The Fat Cell Epigenetic Signature in Post-Obese Women Is Characterized by Global Hypomethylation and Differential DNA Methylation of Adipogenesis Genes

ORIGINAL ARTICLE The fat cell epigenetic signature in post-obese women is characterized by global hypomethylation and differential DNA methylation of adipogenesis genes

I Dahlman1, I Sinha2, H Gao2, D Brodin2, A Thorell3,4, M Rydén1, DP Andersson1, J Henriksson2,5,APerﬁlyev6, C Ling6, K Dahlman-Wright2,7 and P Arner1

BACKGROUND/OBJECTIVES: Obese subjects have increased number of enlarged fat cells that are reduced in size but not in number in post-obesity. We performed DNA methylation proﬁling in fat cells with the aim of identifying differentially methylated DNA sites (DMS) linked to adipose hyperplasia (many small fat cells) in post-obesity. SUBJECTS/METHODS: Genome-wide DNA methylation was analyzed in abdominal subcutaneous fat cells from 16 women examined 2 years after gastric bypass surgery at a post-obese state (body mass index (BMI) 26 ± 2 kg m–2, mean ± s.d.) and from 14 never-obese women (BMI 25 ± 2 kg m–2). Gene expression was analyzed in subcutaneous adipose tissue from nine women in each group. In a secondary analysis, we examined DNA methylation and expression of adipogenesis genes in 15 and 11 obese women, respectively. RESULTS: The average degree of DNA methylation of all analyzed CpG sites was lower in fat cells from post-obese as compared with never-obese women (P = 0.014). A total of 8504 CpG sites were differentially methylated in fat cells from post-obese versus never-obese women (false discovery rate 1%). DMS were under-represented in CpG islands and surrounding shores. The 8504 DMS mapped to 3717 unique genes; these genes were over-represented in cell differentiation pathways. Notably, 27% of the genes linked to adipogenesis (that is, 35 of 130) displayed DMS (adjusted P =10− 8) in post-obese versus never-obese women. Next, we explored DNA methylation and expression of genes linked to adipogenesis in more detail in adipose tissue samples. DMS annotated to adipogenesis genes were not accompanied by differential gene expression in post-obese compared with never-obese women. In contrast, adipogenesis genes displayed differential DNA methylation accompanied by altered expression in obese women. CONCLUSIONS: Global CpG hypomethylation and over-representation of DMS in adipogenesis genes in fat cells may contribute to adipose hyperplasia in post-obese women. International Journal of Obesity (2015) 39, 910–919; doi:10.1038/ijo.2015.31

INTRODUCTION obese women.5 The concept of adipose hyperplasia—that is, In obese subjects, weight reduction has a beneficial impact on larger number but smaller fat cells than expected for a specific 6 health, including reversal of type 2 diabetes as reviewed.1 total body fat mass—is established. Whether hyperplasia in the However, an increased risk of diabetes and other chronic diseases, post-obese state is a primary disturbance in obese-prone subjects as well as higher mortality, persist in post-obese patients as or an adaptation to weight loss is unknown, as no prospective compared with the general population.2 In addition, weight regain studies have been performed. However, an important role of fat following successful weight reduction, which occurs even after cell size and number for obesity development is supported by the bariatric surgery, remains a challenge and contributes to an following findings: (a) the obese have a higher absolute unfavorable prognosis.3 production of new fat cells and a higher number of fat cells in The increase in white adipose tissue (WAT) mass in conjunction total; (b) fat cell number is set already in adolescence, whereas with obesity is accompanied by fat cell metabolic disturbances many patients develop obesity later in life; and (c) absolute such as increased fat cell size, spontaneous lipolytic activity and production of new fat cell and cell number does not change in the secretion of inflammatory mediators, which probably contribute obese after weight reduction.7 to secondary complications including insulin resistance and type 2 The causes of weight regain are unclear. Epigenetic mechanisms diabetes.4 Whereas most of these metabolic disturbances are such as DNA methylation are important in phenotype transmission normalized upon weight loss, fat cell size is not. Instead post- and in the development of various states of disease.8 DNA obese subjects display smaller fat cells and increased fat cell methylation mainly occurs in the context of CG dinucleotides number (hyperplasia) in their adipose tissue compared with never- (CpGs) and has traditionally been associated with gene

