Sex-specific epigenetic alterations of the hypothalamic Agrp-Pomc system do not explain 'diabesity' in the offspring of high-fat diet (HFD) overfed maternal rats.

Maternal high-fat diet (HFD) overfeeding pre- and during pregnancy and lactation may 'program' a 'diabesity' predisposition in the offspring, for inconclusive reasons. Acquired alterations of the hypothalamic promoter methylation and mRNA expression of the satiety neurohormone Pomc are possibly of critical importance here. We investigated within one developmental approach, including male and female rats, the sex-specific DNA methylation pattern and corresponding mRNA expression of both Pomc and its endogenous functional antagonist Agrp in the hypothalamus of adult HFD offspring. Obesity and diabetic disturbances occurred in both male and female HFD offspring, accompanied by altered Pomc promoter methylation pattern. However, this was not related to significant Pomc mRNA expression alterations. In contrast, male-specific alterations of Agrp promoter methylation were found, even associated with reduced mRNA expression of this orexigenic/anabolic Pomc antagonist. In conclusion, acquired epigenetic alterations of the hypothalamic Agrp-Pomc system hardly explain the 'diabesity' phenotype in HFD offspring, while distinct vulnerability and functionality of Agrp promoter and related genomic regions methylation should be further investigated.

Hypothalamic insulin receptor expression and DNA promoter methylation are sex-specifically altered in adult offspring of high-fat diet (HFD)-overfed mother rats.

Maternal overnutrition around reproduction has been shown to increase the offspring's risk for "diabesity," mediated by altered hypothalamic neuropeptide expression. In this report, a possible contribution of altered hypothalamic sensing capacity for the peripheral satiety signals glucose, insulin and leptin will be addressed, taking into account potential sex differences. Specifically, we evaluated the effects a maternal high-fat diet (HFD) overfeeding has in rats pre- and during pregnancy and lactation on the hypothalamic gene expression patterns of insulin and leptin receptors (InsR, ObRb) and glucose transporter 3 (Glut3) as well as DNA methylation in the offspring at adult age (day 200 of life). Maternal HFD consumption resulted in a metabolic syndrome phenotype, i.e., obesity, hyperleptinemia, hyperinsulinemia, impaired glucose tolerance and increased homeostatic model assessment of insulin resistance. Interestingly, in turn, insulin resistance was more pronounced in male offspring, accompanied by decreased hypothalamic InsR-mRNA. This was linked with hypermethylation of an activating transcription factor binding site within the hypothalamic InsR promoter. The degree of methylation correlated inversely with respective InsR expression, while InsR expression itself was inversely related to phenotypic "diabesity." Expression of ObRb and Glut3 mRNA was not significantly changed. In conclusion, sex-specific alterations of hypothalamic InsR expression and DNA promoter methylation in adult offspring of HFD-overfed dams may lead to hypothalamic insulin resistance and "diabesity," with males predisposed to this epigenetic malprogramming.

Short-term regulation of the hypothalamic melanocortinergic system under fasting and defined glucose-refeeding conditions in rats: a laser capture microdissection (LMD)-based study.

It is well established that under fasting conditions the expression of the orexigenic neuropeptide agouti-related peptide (AGRP) is up-regulated in the hypothalamic arcuate nucleus (ARC), while inconsistent data exist regarding fasting regulation of the anorexigenic neurohormone proopiomelanocortin (POMC). Inconsistencies might have methodological reasons, especially concerning neuromorphological and/or experimental (nutritional) specificity. We analyzed the expression of both neuropeptides in ARC neurons, using lasercapture microdissection (LMD) and real-time PCR in 12h fasted vs. fed Wistar rats as well as after a standardized glucose load, i.e., under clinically relevant conditions in terms of diagnosing glucose intolerance in the human. Under fasting conditions, clear up-regulation of AGRP was observed, with increasing magnitude in ARC single neurons (SNP) as compared to ARC cell layers (+125% vs. +23%, resp.), closely correlated to hypoinsulinemia and hypoleptinemia. Surprisingly, in the fasting state POMC was not found to be down-regulated, neither in ARC cell layers nor in ARC single neurons (+9% vs. +6%). However, glucose-refeeding under diagnostically relevant conditions led to strong neuronal up-regulation of POMC expression in ARC SNP (+128%), and AGRP down-regulation (-50%). In conclusion, experimentally, topographically, and analytically specific and standardized conditions confirmed AGRP in ARC neurons as being neuronally up- and down-regulated, resp., depending on the general nutritional state, while POMC was found to be (up-) regulated only after peripheral glucose load. Findings suggest that POMC in ARC neurons acts glucose-mediated as an "anti-orexigenic" neurohormone, specifically responding to hyperglycemia.

