcripts located in the in T2D. The vast majority of the significantly differentially
genomic region around the 251 CpG sites differentially methylated CpG sites (94%) showed decreased DNA
methylated in diabetic liver (transcripts with significant methylation in the liver from the T2D compared with the
CpG sites within the cis distance 500 kb upstream and 100 nondiabetic subjects. Intriguingly, we have previously ob-

Figure 2. Altered mRNA expression of a gene with differential DNA methylation. For RIPK4, both DNA methylation (A) and mRNA expression (B)
differed between T2D subjects and nondiabetic controls. The RIPK4 mRNA expression data were validated with qPCR in the full cohort (C). Data
are presented as mean ⫾ SD for methylation and mean ⫾ SEM for expression. a.u., arbitrary units. #, Q ⬍ .05; *, P ⬍ .05.

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E1498 Nilsson et al Epigenetic Alterations in Human T2D Liver J Clin Endocrinol Metab, November 2015, 100(11):E1491–E1501

served a similar pattern in pancreatic islets, in which 97% diabetic subjects, 86 individual CpG sites had an absolute
of differentially methylated CpG sites showed decreased difference in methylation of 5%–18.5% points, represent-
DNA methylation in diabetic compared with nondiabetic ing a fold change between 6% and 54% in T2D compared
islets (2). This may be explained by changed expression with nondiabetic liver. The differentially methylated sites
and/or activity of proteins controlling DNA methylation. include CpG sites in genes with previous known functions
However, we found no significant difference in the gene in liver and/or in the development of T2D such as GRB10,
expression of DNA methylation regulators such as DNA encoding the growth factor receptor-bound protein 10.
methyltransferases or ten-eleven translocation enzymes Common variants in the GRB10 have in GWAS been as-
between T2D and nondiabetic subjects in this study (data sociated with increased risk of T2D and impaired insulin
not shown). secretion (38, 57). In the present study, subjects with T2D
Hypomethylation may also have other explanations had lower methylation of GRB10 in liver compared with
such as a methyl donor supply-consumption imbalance nondiabetic controls. Interestingly, in a previous study, we
(51). Folate is a methyl donor in the methylation cycle, found lower DNA methylation of GRB10 in skeletal mus-
which maintains adequate cellular levels of S-adenosyl- cle from first-degree relatives of T2D compared with sub-
methionine for biological methylation reactions, includ- jects without any family history of the disease (58).
ing DNA methylation. Interestingly, we observed signifi- We have also shown that a risk SNP for elevated glu-
cantly reduced circulating folate levels in the T2D cose/T2D is associated with differential DNA methylation
compared with the nondiabetic subjects. The reduced fo- of GRB10 in human pancreatic islets (59). Together these
late levels may potentially explain why most significant studies support that altered DNA methylation of GRB10
sites in diabetic subjects are hypomethylated. Indeed, the in primary tissues for T2D affect the disease. GRB10 is
average DNA methylation of the 236 hypomethylated
imprinted in a parent-of-origin fashion in different tissues.
sites correlated (nominally significant, P ⫽ .051) with sub-
Recent gene knockouts in mice have established that
jects folate levels. Our human data are in line with a pre-
Grb10 acts as an inhibitor of intracellular signaling path-
vious rodent study, in which the reduced folate levels were
ways regulating growth and metabolism. Ablation of
associated with reduced global DNA methylation in the
Grb10 impacts on hepatic glycogen synthesis and proba-
rat liver (52). In addition, folate intake and folate levels
bly on glucose homoeostasis (60). Grb10 has also been
have been associated with liver lipid metabolism in animal
found to affect the proportions of lean and fat tissue dur-
studies (53–56). It is difficult to draw conclusions about
ing embryonic development, thereby influencing energy
causality in case-control cohorts, and the ideal study de-
homeostasis in the adult (61).