1Department of Medicine, Karolinska Institutet, Stockholm, Sweden; 2Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden; 3Department of Clinical Sciences, Danderyds Hospital, Karolinska Institutet., Stockholm, Sweden; 4Department of Surgery, Ersta Hospital, Karolinska Institutet, Stockholm, Sweden; 5European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, UK; 6Department of Clinical Sciences, Epigenetics and Diabetes, Lund University Diabetes Centre, CRC, Scania University Hospital, Malmo, Sweden and 7SciLifeLab, Science for Life Laboratory, Solna, Sweden. Correspondence: Dr I Dahlman, Department of Medicine, Karolinska Institutet, Huddinge C2 94, S-141 86 Stockholm, Sweden. E-mail: [email protected] Received 19 November 2014; revised 16 February 2015; accepted 22 February 2015; accepted article preview online 18 March 2015; advance online publication, 28 April 2015 Fat cell epigenetics in post-obesity I Dahlman et al 911 repression.9 The dynamics of genomic DNA methylation is poorly specimens, limited to genes and pathways with DMS, was conducted on understood; the epigenetic pattern is influenced by age.10 18 of the above patients (9 post-obese and 9 never-obese). For the The global variation in DNA-methylation is small between remaining subjects included in this study we did not have WAT samples for individuals, as is the influence of diseases such as type 2 transcriptome analysis. The transcriptome samples comprise a subset of diabetes.11,12 However, the methylation level of specific CpG sites a separate large ongoing study in which mRNA expression in relation to has been reported to be modulated by physical exercise, body weight loss is evaluated (unpublished). In the present study we focused on 11–13 the expression of genes in the vicinity of DMS. mass index (BMI) and type 2 diabetes. In a secondary DNA methylation analysis in fat cells limited to The epigenetic profile varies between organs, but can also differ 14 adipogenesis genes we included 15 obese women who were examined substantially between various cell types within an organ. before gastric bypass surgery (cohort 3). The adipocyte global DNA- In addition to the change in fat cell size and number, obesity is methylome in this group had been inferred in a separate ongoing project associated with altered cellular composition of adipose tissue—for evaluating epigenetic regulation of metabolic disturbances secondary to example, infiltration with inflammatory cells.15 Changes in adipose obesity. Transcriptome data on WAT were available for 11 of the tissue DNA methylation can therefore reflect altered cellular obese women. composition. To avoid this confounder, in this study we performed Four never-obese, four obese and six post-obese women had reached DNA methylation profiling on isolated fat cells, as these cells are menopause stage. Six post-obese women in cohort 1 were treated for hypertension. None had diabetes. All 14 never-obese women in cohort 2 most strongly implicated in metabolic complications of obesity. were healthy. Three of the obese women (cohort 3) had type 2 diabetes, of We studied a unique clinical cohort of never-obese and post- whom two were treated with diet and metformin and one woman with obese women from whom we have obtained isolated fat cells. diet alone. Nine were treated for hypertension. One patient had stable The primary aim of the study was to identify differentially DNA multiple sclerosis and did not receive any drugs. The post-obese women methylated sites (DMS) in isolated fat cells that could be involved received routine vitamin and mineral supplementation. The operated in adipose hyperplasia in post-obese women as compared with women participated in a trial on the effect of bariatric surgery. The Clinical BMI-matched never-obese women. This study design avoids the Trial Registration number is NCT01785134. The study was approved by the identification of DMS that are secondary to obesity and reversible local Ethics Committee and all subjects gave their written informed upon weight loss. In the follow-up analysis, limited to adipogen- consent to participate. esis genes, we further evaluated such reversible events by also examining obese women. Clinical evaluation and adipose sampling Participants were investigated at 0800 hours after an overnight fast. Anthropometric measurements (height, weight, waist and hip circumference, SUBJECTS AND METHODS and blood pressure) were obtained and this was followed by the drawing Subjects and clinical evaluation of a venous blood sample for the determination of blood glucose and blood lipids at the hospital’s routine chemistry laboratory, and insulin Three cohorts of women were investigated. Clinical data are presented in by Radioimmunoassay (Pharmacia, Uppsala, Sweden) as previously Table 1. Women were recruited in association with planned visits to our described.5 Biopsies from the subcutaneous abdominal WAT were surgical units for gastric bypass surgery because of obesity or through local obtained by needle aspiration under local anesthesia. Adipose samples advertisement for the purpose of studying adipose factors regulating body were rinsed in saline (NaCl 9 mg ml–1). weight. In the primary fat cell DNA methylation analysis we included 16 women who were examined 2 years after gastric bypass surgery when they had reached a non-obese state (post-obese group, cohort 1) and 14 never- Handling of adipose tissue samples and isolation of fat cells obese women (cohort 2). The patients examined 2 years post gastric From WAT samples we isolated the fat cell fraction according to the bypass surgery had reached their lowest post-surgery weight and had collagenase procedure as described.16 Mean fat cell volume was been weight stable at this weight for 46 months. Their mean BMI was determined as previously described.17 Briefly, in adipocyte suspensions 16 kg m–2 lower as compared with before surgery. The never-obese had we measured cell sizes by direct microscopy, and the mean adipocyte also been weight stable for 46 months. Transcriptome analysis on WAT diameter was calculated from measurements of 100 cells. Because

Table 1. Clinical characteristics of the patients

P-valuec post-obese Post-obese (cohort 1)a Never-obese (cohort 2) Obese (cohort 3)b vs never-obese

n 16 14 15 Age (years) 48 ± 11 45 ± 11 46 ± 11 0.55 Weight (kg) 72 ± 10 69 ± 7 115 ± 11 0.50 BMI (kg m–2) 25.7 ± 2.3 25.2 ± 2.5 41.4 ± 4.5 0.58 Waist to hip ratio 0.88 ± 0.05 0.85 ± 0.06 0.98 ± 0.06 0.17 Systolic blood pressure (mm Hg) 126 ± 16 123 ± 19 138 ± 22 0.62 Diastolic blood pressure (mm Hg) 75 ± 10 74 ± 685± 9 0.59 P-glucose (mmol l–1) 4.7 ± 0.6 5.1 ± 0.4 5.7 ± 1.2 0.06 P-insulin (mU l–1) 4.5 ± 1.8 4.6 ± 2.3 16.0 ± 10.3 0.94 P-cholesterol (mmol l–1) 4.2 ± 0.9 4.7 ± 1.0 4.9 ± 0.7 0.19 P-HDL cholesterol (mmol l–1) 1.7 ± 0.5 1.5 ± 0.4 1.1 ± 0.3 0.43 P-triglycerides (mmol l–1) 1.02 ± 0.40 0.86 ± 0.72 1.67 ± 0.92 0.43 P-NEFA (mmol l–1) 0.70 ± 0.34 0.57 ± 0.17 0.83 ± 0.16 0.24 P-apolipoprotein B (g l–1) 0.80 ± 0.13 0.83 ± 0.25 0.96 ± 0.25 0.67 P-apolipoprotein A1 (g l–1) 1.54 ± 0.39 1.39 ± 0.22 1.19 ± 0.24 0.22 Mean fat cell volume (pl) 370 ± 128 443 ± 169 994 ± 184 0.20 aSix post-obese women were treated for hypertension and one patient had stable multiple sclerosis and did not receive any drugs. None had diabetes. bThree of the obese women (cohort 3) had type 2 diabetes, of whom two were treated with diet plus metformin, and one woman with diet alone. Nine were treated for hypertension and one patient had stable multiple sclerosis and did not receive any drugs. cComparison of control group and post-obese with unpaired t- test. Values are mean ± s.d.

