Young men with low birthweight exhibit decreased plasticity of genome-wide muscle DNA methylation by high-fat overfeeding
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The association between low birthweight (LBW) and risk of developing type 2 diabetes may involve epigenetic mechanisms, with skeletal muscle being a prime target tissue. Differential DNA methylation patterns have been observed in single genes in muscle tissue from type 2 diabetic and LBW individuals, and we recently showed multiple DNA methylation changes during short-term high-fat overfeeding in muscle of healthy people. In a randomised crossover study, we analysed genome-wide DNA promoter methylation in skeletal muscle of 17 young LBW men and 23 matched normal birthweight (NBW) men after a control and a 5 day high-fat overfeeding diet.
DNA methylation was measured using Illumina’s Infinium BeadArray covering 27,578 CpG sites representing 14,475 different genes.
After correction for multiple comparisons, DNA methylation levels were found to be similar in the LBW and NBW groups during the control diet. Whereas widespread DNA methylation changes were observed in the NBW group in response to high-fat overfeeding, only a few methylation changes were seen in the LBW group (χ2, p < 0.001).
Our results indicate lower DNA methylation plasticity in skeletal muscle from LBW vs NBW men, potentially contributing to understanding the link between LBW and increased risk of type 2 diabetes.
KeywordsDNA methylation Epigenetics High-fat overfeeding Insulin resistance Low birthweight Skeletal muscle Type 2 diabetes
Epigenetics has been proposed as a link between low birthweight (LBW) and increased risk of metabolic disease and type 2 diabetes . The involvement of tissue-specific DNA methylation in the pathogenesis of type 2 diabetes is supported by several studies [2, 3, 4]. Maternal diet and/or the intrauterine environment may influence DNA methylation in the offspring, possibly affecting phenotypes and disease risk , as exemplified by our finding of increased DNA methylation of PPARGC1A in skeletal muscle of LBW individuals .
Fat overload may induce and/or prolong the presence of methyl groups attached to DNA [2, 7], and we have shown that high-fat overfeeding introduced widespread DNA methylation changes in young men . In our study of PPARGC1A, we found that LBW individuals, in contrast with normal birthweight (NBW) controls, did not exhibit methylation changes in skeletal muscle during overfeeding .
Extending our previous epigenetic findings [6, 7], we investigated whether genome-wide DNA methylation levels are different in skeletal muscle of LBW individuals compared with NBW controls, and whether a high-fat overfeeding challenge reveals a differential DNA methylation response in LBW individuals.
Study design and participants
The data are part of a study investigating the metabolic effects of high-fat overfeeding in healthy LBW and NBW individuals, as previously described [6, 8]. Briefly, 20 LBW (birthweight ≤10th percentile) and 26 matched NBW men born at term were recruited. All participants received a 5 day high-fat overfeeding diet and a control diet in a randomised order separated by 6–8 weeks. The study was designed to ensure identical conditions during interventions and laboratory procedures. The protocol conformed to the Declaration of Helsinki and was approved by the ethics committee of Copenhagen County. All participants gave informed consent.
Detailed clinical descriptions have previously been published [6, 8]. Muscle biopsies and blood samples were taken after an overnight fast following both diets. Biopsies were successfully obtained from the vastus lateralis muscle of 17 LBW and 23 NBW participants; among these, there were 14 paired LBW and 21 paired NBW samples, of which eight LBW and 11 NBW men received the control diet first.
DNA methylation profiling
Genomic DNA was extracted using the DNeasy Kit (Qiagen, Valencia, CA, USA), and 600 ng DNA was bisulphite-treated with the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA). DNA methylation was assessed at 27,578 CpG sites close to transcription start in 14,475 genes using Illumina’s Infinium 27 k BeadArray (Illumina, San Diego, CA, USA) employing previously described procedures . Each array had an even distribution of LBW and NBW participants and of control and overfeeding samples. The batch effect of the dataset was examined using a principal components analysis plot. Since no batch effect was found, and as data processing beyond the BeadStudio software was not recommended by Illumina, no further normalisation was performed. Technical validation of the BeadArray data was performed for six CpG sites/genes using pyrosequencing of bisulphite-treated DNA in the PyroMark Q96ID instrument (Qiagen) with primers designed using PyroMark Assay Design 2.0. The data were analysed using PyroMark software v.2.5.7.
