Impact of obesity on global DNA methylation patterns in hASCs and differentiated adipocytes

As a first step to investigate the potential contribution of obesity to DNA methylation and adipocyte differentiation, hASCs were isolated from the SAT compartment of lean and obese subjects (n = 6; as described in Materials and methods). Isolation and differentiation of hASCs to mature adipocytes was performed as described [6, 9]. We extracted genomic DNA from cells before and after differentiation and performed EWAS on the Illumina platform using the Infinium Human-Methylation450K BeadChip array (Fig. 1a). We established a threshold of log 2-fold change with a p value < 0.0001 to discern DMSs between the conditions. At the global level, only 0.01% of the CpG sites studied displayed changes between differentiation states, whereas this rose to 0.2% when lean and obese conditions were considered. We identified 650 DMSs between hASCs from lean and obese subjects and only 206 DMSs between equivalent mature adipocytes (Fig. 1a, b), with a total of 64 DMSs in common. In lean subjects, 43 DMSs were identified between hASCs and differentiated adipocytes, whereas 34 DMSs were identified in obese subjects (Fig. 1a). Overall, these data show that the methylome is comparatively stable between hASCs and mature adipocytes, and also that the majority of the differences detected are due to the obese environment already established in the hASC niche. The apparent discordance between the number of DMSs in hASCs and adipocytes according to phenotype (lean vs. obese) can be explained because of the use of an arbitrary cutoff (see Materials and methods), which could have led to small changes not identified as statistically different DMSs. Nevertheless, β value differences between lean and obese donors showed a strong correlation for DMSs both in ASCs (n = 650) and in adipocytes (n = 206), but not for a subset of randomly selected probes (n = 1000) (Supplementary Figure S1). This indicates that DMSs between lean and obese are largely maintained in both ASCs and adipocytes, even when the p value does not reach the threshold (0.0001). Also, since global methylation is known to increase during differentiation, this may have conceivably masked small changes occurring during the differentiation of hASCs to mature counterparts. Interestingly, global methylation levels were significantly lower in obese-derived hASCs than in lean-derived counterparts (Fig. 1c). Moreover, 88.37% (38 from 43 DMSs) of the methylation changes that occurred during adipogenesis were hypermethylation events. We next analyzed hierarchically all the CpG methylation differences observed between lean and obese states. Supervised cluster analysis and heat map analysis revealed that the CpG sites analyzed segregated into two clusters, and indicated that only a small proportion of the CpG sites had a low degree of methylation (Fig. 1d).

Fig. 1 Obesity modifies the hASC methylome. a Schematic representation of the approach performed and the main results obtained (p < 0.0001). b Manhattan plots (−Log 10 (p) genome-wide association plot). All DMSs achieving the significance threshold are colored in green (p < 0.0001). c Global methylation status of ASCs and mature counterparts. Results are shown as mean ± SEM. t test; *p < 0.05, ***p < 0.001 vs. lean-derived hASCs; ###p < 0.001 vs. obese-derived hASCs. d Supervised cluster analysis and heat map showing the differential groups of CpGs according to their methylation profile in all analyzed samples. n = 6, patients per group. hASCs human adipose-derived stem cells, AD adipocyte, DMS differentially methylated site (color figure online) Full size image