© 2015 Macmillan Publishers Limited International Journal of Obesity (2015) 910 – 919 Fat cell epigenetics in post-obesity I Dahlman et al 912 adipocytes are spherical in shape, cell volume can be estimated from the Statistical analysis diameter. From adipose specimens 200 μl of packed isolated fat cells and/ We used the Bioconductor package Limma on methylation M-values to or 300 mg unfractionated WAT pieces was frozen in liquid nitrogen and identify DMS between post-obese and never-obese women, adjusting for kept at − 70 °C for subsequent DNA (cells) or RNA (tissue) preparation. age, which is known to influence DNA methylation.22 We used the unpaired t-test to compare average global DNA methylation and specific DNA preparation gene expression between the groups, and in secondary analysis of DNA methylation in adipogenesis genes between obese and never- Genomic DNA was prepared from fat cells using the QiAamp DNA Mini kit (cat obese women. no. 51304, Qiagen, Hilden, Germany). The DNA purity and quality was confirmed by A260/280 ratio 41.8 on a Nanodrop ND-1000 Spectro- fi photometer (Thermo Fisher Scienti c Inc., Waltham, MA, USA). The DNA RESULTS concentration was measured by Qubit (Life technologies, Stockholm, Sweden). Clinical characteristics of the three cohorts DNA methylation microarray assay Clinical characteristics of the cohorts are presented in Table 1. The fi DNA methylation was analyzed for DNA extracted from fat cells using the post-obese group (cohort 1) did not differ signi cantly in age, BMI Infinium Human Methylation 450 BeadChip assay (Illumina, San Diego, CA, or fasting levels of glucose, insulin and plasma lipids as compared USA). The methylation assays were conducted at BEA (www.bea.ki.se). with the never-obese women (cohort 2). Although mean fat cell Genomic DNA (500 ng) was bisulfite treated using the EZ DNA methylation volume was smaller (370 pl) in the post-obese than in the never- kit (Zymo Research, Orange, CA, USA) with alternative incubation obese women (443 pl), this difference was not statistically conditions recommended when using the infinium methylation assay. significant. The methylation assay was performed on 4 μl bisulfite-converted genomic –2 –1 The obese group (cohort 3) had a mean BMI 16 kg m higher as DNA at 50 ng μl according to the Infinium HD Methylation Assay protocol compared with the never-obese and post-obese women. The (Part #15019519, Illumina). We have previously shown that DNA obese women had higher glucose and insulin levels as compared methylation assayed in this way is technically valid as compared with results from PyroSequencing.18 DNA methylation data have been with the other groups as a sign of insulin resistance, and had two- deposited in the National Center for Biotechnology Information Gene fold larger mean fat cell volume (994 pl). Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible using Gene Expression Omnibus series accession number GSE58622. Global pattern of adipocyte CpG methylation in never-obese and post-obese women Transcriptome microarray assay The average degree of DNA methylation—that is, the average WAT specimens (100 mg) were disrupted mechanically. From high-quality beta-value of 129,900 analyzed probes—was lower in fat cells total RNA we prepared and hybridized biotinylated complementary RNA to from post-obese women (cohort 1) as compared with never-obese Gene 1.1 ST Arrays, and then washed, stained and scanned the slides using women (cohort 2) (P=0.014, Figure 1). We next calculated, for standardized protocols (Affymetrix Inc., Santa Clara, CA, USA). The these probes, the average level of DNA methylation stratified on microarray hybridizations were performed at BEA (www.bea.ki.se). the genome region in relation to CpG content (Figure 1a) and Subsequent data analyses were performed using the Affymetrix Expression functional parts of genes (Figure 1b). The average DNA methylation Console version 1.1. The Robust Multi-array analysis algorithm was used for fi data normalization and calculation of gene expression. To allow of CpG sites located in open sea and shelf regions was signi cantly comparisons of transcript levels between samples, all samples were lower in post-obese compared with never-obese women, whereas subjected to an all-probe set scaling-to-target signal of 100. When multiple no significant difference was observed in CpG islands and probe sets represented the same gene, we took the one with the highest surrounding shore regions (Figure 1a). The average DNA signal forward for subsequent phenotypic analysis. We only used the methylation of CpG sites located in 5′ regions and gene bodies subset of microarray data that was relevant for the DMS in the present was significantly lower in post-obese as compared with never analysis. obese women, whereas there was no significant difference in the 3′ untranslated region. Bioinformatic analyses We used Limma to identify 8504 of 129 900 analyzed CpG sites The BeadChip images were captured using the Illumina iScan (Illumina). significantly differentially methylated in fat cells from post-obese The raw methylation score for each probe represented as methylation compared with never-obese women by applying a false discovery beta-values was calculated using GenomeStudio Methylation module rate (FDR) o1%. The resulting significant DMS displayed 19 software (Illumina) (2010.3). All included samples showed high-quality a minimum difference in beta-value of around 0.05 between the fi bisul te conversion according to Zymo-control samples and also passed all post-obese and never-obese groups. The genomic distribution of GenomeStudio quality control steps based on built-in control probes for the 8504 DMS between post-obese and never-obese women, as staining, hybridization, extension and specificity. We next applied the Bioconductor Lumi package to perform color and quantitative normal- compared with all 129 900 analyzed probes, is shown in Figure 2 ization of the DNA methylation data.20 The BMIQ package was used to in relation to CpG content (Figure 2a) and the functional part of adjust the beta-values of type 2 design microarray probes into a statistical genes (Figure 2b). DMS are under-represented in and near CpG distribution characteristic of type 1 probes. Beta-values were converted to islands and surrounding shores, and over-represented in open sea M-values (M = log2(beta/(1-beta))), a statistically more valid method for regions. DMS are under-represented in the 5′ region of genes conducting differential methylation analysis. As the beta-value is easier to (TSS1500, TSS200). The 248 genes with DMS with difference in interpret biologically, M-values were reconverted to beta-values when beta-value 40.20 between post-obese and never-obese groups describing the results. fi are listed in Supplementary Table 1. These include DMS annotated The In nium Human Methylation 450 BeadChip array contains 485 577 to IGF1R, IRS2, FASN, and RGS3, TGFB2. probes, which cover 21 231 (99%) of RefSeq genes. Probes overlapping single nucleotide polymorphisms can interfere with hybridization. A total of 88 464 probes containing single nucleotide polymorphisms with minor Adipogenesis genes are enriched for DMS in post-obese and allele frequency 410% according to Illumina were therefore excluded, obese women leaving 397 113 probes for subsequent analysis. We limited subsequent To evaluate potential biological functions, the 8504 DMS in fat analysis to probes displaying the largest variation in beta-value signal between subjects. We filtered for the 129 900 probes (1/3 of all probes) cells between post-obese and never-obese women were mapped that passed the threshold variance 0.1 among post-obese and never-obese to genes. In total, 5577 DMS could be mapped to 3717 unique women, which were taken forward for phenotypic analysis (Qlucore, www. genes based on Illumina annotation. These genes were then qlucore.com). We used Webgestalt to identify pathways over-represented analyzed for over-representation of specific WikiPathways, as with DMS as compared with all genes in the human genome.21 compared with all genes in the human genome. Most significantly

