Maternal diet, rather than obesity itself, has a main influence on milk triacylglycerol profile in dietary obese rats.

Triacylglycerols (TG) in milk derive from different sources, and their composition may be influenced by both maternal diet and obesity. We used two rat models to ascertain potential changes in TG composition in milk associated to maternal intake of an obesogenic diet during lactation and to distinguish them from the effects attributable to maternal adiposity. Milk samples were obtained from dams fed a cafeteria diet during lactation (CAF) and from dams made obese by cafeteria diet feeding, with dietary normalization before gestation (PCaf). Levels of specific TG species in milk collected at different time points of lactation were determined by shotgun lipidomics. CAF and PCaf dams presented a greater adiposity than their respective controls. The principal component analysis of TG peaks showed a clear separation between milk from CAF dams and milk from control and Pcaf dams, already evident at 5 days of lactation. Milk from CAF dams was enriched with TG species with greater number of carbons and double bonds and reduced in TG with lower number of carbons. TG composition of milk from Pcaf dams was similar to controls, although specific differences were observed at day 5 of lactation. Thus, the intake of a cafeteria diet during lactation, rather than maternal adiposity, alters milk composition. This effect is avoided with dietary normalization before gestation, although the remaining fat reserves may also influence TG composition at initial stages of lactation. Therefore, normalization of maternal diet prior to pregnancy should be considered as a strategy for achieving optimal milk composition.

Dietary vitamin A impacts DNA methylation patterns of adipogenesis-related genes in suckling rats.

We previously showed that vitamin A supplementation in early life impacts white adipose tissue (WAT) biology. We here studied the vitamin's effects on DNA methylation of genes crucial for WAT cell development, determination and metabolism. CpG promoter methylation and mRNA expression of Pparg, Zfp423, Pcna, and Rbp4 was compared in inguinal WAT of 21-day-old rats supplemented during the suckling period with vehicle (controls) or an emulsion of vitamin A as retinyl ester (RE) or ß-carotene (BC). The methylation profile of promoters was affected by vitamin A supplementation with pronounced differences between the RE and BC groups. In the RE group, hypermethylation of the Rbp4 (at multiple CpGs) and the Pparg2 (at a specific CpG) promoters and hypomethylation of the Pcna promoter (at multiple CpGs) was observed, together with inverse changes in gene expression levels. In the BC group, hypomethylation of the Rbp4 and hypermethylation of the Pcna promoter at distinct CpGs was observed, with no effects on gene expression. In both supplemention groups, hypomethylation and increased expression was found for Zfp423. Thus, modest vitamin A supplementation in early postnatal life impacts methylation marks in developing WAT. Differential epigenetic effects of RE and BC in early life may affect adipose tissue programming activity.

Transcriptome analysis in blood cells from children reveals potential early biomarkers of metabolic alterations.

OBJECTIVES: The development of effective strategies to prevent childhood obesity and its comorbidities requires new, reliable early biomarkers. Here, we aimed to identify in peripheral blood cells potential transcript-based biomarkers of unhealthy metabolic profile associated to overweight/obesity in children. METHODS: We performed a whole-genome microarray analysis in blood cells to identify genes differentially expressed between overweight and normal weight children to obtain novel transcript-based biomarkers predictive of metabolic complications. RESULTS: The most significant enriched pathway of differentially expressed genes was related to oxidative phosphorylation, for which most of genes were downregulated in overweight versus normal weight children. Other genes were involved in carbohydrate metabolism/glucose homoeostasis or in lipid metabolism (for example, TCF7L2, ADRB3, LIPE, GIPR), revealing plausible mechanisms according to existing biological knowledge. A set of differentially expressed genes was identified to discriminate in overweight children those with high or low triglyceride levels. CONCLUSIONS: Functional microarray analysis has revealed a set of potential blood-cell transcript-based biomarkers that may be a useful approach for early identification of children with higher predisposition to obesity-related metabolic alterations.

Maternal consumption of a cafeteria diet during lactation in rats leads the offspring to a thin-outside-fat-inside phenotype.