We next segmented the four methylomes into three classes: fully methylated regions (FMRs; >50% methylated CpGs), low methylated regions (LMRs; <13.9–50%) and unmethylated regions (UMRs; <13.9%), as previously described [22, 23]. The FMR class comprised over 50% of the methylome in all samples (Fig. 2a), and the global percentage of methylation was stable between lean and obese hASCs. By contrast, when we compared adipocytes differentiated from hASCs of lean and obese subjects, we found a global increase in methylation in the latter, with an important gain in FMRs (overall increase of 16%) and a decrease in the number of UMRs. Several studies suggest that the effects of DNA methylation on gene expression are highly dependent on the genomic location of the CpG dinucleotide [22, 24]. To assess whether differential methylation is preferentially allocated at specific genomic regions, we classified all DMSs into seven categories of genome annotations: located in promoters–transcription start site (TSS) (a) −200 bp (TSS200) and (b) −1500 bp (TSS1500); (c) intergenic region (IGR); located in intragenic regions (d) 1st exon, (e) gene body, (f) 3′UTR, and (g) 5′UTR. The majority of changes found in hASCs were those located in transcribed regions (body), which have been shown to positively correlate with transcribed genes [24] (Fig. 2b, left panel). While a similar tendency was observed in mature adipocytes, we detected an approximate twofold increase in the location of DMSs at distal promoters (TSS1500) where DNA methylation is normally negatively correlated with gene expression (Fig. 2b, left panel). The DMS distribution within a CpG island (island>shelf>shore>open sea) is depicted in Fig. 2b, right panels. In both settings, 67% of the significant changes were found in the most distal areas from a CpG island (shore and open sea).

Fig. 2 DNA methylation changes during adipocyte differentiation. a The percentage of DMSs distributed into three classes: fully methylated regions (FMRs > 50% methylated CpGs); low methylated regions (LMR, 13.9–50%); and unmethylated regions (UMRs < 13.9%). b DMS distribution and CpG allocation. c Pathway analysis for genes associated with DMSs in hASCs Full size image

Overall, the above data show that hASCs are obesity-conditioned through the accumulation of epigenetic modifications, at least at the level of DNA methylation, which might underlie the altered plasticity of obese-derived hASCs as previously reported by us [6, 9] and others [8, 25]. In an attempt to discern which molecular pathways might be influencing the hASC niche in an obese environment, we performed a functional analysis by GO enrichment using the Ingenuity database for all genes containing DMSs. Since we were specifically interested in the functionality of adipocyte precursors as key determinants of adipose tissue expansion, we focused on changes in the hASC compartment. As anticipated, GO analysis identified gene functions associated with adipogenesis (Fig. 2c). Other differentially enriched biological pathways detected included inflammation (LPS/IL-1) and immunosuppression/repair (TFG-β). These findings are in agreement with our earlier studies showing that hASCs from obese patients have a heightened inflammatory profile and reduced immunosuppressive properties [6, 9], and are consistent with results from other independent studies [26, 27].

Influence of epigenetic signature on gene expression in hASCs

To explore in more detail the methylation changes in lean- and obese-derived hASCs, we first used a more restrictive cutoff to exclude all sites with less than 20% methylation change and to give us a more manageable number, which reduced the list from 650 DMSs to 60. The top ten genes with the greatest DNA methylation changes (hyper- and hypomethylated) are shown in Table 1. DNA methylation is most often linked to gene silencing. From the eight genes screened to evaluate the correlation between methylation status and mRNA expression, the expected inverse relationship between gene expression and methylation level was established for five of them: TBX15, PRDM16, ACLY, GLI2, and LSP1 (Fig. 3a). In the remaining three genes (POU3F3, CCL4, CDKN1A), an increase in methylation in obese hASCs was associated with higher mRNA levels (Fig. 3a). As the genomic location of DMSs might contribute to explain these divergences, we next examined this finding that DNA methylation around promoter and intragenic regions negatively correlated with gene expression (Table 1 and Fig. 3a). For example, TBX15 was demethylated to different degrees in obese hASCs as compared with lean counterparts, which was accompanied by upregulation at the mRNA level. By contrast, DMSs around the IGR did not trigger a direct modification at the mRNA level. This was the pattern observed for CCL4, CDKN1A, and POU3F3 in which the occurrence of methylated DMSs located in the IGR (Table 1) did not correlate with downregulation of the mRNA (Fig. 3a). For the transcription factor GLI2, we detected an increase in methylation above 20% within an IGR; nevertheless, expression of this gene was significantly decreased in obese hASCs. This could be explained by the finding that GLI2 possesses other significant DMSs in the gene body whose DNA methylation levels were in agreement with its pattern of mRNA expression (Supplementary Table S2).