Gene expression analysis
Gene expression of DNMT1, DNMT3A and DNMT3B was determined by quantitative PCR using gene-specific primer/probe pairs and the ABI 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to the same procedure as described previously . DNMT gene expression data are presented in the electronic supplementary material (ESM [ESM text: Skeletal muscle gene expression of DNA methyltransferases and ESM Tables 1 and 2]).
Statistics were performed using R v.2.9.0 (http://www.r-project.org) . Differences between methylation percentages are absolute percentage points (pp). Significance levels between diet intervention and birthweight groups were evaluated using paired and unpaired Student’s t tests, respectively (p < 0.05). Deviations from expected distributions were estimated with χ2 tests. Wilcoxon’s rank sum tests were used to compare the absolute, individual diet-induced methylation response in LBW compared with NBW men, and unpaired Student’s t tests were used to compare the average, diet-induced methylation response in the birthweight groups on all 27,578 sites simultaneously (delta analyses). The Benjamini–Hochberg procedure was applied to correct for multiple testing where q < 0.10 was considered statistically significant. Self-organising maps were employed to investigate the diet-order impact using MeV software v.4.8 (http://www.tm4.org). The dataset is available at NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo), accession number GSE40798.
Clinical and metabolic characteristics of the NBW (n = 23) and LBW (n = 17)a groups after the control and the 5 day high-fat overfeeding diets
3,903 ± 206
2,677 ± 272
24.6 ± 1.1
24.1 ± 0.5
1.83 ± 0.1
1.77 ± 0.1
78.6 ± 9.6
77.3 ± 11.5
78.9 ± 10
76.9 ± 11.9
Fat mass (kg)
14.4 ± 6.2
17.3 ± 9.5
14.2 ± 6.3
15.6 ± 9.1
61.3 ± 6.1
58.0 ± 5.1
58.2 ± 14.0
58.1 ± 5.4
23.4 ± 2.4
24.6 ± 3.9
23.2 ± 2.5
24.7 ± 4.0
Fasting plasma glucose (mmol/l)
4.63 ± 0.47
4.86 ± 0.66
5.04 ± 0.39
5.03 ± 0.54
Fasting serum insulin (pmol/l)
31.0 ± 15.1
40.3 ± 16.0
41.2 ± 27.4
44.6 ± 18.6
Fasting plasma NEFA (mmol/l)
348 ± 134
411 ± 201
211 ± 80
196 ± 93
Basal fat oxidation (mg kg−1 FFM−1 min−1)
0.98 ± 0.39
1.06 ± 0.51
1.04 ± 0.65
1.14 ± 0.32
Insulin-stimulated glucose uptake (mg kg−1 FFM−1 min−1)
13.1 ± 3.7
12.9 ± 2.1
12.7 ± 4.4
11.5 ± 2.8
Only 404 CpG sites in 393 (3%) genes showed differential methylation between the groups (p < 0.05) during the control diet, as opposed to 9% of the genes during overfeeding; both non-significant after correction for multiple comparisons (q > 0.10).
Among 11 candidate genes relevant to intrauterine growth restriction (four imprinted/seven non-imprinted) and 38 type 2 diabetes candidate genes (ESM [text: Significant DNA methylation changes of candidate genes relevant to intrauterine growth restriction and type 2 diabetes]) [5, 7, 9], we found significant differences (p < 0.05, uncorrected) between the NBW and LBW men in two genes for intrauterine growth restriction (IGF2R, TNF) and in three genes for type 2 diabetes (CDKN2B, KCNJ11, KCNQ1) (ESM [Table 3]).
We have previously validated the BeadArray data in the NBW participants after overfeeding in 12 selected CpG sites/genes . Additionally, we applied pyrosequencing to validate the DNA methylation responses in LBW men in GABRA3, UGT2B7, FOLH1, FUT1, NDUFS2 and FAP (ESM [text: Technical validation of DNA methylation BeadArray data with pyrosequencing and ESM Table 4]), and indeed these validations supported the BeadArray data.
