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.

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Disturbances in the prenatal period are linked to metabolic disorders in adulthood, implying the hypothalamic systems of appetite and energy balance regulation. In order to analyze the central effects of a high-fat-sucrose (HFS) diet in prenatally stressed (PNS) female adult rats, Wistar dams were exposed to chronic-mild-stress during the third week of gestation and were then compared with unstressed controls. Adult female offspring were fed a chow or HFS diet for 10 weeks. Changes in body weight, adiposity as well as expression and methylation levels of selected hypothalamic genes were analyzed. PNS induced lower birthweight and body length with no changes in body fat mass. After the HFS diet, the expected overweight model was observed accompanied by higher adiposity and insulin resistance, which was worsened by PNS. The stress model induced higher energy intake in adulthood. Hypothalamic gene expression analysis revealed that the HFS diet decreased Slc6a3 (dopamine active transporter), NPY (neuropeptide Y) and IR (insulin receptor) and increased POMC (pro-opiomelanocortin). Hypothalamic DNA methylation levels in the promoter region of Slc6a3 revealed that Slc6a3 was hypermethylated by the HFS diet in CpG site –53 bp to the transcription start site. HFS diet also hypermethylated CpG site –167 bp of the POMC promoter only in nonstressed animals. No correlations were found between gene expression and DNA methylation levels. These results imply that early-life stress in females increased predisposition to diet-induced obesity in adulthood.

Stress during pregnancy can induce metabolic disorders in adult offspring. To analyze the possible differential response to a high-fat-sucrose (HFS) diet in offspring affected by prenatal stress (PNS) or not, pregnant Wistar rats (n=11) were exposed to a chronic-mild-stress during the third week of gestation. This aimed to model a chronic depressive-like state that develops over time in response to stress, involving exposure of rats to a series of mild and unpredictable stressors. Control dams (n=11) remained undisturbed. Adult offspring were fed chow or HFS diet (20% protein, 35% carbohydrate, 45% fat) for 10 weeks. Changes in adiposity, biochemical profile and retroperitoneal adipose tissue gene expression by real-time polymerase chain reaction were analyzed. An interaction was observed between HFS and PNS concerning visceral adiposity, with higher fat mass in HFS-fed stressed rats, although this reached statistical significance only in females. HFS modified lipid profile and increased insulin resistance biomarkers, while PNS reduced insulin levels and the HOMA index. HFS diet intake increased gene (mRNA) expression of leptin and apelin and decreased cyclin-dependent kinase inhibitor 1A (Cdkn1a) and fatty-acid synthase (Fasn), whereas PNS increased fasn and stearoylCoA desaturase1 (Scd1). An interaction between diet and PNS was observed for adiponutrin (Adpn) and peroxisome proliferator-activated receptor-coactivator1-(Ppargc1a) gene expression: Adpn was increased by the PNS only in HFS-fed rats, whereas Ppargc1a was increased by PNS only in chow-fed rats. With these results it can be concluded that experience of maternal stress during intrauterine development can enhance predisposition to obesity induced by a HFS diet intake.

Adverse early life events are associated with altered stress responsiveness and metabolic disturbances in the adult life. Dietary methyl donor supplementation could be able to reverse the negative effects of maternal separation by affecting DNA methylation in the brain. In this study, maternal separation during lactation reduced body weight gain in the female adult offspring without affecting food intake, and altered total and HDLcholesterol levels. Also, maternal separation induced a cognitive deficit as measured by NORT and an increase in the immobility time in the Porsolt forced swimming test, consistent with increased depression-like behaviour. An 18-week dietary supplementation with methyl donors (choline, betaine, folate and vitamin B12) from postnatal day 60 also reduced body weight without affecting food intake. Some of the deleterious effects induced by maternal separation, such as the abnormal levels of total and HDL-cholesterol, but especially the depression-like behaviour as measured by the Porsolt test, were reversed by methyl donor supplementation. Also, the administration of methyl donors increased total DNA methylation (measured by immunohistochemistry) and affected the expression of insulin receptor in the hippocampus of the adult offspring. However, no changes were observed in the DNA methylation status of insulin receptor and corticotropin-releasing hormone (CRH) promoter regions in the hypothalamus. In summary, methyl donor supplementation reversed some of the deleterious effects of an early life-induced model of depression in rats and altered the DNA methylation profile in the brain.