BACKGROUND AND OBJECTIVE: The suckling period is a critical phase of development, in which maternal overnutrition may program the susceptibility of developing chronic diseases and disorders, such as obesity and metabolic alterations, in adult life. Here, we questioned whether the consumption of a cafeteria diet throughout lactation in rats affects the macronutrient composition of milk and whether it results in permanent metabolic effects in the offspring. METHODS: Nursing rats were fed a control diet or a cafeteria diet during lactation. Milk was obtained at different time points of lactation. Offspring (males and females) were weaned onto a control diet until the age of 6 months. Circulating parameters were measured under ad libitum feeding and under 12-h fasting conditions at weaning and at 3 and 6 months of age. An oral glucose tolerance test (OGTT) was performed at 3 and 6 months of age. RESULTS: Rats fed a cafeteria diet during lactation consumed an unbalanced diet, with lower protein and higher fat content versus controls, which was reflected in the composition of the milk. The offspring of rats fed a cafeteria diet during lactation showed lower body weight and lower lean mass, but greater fat accumulation, compared with controls. They also displayed hyperleptinaemia, altered lipid profile and impaired response to an OGTT. CONCLUSION: Maternal consumption of a cafeteria diet throughout lactation in rats produces lasting effects in the metabolic health of their offspring, which are not associated with a higher body weight but with a greater fat accumulation, similarly to the thin-outside-fat-inside phenotype.

Oral leptin supplementation throughout lactation in rats prevents later metabolic alterations caused by gestational calorie restriction.

OBJECTIVES: Calorie-restriction during gestation in rats has been seen to produce lasting detrimental effects in the offspring, affecting energy balance control and other related metabolic functions. Our aim was to assess whether leptin supplementation throughout lactation may prevent the dysmetabolic phenotype in adulthood associated with gestational calorie restriction. METHODS: Three groups of male Wistar rats were followed: the offspring of ad libitum fed dams (controls); the offspring of 20% calorie-restricted dams during gestation (CR); and CR rats supplemented with physiological doses of leptin throughout lactation (CR-Leptin). Pups were weaned with a standard diet (SD) until 4 months of age, and then half of the animals of each group were moved to a Western diet (WD) until 6 months of age. Body weight and food intake were recorded. Energy expenditure, locomotive activity, blood parameters, liver triglycerides (TG), and gene expression and specific proteins in liver and white adipose tissue (WAT) were measured in adulthood. RESULTS: Adult CR rats, but not CR-Leptin rats, displayed greater adiposity index and feed efficiency (both under SD) than controls, along with lower locomotive activity and energy expenditure, higher HOMA-IR index and greater circulating TG and leptin levels. CR animals also exhibited increased values of the respiratory exchange ratio and more severe signs of hepatic steatosis under WD than CR-Leptin animals. Gene expression analysis revealed that CR, but not CR-Leptin, animals displayed indicators of lower capacity for WAT expansion, along with decreased lipogenesis and lipolytic capacity under SD, and impaired lipogenic response of the liver to WD feeding, in accordance with diminished insulin sensitivity and WAT leptin signaling. CONCLUSIONS: Oral leptin supplementation in physiological doses throughout lactation in rats prevents most of the detrimental effects on energy homeostasis and metabolic alterations in adulthood caused by inadequate fetal nutrition.

Isocaloric high-fat feeding directs hepatic metabolism to handling of nutrient imbalance promoting liver fat deposition.