Table 1 Top 10 hypo- and hyper-methylated sites with more of 20% of change Full size table

Fig. 3 Differentially methylated sites have a differential effect on mRNA expression. a Methylation and mRNA levels of TBX15, PRDM16, ACLY, GLI2, LSP1, POU3F3, CCL4, and CDKN1A genes in hASCs. Significant DMSs (#p < 0.0001) identified by EWAS are shown (n = 6 patients per group). Verification of mRNA levels of genes associated with the DMSs identified (n = 4 patients per group). Results are shown as mean ± SEM. t test; *p < 0.05, **p < 0.01 vs. lean hASCs. Gene name in green (decreased methylation and increased mRNA expression), in orange (increased methylation and decreased mRNA expression), and in blue (methylation and mRNA expression incongruity) (color figure online) Full size image

Hypomethylation of mesodermal developmental gene TBX15 in obese hASCs is associated with mitochondrial function in mature adipocytes

We focused on TBX15 since it was highly regulated at the epigenetic level, containing a total of 13 DMSs (Table 1 and Supplementary Figure S2). TBX15 belongs to the T-box family of homeodomain transcription factors, which are essential for many developmental processes. The methylation levels of TBX15 were further validated by bisulfite sequencing, confirming that the amplicon was demethylated in obese hASCs (Supplementary Figure S3). The high mRNA level of TBX15 in obese hASCs (Fig. 3a) was confirmed at the protein level (Fig. 4a). Intriguingly, whereas the protein levels of TBX15 were higher in obese hASCs than in lean hASCs, its expression significantly decreased throughout the adipogenic differentiation process both in obese- and lean-derived cells, suggesting a developmental role for TBX15 in WAT adipogenesis. Even so, TBX15 protein levels remained higher in mature adipocytes from obese-derived hASCs than from those of lean subjects. A previous study in murine adipocyte precursors indicated that Tbx15 regulates brown but not white adipocyte differentiation [17], whereas another study described Tbx15 as a negative regulator of mitochondrial mass and adipogenesis in murine 3T3-L1 pre-adipocytes [15]. To explore the effects of TBX15 on adipocyte precursors, we first transfected obese-derived hASCs with a pool of three target-specific siRNAs against TBX15 to silence its expression. TBX15-silenced cells showed a significant decrease in the expression of beige adipocyte markers (including TBX1 and TMEM26), catalytic components of mitochondrial complex I (NDUFA9) and II (SDHA), and mitochondrial fusion markers (including MFN2 and OPA1) (Fig. 4b), in part, supporting the proposed role of TBX15 in the beiging of SAT [17]. Remarkably, most of these genes were upregulated in obese ASCs as compared with equivalent lean cells (Supplementary Figure S4) which, considering the higher levels of TBX15 in obese ASCs, supports its instructive role in the mitochondrial phenotype of adipocytes. Additionally, we detected a decrease in the expression of genes encoding proteins for fatty acid transport and oxidation (ACAA1, CPT1A, CPT1B, SLC25A20), glucose uptake (GLUT4), glycolysis (LDHA, LDHB) and the TCA cycle (OGDH, PDH) in TBX15-silenced obese ASCs (Supplementary Figure S5). Interestingly, most of these genes were also found increased in obese compared with lean ASCs [9]. Overall, these results suggest a potential role for TBX15 in the mitochondrial and/or metabolic phenotype of mature adipocytes in an obesity context. Accordingly, downregulation of TBX15 expression in obese precursors before inducing differentiation resulted in a decrease in the mitochondrial mass of mature adipocytes, pointing to a possible reversal of the obese phenotype (Fig. 4c). Further, silencing TBX15 expression did not affect adipocyte differentiation, at least in terms of lipid content (Fig. 4d) and expression of typical adipogenic markers (Fig. 4e). Conversely, when we increased TBX15 expression in lean-derived ASCs with an adenovirus vector, cells showed an upregulation of mitochondrial marker expression (Supplementary Figure S6), and this was accompanied by an increase in mitochondrial mass in lean-derived differentiated adipocytes (Fig. 4c). Although we failed to detect significant changes in mitochondrial mass or respiratory capacity between obese- and lean-derived hASCs (Supplementary Figure S7), mature adipocytes from obese-derived hASCs exhibited a higher mitochondrial respiratory capacity than those from equivalent lean-derived cells (Fig. 4f). Overall, these results indicate that TBX15 expression in human adipocyte precursors establishes the mitochondrial phenotype of mature cells.

