Christine Laborie

Prenatal morphine exposure affects sympathoadrenal axis activity and serotonin metabolism in adult male rats both under basal conditions and after an ether inhalation stress

We have previously shown that prenatal morphine exposure inhibited the hypothalamo-pituitary-adrenal (HPA) axis and altered the hypothalamic metabolism of serotonin during the early postnatal period in the rat and induced a chronic sympathoadrenal hyperactivity under resting conditions in adult male rats. In this study, we examined the effects of prenatal morphine exposure on the responsiveness to an acute ether inhalation stress of the sympathoadrenal and HPA axis and the hippocampal and hypothalamic concentrations of serotonin (5HT) and 5-hydroxylindoleacetic acid (5HIAA) in 3-month-old male rats. The plasma levels of adrenocorticopic hormone (ACTH) and corticosterone (B) did not differ between the two groups both under resting conditions and after ether exposure. Ether inhalation increased adrenal tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNA expression as well as adrenal epinephrine (E) concentration in control rats but not in prenatally morphine-exposed (PM) animals. Under basal conditions, hypothalamic concentrations of 5HT and 5HIAA increased in PM animals. In contrast to control animals, PM rats showed, in response to stress, an increased level of 5HT and 5HIAA in both the hypothalamus and in the hippocampus. In conclusion, prenatal morphine exposure produces long-lasting alterations in brain serotonin transmission and in the sympathoadrenal responsiveness to an acute systemic stress.

Maternal Perinatal Undernutrition has Long-Term Consequences on Morphology, Function and Gene Expression of the Adrenal Medulla in the Adult Male Rat

Epidemiological studies suggest that maternal undernutrition sensitises to the development of chronic adult diseases, such as type 2 diabetes, hypertension and obesity. Although the physiological mechanisms involved in this ‘perinatal programming’ remain largely unknown, alterations of stress neuroendocrine systems such as the hypothalamic‐pituitary‐adrenal (HPA) and sympathoadrenal axes might play a crucial role. Despite recent reports showing that maternal perinatal undernutrition disturbs chromaffin cells organisation and activity in male rats at weaning, its long‐term effects on adrenal medulla in adult animals are unknown. Using a rat model of maternal perinatal 50% food restriction (FR50) from the second week of gestation until weaning, histochemistry approaches revealed alterations in noradrenergic chromaffin cells aggregation and in cholinergic innervation in the adrenal medulla of 8‐month‐old FR50 rats. Electron microscopy showed that chromaffin cell granules exhibited ultrastructural changes in FR50 rats. These morphological changes were associated with reduced circulating levels and excretion of catecholamines. By contrast, catecholamine plasma levels were significantly increased after a 16 or 72 h of fasting, indicating that the responsiveness of the sympathoadrenal system to food deprivation was accentuated in FR50 adult rats. Among 384 pituitary adenylate cyclase‐activating polypeptide‐sensitive genes, we identified 129 genes (33.6%) that were under expressed (ratio < 0.7) in FR50 animals. A large number of these genes are involved in cytoskeleton remodelling and vesicle trafficking. Taken together, our results show that maternal perinatal undernutrition programmes adrenomedullary function and gene expression in adult male rats. Because catecholamines contribute to metabolic homeostasis, as well as arterial blood pressure regulation, the alterations observed in the adrenal medulla of adult male FR50 rats may participate in the programming of chronic adult diseases.

The hypothalamic POMC mRNA expression is upregulated in prenatally undernourished male rat offspring under high-fat diet

Epidemiological studies demonstrated that adverse environmental factors leading to intrauterine growth retardation (IUGR) and low birth weight may predispose individuals to increased risk of metabolic syndrome. In rats, we previously demonstrated that adult male IUGR offspring from prenatal 70% food-restricted dams throughout gestation (FR30) were predisposed to energy balance dysfunctions such as impaired glucose intolerance, hyperleptinemia, hyperphagia and adiposity. We investigated whether postweaning moderate high-fat (HF) diet would amplify the phenotype focusing on the hypothalamus gene expression profile. Prenatally undernourished rat offspring were HF-fed from weaning until adulthood while body weight and food intake were measured. Tissue weights, glucose tolerance and plasma endocrine parameters levels were determined in 4-month-old rats. Hypothalamic gene expression profiling of adult FR30 rat was performed using Illumina microarray analysis and the RatRef-12 Expression BeadChip that contains 21,792 rat genes. Under HF diet, contrary to C animals, FR30 rats displayed increased body weight. However, most of the endocrine disorders observed in chow diet-fed adult FR30 were alleviated. We also observed very few gene expression changes in hypothalamus of FR30 rat. Amongst factors involved in hypothalamic energy homeostasis programming system, only the POMC and transthyretin mRNA expression levels were preferentially increased under HF diet. Both elevated gene expression levels may be seen as adaptive mechanisms counteracting against deleterious effects of HF feeding in FR30 animals. This study shows that the POMC gene expression is a key target of long-term developmental programming in prenatally undernourished male rat offspring, specifically within an obesogenic environment.