BACKGROUND/OBJECTIVES: Consumption of fat-rich foods is associated with obesity and related alterations. However, there is a group of individuals, the metabolically obese normal-weight (MONW) subjects, who present normal body weight but have metabolic features characteristic of the obese status, including fat deposition in critical tissues such as liver, recognized as a major cause for the promotion of metabolic diseases. Our aim was to better understand metabolic alterations present in liver of MONW rats applying whole genome transcriptome analysis. METHODS: Wistar rats were chronically fed a high-fat diet isocaloric relative to Control animals to avoid the hyperphagia and overweight and to mimic MONW features. Liver transcriptome analysis of both groups was performed. RESULTS: Sustained intake of an isocaloric high-fat diet had a deep impact on the liver transcriptome, mainly affecting lipid metabolism. Although serum cholesterol levels were not affected, circulating triacylglycerols were lower, and metabolic adaptations at gene expression level indicated adaptation toward handling the increased fat content of the diet, an increased triacylglycerol and cholesterol deposition in liver of MONW rats was observed. Moreover, gene expression pointed to increased risk of liver injury. One of the top upregulated genes in this tissue was Krt23, a marker of hepatic disease in humans that was also increased at the protein level. CONCLUSION: Long-term intake of a high-fat diet, even in the absence of overweight/obesity or increase in classical blood risk biomarkers, promotes a molecular environment leading to hepatic lipid accumulation and increasing the risk of suffering from hepatic diseases.

XV Congreso SEINAP. La reglamentación europea sobre declaraciones de salud en los alimentos y su aplicación a la población infantil, niños y adolescentes / XV Congreso SEINAP. European regulation on health declarations in foods and their application in the infant, child and adolescent population

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TAS1R3 and UCN2 Transcript Levels in Blood Cells Are Associated With Sugary and Fatty Food Consumption in Children.

CONTEXT: New types of dietary exposure biomarkers are needed to implement effective strategies for obesity prevention in children. Of special interest are biomarkers of consumption of food rich in simple sugars and fat because their intake has been associated with obesity development. Peripheral blood cells (PBCs) represent a promising new tool for identifying novel, transcript-based biomarkers. OBJECTIVE: This study aimed to study potential associations between the transcripts of taste receptor type 1 member 3 (TAS1R3) and urocortin II (UCN2) genes in PBCs and the frequency of sugary and fatty food consumption in children. DESIGN, SETTING, AND PARTICIPANTS: Four hundred sixty-three children from the IDEFICS cohort were selected to include a similar number of boys and girls, both normal-weight and overweight, belonging to eight European countries. MAIN OUTCOME MEASURES: Anthropometric parameters (measured at baseline and in a subset of 193 children after 2 years), food consumption frequency and transcript levels of TAS1R3 and UCN2 genes in PBCs were measured. RESULTS: Children with low-frequency consumption of sugary foods displayed higher TAS1R3 expression levels with respect to those with intermediate or high frequency. In turn, children with high-frequency consumption of fatty foods showed lower UCN2 expression levels with respect to those with low or intermediate frequency. Moreover, transcripts of TAS1R3 were related with body mass index and fat-mass changes after a 2-year follow-up period, with low expression levels of this gene being related with increased fat accumulation over time. CONCLUSION: The transcripts of TAS1R3 and UCN2 in PBCs may be considered potential biomarkers of consumption of sugary and fatty food, respectively, to complement data of food-intake questionnaires.

The intake of high-fat diets induces the acquisition of brown adipocyte gene expression features in white adipose tissue.

BACKGROUND/OBJECTIVE: White-to-brown adipose tissue remodeling (browning) in response to different stimuli constitutes an active research area for obesity treatment. The emergence in traditional white adipose tissue (WAT) depots of multilocular adipocytes that express uncoupling protein 1 (UCP1) and resemble brown adipocytes, the so called 'brite' adipocytes, could contribute to increased energy expenditure. In rodents, obesogenic stimuli such as the intake of hyperlipidic diets can increase brown adipose tissue (BAT) thermogenic capacity and contribute to maintaining body weight. The aim of this study was to investigate the potential of two different hyperlipidic diets, a commercial high-fat (HF) diet and a highly palatable cafeteria (CAF) diet, to induce WAT browning. METHODS: We analyzed gene expression of a wide number of brown/brite adipocyte markers in different WAT depots, in BAT and in peripheral blood mononuclear cells (PBMCs) increasingly being used in nutrition studies as a potential source of biomarkers of physiological effects. We also performed morphological analysis of adipose tissue. RESULTS: Both HF diets studied were able to increase the expression of the markers studied in WAT in a depot-specific manner, as well as in BAT; some of these changes were also reflected in PBMCs. This increased browning capacity was translated into the appearance of UCP1- and CIDE-A (cell death-inducing DFFA-like effector A)-positive brite adipocytes in retroperitoneal WAT. Administration of the CAF diet, associated with higher adiposity, produced the strongest impact on the parameters studied while its withdrawal restored basal conditions. CONCLUSIONS: Acquisition of brown adipocyte features in WAT could evidence an adaptation to try to counteract increased adiposity due to the intake of HF diets. Additionally, PBMCs could constitute an interesting easily obtainable material to assess the effect of nutritional interventions on browning capacity.

