**3.4. Epigenetics and intervention studies**

of a methylome map within Visceral adipose tissue (VAT) that may predispose individuals to

Family studies are beneficial as they have the potential to identify causative candidate regions of differential DNA methylation. A study by Ali et al. (2016) used a discovery approach in 192 subjects from seven families from the Take off Pounds Sensibly Family Study of Epigenetics (TFSE), and then validated the initial differential methylation marks in an extended cohort of 1,052 subjects [39]. They identified and replicated three loci where methylation status was associated with BMI%, and these were located in the body of suppressor of cytokine signalling (*SOCS3*), 3′ untranslated region of the zinc finger protein 771 (*ZNF771*) and at the transcription start site of LIM domain containing 2 (*LIMD2*) gene. Functional analyses were then

Examining promoter methylation patterns is thought to be a promising strategy for the use of early detection of disease (Laird 2003). Given that TNF*a* is pro‐inflammatory cytokine elevated in obese subjects, Campion et al. (2009) investigated methylation patterns in the promoter region of TNF*a*. Methylation was measured in baseline peripheral blood mononuclear cells (PBMCs) from 24 patients who were put on an 8‐week calorie restricted diet. Two differentially methylated promoter regions were assessed according to previous literature. The group of men who lost weight with the low calorie diet showed a significantly lower TNF*a* promoter methylation and that their baseline circulating TNF*a* levels were positively associated with total promoter methylation (Campion, Milagro et al. 2009). This demonstrates that examining promoter methylation may well be an important aspect in early detection and prognosis of

Despite progress in the epigenetic marks of obesity, there still remain some controversy in the use of peripheral blood mononuclear cells (PBMCs) as a surrogate for identification of epigenetic marks that may contribute to obesity development as there may be site‐specific marks that may be missed. However, it is important to consider that with human studies, we are limited to tissues that we can readily access and the most commonly and easily accessible tissue to examine epigenetic modifications in humans is whole blood or PBMCs. This has been thought to have limitations in itself as it may not explain tissue‐specific modifications that may be present. In light of this, many recent studies have examined epigenetic profiles in both PBMCs as well as subcutaneous adipose tissue (SAT). Once such study was from Arner et al. 2016, where they performed and EWAS using PBMCs, SAT and VAT in 80 obese women of which 40 insulin resistant and 40 insulin sensitive (Arner, Sahlqvist et al. 2016). They wanted to determine similarities of methylation status between the tissues and to determine whether PBMCs could be used as a marker of systemic IR. They were not able to show similar methylation sites between the PBMCs and adipose tissue and therefore concluded that CpG methylation in PBMCs does not reflect differential methylation sites in white adipose tissue (WAT). These data suggest that PBMCs may not be a suitable tissue for metabolic phenotyping of obese individuals. However, another study by Demarath et al. (2015) (described above) also measured methylation markers in SAT from their ARIC cohort and replicated 16 of these BMI

performed in 330 subjects from the methylation analyses [39].

**3.3. Can PBMCs be used as a surrogate for epigenetic studies?**

IR and T2D [38].

28 Adiposity - Omics and Molecular Understanding

disease.

The variation in epigenetic patterns with obesity has raised some interest in the field. There have been several intervention studies that have investigated the role of obesity and weight‐ loss interventions on global and promoter specific DNA methylation patterns. A 6‐month exercise intervention study using adipose tissue from 23 healthy men with a family history (or not) of T2D demonstrated a global increase of adipose tissue DNA methylation in response to the exercise intervention (Ronn, Volkov et al. 2013). Interestingly, the subjects with a family history of T2D had less CpG sites with a significant difference in methylation patterns in obesity‐related genes in response to the 6‐month exercise intervention (Ronn, Volkov et al. 2013). Another study examined methylation changes in subjects that underwent gastric band surgery and were able to demonstrate significant effects on promoter methylation. More interestingly, before the patients underwent surgery, they observed significant methylation changes in genes involved in metabolic pathways and mitochondrial function, however after surgery and subsequent weight loss, the expression of these same genes normalised to similar levels to non‐obese subjects (Barres, Kirchner et al. 2013). An additional study examined epigenetic changes in a normal weight, obese and successful weight loss maintainers (maintained weight for 9 years) that consisted of 48 males and females and demonstrated that the successful weight‐loss maintainer group had methylation patterns that resembled the normal weight group rather than the obese group, which shows that methylation changes can be reversed (Huang, Maccani et al. 2015).

In summary, these studies collectively validate an important role for epigenetic regulation in metabolic processes. Furthermore, epigenetic marks coupled with gene expression in candidate genes may offer new pharmacological targets to counteract these modifications and potentially help avert obesity and associated diseases.