Figure 1. DNA methylation landscape in post-obese and never-obese women. The 129 900 CpG probes with the largest variance between subjects were mapped to genome regions based on Illumina annotation. We calculated the average level of DNA methylation within each of the post-obese (striped bars) and never-obese (black bars) groups stratiﬁed on genome region in relation to CpG content (a) and functional gene regions (b). TSS1500: within 1500 basepairs of transcriptional start site (TSS). TSS200: within 200 basepairs of TSS. There was a signiﬁcant difference in mean DNA methylation between groups in all genome regions except CpG islands and surrounding shores (Po0.01).

Figure 2. Genomic distribution of DMS between post-obese and never-obese women in relation to CpG content (a) and the functional part of genes (b). The genomic distribution of 8504 DMS between post-obese and never-obese (black bars) women (FDR 1%) was compared with all 129 900 analyzed CpG probes (hatched bars). The numbers are in open sea (a) (0.60 and 0.45%, respectively) and in gene bodies (b) (0.40% and 0.38%, respectively). TSS1500; within 1500 basepairs of transcriptional start site (TSS). TSS200; within 200 basepairs of TSS.

over-represented pathways include calcium regulation, focal such as obesity. We therefore studied the expression and DNA adhesion, actin cytoskeleton and G-protein-coupled receptors, methylation of adipogenesis genes in adipose samples from pathways linked to cell differentiation—that is, Wnt signaling and a group of obese women (cohort 3). DNA methylation had been adipogenesis, as well as apoptosis and autophagy. Of particular inferred in isolated fat cells from 15 obese women, and gene interest, 35 of 130 (27%) genes linked to ‘adipogenesis’ displayed expression in WAT pieces from 11 of these obese women. The DMS (adjusted P = 1.02 × 10 − 8) (Table 2 and Table 3). analysis of DNA methylation in the obese group was limited to the We next evaluated whether DMS in the 35 genes linked to CpG sites in the 35 adipogenesis genes that were differentially adipogenesis were accompanied by differential gene expression methylated between never-obese and post-obese women. in WAT from post-obese versus never-obese women (Table 3). All inferred CpG sites in 32 of 35 adipogenesis genes displayed Among adipogenesis genes with DMS, only GATA2 and PPARD significantly differential methylation between never-obese and were differentially expressed between post-obese and never- obese women (exceptions were PRLR, NAMPT and HMGA1); the obese women. Thus, the majority of DMS annotated to effects were in a consistent direction with the never-obese versus adipogenesis genes in post-obese as compared with never- post-obese comparison (Table 3). Thus, DNA methylation of obese women are not accompanied by differential gene adipogenesis genes in post-obese and obese women was similar, expression. and differed in comparison with the never-obese group. We next hypothesized that epigenetic changes in adipogenesis Furthermore, numerous adipogenesis genes displayed differential genes could influence gene expression under specific conditions, DNA methylation in fat cells accompanied by altered expression in

Table 2. WikiPathways enriched for DMS in post-obese versus never-obese women

Pathway Genes in pathway Observed genes with DMS Expected genes with DMS Adjusted P-value

Calcium regulation in the cardiac cell 151 55 12 4.10 × 10-20 Focal adhesion 185 53 15 1.36 × 10-14 Regulation of actin cytoskeleton 157 44 13 7.77 × 10-12 G-protein coupled receptors, class A rhodopsin-like 259 58 21 4.29 × 10-11 Endochondral ossification 69 25 6 1.51 × 10-9 Wnt signaling pathway 50 21 4 1.68 × 10-9 Apoptosis 92 28 7 9.51 × 10-9 Adipogenesis 130 35 11 1.02 × 10-8 Insulin signaling 163 39 13 1.02 × 10-8 MAPK signaling pathway 165 38 14 4.20 × 10-8 EGF–EGFR signaling pathway 172 39 14 4.20 × 10-8 Signaling pathways in glioblastoma 84 24 7 2.93 × 10-7 Senescence and autophagy 120 25 10 4.70 × 10-5 Abbreviations: DMS, differentially methylated sites; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase. We examined whether 3717 genes with DMS were over-represented for specific WikiPathways as compared with all genes in the human genome applying a hypergeometric test, and threshold minimum of five genes with DMS in the pathway.