Leptin intake in suckling rats restores altered T3 levels and markers of adipose tissue sympathetic drive and function caused by gestational calorie restriction.

BACKGROUND: Maternal calorie restriction during gestation in rats has been associated with altered white adipose tissue (WAT) sympathetic innervation and function in offspring. Here, we aimed to investigate whether supplementation with oral leptin (a breast milk component) throughout the lactation period may revert the aforementioned adverse programming effects. METHODS: Three groups of male and female rats were studied at the postnatal day 25: the offspring of control dams, the offspring of 20% calorie-restricted dams during pregnancy (CR) and CR rats supplemented with physiological doses of leptin throughout lactation (CR-Leptin). Tyrosine hydroxylase (TH) levels and its immunoreactive area, and mRNA expression levels of lipid metabolism-related genes and of deiodinase iodothyronine type II (Dio2) were determined in WAT. Triiodothyronine (T3) levels were determined in the blood. RESULTS: In CR males, leptin treatment restored the decreased TH levels and its immunoreactive area in WAT, and partially normalized expression levels of genes related to lipolysis and fatty acid oxidation (adipose triglyceride lipase, hormone-sensitive lipase, carnitine palmitoyltransferase 1b and peroxisome proliferator-activated receptor gamma coactivator 1-alpha). Leptin treatment also reverted the decreased T3 plasma levels and WAT lipoprotein lipase mRNA levels occurring in CR males and females, and the decreased Dio2 mRNA levels in CR females. CONCLUSIONS: Leptin supplementation throughout the lactation period reverts the malprogrammed effects on WAT structure and function induced by undernutrition during pregnancy. These findings support the relevance of the intake of leptin during lactation, bearing clear characteristics of essential nutrient, and provide a strategy to treat and/or prevent the programmed trend to obesity acquired by inadequate fetal nutrition.

Cafeteria diet overfeeding in young male rats impairs the adaptive response to fed/fasted conditions and increases adiposity independent of body weight.

OBJECTIVE: We analyzed the effects of a short exposure to a cafeteria diet during early infancy in rats on their metabolic response to fed/fasting conditions in key tissues involved in energy homeostasis. METHODS: Ten-day-old male pups were fed a control or a cafeteria diet for 12 days and then killed under ad libitum feeding conditions or 12 h fasting. The expression of key genes related to energy metabolism in liver, retroperitoneal white adipose tissue (WAT) and hypothalamus were analyzed. RESULTS: Despite no differences in body weight, cafeteria-fed animals had almost double the fat mass of control rats. They also showed higher food intake, higher leptinemia and altered hypothalamic expression of Neuropetide Y, suggesting a dysfunction in the control of food intake. Unlike controls, cafeteria-fed animals did not decrease WAT expression of Pparg, sterol regulatory element binding transcription factor 1 or Cidea under fasting conditions, and displayed lower Pnpla2 expression than controls. In liver, compared with controls, cafeteria animals presented: (i) lower expression of genes related with fatty acid uptake and lipogenesis under ad libitum-fed conditions; (ii) higher expression of fatty acid oxidation-related genes and glucokinase under fasting conditions; (iii) greater expression of leptin and insulin receptors; and higher protein levels of insulin receptor and the pAMPK/AMPK ratio. CONCLUSION: A short period of exposure to a cafeteria diet in early infancy in rat pups is enough to disturb the metabolic response to fed/fasting conditions in key tissues involved in energy homeostasis, particularly in WAT, and hence induces an exacerbated body fat accumulation and increased metabolic risk, with no apparent effects on body weight.

