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-κB) 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.

Early postnatal overnutrition is a risk factor for obesity in juvenile and adult life. Underlying pathophysiological mechanisms are still unclear. Hypothalamic neuropeptides are decisively involved in the regulation of body weight and food intake. In this study, we investigated consequences of early postnatal overnutrition, as compared to normo-and undernutrition, on NPY within the arcuate nucleus and paraventricular nucleus (PVN). The normal litter size of Wistar rats was adjusted on the third day of life from 10 pups (normal litters, NL; normonutrition) to only three newborns (small litters, SL; overnutrition) or 18 pups per mother (large litters, LL; undernutrition). SL rats developed clear overweight until the day 21 of life (P<0.0001), as well as hyperleptinaemia (P<0.001), and hyperinsulinaemia (P<0.01). LL rats were underweight and had decreased leptin and insulin concentrations. Using radioimmunoassay, NPY contents were determined in hypothalamic micropunches, and immunocytochemistry for NPY was performed in serial hypothalamic sections on day 21 of life. While in the underweight, hypoleptinaemic, and hypoinsulinaemic LL rats increased concentrations of NPY in the arcuate nucleus and PVN were observed, no decrease in NPY content was found in the overweight, hyperleptinaemic, and hyperinsulinaemic SL rats. Moreover, the percentage of NPY-immunopositive neurones per total number of neurones was increased not only in the LL rats, but also in the SL rats. Since the NPY system is functionally mature already at this age, these findings might indicate an acquired resistance of the hypothalamic NPY system to increased levels of insulin and/or leptin in early postnatally overfed SL rats.

Maternal low protein malnutrition during gestation and lactation (LP) is an animal model frequently used for the investigation of long-term deleterious consequences of perinatal growth retardation. Both perinatal malnutrition and growth retardation at birth are risk factors for diabetic and cardiovascular disturbances in later life. The pathophysiologic mechanisms responsible are unknown. Hypothalamic nuclei are decisively involved in the central nervous regulation of food intake, body weight and metabolism. We investigated effects of a low protein diet (8% protein; control diet, 17% protein) during gestation and lactation in rat dams on the organization of hypothalamic regulators of body weight and metabolism in the offspring at weaning (d 20 of life). LP offspring had significantly lower body weight than control offspring (CO; P: < 0.001), associated with hypoglycemia and hypoinsulinemia (P: < 0. 005) on d 20 of life. This was accompanied by a greater relative volume of the ventromedial hypothalamic nucleus (P: < 0.01) and a greater numerical density of Nissl-stained neurons in this nucleus (P: < 0.01) as well as in the paraventricular hypothalamic nucleus (PVN; P: < 0.001). In contrast, no significant differences in neuronal densities were observed generally in the lateral hypothalamic area, arcuate hypothalamic nucleus (ARC), and dorsomedial hypothalamic nucleus between LP offspring and CO offspring. On the other hand, LP offspring displayed fewer neurons immunopositive for neuropeptide Y in the ARC (P: < 0.05), whereas in the PVN, lower neuronal densities of neurons immunopositive for galanin were found in LP offspring compared with CO offspring (P: < 0.001). On the contrary, in the PVN, no significant group difference in the numerical density of cholecystokinin-8S-positive neurons was present. A long-term effect of these specific hypothalamic alterations on body weight and metabolism in LP offspring during later life is suggested.