WAT in the obese versus never-obese group comparison (Table 3). Other genes. Other interesting genes with DMS between post- A total of 18 of the adipogenesis genes were differentially obese and never-obese women include HMGA2, which has been expressed between obese and never-obese women, of which linked to lipoma in humans.35 12 displayed inverse relationship between methylation and expression—namely, IGF1, NCOR2, RARA, MEF2D, SREBF1, NRIP1, DMS between never obese and post-obese women in susceptibility LIFR, EBF1, GDF10, KLF5, AHR and PLIN2. gene loci for obesity. Finally, we evaluated whether reported Among 28 apoptosis and 25 autophagy genes with DMS only susceptibility gene loci for obesity and fat distribution from GWAS were associated with DMS in fat cells between post-obese and four and two genes, respectively, were differentially expressed 36 between post-obese and never-obese women (results not shown). never-obese women. Of 54 genes in susceptibility loci for obesity and fat distribution according to the original publications, 12 genes contained one or more DMS (Table 5). Expression of genes linked to DMS between post-obese and never-obese women DISCUSSION We subsequently performed a more comprehensive analysis of all 8504 DMS between post-obese and never-obese women in relation The methylome of isolated human fat cells has not been described before. We compared global DNA methylation profiles in fat cells to WAT gene expression. Of 3717 genes with DMS, 391 displayed from post-obese and BMI-matched never-obese women. We nominally significant difference (with Po0.05) in expression found that the post-obese state is characterized by significant between post-obese and never-obese women (Supplementary global DNA hypomethylation. Furthermore, adipogenesis genes Table 2), of which 224 genes displayed inverse association between were enriched for DMS in post-obese compared with never-obese fat cell DNA methylation and WAT gene expression. A selection of women and were associated with differential gene expression in the genes of potential importance for obesity and fat accumulation obese but not post-obese women. are described below and listed in Table 4. The global pattern of DMS in the genome reported here is in agreement with what has been previously reported for human Adipogenesis. DMS accompanied by differential gene expression adipose tissue, which contains a number of additional cell types between post-obese and never-obese women were observed in a besides the fat cells. The small magnitude of significant differences few genes with pleiotrophic functions, including an impact on in beta-values between groups, and the genomic distribution of adipogenesis—for example, IGF1R,23 TGFB2,24 LRP5,25 LRP1B,26 DMS, that is, relatively few near CpG islands and in the promoter MBD6,27 MAP4K428 and WISP2.25 Of note, these genes were regions, and over-representation in open sea regions, is in not included in the WikiPathway adipogenesis pathway agreement with what has been reported.11,12,37 Recent studies in described above. human skeletal muscle support that such small changes in beta- value can indeed be functionally important.38,39 On the other hand, fi Lipid turnover and metabolism. Other genes with DMS and the speci c fat cell DMS in post-obese women reported here display limited overlap with WAT DMS associated with BMI or responding to differential gene expression included ABCG1, which controls LPL 11,13,37 activity and promotes lipid accumulation,29 and AGPAT4, which is exercise. There are a number of plausible explanations for the discrepancies between our and previous studies; for example, most implicated in lipid synthesis.30 ACAD931 has been shown to BMI-associated changes in adipose tissue are secondary to obesity regulate oxidative phosphorylation. and reversed upon weight loss; such features may not be picked up in our comparison of never-obese and post-obese subjects. Obesity. Multiple DMS accompanied by altered gene expression Furthermore, previous studies were performed on WAT pieces, between post-obese and never-obese women were observed in the and reported DMS may therefore represent changes in adipose SNTG2 gene, which is located in a chromosome 2p region cellular composition or in non-adipose cells. 32 implicated in early-onset obesity. The human obesity gene MRAP2 Obese individuals have a higher number of fat cells compared contains a CpG site in the 3′ untranslated region, that is with non-obese individuals, and this higher number does not differentially methylated in post-obese women.33 The RCAN2 gene decrease following weight loss.7 Furthermore, we previously is involved in experimental obesity and there are DMS in the 5′ reported that post-obese subjects, as compared with age and region of the gene between post-obese and never-obese women.34 BMI-matched never-obese women, have smaller fat cells, suggesting

International Journal of Obesity (2015) 910 – 919 © 2015 Macmillan Publishers Limited 05McilnPbihr iie nentoa ora fOeiy(05 910 (2015) Obesity of Journal International Limited Publishers Macmillan 2015 ©

Table 3. Adipogenesis genes with DMSa

DNA methylation Expression

Gene Probeset Region Obeseb Post-obese Never-obese PO-NOa Obese Post-obese Never-obese O/NO P-value Beta-value (mean) Mean ± s.d. PO vs NO O vs NO

Growth factors TGFB1 cg09926389 Body 0.83 0.79 0.66 0.13 144 ± 21 87 ± 14 78 ± 12 1.85 0.17 2×10-07 INS cg20254598 TSS200 0.79 0.80 0.86 − 0.07 Not applicable cg00613255 5'UTR 0.68 0.64 0.72 − 0.08 cg24522478 Body 0.61 0.59 0.66 − 0.07 IGF1 cg20874044 TSS1500 0.31 0.23 0.10 0.13 790 ± 201 963 ± 141 1054 ± 173 0.75 0.24 0.0062 PRLR cg24070673 3'UTR 0.55 0.46 0.61 − 0.15 10 ± 18± 11± 1 1.09 0.18 0.071

TF/modulators NCOR2 cg23021584 Body 0.45 0.46 0.63 − 0.18 159 ± 11 156 ± 44 142 ± 10 1.13 0.34 0.0015 cg06942010 Body 0.67 0.62 0.73 − 0.11 RARA cg25362050 5'UTR 0.68 0.69 0.77 − 0.09 94 ± 978± 12 73 ± 9 1.29 0.41 5×10-05 cg00442282 5'UTR 0.11 0.09 0.15 − 0.06 cg07944862 Body 0.34 0.33 0.47 − 0.14 cg19314352 Body 0.14 0.14 0.20 − 0.07 MEF2D cg04298847 Body 0.48 0.33 0.43 − 0.09 111 ± 6 111 ± 14 117 ± 5 0.95 0.24 0.025 cg06034998 3'UTR 0.81 0.8 0.72 0.08 RORA cg06100161 Body 0.89 0.87 0.95 − 0.08 116 ± 9 136 ± 12 125 ± 17 0.93 0.13 0.13 cg27109006 Body 0.87 0.83 0.74 0.09 cg22412536 Body 0.47 0.37 0.55 − 0.18 − cg14720274 Body 0.83 0.77 0.86 0.09 Dahlman post-obesity I in epigenetics cell Fat cg02458188 Body 0.22 0.11 0.20 − 0.10 PPARD cg15611037 Body 0.91 0.9 0.82 0.08 120 ± 9 132 ± 10 119 ± 6 1.01 0.01 0.73 SREBF1 cg09494646 TSS200 0.85 0.84 0.69 0.14 142 ± 25 159 ± 33 189 ± 58 0.75 0.19 0.024

