Asaf Marco

Overweight and CpG methylation of the Pomc promoter in offspring of high-fat-diet-fed dams are not “reprogrammed” by regular chow diet in rats

This study aimed to determine whether epigenetic malprogramming induced by high‐fat diet (HFD) has an obesogenic effect on nonmated and mated female rats and their offspring. Further, it aimed to reprogram offsprings epigenetic malprogramming and phenotype by providing normal diet after weaning. Body weight (BW) was measured, and plasma and hypothalamic arcuate nuclei were collected for analysis of hormones, mRNA, and DNA CpG methylation of the promoter of Pomc, a key factor in control of food intake. In nonmated females, HFD decreased Pomc/leptin ratio by ~38%. This finding was associated with Pomc promoter hypermethylation. While heavier during pregnancy, during lactation HFD dams showed sharper BW decrease (2.5‐fold) and loss of Pomc promoter hypermethylation. Moreover, their weight loss was correlated with demethylation (r=‐0.707) and with gadd45b mRNA expression levels (r=0.905). Even though offspring of HFD dams ate standard chow from weaning, they displayed increased BW, Pomc promoter hypermethylation, and vulnerability to HFD challenge (3‐fold kilocalorie intake increase). These findings demonstrate a long‐term effect of maternal HFD on CpG methylation of the Pomc promoter in the offspring, which was not reprogrammed by standard chow from weaning. Further, the results suggest a possible mechanism of demethylation of the Pomc promoter following pregnancy and lactation.—Marco, A., Kisliouk, T., Tabachnik, T., Meiri, N., Weller, A. Overweight and CpG methylation of the Pomc promoter in offspring of high‐fat‐diet‐fed dams are not “reprogrammed” by regular chow diet in rats. FASEB J. 28, 4148‐4157 (2014). www.fasebj.org

High fat diet induces hypermethylation of the hypothalamic Pomc promoter and obesity in post-weaning rats

Impaired response of the brain to the leptin signal leads to a persisting dysregulation of food intake and energy balance. High plasma leptin or insulin should activate proopiomelanocortin (POMC), the precursor of the anorexigenic neuropeptide α-melanocyte-stimulating hormone (α-MSH) in the hypothalamic arcuate nucleus (ARC). Nevertheless, in obesity, this signal transduction pathway might be impaired. In this study we investigated whether chronic high fat (HF) diet consumption from post-weaning to adulthood increases CpG methylation of the Pomc promoter. The hypothesis that this would disrupt the essential binding of the transcription factor Sp1 to the Pomc promoter was tested. Male rats were raised from postnatal day 21 till 90 on either HF or standard diet. As a result HF fed rats were significantly heavier, with high leptin and insulin levels in their plasma but almost no changes in ARC mRNA expression levels of Pomc. The Pomc promoter area in the HF-treated rats was found to be hypermethylated. Furthermore, there was a direct correlation in individual rats between CpG methylation at specific sites that affect Sp1 binding and plasma leptin levels and/or body weight. Although, as expected the HF diet resulted in up-regulation of Sp1, the binding of Sp1 to the hypermethylated Pomc promoter was significantly reduced. Therefore, we suggest that hypermethylation on the promoter region of the Pomc gene can emerge at post-lactation periods and interfere with transcription factor binding, thus blocking the effects of high leptin levels, leading to obesity.

DNA CpG Methylation (5-Methylcytosine) and Its Derivative (5-Hydroxymethylcytosine) Alter Histone Posttranslational Modifications at the Pomc Promoter, Affecting the Impact of Perinatal Diet on Leanness and Obesity of the Offspring

A maternal high-fat diet (HFD) alters the offsprings feeding regulation, leading to obesity. This phenomenon is partially mediated by aberrant expression of the hypothalamic anorexigenic neuropeptide proopiomelanocortin (POMC). Nevertheless, although some individual offspring suffer from morbid obesity, others escape the malprogramming. It is suggested that this difference is due to epigenetic programming. In this study, we report that in lean offspring of non-HFD–fed dams, essential promoter regions for Pomc expression were enriched with 5-hydroxymethylcytosine (5hmC) together with a reduction in the level of 5-methylcytosine (5mC). Moreover, 5hmC was negatively correlated whereas 5mC was positively correlated with body weight in offspring from both HFD- and control-fed dams. We further found that Pomc expression in obese offspring is determined by a two-step epigenetic inhibitory mechanism in which CpG methylation is linked with histone posttranslational modifications. An increase in CpG methylation at the Poxmc promoter enables binding of methyl-binding domain 1 (MBD1) to 5mC, but not to its derivative 5hmC. MBD1 then interacts with SET domain bifurcated 1 methyltransferase to promote bimethylation on the histone 3 lysine 9 residue, reducing Pomc mRNA expression. These results suggest an epigenetic regulatory mechanism that affects obesity-prone or resilient traits.

Microstructural pattern of palatable food intake from weaning to adulthood in male and female OLETF rats.