Epigenetic malprogramming of the insulin receptor promoter due to developmental overfeeding.

AIM: Prenatal and neonatal overfeeding programs a permanent obesity and diabetes disposition, e.g., due to induction of hypothalamic insulin resistance. We investigated acquired alterations of the DNA methylation pattern of the hypothalamic insulin receptor promoter (IRP) which might be an underlying molecular mechanism. METHODS: Neonatal overfeeding was induced by rearing Wistar rats in small litters (SL). Methylation of CpG-dinucleotides of the hypothalamic IRP was mapped using bisulfite sequencing. RESULTS: Neonatal overfeeding led to rapid early weight gain, resulting in a metabolic syndrome phenotype, i.e., obesity, hyperleptinemia, hyperglycemia, hyperinsulinemia, and increased insulin/glucose-ratio. The proportion of animals carrying any methylated CpG residue in the 322 bp CpG island of the IRP was increased in neonatally overfed SL rats (n=8), as compared to controls (n=8; P=0.04). Moreover, the mean percentage of methylated CpG positions was also higher in SL rats (P=0.01). Over both groups, neonatal blood glucose levels were positively correlated to the extent of promoter methylation (r=0.52; P=0.04). CONCLUSIONS: This study characterizes for the first time the IRP epigenomically in any species and tissue. Our data reveal that the IRP is vulnerable to hypermethylation due to overnutrition, probably especially glucose-dependent in a dose-response manner. This paradigmatically indicates the impact of nutrient-dependent epigenetic malprogramming, leading to a "diabesity" disposition which may become pathogenic throughout life.

Hypothalamic proopiomelanocortin promoter methylation becomes altered by early overfeeding: an epigenetic model of obesity and the metabolic syndrome.

Pre- and neonatal overfeeding programmes a permanent obesity disposition and accompanying diabetic and cardiovascular disorders, by unknown mechanisms. We proposed that early overfeeding may alter DNA methylation patterns of hypothalamic promoter regions of genes critically involved in the lifelong regulation of food intake and body weight. We induced neonatal overfeeding by rearing Wistar rats in small litters (SL) and thereafter mapped the DNA methylation status of CpG dinucleotides of gene promoters from hypothalamic tissue, using bisulfite sequencing. Neonatal overfeeding led to rapid early weight gain, resulting in a metabolic syndrome phenotype, i.e. obesity, hyperleptinaemia, hyperglycaemia, hyperinsulinaemia, and an increased insulin/glucose ratio. Accompanying, without group difference to controls, the promoter of the main orexigenic neurohormone, neuropeptide Y, was methylated at low levels (i.e. < 5%). In contrast, in SL rats the hypothalamic gene promoter of the main anorexigenic neurohormone, proopiomelanocortin (POMC), showed hypermethylation (P < 0.05) of CpG dinucleotides within the two Sp1-related binding sequences (Sp1, NF-kappaB) which are essential for the mediation of leptin and insulin effects on POMC expression. Consequently, POMC expression lacked upregulation, despite hyperleptinaemia and hyperinsulinaemia. Accordingly, the extent of DNA methylation within Sp1-related binding sequences was inversely correlated to the quotients of POMC expression/leptin (P = 0.02) and POMC expression/insulin (P < 0.001), indicating functionality of acquired epigenomic alterations. These data for the first time demonstrate a nutritionally acquired alteration of the methylation pattern and, consequently, the regulatory 'set point' of a gene promoter that is critical for body weight regulation. Our findings reveal overfeeding as an epigenetic risk factor of obesity programming and consecutive diabetic and cardiovascular disorders and diseases, in terms of the metabolic syndrome.