To the best of our knowledge, fasting as an intervention has not previously been investigated with regard to effects on DNA methylation changes, either in animals or in humans. This study shows that the promoter DNA methylation of two metabolically important adipokine genes, LEP and ADIPOQ, are affected by 36 h fasting in SAT only in NBW subjects and not LBW subjects. Furthermore, we show that LBW subjects have higher baseline methylation degree (after overnight fast) of both gene promoters investigated. However, our results do not support that the promoter LEP and ADIPOQ DNA methylation degree is directly involved in regulating gene expression under a short-term fasting intervention.

The literature investigating effects of nutritional challenges on DNA methylation in humans is limited and includes studies addressing effects of high-calorie diets [6–9]. We have previously found that LBW subjects, when undergoing a high-fat feeding, exhibit less changeability in muscle tissue DNA methylation than matched NBW controls [33]. Together with our current findings showing that only NBW subjects increase DNA methylation with fasting, it leads us to speculate that LBW subjects may be more inflexible in altering the DNA methylation status when metabolically challenged. The extent to which such metabolic inflexibility in terms of acute regulation of DNA methylation may contribute to an increased risk of metabolic disease in LBW subjects remains uncertain.

The shown changes in DNA methylation among NBW subjects occurred mostly at CpG sites located closer to the transcription start site, which was particularly prominent in the LEP promoter. These results suggest that the proximal LEP promoter region including the TATA box (−30 to −25) and a transcription factor-binding site for CCAAT-enhancer-binding proteins (C/EBP) (covering CpG −51), which previously was shown to be important for regulation of gene expression in rat adipocytes [34], is more receptive to changes and might be an important region in epigenetic regulation. With respect to the observed significant changes in DNA methylation, it cannot be excluded that short-term diet or fasting-induced methylation changes, in contrast to long-term and more constitutive methylation changes, may represent some beneficial cellular effects as a part of a normal physiological response to diet changes. For example, short-term, fasting-induced methylation (and potentially gene expression) changes of LEP could relate to a feedback mechanism to decrease the inhibiting effects of leptin on appetite and stimulate the subject to seek refeeding to compensate for the decreased nutrient supply. Brøns et al. demonstrated that when subjected to a 5-day high-fat diet, LBW subjects in contrast to NBW subjects did not increase p-leptin levels [26]. The lacking p-leptin response to high-fat diet may contribute to a relatively lower induction of satiety in LBW subjects. In the current study, we found a higher baseline level of p-leptin among LBW subjects, which also may affect satiety among the LBW men. Moreover, p-leptin decreased with fasting in both birth weight groups.

Despite the decrease in p-leptin with fasting, which is in agreement with previous findings in fasting humans and animals [35–37], we found no effect of fasting on LEP gene expression. Epigenetic influences on gene expression have in several studies been proposed to be involved in the pathogenesis of insulin resistance and T2D [4, 10–12, 38, 39]. Indeed LEP gene expression has been shown to be switched on by demethylation of specific CpG sites located in the proximal LEP promoter, during the differentiation of adipocytes [30]. Inspired by this study, we recently examined LEP DNA methylation in differentiating adipocytes isolated from LBW and NBW subjects, and found an increased LEP DNA methylation and a decreased expression in mature adipocytes from LBW subjects compared to NBW subjects, further reinforcing the hypothesis that LBW subjects are less capable of regulating leptin secretion [40]. The decreased secretion of leptin in the differentiated adipocytes is not consistent with the increased baseline levels of p-leptin among the LBW men described in the present work, but could be explained by an intrinsic functional impairment in LBW adipocytes, causing a reduced leptin secretion. This may be compensated by an increased adiposity in vivo resulting in increased p-leptin levels. In our current study, no significant differences were found in BMI and BF%, but indeed this has been observed in previous studies of LBW subjects [6, 23].

Regarding adiponectin, previous studies have consistently reported lower circulating adiponectin levels in LBW subjects [41–43], but this association was not replicated here, possibly due to the younger age and matched study participants included in this study. An increase in ADIPOQ gene expression levels was observed after fasting among LBW subjects only. This was unexpected but could relate to unknown compensatory mechanisms associated to the fasting-induced insulin resistance. Only a few studies have previously investigated the effects of fasting on ADIPOQ expression and the results are of contradictory nature [44–47]. Nevertheless, we speculate that an increased degree of DNA methylation during fasting may contribute to the fasting-induced insulin resistance.

Recently BMI was reported to be associated with methylation degree in SAT, suggesting that obesity may involve pathological pathways that to some extent are epigenetically regulated [3]. In addition, Houde et al. reported that DNA methylation levels of ADIPOQ in SAT were positively associated with BMI in severely obese patients [48]. In our study, total BF% was found to be positively associated with both LEP and ADIPOQ promoter methylation in SAT. This further enforces that changes in SAT DNA methylation patterns are associated with obesity and could be involved in energy homeostasis, but to which degree needs to be further studied. Interestingly, LBW subjects had a higher average degree of DNA methylation in both LEP and ADIPOQ compared to NBW subjects, in the control study. In the same study cohort we also showed that DNA methylation of the metabolic master regulator PPARGC1A was higher in the LBW compared to the NBW subjects after 36 h fasting [27]. These results are in line with a previous finding of increased LEP promoter DNA methylation in blood from adult subjects whose mothers were exposed to famine during the peri-conceptional period [49].

In both birth weight groups, p-leptin level was positively associated with BMI and total BF%, corresponding well with the theory and previous studies [14, 15]. Additionally, although a significant negative correlation between plasma adiponectin levels and BMI only was observed among the NBW subjects, negative relations were shown at both body fat parameters in both groups. Still, this could indicate that LBW subjects regulate adiponectin levels differentially and not as directly associated to the amount of body fat, as NBW subjects.

There are limitations to this study, i.e., we use birth weight as a “proxy” for fetal environment. Additionally, we do not have data on the reasons for the low birth weight, e.g., maternal smoking in pregnancy or parental height and cannot exclude possible causes.

The methylation differences that we report here in SAT are slightly smaller than we observed in our previous study of PPARGC1A in muscle between LBW and NBW men after fasting (4.5%) [27]. However, overall the magnitude of birth weight on DNA methylation seems to be modest [9]. Furthermore, a 36-h fasting intervention is a quite short time period, compared to a previous 6-month exercise study where larger effects in DNA methylation changes were observed [4]. Still, the magnitude of the methylation differences we observe in the fasting study is similar to the 5-day overfeeding study in both muscle [8] and SAT [25]. Moreover, we are only studying two genes and cannot exclude that other genes may show bigger differences in DNA methylation between the birth weight groups or with fasting. LBW is a risk factor for complex, polygenic diseases including T2D and it is well established that numerous genes with modest effect sizes in methylation changes contribute to this disease [11, 12]. However, we cannot exclude that the DNA methylation measured in our study could be affected by a stochastic variation in methylation, as earlier suggested [50].