Ontogenetic trajectories from weaning to adulthood and sex differences in feeding patterns were examined in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of early onset overeating-induced obesity, and a natural cholecystokinin-1 receptor knockout. Overnight patterns of licking a palatable liquid diet (Ensure) were analyzed on Postnatal Days 22, 38, 60, and 90. Because different microstructure profiles may reflect alterations in the influence of positive and negative signals, we examined meal parameters to uncover developing mechanisms underlying eating behavior in this strain. OLETF rats displayed significantly greater caloric intake, larger meals (in number of licks), and more (within-meal) clusters of feeding (which were shorter in duration and contained fewer licks per cluster) than did Long-Evans Tokushima Ohtsuka (LETO) strain controls. OLETF rats also had significantly lower satiety ratios than LETO rats. Moreover, we identified sex differences in the age of emergence of microstructural patterns of obesity-related overeating, suggesting that systems other than cholecystokinin may be disrupted, possibly worsening the OLETF strains obesity phenotype.

Thyroid Hormone-Dependent Epigenetic Regulation of Melanocortin 4 Receptor Levels in Female Offspring of Obese Rats

Maternal obesity is a risk factor for offspring obesity. The melanocortin 4 receptor (Mc4r) is one of the mediators of food intake and energy balance. The present study examined the epigenetic mechanisms underlying altered Mc4r levels in the hypothalamic paraventricular nucleus in the offspring of high-fat diet (HFD)-induced obese dams and sought to elucidate the role of thyroid hormones in epigenetic regulation and tagging of their nucleosome at the Mc4r promoter. Female Wistar rats were fed an HFD or standard chow from weaning through gestation and lactation. Epigenetic alterations were analyzed in the offspring on postnatal day 21 at the Mc4r promoter using chromatin immunoprecipitation and bisulfite sequencing. To study the role of triiodothyronine (T3) in Mc4r downregulation, dams received methimazole (MMI), an inhibitor of thyroid hormone production. Offspring of HFD-fed dams had a greater body weight, elevated plasma T3 concentrations, and lower Mc4r messenger RNA levels than controls. At the Mc4r promoter, offspring of HFD-fed mothers demonstrated increased histone 3 lysine 27 acetylation (H3K27ac) with a greater association to thyroid hormone receptor-β (TRβ), an inhibitor of Mc4r transcription. Moreover, TRβ coimmunoprecipitated with H3K27ac, supporting their presence in the same complex. Maternal MMI administration prevented the HFD reduction in Mc4r levels, the increase in TRβ, and the increase in the TRβ-H3K27ac association, providing further support for the role of T3 in downregulating Mc4r levels. These findings demonstrate that a perinatal HFD environment affects Mc4r regulation through a T3 metabolic pathway involving histone acetylation of its promoter.

Trait and state binge eating predispose towards cocaine craving

Binge eating (BE) and drug seeking share similar behavioral features, including loss of control over consumption and compulsive seeking of the craved substance. Previous studies in animal models have demonstrated a complex interaction between ‘state’ BE, produced by intermittent access to a palatable diet, and ‘trait’ BE, a phenotypical proneness towards overeating. In the present study, we examined the relationship between state and trait BE and cocaine seeking. We used Otsuka Long Evans Tokushima Fatty rats, a genetic model for obesity that demonstrates BE‐like behavior, and Long Evans Tokushima Otsuka controls. They received a schedule of limited access to a palatable diet (3 days/week or 5 days/week access to Ensure for a month). Next, they underwent cocaine self‐administration training (1 mg/kg, 1 hour/day for 10 days) followed by extinction sessions (7 days). We found that the degree of BE‐like behavior and the state and trait BE combination predicted cocaine craving patterns. Lower levels of dopamine D2 receptors in the prefrontal cortex were correlated with increased drug craving. Moreover, restricted access to an attractive diet was found to be a risk factor for heightened cocaine craving, particularly in trait binge eaters, as rats on the 3 days/week access schedule persistently failed to cease cocaine seeking throughout extinction. Hence, we postulate a joint role of state and trait BE as risk factors for heightened cocaine craving.

Epigenetic Programming of Hypothalamic Pomc Regulates Feeding and Obesity

The environment can have a long-lasting influence on an individual’s physiology and behavior. While some environmental conditions can be beneficial and result in adaptive responses, others can lead to pathological behaviors (Franklin et al. Neuron 75:747–761, 2012). The period of perinatal development is one of the most critical windows during which adverse conditions can influence the growth and development of the fetus, as well as the offspring’s postnatal health and behavior (Franklin et al. Neuron 75:747–761, 2012). Moreover, recent evidence points to the possibility that changes which occur in the individual can sometimes pass between generations even if the offspring are not directly exposed to the stimulus (Gapp et al. Biology 36:491–502, 2014). Epigenetic alterations are prime candidates for the major molecular mechanism acting at the interface between genetic and environmental factors. Different studies showed that environmental factors, such as fetal alcohol exposure, maternal stress, under or overnutrition, or smoking exposure during sensitive periods affect gene expression in the offspring via altering epigenetic mechanisms, sometimes even across multiple generations (Begum et al. Endocrinology 154:4560–4569, 2013; Blaze and Roth Int J Dev Neurosci 31:804–810, 2013; Laufer et al. Dis Model Mech 6:977–992, 2013; Novakovic et al. Epigenetics 9, 2013). In this review, we discuss the involvement of the proopiomelanocortin (POMC) system, one of the most important regulators of energy balance, and describe how epigenetic changes such as histone modifications and DNA methylation modulate Pomc gene expression and function. We also summarize the recent findings from animal models which show that both diet-induced obesity (DIO) and malnutrition program the POMC system of subsequent generations via epigenetic mechanisms.