Moderate calorie restriction during gestation programs offspring for lower BAT thermogenic capacity driven by thyroid and sympathetic signaling.

BACKGROUND: Maternal calorie restriction during pregnancy programs offspring for later overweight and metabolic disturbances. Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis and has recently emerged as a very likely target for human obesity therapy. OBJECTIVE: Here we aimed to assess whether the detrimental effects of undernutrition during gestation could be related to impaired thermogenic capacity in BAT and to investigate the potential mechanisms involved. METHODS: Offspring of control and 20% calorie-restricted rats (days 1-12 of pregnancy) (CR) were studied at the age of 25 days. Protein levels of uncoupling protein 1 (UCP1) and tyrosine hydroxylase (TyrOH); mRNA levels of lipoprotein lipase (LPL), carnitine palmitoyltransferase 1 (CPT1) and deiodinase iodothyronine type II (DIO2) in BAT; and blood parameters including thyroid hormones, were determined. The response to 24-h cold exposure was also studied by measuring body temperature changes over time, and final BAT UCP1 levels. RESULTS: Compared with controls, CR animals displayed in BAT lower UCP1 and TyrOH protein levels and lower LPL and CPT1 mRNA levels; they also showed lower triiodothyronine (T3) plasma levels. CR males, but not females, revealed lower DIO2 mRNA levels than controls. When exposed to cold, CR rats experienced a transient decline in body temperature, but the values were reestablished after 24 h, despite having lower UCP1 levels than controls. CONCLUSIONS: These results suggest that BAT thermogenic capacity is diminished in CR animals, involving impaired BAT sympathetic innervation and thyroid hormone signaling. These alterations make animals more sensitive to cold and may contribute to long-term outcomes of gestational calorie restriction in promoting obesity and related metabolic alterations.

Influence of breastfeeding on blood-cell transcript-based biomarkers of health in children.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: The expression of specific genes in peripheral blood cells (PBCs) may be used as biomarkers of the metabolic status. High levels of expression of CPT1A, SLC27A2, INSR, LEPR, FASN and PPARα in PBCs are indicative of a lower risk for the insulin resistant or dyslipidaemic state associated with obesity in children. Breastfeeding seems to confer protective effects against obesity and its related metabolic problems. WHAT THIS STUDY ADDS: Children who had been breastfed showed higher expression levels of SLC27A2, FASN, PPARα and INSR in PBCs compared with formula-fed subjects. The relationship of the PBC transcript levels of SLC27A2, INSR, FASN and PPARα with insulin resistance and dyslipidaemia may be dependent on the type of infant feeding (breast vs. formula). The transcript levels of the mentioned biomarkers could be useful to distinguish the formula-fed children who are at higher risk of metabolic alterations. BACKGROUND: Blood-cell transcripts have showed to be good biomarkers of metabolic alterations and their use in early detection and prevention of future disorders is promising. OBJECTIVE: This study aimed to examine the relation between previously proposed transcriptional biomarkers of metabolic health (SLC27A2, CPT1A, FASN, PPARα, INSR, LEPR) in peripheral blood cells and the type of infant feeding in a subset of children from the IDEFICS (Identification and Prevention of Dietary- and Lifestyle-Induced Health Effects in Children and Infants) cohort. SUBJECTS: A total of 237 children aged 2-9 years from eight European countries were studied. RESULTS: Breastfed children showed higher expression levels of SLC27A2, FASN, PPARα and INSR, and lower risk of being overweight and of having high plasma triglyceride levels vs. formula-fed children. Besides, overweight formula-fed children presented higher HOMA-index than overweight breastfed children (1.90 vs. 1.62); however, this negative effect was absent in formula-fed children with high expression of SLC27A2. Moreover, formula-fed children with low expression of SLC27A2, FASN, PPARα and INSR presented higher triglyceride levels than subjects with high expression of these genes (77.7 mg dL(-1) vs. 44.8 mg dL(-1) ). This difference was absent in breastfed children. CONCLUSIONS: Protective effects of breastfeeding are reflected in higher expression levels of SLC27A2, FASN, PPARα and INSR in blood cells. These biomarkers may also serve to discriminate the formula-fed children that are at higher risk of metabolic alterations.