sign would be to longitudinally assess changes in folate
One should be aware of that this study is performed in
levels during individuals’ transition into disease. Never-
very obese subjects and do not necessary reflect T2D in
theless, we found that folate levels correlated negatively
general across the range of BMI. Also, in addition to hepa-
with fasting glucose levels already in nondiabetic subjects,
suggesting that reduced circulating folate levels may con- tocytes, liver tissue comprises a mixture of different cell
tribute to the development of T2D. It should further be types. Differences in the cell type composition between
noted that 89% of subjects with T2D were treated with T2D and nondiabetics could therefore potentially be re-
metformin, and hence, we cannot exclude that this treat- sponsible for some of the observed differences in the DNA
ment affected the degree of methylation in the liver. Nev- methylation and mRNA expression. Based on the histo-
ertheless, persons with diabetes treated with metformin logical assessment of liver biopsy samples, simple steatosis
had no difference in the methylation of the 251 CpG sites or NASH could be detected in several subjects included in
identified in this study compared with persons with dia- this study. Importantly, these phenotypes were taken into
betes without this treatment, suggesting a limited effect of consideration when identifying differences in DNA meth-
metformin on the degree of methylation of these sites. ylation and mRNA expression in the liver from diabetic vs
In complex polygenic diseases like T2D, it is believed nondiabetic subjects in this study. Only a modest propor-
that multiple modest changes may interact to drive a phys- tion of our significant CpG sites were associated with both
iological response and disease development. Several af- T2D and NASH. Additionally, all of the 251 CpG sites
fected CpG sites may in combination potentially contrib- were differentially methylated (Q ⬍ .05) in T2D com-
ute to diabetes. We identified 251 individual CpG sites pared with nondiabetic subjects when excluding those
that exhibit differential DNA methylation in liver ob- with NASH. Moreover, the level of lobular inflammation
tained from diabetic compared with nondiabetic subjects. based on histology was not associated with an altered
Although many of these sites display absolute differences methylation of our significant CpG sites. Therefore, we
in methylation less than 5% points between T2D and non- believe that most our significant results represent inde-

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doi: 10.1210/jc.2015-3204 press.endocrine.org/journal/jcem E1499

pendent effects of T2D on DNA methylation in the human Author contributions include the following: E.N. researched
liver. the data and wrote/reviewed the manuscript, A.M performed the
validation experiments, collected clinical data, and reviewed the
By combining our genome-wide DNA methylation
manuscript, V.D.d.M. and P.K. collected the clinical data and
data with transcriptome profiles, we identified 29 genes reviewed the manuscript, A.P. analyzed the data and reviewed
that exhibit both differential DNA methylation and gene the manuscript, J.P. designed the study, collected the clinical
expression in T2D liver. These include H19 and RIPK4, data, and reviewed the manuscript, and C.L. designed the study
which both showed a decreased methylation and an in- and wrote/reviewed the manuscript. E.N. and C.L. are guaran-
creased expression in the diabetic liver. H19 is a paternally tors of this work and, as such, had full access to all of the data in
imprinted gene, and studies in mice have shown that ma- the study and take responsibility for the integrity of the data.
This work was supported by grants from the Academy of
ternal diabetes leads to differential methylation of this Finland (Contracts 120979, 138006, and 131593), the Finnish
gene in the offspring (48). RIPK4 encodes an activator of Diabetes Research Foundation, the Finnish Cultural Founda-
nuclear factor-␬B. Nuclear factor-␬B activation results in tion, and the Kuopio University Hospital EVO and VTR fund-
increased levels of proinflammatory mediators leading to ing. This work was also supported by grants from the Swedish
decreased insulin sensitivity in liver (39). It should be Research Council, Region Skåne, the Knut and Alice Wallenberg
noted that the expression data were nominally significant Foundation, the Novo Nordisk Foundation, the EFSD/Lilly Fel-
lowship, the Söderberg Foundation, The Swedish Diabetes
after correction for multiple testing. However, we man-
Foundation, the Påhlsson Foundation, EXODIAB, and a Linné
aged to validate expression data by qPCR in a larger num- grant.
ber of samples. Disclosure Summary: The authors have nothing to disclose.
