High fat diet-induced obesity modifies the methylation pattern of leptin promoter in rats

Abstract

Leptin is an adipokine involved in body weight and food intake regulation whose promoter region presents CpG islands that could be subject to dynamic methylation. This methylation process could be affected by environmental (e.g. diet) or endogenous (e.g., adipocyte differentiation, inflammation, hypoxia) factors, and could influence adipocyte leptin gene expression. The aim of this article was to study whether a high-energy diet may affect leptin gene promoter methylation in rats. A group of eleven male Wistar rats were assigned into two dietary groups, one fed on a control diet for 11 weeks and the other on a high-fat cafeteria diet. Rats fed a high-energy diet become overweight and hyperleptinemic as compared to the controls. DNA isolated from retroperitoneal adipocytes was treated with bisulfite and a distal portion of leptin promoter (from -694 to -372 bp) including 13 CpG sites was amplified by PCR and sequenced. The studied promoter portion was slightly more methylated in the cafeteria-fed animals, which was statistically significant (p < 0.05) for one of the CpG sites (located at the position -443). In obese rats, such methylation was associated to lower circulating leptin levels, suggesting that this position could be important in the regulation of leptin gene expression, probably by being a target sequence of different transcription factors. Our findings reveal, for the first time, that leptin methylation pattern can be influenced by diet-induced obesity, and suggest that epigenetic mechanisms could be involved in obesity by regulating the expression of important epiobesigenic genes.

Leptin and TNF-alpha promoter methylation levels measured by MSP could predict the response to a low-calorie diet

“…It is overexpressed in obese subjects and its plasma levels are related with appetite and energy metabolism [21]. The epigenetic control of this molecule has been studied previously and differences in promoter specific CpG methylation levels associated with diet-induced obesity in rats were found, together with increased plasma and mRNA leptin expression levels [31]. Contrariwise, Okada et al did not found a relationship between increased mRNA leptin expression in white adipose tissue from obese mice and methylation levels of this gene in 3T3-L1 cells [34].…”

“…DNA was amplified with a methyl-specific primer set and with another unmethyl-specific primer set (figure 1) for TNF-alpha and leptin promoters that were designed with MethPrimer [24]. Primers included CpG site -454 for leptin (equivalent to -443 CpG in rats) [31] and -245 and -239 CpG sites for TNF-alpha [11] and amplicons were 108 and 120 bp lenght respectively. PCR conditions were as follows: initial denaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing at 57°C for 1 min, and extension at 72°C for 1 min for 38 cycles, followed by stabilization for 7 min at 72°C.…”

“…Thus, changes in the DNA methylation pattern as a result of dietary treatments or conditions have been studied in the last years, comparing vegetarians with omnivores [39], the effects of different obesogenic diets [26,31] or different dietary treatments to lose weight [7]. Interventional studies are showing that the DNA methylation profile previous to the treatment might be able to predict the response to the low calorie diet.…”

“…Previous studies by bisulfite sequencing PCR (BSP, a time-consuming technique) showed that leptin promoter methylation in rat adipocytes was modified by a high fat diet [31]. On the other hand, the cytokine TNF-alpha is a recognized marker of the obesity-related inflammatory state [33], and DNA methylation pattern of its promoter in blood cells is able to predict the response to a hypocaloric treatment in humans [12].…”

Effects of short-term high-fat overfeeding on genome-wide DNA methylation in the skeletal muscle of healthy young men

“…It is well established that diets rich in genistein [17] and methyl donors [12] are able to modulate DNA methylation patterns in the rodent offspring of mothers consuming such diets, influencing the offspring's incidence of obesity, diabetes and cancer in a potentially transgenerational manner [1,12,17]. Diets high in fat have been shown to increase DNA methylation of the leptin promoter [18] and to prolong the presence of general DNA methylation induced by treatment with carcinogenic agents in rats [19,20]. Limited data are available in humans.…”