Fig. 4 Obesity impacts mitochondrial functionality in hASCs and in differentiated adipocytes. a TBX15 protein levels in hASCs from lean and obese subjects and in differentiated adipocytes (AD) (n = 3 patients per group). t test; #p < 0.05 vs. hASCs; *p < 0.05 vs. lean cells. b hASCs isolated from obese subjects were transfected with 100 nM of siRNAs against TBX15 or control, followed by quantitative PCR (qPCR) analysis of the expression of brown and beige markers, mitochondrial redox carriers and mitochondrial fusion genes (n = 4 patients per group). t test; #p < 0.001 and *p < 0.05 vs. control cells. c Mature adipocytes derived from obese TBX15-silenced hASCs or lean hAScs overexpressing TBX15 were analyzed using Mitotracker staining by flow cytometry; representative images are shown (n = 3) t test; **p < 0.01 vs. obese control; #p < 0.05 vs. lean control. d Quantification and representative intracellular lipid enrichment in adipocytes derived from obese, obese TBX15-silenced and lean individuals (magnification, ×20). t test; *p < 0.05 vs. control obese cells. e Gene expression analysis of adipogenic markers by qPCR in mature obese adipocytes (n = 4 patients per group). t test; #p < 0.001 vs. control obese cells. f Oxygen consumption in intact adipocytes was measured by respirometry (n = 6 patients per group). Results are shown as mean ± SEM. t test, *p < 0.05 vs. lean Full size image

Mitochondrial phenotype of human subcutaneous adipose tissue in obesity

To extend this analysis, we examined TBX15 expression in SAT from lean and obese subjects. Several studies have demonstrated obesity-triggered mitochondrial dysfunction in WAT, both in clinical subjects and experimental models [28, 29], but whether this is a cause or a consequence of the disease process is unclear. Moreover, contrasting reports have been published regarding depot-specific and obesity-dependent expression of TBX15 [15, 16, 30], and its potential modulation of mitochondrial metabolism [15, 16, 30]. Expression analysis of TBX15 in SAT from individuals classified according to BMI (clinical and laboratory data summarized in Supplementary Table S3) revealed significantly higher TBX15 protein levels in obese than in lean subjects (Fig. 5a). Consistent with the ability of TBX15 to regulate the mitochondrial phenotype (Fig. 4), higher TBX15 expression in obese SAT correlated with a significant increase in the expression of several mitochondria-related genes such as the oxidative phosphorylation subunits NDUFA9 (complex I), SDHA (complex II) and COX4–1 (complex IV) (Fig. 5b). Interestingly, the expression of the mitochondrial fusion proteins MFN2 and OPA1 was also higher in mitochondria fractions of SAT from obese subjects (Fig. 5b). We also detected a significant increase in the expression of porin, a mitochondrial mass indicator. Accordingly, expression of these proteins was positively associated with BMI (Fig. 5c). Finally, we examined mitochondrial morphology in SAT samples by transmission electron microscopy (Fig. 5d). Results showed that adipocytes from obese subjects had significant alterations in mitochondrial morphology and size as compared with those of lean individuals. Specifically, mitochondrial number and area were significantly higher in obese-derived SAT than in lean-derived SAT, whereas mitochondrial size was significantly smaller (Fig. 5e). Since mitochondrial function is closely linked both to mitochondrial shape and the intracellular distribution of mitochondria, these changes detected in obese WAT might be directly associated with adipocyte energy disturbances linked to obesity.