Our results indicate that LBW and NBW individuals exhibit similar DNA methylation patterns in skeletal muscle on a control diet. Interestingly, when exposed to 5 days’ high-fat overfeeding, the LBW group displayed an impairment of their capability to change DNA methylations compared with the NBW controls .
We are, to the best of our knowledge, the first to perform genome-wide examination of DNA methylations in a primary insulin-sensitive tissue of LBW individuals at increased risk of developing type 2 diabetes. We found no differences between the groups at baseline nor during overfeeding after correction for multiple testing, as well as no major differences in skeletal muscle DNA methylation among selected candidate genes associated with intrauterine growth restriction  or type 2 diabetes susceptibility [7, 9]. Importantly, with the applied BeadArray platform that only covers 0.1% of the CpG sites in the genome and predominantly includes CpG sites in promoter regions and CpG islands, we cannot rule out that significant differences in DNA methylations at other intra- and intergenic locations might have been overlooked.
Although the statistical power to detect diet-induced methylation changes appears to have been slightly lower in the LBW group, our additional delta analyses of all array CpG sites signifies that diet-induced differences are truly less abundant in LBW individuals. Furthermore, the present epigenome-wide data are in line with our previous study of PPARGC1A , where DNA methylation changed only in NBW individuals after overfeeding.
Our finding that the lower DNA methylation response included changes in both positive and negative directions underscores that decreased plasticity and not unidirectional preferential changes characterise LBW individuals (Fig. 1b, c). It appears reasonable to speculate that short-term changes to DNA methylations represent a normal systemic response to lifestyle and metabolic factors including both overfeeding and physical activity . In support of this, a recent study reported that obesity and weight loss induced by bariatric surgery exhibited dynamic effects on skeletal muscle DNA methylation . We hypothesise that the decreased plasticity of muscle DNA methylation changes in LBW individuals may adversely affect protective gene functions of various pathways, including inflammation . This may increase their susceptibility to metabolic disease following fat overloading, especially if sustained over longer time periods. Theoretically, this could have paradigm changing consequences in our attempts to understand fetal programming of metabolic diseases by epigenetic mechanisms .
The increase in DNA methyltransferases among NBW controls exposed first to the control diet  was not observed among the LBW group (ESM [Tables 1 and 2]), which to some extent supports the possibility that differential overfeeding responses between LBW and NBW individuals may be caused by differential de novo DNA methylations. However, direct measurements of the activities of these enzymes are needed to substantiate this idea.
In conclusion, no major absolute skeletal muscle DNA methylation differences in LBW vs NBW controls at baseline or after 5 days’ high-fat overfeeding were observed using a random array approach. We did, however, discover indications of decreased plasticity of muscle DNA methylation dynamics in LBW men after overfeeding, which may contribute to the risk of developing insulin resistance and type 2 diabetes in those born with LBW.
We thank M. Modest and L.S. Koch from the Steno Diabetes Center, Gentofte, Denmark, and L. Moreno and R. Alonso from the Spanish National Cancer Research Centre, Madrid, Spain, for excellent assistance in the laboratory. We also thank the study participants for taking part in the study.
This work was supported by The Danish Council for Independent Research—Medical Sciences (FSS), the Danish Council for Strategic Research, the Programme Commission on Food and Health (FØSU), the Danish Diabetes Association, the European Foundation for the Study of Diabetes (EFSD), the EU 6th Framework EXGENESIS Grant, Augustinus Fonden, The Novo Nordisk Foundation, and the Aase and Ejnar Danielsen Foundation.
Duality of interest
SCJ and PP are shareholder and employee at Novo Nordisk A/S; AV is a shareholder and lecturer at Novo Nordisk A/S; CB and JB-J are shareholders at Novo Nordisk A/S. Other authors declare that there is no duality of interest in correction with their involvement in this study.
SCJ and LG acquired, analysed and interpreted the data, and drafted the manuscript. CB designed the study and analysed and interpreted the data. JB-J, RR-M, MFF, CL and PP analysed and interpreted the data. AFF, EL and VC acquired the data. AV designed the study and interpreted the data, and is responsible for the integrity of the work as a whole. All authors critically revised and approved the final version of the manuscript.
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