Identification of early transcriptome-based biomarkers related to lipid metabolism in peripheral blood mononuclear cells of rats nutritionally programmed for improved metabolic health.

Moderate maternal calorie restriction during lactation protects rat offspring against obesity development in adulthood, due to an improved ability to handle and store excess dietary fuel. We used this model to identify early transcriptome-based biomarkers of metabolic health using peripheral blood mononuclear cells (PBMCs), an easily accessible surrogate tissue, by focusing on molecular markers of lipid handling. Male and female offspring of control and 20 % calorie-restricted lactating dams (CR) were studied. At weaning, a set of pups was killed, and PBMCs were isolated for whole-genome microarray analysis. The remaining pups were killed at 6 months of age. CR gave lower body weight, food intake and fat accumulation, and improved levels of insulin and leptin throughout life, particularly in females. Microarray analysis of weaned rat PBMCs identified 278 genes significantly differentially expressed between control and CR. Among lipid metabolism-related genes, expression of Cpt1a, Lipe and Star was increased and Fasn, Lrp1 and Rxrb decreased in CR versus control, with changes fully confirmed by qPCR. Among them, Cpt1a, Fasn and Star emerged as particularly interesting. Transcript levels of Cpt1a in PBMCs correlated with their levels in WAT and liver at both ages examined; Fasn expression levels in PBMCs at an early age correlated with their expression levels in WAT; and early changes in Star expression levels in PBMCs correlated with their expression levels in liver and were sustained in adulthood. These findings reveal the possibility of using transcript levels of lipid metabolism-related genes in PBMCs as early biomarkers of metabolic health status.

Vitamin A supplementation in early life affects later response to an obesogenic diet in rats.

OBJECTIVE: To assess the influence of supplementation with a moderate dose of vitamin A in early life on adipose tissue development and the response to an obesogenic diet later in life. METHODS: During the suckling period, rat pups received a daily oral dose of retinyl palmitate corresponding to three times the vitamin A ingested daily from maternal milk. Control rats received the vehicle (olive oil). Short-term effects of treatment on gene expression and morphology of white adipose tissue (WAT) were analyzed in animals on the day after weaning (day 21). To study long-term effects, control and vitamin A-treated rats were fed, after weaning, a normal fat or a high-fat (HF) diet for 16 weeks. RESULTS: WAT of vitamin A-treated young rats (day 21) was enriched in small adipocytes with a reduced expression of adipogenic markers (peroxisome proliferator-activated receptor γ and lipoprotein lipase) and an increased cell proliferation potential as indicated by increased expression of proliferating cell nuclear antigen. Increased retinoic acid (RA)-induced transcriptional responses were present in the tissues of vitamin A-treated young rats (day 21) including WAT. Vitamin A-treated rats developed higher adiposity than control rats on a HF diet as indicated by body composition analysis and increased WAT depot mass, adipocyte diameter, WAT DNA content, leptinemia and adipose leptin gene expression. Excess adiposity gain in vitamin A-treated rats developed in the absence of changes in body weight and was attributable to excess adipocyte hyperplasia. No differences in adiposity were observed between vitamin A-treated rats and control rats on a normal fat diet. Total retinol levels in WAT of vitamin A-treated rats were elevated at weaning (day 21) and normalized by day 135 of age. CONCLUSION: Vitamin A intake in the early stages of postnatal life favors subsequent HF diet-induced adiposity gain through mechanisms that may relate to changes in adipose tissue development, likely mediated by RA.

Diet-induced obesity affects expression of adiponutrin/PNPLA3 and adipose triglyceride lipase, two members of the same family.