Mild Gestational Hyperglycemia in Rat Induces Fetal Overgrowth and Modulates Placental Growth Factors and Nutrient Transporters Expression

Mild gestational hyperglycemia is often associated with fetal overgrowth that can predispose the offspring to metabolic diseases later in life. We hypothesized that unfavorable intrauterine environment may compromise the development of placenta and contribute to fetal overgrowth. Therefore, we developed a rat model and investigated the effects of maternal dysglycemia on fetal growth and placental gene expression. Female rats were treated with single injection of nicotinamide plus streptozotocin (N-STZ) 1-week before mating and were studied at gestational day 21. N-STZ pregnant females displayed impaired glucose tolerance that is associated with a lower insulin secretion. Moderate hyperglycemia induced fetal overgrowth in 40% of newborns, from pregnancies with 10 to 14 pups. The incidence of macrosomia was less than 5% in the N-STZ pregnancies when the litter size exceeds 15 newborns. We found that placental mass and the labyrinthine layer were increased in macrosomic placentas. The expression of genes involved in placental development and nutrient transfer was down regulated in the N-STZ placentas of macrosomic and normosomic pups from pregnancies with 10 to 14 ones. However, we observed that lipoprotein lipase 1 (LPL1) gene expression was significantly increased in the N-STZ placentas of macrosomic pups. In pregnancies with 15 pups or more, the expression of IGFs and glucose transporter genes was also modulated in the control placentas with no additional effect in the N-STZ ones. These data suggest that placental gene expression is modulated by gestational conditions that might disrupt the fetal growth. We described here a new model of maternal glucose intolerance that results in fetal overgrowth. We proposed that over-expression of LPL1 in the placenta may contribute to the increased fetal growth in the N-STZ pregnancies. N-STZ model offers the opportunity to determinate whether these neonatal outcomes may contribute to developmental programming of metabolic diseases in adulthood.

Maternal obesity programs increased leptin gene expression in rat male offspring via epigenetic modifications in a depot-specific manner

Objective According to the Developmental Origin of Health and Disease (DOHaD) concept, maternal obesity and accelerated growth in neonates predispose offspring to white adipose tissue (WAT) accumulation. In rodents, adipogenesis mainly develops during lactation. The mechanisms underlying the phenomenon known as developmental programming remain elusive. We previously reported that adult rat offspring from high-fat diet-fed dams (called HF) exhibited hypertrophic adipocyte, hyperleptinemia and increased leptin mRNA levels in a depot-specific manner. We hypothesized that leptin upregulation occurs via epigenetic malprogramming, which takes place early during development of WAT. Methods As a first step, we identified in silico two potential enhancers located upstream and downstream of the leptin transcription start site that exhibit strong dynamic epigenomic remodeling during adipocyte differentiation. We then focused on epigenetic modifications (methylation, hydroxymethylation, and histone modifications) of the promoter and the two potential enhancers regulating leptin gene expression in perirenal (pWAT) and inguinal (iWAT) fat pads of HF offspring during lactation (postnatal days 12 (PND12) and 21 (PND21)) and in adulthood. Results PND12 is an active period for epigenomic remodeling in both deposits especially in the upstream enhancer, consistent with leptin gene induction during adipogenesis. Unlike iWAT, some of these epigenetic marks were still observable in pWAT of weaned HF offspring. Retained marks were only visible in pWAT of 9-month-old HF rats that showed a persistent “expandable” phenotype. Conclusions Consistent with the DOHaD hypothesis, persistent epigenetic remodeling occurs at regulatory regions especially within intergenic sequences, linked to higher leptin gene expression in adult HF offspring in a depot-specific manner.