cg25999891 Body 0.59 0.54 0.40 0.15 al et NRIP1 cg07543138 5'UTR 0.38 0.34 0.22 0.12 453 ± 70 734 ± 186 933 ± 249 0.49 0.07 8× 10-06 ID3 cg20485144 TSS200 0.36 0.33 0.24 0.09 99 ± 18 91 ± 11 88 ± 21 1.12 0.74 0.25 LIFR cg04444661 5'UTR 0.89 0.87 0.81 0.06 594 ± 81 810 ± 158 696 ± 86 0.85 0.08 0.014

Inhibition of pre-adipocyte differentiation WNT cg11701621 TSS1500 0.15 0.14 0.2 − 0.07 23 ± 327± 326± 3 0.87 0.31 0.029 GATA2 cg27199494 TSS1500 0.64 0.63 0.72 − 0.09 36 ± 645± 735± 3 1.03 0.003 0.64 DLK1 cg09865386 TSS1500 0.74 0.75 0.82 − 0.06 19 ± 423± 420± 2 0.94 0.12 0.47 cg18121862 Body 0.73 0.74 0.82 − 0.08 cg12193817 Body 0.66 0.59 0.68 − 0.09 cg21902964 Body 0.82 0.79 0.85 − 0.05

Miscellaneous EBF1 cg18370682 Body 0.65 0.69 0.60 0.09 1379 ± 132 1626 ± 216 1701 ± 156 0.81 0.41 9 × 10-5 EPAS1 cg07901411 Body 0.41 0.36 0.23 0.13 2475 ± 144 2398 ± 142 2403 ± 171 1.03 0.95 0.32 cg19311375 Body 0.14 0.08 0.16 − 0.07 GDF10 cg13565300 Body 0.8 0.83 0.92 − 0.09 73 ± 14 63 ± 15 56 ± 10 1.32 0.21 0.0052 CNTFR cg08906194 5'UTR 0.14 0.15 0.23 − 0.08 374 ± 57 673 ± 119 694 ± 156 0.54 0.74 5×10-06 SCD cg00699831 Body 0.84 0.83 0.90 − 0.06 4452 ± 540 4919 ± 399 4838 ± 361 0.92 0.66 0.08 STAT5B cg05891054 Body 0.34 0.32 0.21 0.11 468 ± 47 447 ± 41 485 ± 44 0.96 0.08 0.40 MBNL1 cg17185710 TSS1500 0.56 0.46 0.65 − 0.20 1771 ± 72 1697 ± 127 1765 ± 126 1.00 0.27 0.90

– cg02061820 Body 0.17 0.13 0.29 − 0.16 919 SMAD3 cg05438378 Body 0.12 0.1 0.17 − 0.07 279 ± 35 247 ± 40 266 ± 35 1.05 0.30 0.42 915 916 nentoa ora fOeiy(05 910 (2015) Obesity of Journal International

Table. 3. (Continued ) a eleieeisi post-obesity in epigenetics cell Fat DNA methylation Expression – 1 05McilnPbihr Limited Publishers Macmillan 2015 © 919

Gene Probeset Region Obeseb Post-obese Never-obese PO-NOa Obese Post-obese Never-obese O/NO P-value Beta-value (mean) Mean ± s.d. PO vs NO O vs NO

SOCS3 cg22749855 3'UTR 0.58 0.52 0.67 − 0.15 198 ± 136 161 ± 30 151 ± 87 1.31 0.77 0.39 Dahlman I WWTR1 cg06728055 Body 0.30 0.32 0.53 − 0.21 436 ± 41 433 ± 51 418 ± 35 1.04 0.47 0.30 KLF5 cg01645401 Body 0.92 0.93 0.87 0.06 33 ± 443± 745± 9 0.73 0.52 0.0013 cg04339360 Body 0.78 0.78 0.84 − 0.07

KLF7 cg06916059 Body 0.51 0.45 0.34 0.11 692 ± 70 695 ± 135 622 ± 69 1.11 0.16 0.038 al et AHR cg07156115 Body 0.90 0.88 0.95 − 0.07 800 ± 141 634 ± 98 543 ± 99 1.47 0.07 0.00022 HMGA1 cg20294304 TSS1500 0.63 0.75 0.64 − 0.09 30 ± 631± 532± 7 0.94 0.58 0.53 Differentiated adipocyte markers PLIN2 cg03885527 Body 0.84 0.82 0.9 − 0.08 506 ± 102 357 ± 73 408 ± 53 1.24 0.11 0.017

Lipodystropy genes LPIN1 cg10142874 Body 0.92 0.93 0.85 0.08 285 ± 28 500 ± 143 611 ± 262 0.47 0.28 0.00064 cg20323962 Body 0.68 0.66 0.46 0.2

Insulin action genes IRS2 cg12085119 Body 0.53 0.50 0.34 0.16 115 ± 19 160 ± 25 184 ± 28 0.62 0.07 3x10-06 cg20445402 Body 0.76 0.66 0.44 0.22