BACKGROUND: Adiponutrin/PNPLA3 and adipose triglyceride lipase (ATGL) are proteins highly expressed in adipose tissue which have apparently different roles (lipogenic/lipolytic). Gene expression of both proteins and their nutritional regulation have been described to be altered in genetically obese animals. METHODS: We studied adiponutrin and ATGL expression in 6-month-old rats made obese by cafeteria diet feeding, submitted to different feeding conditions (feeding/fasting/re-feeding), compared with normoweight animals. Adiponutrin and ATGL mRNA levels were determined in white adipose tissue depots (subcutaneous and visceral) and in interscapular brown adipose tissue, and ATGL protein levels in selected depots. In addition, basal adiponutrin and ATGL expression levels were compared between 6- and 3-month-old animals. RESULTS: Obesity decreased adiponutrin and ATGL expression in different adipose depots. For adiponutrin, a tendency to lower mRNA levels was observed in the white adipose depots studied in obese animals, although the decrease was only significant in the subcutaneous depot. For ATGL, a generalized and significant lower expression was found in white and brown adipose tissue of cafeteria-obese rats. When considering nutritional regulation, according to a lipogenic role, adiponutrin mRNA expression decreased with fasting and was recovered by re-feeding in normoweight animals; this regulation was lost in obese rats. Expression of the lipolytic ATGL (mRNA and protein levels) was increased by fasting in normoweight animals in the mesenteric adipose depot, while no change was evident in obese rats. Moreover, adiponutrin and ATGL nutritional regulation was affected by age, and we report a downregulation of adiponutrin mRNA basal levels with age in internal adipose depots. CONCLUSIONS: Cafeteria diet-induced obesity and age alter adiponutrin and ATGL expression and their regulation by feeding conditions. These results reinforce the importance of a proper expression and regulation of both proteins for body weight maintenance and their role in energy metabolism.

Protective effects of leptin during the suckling period against later obesity may be associated with changes in promoter methylation of the hypothalamic pro-opiomelanocortin gene.

Leptin supplementation of neonatal rats during the suckling period protects against being overweight in adulthood and ameliorates the control of food intake. This was associated with changes in the expression of hypothalamic genes involved in the central action of leptin: pro-opiomelanocortin (Pomc), leptin receptor (Lepr) and suppressor of cytokine signalling (Socs3). The purpose of the present study was to determine the methylation status within the promoter regions of these genes and to assess whether the observed changes in the expression levels of these genes could be explained by changes in their methylation status. Male rats were treated daily with an oral physiological dose of leptin or vehicle during the suckling period. After weaning, animals were fed with a normal-fat or a high-fat (HF) diet until aged 6 months. DNA was extracted from the hypothalamus and methylation within the promoter regions of the gene panel was measured by pyrosequencing. Pomc promoter methylation increased in control animals fed the HF diet but decreased in leptin-treated animals. In addition, there was a weak negative correlation between DNA methylation and POMC mRNA levels (P = 0·075). There were no changes in the methylation status of the CpG sites studied within the promoter regions of Lepr and Socs3 in response to leptin or HF treatments. This is the first demonstration that leptin treatment during lactation may programme methylation of an appetite-related gene in the hypothalamus of animals fed HF diets, with possible implications for gene expression and protection against the development of obesity.

Moderate caloric restriction during gestation results in lower arcuate nucleus NPY- and alphaMSH-neurons and impairs hypothalamic response to fed/fasting conditions in weaned rats.

AIM: We aimed to characterize the developmental programming effects of moderate caloric restriction during early pregnancy on factors involved in hypothalamic control of energy balance. METHODS: Twenty-five-days-old offspring Wistar rats from 20% caloric restricted dams (from 1 to 12 days of pregnancy) (CR) and from control dams were studied under fed and 12 h fasting conditions. Morphometric studies on arcuate nucleus (ARC) and determinations of circulating parameters and hypothalamic levels of neuropeptide Y (NPY), proopiomelanocortin (POMC), long-form leptin receptor (ObRb), insulin receptor (InsR) and suppressor of cytokine signalling-3 (SOCS-3) mRNA were performed. RESULTS: CR animals did not show different body weight with respect to their controls, but presented higher food intake. They exhibited lower neuropeptide Y- and alpha-melanocyte-stimulating hormone-neurons (decreases of 18 and 13% in males, and 10 and 18% in females respectively) and lower total cells (decrease of 3% in males and 18% in females) in ARC. Under fed conditions, CR animals presented lower circulating leptin and ghrelin levels (decreases of 37 and 43% in males, and 15 and 34% in females respectively); furthermore, hypothalamic POMC, NPY (only in females), ObRb and InsR mRNA levels were reduced (39, 16 and 26% in males, and 112, 33, 61 and 56% in females), and those of SOCS-3 were increased (86% in males and 74% in females). Unlike control animals, under fasting conditions, ObRb, InsR and POMC mRNA levels did not decrease in CR females, and NPY mRNA decreased instead of increase in CR males. CONCLUSIONS: Moderate caloric restriction during gestation affects offspring hypothalamic structure and function, impairing its response to fed/fasting conditions, which suggests a predisposition to insulin and leptin resistance.