DNA methylation was initially thought to be a silencing
mark and increased DNA methylation in promoter re-
gions has been associated with decreased gene expression References
(45). However, recent studies suggest that DNA methyl-
ation also affects additional biological processes such as 1. Groop L, Pociot F. Genetics of diabetes—are we missing the genes
or the disease? Mol Cell Endocrinol. 2014;382:726 –739.
the expression of noncoding RNAs, transcriptional elon- 2. Dayeh T, Volkov P, Salo S, et al. Genome-wide DNA methylation
gation, splicing events, and the overall genomic stability, analysis of human pancreatic islets from type 2 diabetic and non-
which may depend on the genomic location of the meth- diabetic donors identifies candidate genes that influence insulin se-
cretion. PLoS Genet. 2014; 10:e1004160.
ylated CpG sites (45). Therefore, altered DNA methyl-
3. Nilsson E, Jansson PA, Perfilyev A, et al. Altered DNA methylation
ation in the diabetic liver may affect other biological pro- and differential expression of genes influencing metabolism and in-
cesses not studied here, and it may be positively associated flammation in adipose tissue from subjects with type 2 diabetes.
with gene expression. In fact, we found an altered DNA Diabetes. 2014;63:2962–2976.
4. Ribel-Madsen R, Fraga MF, Jacobsen S, et al. Genome-wide analysis
methylation of CpG sites annotated to miRNAs, including of DNA methylation differences in muscle and fat from monozygotic
miR-548d2, miR-148b, and miR-30a. twins discordant for type 2 diabetes. PLoS One. 2012;7:e51302.
In conclusion, we find that subjects with T2D exhibit 5. Barres R, Osler ME, Yan J, et al. Non-CpG methylation of the
PGC-1␣ promoter through DNMT3B controls mitochondrial den-
epigenetic and transcriptional changes in the liver relevant
sity. Cell Metab. 2009;10:189 –198.
to the development of the disease together with reduced 6. Volkmar M, Dedeurwaerder S, Cunha DA, et al. DNA methylation
folate levels. Our data support the role of epigenetic vari- profiling identifies epigenetic dysregulation in pancreatic islets from
ation in the pathogenesis of T2D and suggest that future type 2 diabetic patients. EMBO J. 2012;31:1405–1426.
7. Crider KS, Yang TP, Berry RJ, Bailey LB. Folate and DNA meth-
studies should test whether folic acid supplementation ylation: a review of molecular mechanisms and the evidence for
may prevent and/or treat the disease. folate’s role. Adv Nutr. 2012;3:21–38.
8. Horvath S, Erhart W, Brosch M, et al. Obesity accelerates epigenetic
aging of human liver. Proc Natl Acad Sci USA. 2014;111:15538 –
15543.
Acknowledgments 9. Pirola CJ, Gianotti TF, Burgueno AL, et al. Epigenetic modification
of liver mitochondrial DNA is associated with histological severity
We thank Päivi Turunen, Tiina Sistonen, and Matti Laitinen for of nonalcoholic fatty liver disease. Gut. 2013;62:1356 –1363.
their technical assistance in the KOBS study. We also thank the 10. Zhang H, Chu X, Huang Y, et al. Maternal vitamin D deficiency
Swegene Center for Integrative Biology at Lund University during pregnancy results in insulin resistance in rat offspring, which
Genomics Facility for help with the DNA methylation and is associated with inflammation and I␬b␣ methylation. Diabetolo-
gia. 2014;57:2165–2172.
mRNA expression analyses.
11. Chang X, Yan H, Fei J, et al. Berberine reduces methylation of the
MTTP promoter and alleviates fatty liver induced by a high-fat diet
Address all correspondence and requests for reprints to: in rats. J Lipid Res. 2010;51:2504 –2515.