Adipocyte secretory products IL6 cg01770232 TSS1500 0.14 0.12 0.19 − 0.07 106 ± 143 34 ± 14 25 ± 8 4.20 0.12 0.11 NAMPT/PBEF cg05004518 Body 0.55 0.42 0.57 − 0.15 1131 ± 377 933 ± 275 1024 ± 270 1.10 0.49 0.49 Abbreviations: DMS, differentially methylated sites; FDR, false discovery rate; NO, never-obese; O, obese; PO, post-obese; TSS, transcriptional start site; UTR, untranslated region. aAll probe sets are differentially methylated between post-obese and never-obese women according to Limma adjusted for age (FDR 1%). bAll probe sets except for those in PRLR, NAMPT and HMGA1 are differentially methylated between obese and never-obese women (P = 0.05). Fat cell epigenetics in post-obesity I Dahlman et al 917

Table 4. Fat cell DMS between post-obese and never-obese accompanied by differential expression in adipose tissue

CpG Methylation Expression

Gene Target ID PO (μ) PO (s.d.) NO (μ) NO (s.d.) PO_vs_NO PO (μ) PO (s.d.) NO (μ) NO (s.d.) PO/NO P-value

ABCG1 cg10192877 Body 0.82 0.05 0.88 0.03 − 0.06 76 19 53 13 1.42 0.011 Inverse ACAD9 cg09447658 Body 0.57 0.08 0.68 0.05 − 0.12 361 43 404 28 0.89 0.023 — AGPAT4 cg14840850 Body 0.58 0.14 0.79 0.09 − 0.21 50 5 43 4 1.15 0.007 Inverse AGPAT4 cg09043403 Body 0.68 0.07 0.83 0.06 − 0.15 50 5 43 4 1.15 0.007 Inverse HMGA2 cg12141052 Body 0.52 0.08 0.65 0.04 − 0.13 15 4 12 2 1.30 0.018 Inverse HMGA2 cg04890607 Body 0.2 0.08 0.37 0.09 − 0.17 15 4 12 2 1.30 0.018 Inverse HMGA2 cg02123091 Body 0.15 0.11 0.29 0.07 − 0.14 15 4 12 2 1.30 0.018 Inverse HMGA2 cg25161912 Body 0.34 0.05 0.47 0.10 − 0.14 15 4 12 2 1.30 0.018 Inverse IGF1R cg27139419 Body 0.23 0.07 0.35 0.07 − 0.12 263 26 218 26 1.21 0.002 Inverse IGF1R cg12486493 Body 0.55 0.11 0.78 0.04 − 0.24 263 26 218 26 1.21 0.002 Inverse IGF1R cg18158670 Body 0.28 0.08 0.42 0.08 − 0.13 263 26 218 26 1.21 0.002 Inverse LRP1B cg18446140 Body 0.77 0.07 0.86 0.03 − 0.09 13 5 35 24 0.36 0.014 — LRP1B cg16671674 Body 0.13 0.05 0.2 0.05 − 0.07 13 5 35 24 0.36 0.014 — LRP5 cg03064005 Body 0.89 0.04 0.81 0.03 0.08 327 31 359 33 0.91 0.049 Inverse LRP5 cg05984508 Body 0.26 0.08 0.16 0.04 0.11 327 31 359 33 0.91 0.049 Inverse LRP5 cg26878836 Body 0.7 0.08 0.56 0.05 0.13 327 31 359 33 0.91 0.049 Inverse LRP5 cg22151881 Body 0.28 0.06 0.16 0.05 0.11 327 31 359 33 0.91 0.049 Inverse MAP4K4 cg04217929 Body 0.62 0.06 0.71 0.03 − 0.09 352 32 305 32 1.15 0.007 Inverse MBD2 cg25142283 3'UTR 0.81 0.05 0.88 0.03 − 0.07 605 42 669 43 0.90 0.006 — MRAP2 cg02129814 3'UTR 0.23 0.06 0.32 0.04 − 0.08 11 2 10 1 1.14 0.037 Inverse NEGR1 cg24940889 Body 0.56 0.14 0.82 0.06 − 0.26 258 50 208 25 1.24 0.016 Inverse RCAN2 cg04652496 TSS1500 0.72 0.10 0.58 0.07 0.13 151 60 100 26 1.51 0.033 — RCAN2 cg06665622 TSS200 0.19 0.06 0.28 0.05 − 0.09 151 60 100 26 1.51 0.033 Inverse SNTG2 cg09294347 Body 0.22 0.05 0.3 0.05 − 0.08 53 11 65 12 0.82 0.046 — SNTG2 cg00148862 Body 0.19 0.06 0.27 0.05 − 0.07 53 11 65 12 0.82 0.046 — SNTG2 cg09062961 Body 0.86 0.10 0.93 0.02 − 0.08 53 11 65 12 0.82 0.046 — SNTG2 cg13049247 Body 0.9 0.05 0.94 0.01 − 0.04 53 11 65 12 0.82 0.046 — SNTG2 cg25481636 Body 0.84 0.05 0.92 0.02 − 0.08 53 11 65 12 0.82 0.046 — SNTG2 cg24982556 Body 0.73 0.04 0.8 0.03 − 0.07 53 11 65 12 0.82 0.046 — SNTG2 cg05978707 Body 0.71 0.07 0.83 0.03 − 0.11 53 11 65 12 0.82 0.046 — SNTG2 cg05299774 Body 0.63 0.04 0.7 0.04 − 0.07 53 11 65 12 0.82 0.046 — SNTG2 cg08019798 Body 0.4 0.09 0.57 0.08 − 0.16 53 11 65 12 0.82 0.046 — SNTG2 cg07609206 Body 0.68 0.04 0.8 0.04 − 0.13 53 11 65 12 0.82 0.046 — SNTG2 cg15602677 Body 0.56 0.07 0.67 0.06 − 0.11 53 11 65 12 0.82 0.046 — TGFB2 cg25132662 Body 0.14 0.06 0.07 0.02 0.07 62 19 43 11 1.46 0.017 — TGFB2 cg16967578 Body 0.86 0.07 0.66 0.10 0.2 62 19 43 11 1.46 0.017 — TGFB2 cg20698667 Body 0.63 0.13 0.42 0.09 0.21 62 19 43 11 1.46 0.017 — WISP2 cg21425802 TSS200 0.4 0.13 0.6 0.12 − 0.21 398 68 312 79 1.27 0.026 Inverse WISP2 cg03562120 1stExon 0.17 0.06 0.36 0.12 − 0.18 398 68 312 79 1.27 0.026 Inverse WISP2 cg13093934 3'UTR 0.78 0.04 0.85 0.04 − 0.07 398 68 312 79 1.27 0.026 Inverse Abbreviations: DMS, differentially methylated sites; FDR, false discovery rate; UTR, untranslated region; PO, post-obese; NO, never-obese; TSS, transcriptional start site. Selected DMS between post-obese (PO) and control (NO) women (FDR 1%) with significant difference in gene expression between these groups. that they have retained an increased fat cell number from their cannot be reversed by weight loss. On the other hand, an formerly obese state.5 A similar trend, although not statistically additional challenge, overfeeding or weight gain, may be significant, was observed in the present study. It is therefore necessary to affect the expression of adipogenesis genes. Another intriguing that adipogenesis genes, which are necessary for the explanation is that methylation does affect gene expression generation of fat cells, display over-representation for DMS in differentially in specific cell types of adipose tissue and that this is post-obese women. Unfortunately, to our knowledge there are no concealed when gene expression is measured in RNA from intact data on adipose progenitor cells in post-obese humans. Numerous WAT, as done presently, which contains a mixture of cell types. studies have highlighted the importance of epigenetic regulation Unfortunately, we did not have sufficient amounts of isolated fat of adipogenesis in vitro (reviewed by Musri and Parrizas40). cells to perform RNA expression studies. Several of the DMS in However, to our knowledge this is the first study in which the adipogenesis genes were located in gene bodies. DNA methyla- epigenetic pattern in the vicinity of adipogenesis genes is linked tion of CpG sites in gene bodies often show a positive correlation to a clinical adipose phenotype in humans—that is, hyperplastic with active transcription.49 post-obese adipose tissue (i.e., with many small fat cells). Some Beyond adipogenesis genes, adipocyte DNA methylation in adipogenesis genes with DMS in post-obese women have been obese women was not studied within this project as obesity- reported to be epigenetically regulated—for example, IGF1,41 associated changes in fat cells are primarily secondary to obesity, SREBF1,42 KLF5,43 AHR,44 GDF10,45 TGFB146 EPAS147 and DLK1.48 whereas this project is focused on the phenotypes associated with Differential methylation of adipogenesis genes was accompanied the post-obese state. One limitation of the present study is that by altered expression in the obese, but not in post-obese, women. we did not consider long-range gene regulation—that is, whether One possible explanation for this discrepancy is epigenetic DMS at one location are correlated with gene expression at inflexibility—that is, subjects who are or have been obese display a distant locus. The data set was too small for such an analysis. a stable DNA methylation pattern in adipogenesis genes that Furthermore, we do not know whether our findings are unique to