Dietary l-leucine supplementation of lactating rats results in a tendency to increase lean/fat ratio associated to lower orexigenic neuropeptide expression in hypothalamus.

The aim of this study was to assess the effects of dietary leucine supplementation in lactating dams, particularly on energy homeostasis through signaling mechanisms in the central nervous system. Dams were fed ad libitum with standard diet during pregnancy (control dams) or supplemented with 2% leucine (leucine-supplemented dams) from delivery onwards. Food intake, body weight and composition were periodically recorded. Hypothalamus was collected at the end of lactation, and the expression of neuropeptide Y (NPY), agouti-related protein (AgRP) pro-opiomelanocortin (POMC), cocaine and amphetamine regulated transcript (CART), insulin receptor (InsR), ghrelin receptor (GSHR), melanocortin receptor (MCR4), leptin receptor (Ob-Rb) and suppressor of cytokine signaling 3 (SOCS3) were analyzed. Dietary leucine supplementation to lactating rats increased plasma leucine by 56%, modulated body composition and contributed to a tendency of higher ratio of lean/fat mass content of dams during lactation, without affecting food intake, thermogenesis capacity or body or tissue/organs weights. No differences in body weight of offspring from control and leucine-supplemented dams were found. The expression of orexigenic peptides (NPY and AgRP) decreased in leucine-dams, whereas the expression of anorexigenic peptides (POMC and CART), the hypothalamic receptors of insulin, ghrelin, melanocortin and leptin and SOCS3 did not change by leucine supplementation. In conclusion, increased leucine intake during lactation may contribute to a healthier profile of body composition in dams, without compromising the growth and development of the progeny by a mechanism associated with lower expression of orexigenic neuropeptides in hypothalamus.

Slc27a2 expression in peripheral blood mononuclear cells as a molecular marker for overweight development.

BACKGROUND: Peripheral blood mononuclear cells (PBMC) can be collected easily and repeatedly. Their potential use to reflect the individual's biological status is increasingly explored. Obesity is becoming the most common health problem of the 21st century, being dietary intake an important determinant of this pathology and numerous chronic health conditions. OBJECTIVE: The aim of this study is to identify PBMC genes involved in energy homeostasis, which could be good markers of overweight development. DESIGN: Using whole-genome microarray analysis, we evaluated the gene expression in PBMC of normoweight and diet-induced obese (cafeteria-fed) Wistar rats. RESULTS: Microarray analysis showed 566 genes differentially expressed between normoweight and cafeteria-fed rats. Of these, 35 genes were particularly involved in energy homeostasis. The gene with the biggest fold change was the 'solute carrier family 27 (fatty acid transporter), member 2' (slc27a2), which is implicated in lipid biosynthesis and fatty acid degradation. Scl27a2 was 33-fold overexpressed in cafeteria-fed rats compared with normoweight rats. This result was confirmed by quantitative PCR, although the overexpression was smaller (sixfold). Moreover, the increase in slc27a2 expression in PBMC of cafeteria-fed rats from 2 to 6 months of age paralleled the increase in body weight. CONCLUSION: The progressive overexpression of slc27a2 in PBMC of cafeteria-fed rats as the body weight increases suggests this gene as an early marker of overweight development related to the intake of a hyperlipidic diet.