Emma Nilsson and Charlotte Ling, Department of Clinical Sciences, 12. Mannisto VT, Simonen M, Soininen P, et al. Lipoprotein subclass
Epigenetics and Diabetes Unit, Lund University Diabetes Centre, Jan metabolism in nonalcoholic steatohepatitis. J Lipid Res. 2014;55:
WaldenströmsGata35,CRC91:12,20502Malmö,Sweden. E-mail: 2676 –2684.
emma_a.nilsson@med.lu.se; or charlotte.ling@med.lu.se. 13. Pihlajamaki J, Gronlund S, Simonen M, et al. Cholesterol absorption

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 March 2016. at 00:46 For personal use only. No other uses without permission. . All rights reserved.
E1500 Nilsson et al Epigenetic Alterations in Human T2D Liver
decreases after Roux-en-Y gastric bypass but not after gastric band-
ing. Metabolism. 2010;59:866 – 872.
14. Pihlajamaki J, Kuulasmaa T, Kaminska D, et al. Serum interleukin
1 receptor antagonist as an independent marker of non-alcoholic
steatohepatitis in humans. J Hepatol. 2012;56:663– 670.
15. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of
diabetes mellitus and its complications. Part 1: diagnosis and clas-
sification of diabetes mellitus provisional report of a WHO consul-
tation. Diabet Med. 1998;15:539 –553.
16. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA,
Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and
staging the histological lesions. Am J Gastroenterol. 1999;94:2467–
2474.
17. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation
of a histological scoring system for nonalcoholic fatty liver disease.
Hepatology. 2005;41:1313–1321.
18. Bibikova M, Barnes B, Tsan C, et al. High density DNA methylation
array with single CpG site resolution. Genomics. 2011;98:288 –295.
19. Chen YA, Lemire M, Choufani S, et al. Discovery of cross-reactive
probes and polymorphic CpGs in the Illumina Infinium Human-
Methylation450 microarray. Epigenetics. 2013;8:203–209.
20. Du P, Kibbe WA, Lin SM. lumi: a pipeline for processing Illumina
microarray. Bioinformatics. 2008;24:1547–1548.
21. Du P, Zhang X, Huang CC, et al. Comparison of ␤-value and M-
value methods for quantifying methylation levels by microarray
analysis. BMC Bioinformatics. 2010;11:587.
22. Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open
software development for computational biology and bioinformat-
ics. Genome Biol. 2004;5:R80.
23. Teschendorff AE, Marabita F, Lechner M, et al. A ␤-mixture quan-
tile normalization method for correcting probe design bias in Illu-
mina Infinium 450 k DNA methylation data. Bioinformatics. 2013;
29:189 –196.
24. Johnson WE, Li C, Rabinovic A. Adjusting batch effects in microar-
ray expression data using empirical Bayes methods. Biostatistics.
2007;8:118 –127.
25. Bertola A, Bonnafous S, Anty R, et al. Hepatic expression patterns
of inflammatory and immune response genes associated with obesity
and NASH in morbidly obese patients. PLoS One. 2010;5:e13577.
26. Fox J, Monette G. Generalized collinearity diagnostics. J Am Stat
Assoc. 1992;87:178 –183.
27. Broholm C, Brandt C, Schultz NS, Nielsen AR, Pedersen BK, Scheele
C. Deficient leukemia inhibitory factor signaling in muscle precursor
cells from patients with type 2 diabetes. Am J Physiol Endocrinol
Metab. 2012;303:E283–E292.
28. Ferecatu I, Goncalves S, Golinelli-Cohen MP, et al. The diabetes
drug target MitoNEET governs a novel trafficking pathway to re-
build an Fe-S cluster into cytosolic aconitase/iron regulatory protein
1. J Biol Chem. 2014;289:28070 –28086.
29. Gupta RK, Gao N, Gorski RK, et al. Expansion of adult ␤-cell mass
in response to increased metabolic demand is dependent on HNF-
4␣. Genes Dev. 2007;21:756 –769.