Influence of prenatal undernutrition on the effects of clozapine and aripiprazole in the adult male rats: Relevance to a neurodevelopmental origin of schizophrenia? ☆ ☆☆

Epidemiological and experimental data indicate that maternal undernutrition may sensitize the offspring to the apparition of chronic diseases such as metabolic syndrome and schizophrenia, suggesting that these pathologies may have a developmental origin. To test this hypothesis, we have compared the effects of a 4 weeks treatment of clozapine (30 mg/kg once daily, p.o.) or aripiprazole (10 mg/kg once daily, p.o.) on metabolic and hormonal parameters in 4-month-old male animals from control or 70% prenatally food-restricted mothers (FR30 model). Both neuroleptics did not markedly modify body weight gain and food intake in both controls and FR30 rats. Clozapine decreased insulin secretion in both groups but significantly diminished leptin, corticosterone and glucose plasma levels only in FR30 animals. Aripiprazole decreased corticosterone plasma levels only in FR30 animals. Using quantitative RT-PCR array containing 84 obesity-related genes, we identified several genes involved in energy metabolism regulation whose expression was modified by clozapine or aripiprazole in adult male rat hypothalami. In addition, we demonstrated that expression of some of these genes was differentially affected by each neuroleptic in the hypothalamus of both FR30 and control animals. Although no marked metabolic alterations were observed in both control and FR30 animals after clozapine or aripiprazole treatment, our data indicate that offspring from undernourished mothers exhibit a modified sensitivity to atypical neuroleptics. Our results do not rule out a putative developmental origin of schizophrenia and may help to understand the way by which atypical neuroleptics, such as clozapine, sensitize schizophrenic patients to the development of metabolic disorders.

Effect of diet in females (F1) from prenatally undernourished mothers on metabolism and liver function in the F2 progeny is sex-specific

PurposePoor maternal nutrition sensitises to the development of metabolic diseases and obesity in adulthood over several generations. The prevalence increases when offspring is fed with a high-fat (HF) diet after weaning. This study aims to determine whether such metabolic profiles can be transmitted to the second generation and even aggravated when the mothers were exposed to overnutrition, with attention to potential sex differences.MethodsPregnant Wistar rats were subjected to ad libitum (control) or 70% food-restricted diet (FR) during gestation (F0). At weaning, F1 females were allocated to three food protocols: (1) standard diet prior to and throughout gestation and lactation, (2) HF diet prior to and standard diet throughout gestation and lactation, and (3) HF diet prior to and throughout gestation and lactation. F2 offspring was studied between 16 and 32 weeks of age.ResultsFR-F2 offspring on standard diet showed normal adiposity and had no significant metabolic alterations in adulthood. Maternal HF diet resulted in sex-specific effects with metabolic disturbances more apparent in control offspring exposed to HF diet during gestation and lactation. Control offspring displayed glucose intolerance associated with insulin resistance in females. Female livers overexpressed lipogenesis genes and those of males the genes involved in lipid oxidation. Gene expression was significantly attenuated in the FR livers. Increased physical activity associated with elevated corticosterone levels was observed in FR females on standard diet and in all females from overnourished mothers.ConclusionsMaternal undernutrition during gestation (F0) improves the metabolic health of second-generation offspring with more beneficial effects in females.

P175: Un régime maternel hypercalorique hyperlidique programme l’expansion du tissu adipeux blanc de la descendance mâle chez le rat