Table 5. DMS near susceptibility genes for obesity and fat mass distribution

Relation to Gene Phenotype Probeset CpG island Gene region Post-obese (μ) Post-obese (s.d.) Never-obese (μ) Never-obese (s.d.) PO vs NO

MSRA WHR cg20679581 Open sea Body 0.66 0.06 0.75 0.05 − 0.09 DNM3 WHR cg06320175 Open sea Body 0.75 0.08 0.88 0.05 − 0.13 cg07802362 Open sea Body 0.77 0.08 0.87 0.04 − 0.10 cg24595586 Open sea Body 0.75 0.06 0.84 0.03 − 0.08 cg26679756 Open sea Body 0.26 0.10 0.42 0.08 − 0.16 cg20420249 Open sea 3'UTR 0.58 0.11 0.72 0.05 − 0.14 FTO BMI cg26651810 Open sea Body 0.34 0.10 0.54 0.09 − 0.20 cg02992067 Open sea Body 0.37 0.11 0.63 0.08 − 0.26 ITPR2 WHR cg23202253 Open sea Body 0.24 0.08 0.40 0.07 − 0.16 cg01088546 Open sea Body 0.55 0.11 0.74 0.04 − 0.19 cg00997853 Open sea Body 0.60 0.06 0.71 0.06 − 0.11 cg08186005 Open sea Body 0.86 0.05 0.92 0.03 − 0.06 cg01354782 Open sea Body 0.20 0.10 0.32 0.08 − 0.13 SSPN WHR cg13909661 Open sea 3'UTR 0.37 0.07 0.54 0.08 − 0.16 KCTD15 BMI cg04566826 S_Shore 5'UTR 0.21 0.05 0.15 0.03 0.06 LRP1B BMI cg18446140 Open sea Body 0.77 0.07 0.86 0.03 − 0.09 cg16671674 Open sea Body 0.13 0.05 0.20 0.05 − 0.07 LY86 WHR cg15553155 Open sea Body 0.42 0.08 0.54 0.04 − 0.12 NEGR1 BMI cg24940889 Open sea Body 0.56 0.14 0.82 0.06 − 0.26 SLC39A8 BMI cg12097340 Open sea Body 0.75 0.07 0.87 0.06 − 0.11 ZNF608 BMI cg08319999 Open sea Body 0.88 0.04 0.77 0.06 0.11 cg01154355 Open sea Body 0.27 0.05 0.19 0.04 0.08 ZNRF3 WHR cg13298682 Island Body 0.44 0.06 0.33 0.06 0.12 cg07127410 S_Shore Body 0.41 0.07 0.60 0.06 −0.19 cg23896685 Open sea 3'UTR 0.73 0.07 0.63 0.04 0.09 Abbreviations: BMI, body mass index; FDR, false discovery rate; UTR, untranslated region; PO, post-obese; NO, never-obese; WHR, waist-hip ratio. aAll probe sets are differentially methylated between post-obese and never-obese women according to Limma adjusted for age (FDR 1%).

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