30. Hayashi Y, Suemitsu E, Kajimoto K, et al. Hepatic monoacylglycerol
O-acyltransferase 1 as a promising therapeutic target for steatosis,
obesity, and type 2 diabetes. Mol Ther Nucleic Acids. 2014;3:e154.
31. Hurov JB, Huang M, White LS, et al. Loss of the Par-1b/MARK2
polarity kinase leads to increased metabolic rate, decreased adipos-
ity, and insulin hypersensitivity in vivo. Proc Natl Acad Sci USA.
2007;104:5680 –5685.
32. Imamura M, Maeda S, Yamauchi T, et al. A single-nucleotide poly-
morphism in ANK1 is associated with susceptibility to type 2 dia-
betes in Japanese populations. Hum Mol Genet. 2012;21:3042–
3049.
33. Kim SJ, Chae S, Kim H, et al. A protein profile of visceral adipose
tissues linked to early pathogenesis of type 2 diabetes mellitus. Mol
Cell Proteomics. 2014;13:811– 822.
34. More VR, Wen X, Thomas PE, Aleksunes LM, Slitt AL. Severe
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 March 2016. at 00:46 For personal use only. No other uses without permission. . All rights reserved.
J Clin Endocrinol Metab, November 2015, 100(11):E1491–E1501
diabetes and leptin resistance cause differential hepatic and renal
transporter expression in mice. Comp Hepatol. 2012;11:1.
35. Morris AP, Voight BF, Teslovich TM, et al. Large-scale association
analysis provides insights into the genetic architecture and patho-
physiology of type 2 diabetes. Nat Genet. 2012;44:981–990.
36. Ohno H, Shinoda K, Spiegelman BM, Kajimura S. PPAR␥ agonists
induce a white-to-brown fat conversion through stabilization of
PRDM16 protein. Cell Metab. 2012;15:395– 404.
37. Piao G, Saito S, Sun Y, et al. A computational procedure for iden-
tifying master regulator candidates: a case study on diabetes pro-
gression in Goto-Kakizaki rats. BMC Syst Biol. 2012;6(suppl 1):S2.
38. Rampersaud E, Damcott CM, Fu M, et al. Identification of novel
candidate genes for type 2 diabetes from a genome-wide association
scan in the Old Order Amish: evidence for replication from diabetes-
related quantitative traits and from independent populations. Dia-
betes. 2007;56:3053–3062.
39. Shoelson SE, Lee J, Goldfine AB. Inflammation and insulin resis-
tance. J Clin Invest. 2006;116:1793–1801.
40. Skalniak L, Mizgalska D, Zarebski A, Wyrzykowska P, Koj A, Jura
J. Regulatory feedback loop between NF-␬B and MCP-1-induced
protein 1 RNase. FEBS J. 2009;276:5892–5905.
41. Wang Q, Zhang X, Wang Y, Deng A, Zhu Z, Feng Y. Significance
and expression of serum and glucocorticoid-inducible kinase in kid-
ney of mice with diabetic nephropathy. J Huazhong Univ Sci Tech-
nolog Med Sci. 2005;25:170 –173.
42. Xu Z, Lefevre GM, Felsenfeld G. Chromatin structure, epigenetic
mechanisms and long-range interactions in the human insulin locus.
Diabetes Obes Metab. 2012;14(suppl 3):1–11.
43. Zbidi H, Lopez JJ, Amor NB, Bartegi A, Salido GM, Rosado JA.
Enhanced expression of STIM1/Orai1 and TRPC3 in platelets from
patients with type 2 diabetes mellitus. Blood Cells Mol Dis. 2009;
43:211–213.
44. Ahrens M, Ammerpohl O, von Schonfels W, et al. DNA methylation
analysis in nonalcoholic fatty liver disease suggests distinct disease-
specific and remodeling signatures after bariatric surgery. Cell
Metab. 2013;18:296 –302.