Introduction et but de l’etude Le concept de l’origine developpe-mentale des maladies (DOHaD) stipule qu’un environnement deletere, incluant une malnutrition maternelle, in utero ou en periode postnatale precoce predispose la descendance a l’âge adulte au developpement d’un syndrome metabolique (diabete de type 2, hypertension et obesite). En particulier, des etudes epidemiologiques chez l’homme et experimentales chez l’animal ont montre que l’obesite maternelle, la surnutrition des nouveau-nes et la croissance postnatale acceleree sensibilisent la descendance a l’adiposite. Le but de notre etude est d’etudier les mecanismes de programmation du tissu adipeux blanc (TAB) chez la descendance mâle issue de meres surnutries. Materiel et methodes Nous avons developpe un modele de surnutrition maternelle chez le rat en alimentant des femelles puberes avec un regime hypercalorique hyperlipidique (60 % de lipides (Kcal)) pendant 4 mois avant la conception, pendant la gestation et la lactation. Au sevrage, la descendance mâle issue de meres surnutries a ete placee sous regime standard jusqu’a l’âge de 9 mois. La caracterisation de la descendance durant cette periode a ete effectuee a l’aide d’approches complementaires physiologiques (suivi des parametres metaboliques, dosages hormonaux par ELISA), moleculaires (RT-PCR quantitative) et histologiques (microscopie optique). Resultats et Analyse statistique Un regime maternel hypercalorique hyperlipidique ne modifie pas le poids de naissance. Cependant, il accelere la croissance postnatale et augmente la masse ponderale de la progeniture au cours de la lactation (periode maximale de l’adipogenese) suggerant un accroissement du tissu adipeux. Des l’âge de 3 mois, les rats mâles issus de meres surnutries ne montrent plus de difference de masse ponderale. A 7 mois, les animaux sont normoglycemiques mais presentent des traits de syndrome metabolique (intolerance au glucose, hyperinsulinemie +21 %, hypercorticosteronemie +89 %). Ils consomment quotidiennement la meme quantite de nourriture que les rats controles, mais leur prise alimentaire au cours du nycthemere est significativement modifiee (augmentation en periode diurne (+176 %) et diminution en periode nocturne). A 9 mois, les rats mâles issus de meres surnutries ne montrent pas de surpoids mais sont predisposes a l’adiposite comme l’atteste l’augmentation de la masse des depots de TAB perigonadique et perirenal (+17 %), du taux de leptine plasmatique (+120 %) et de son niveau d’expression genique adipocytaire (+40 %). De plus, l’analyse histologique montre une hypertrophie globale des adipocytes (+135 %) qui est associee a une augmentation du niveau d’expression genique de facteurs lipogeniques (SREBP-1c +67 %, FAS +50 %) et a une diminution du facteur adi-pogenique PPARg (−29 %). Conclusion En accord avec le concept de programmation precoce des maladies et de la sante, nos resultats confirment que la surnutrition maternelle programme la fonction et l’expansion du TAB de la descendance mâle chez le rat et favorise a l’apparition de troubles metaboliques. En particulier, ces donnees suggerent que la croissance acceleree durant la lactation constituerait un evenement determinant de cette programmation.

P172 - Impact de la programmation nutritionnelle et d’un régime hyperlipidique sur la transmission du risque du diabète chez le rat

Rationnel L’environnement nutritionnel et metabolique au cours des phases precoces du developpement constitue un element cle dans le determinisme de l’obesite et du diabete et entraine des modifications epigenetiques susceptibles d’expliquer la transmission d’une generation a l’autre des pathologies metaboliques Objectif : Evaluer l’impact de la programmation nutritionnelle et d’un regime hyperlipidique sur la croissance et le metabolisme de la descendance. Materiels et methodes Des rats femelles issus de meres (F0) denutries (FR) ou de meres controles (C) nourries ad libitum sont soumises pendant 2 mois a un regime standard (S) ou un regime hyperlipidique (HF). Les femelles de la F1 sont reparties en 6 groupes experimentaux, 3 groupes C et 3 groupes FR recevant pendant la gestation un regime S (CS-S, CHF-S, FRS-S, FRHF-S) ou un regime HF (CHF-HF, FRHF-HF). Resultats Les femelles gestantes CHF-HF, FRS-S, FRHF-S et FRHF-HF presentent une intolerance partielle au glucose en l’absence d’obesite. Nous observons une macrosomie chez pres de 30 % de nouveau-nes des meres FRHF-S associee a une baisse des triglycerides plasmatiques maternels. A l’inverse, nous observons un retard de croissance intra-uterin (RCIU) chez pres de 22 % des nouveau-nes issus de meres CHF-HF et FRHF-HF en rapport avec une augmentation des NEFA plasmatiques maternels. L’analyse moleculaire des genes placentaires montre que la macrosomie est associee a une modification de l’expression des genes impliques dans le metabolisme lipidique, tandis que le RCIU implique l’expression de genes du stress oxydatif. Discussion La denutrition maternelle programme chez les femelles des anomalies de la tolerance au glucose responsables des alterations de la croissance fœtale dans la descendance. La manipulation du regime lipidique des meres conduit a des phenotypes opposes (macrosomie vs RCIU) en relation avec une expression genique placentaire differentielle. Conclusion Il reste a determiner si les nouveau-nes macrosomes et RCIU developpent a l’âge adulte des pathologies similaires.