45. Jones PA. Functions of DNA methylation: islands, start sites, gene
bodies and beyond. Nat Rev Genet. 2012;13:484 – 492.
46. da Costa AV, Calabria LK, de Souza Santos P, Goulart LR, Espin-
dola FS. Glibenclamide treatment modulates the expression and lo-
calization of myosin-IIB in diabetic rat brain. J Neurol Sci. 2014;
340:159 –164.
47. Dasu MR, Devaraj S, Park S, Jialal I. Increased toll-like receptor
(TLR) activation and TLR ligands in recently diagnosed type 2 di-
abetic subjects. Diabetes Care. 2010;33:861– 868.
48. Ge ZJ, Liang QX, Hou Y, et al. Maternal obesity and diabetes may
cause DNA methylation alteration in the spermatozoa of offspring
in mice. Reprod Biol Endocrinol. 2014;12:29.
49. Khan S, Raghuram GV, Bhargava A, et al. Role and clinical signif-
icance of lymphocyte mitochondrial dysfunction in type 2 diabetes
mellitus. Transl Res. 2011;158:344 –359.
50. Taneera J, Fadista J, Ahlqvist E, et al. Identification of novel genes
for glucose metabolism based upon expression pattern in human
islets and effect on insulin secretion and glycemia. Hum Mol Genet.
2015;24:1945–1955.
51. Zhou SS, Zhou YM, Li D, Lun YZ. Dietary methyl-consuming com-
pounds and metabolic syndrome. Hypertens Res. 2011;34:1239 –
1245.
52. Mejos KK, Kim HW, Lim EM, Chang N. Effects of parental folate
deficiency on the folate content, global DNA methylation, and ex-
pressions of FR␣, IGF-2 and IGF-1R in the postnatal rat liver. Nutr
Res Pract. 2013;7:281–286.
53. Akesson B, Fehling C, Jagerstad M, Stenram U. Effect of experi-
mental folate deficiency on lipid metabolism in liver and brain. Br J
Nutr. 1982;47:505–520.
54. Champier J, Claustrat F, Nazaret N, Fevre Montange M, Claustrat
B. Folate depletion changes gene expression of fatty acid metabo-
doi: 10.1210/jc.2015-3204
lism, DNA synthesis, and circadian cycle in male mice. Nutr Res.
2012;32:124 –132.
55. McNeil CJ, Hay SM, Rucklidge GJ, et al. Disruption of lipid me-
tabolism in the liver of the pregnant rat fed folate-deficient and
methyl donor-deficient diets. Br J Nutr. 2008;99:262–271.
56. McNeil CJ, Hay SM, Rucklidge GJ, Reid MD, Duncan GJ, Rees
WD. Maternal diets deficient in folic acid and related methyl donors
modify mechanisms associated with lipid metabolism in the fetal
liver of the rat. Br J Nutr. 2009;102:1445–1452.
57. Prokopenko I, Poon W, Magi R, et al. A central role for GRB10 in
regulation of islet function in man. PLoS Genet. 2014;10:e1004235.
58. Nitert MD, Dayeh T, Volkov P, et al. Impact of an exercise inter-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 March 2016. at 00:46 For personal use only. No other uses without permission. . All rights reserved.
press.endocrine.org/journal/jcem E1501
vention on DNA methylation in skeletal muscle from first-degree
relatives of patients with type 2 diabetes. Diabetes. 2012;61:3322–
3332.
59. Olsson AH, Volkov P, Bacos K, et al. Genome-wide associations
between genetic and epigenetic variation influence mRNA expres-
sion and insulin secretion in human pancreatic islets. PLoS Genet.
2014;10:e1004735.
60. Holt LJ, Siddle K. Grb10 and Grb14: enigmatic regulators of insulin
action—and more? Biochem J. 2005;388:393– 406.
61. Cowley M, Garfield AS, Madon-Simon M, et al. Developmental
programming mediated by complementary roles of imprinted
Grb10 in mother and pup. PLoS Biol. 2014;12:e1001799.