Transcription profiling in the liver of undernourished male rat offspring reveals altered lipid metabolism pathways and predisposition to hepatic steatosis.

Clinical and animal studies have reported an association between low birth weight and the development of nonalcoholic fatty liver disease (NAFLD) in offspring. Using a model of prenatal maternal 70% food restriction diet (FR30) in the rat, we previously showed that maternal undernutrition predisposes offspring to altered lipid metabolism in adipose tissue, especially on a high-fat (HF) diet. Here, using microarray-based expression profiling combined with metabolic, endocrine, biochemical, histological, and lipidomic approaches, we assessed whether FR30 procedure sensitizes adult male offspring to impaired lipid metabolism in the liver. No obvious differences were noted in the concentrations of triglycerides, cholesterol, and bile acids in the liver of 4-mo-old FR30 rats whichever postweaning diet was used. However, several clues suggest that offspring's lipid metabolism and steatosis are modified by maternal undernutrition. First, lipid composition was changed (i.e., higher total saturated fatty acids and lower elaidic acid) in the liver, whereas larger triglyceride droplets were observed in hepatocytes of undernourished rats. Second, FR30 offspring exhibited long-term impact on hepatic gene expression and lipid metabolism pathways on a chow diet. Although the transcriptome profile was globally modified by maternal undernutrition, cholesterol and bile acid biosynthesis pathways appear to be key targets, indicating that FR30 animals were predisposed to impaired hepatic cholesterol metabolism. Third, the FR30 protocol markedly modifies hepatic gene transcription profiles in undernourished offspring in response to postweaning HF. Overall, FR30 offspring may exhibit impaired metabolic flexibility, which does not enable them to properly cope with postweaning nutritional challenges influencing the development of nonalcoholic fatty liver.

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.

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.

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.

Maternal prenatal undernutrition programs adipose tissue gene expression in adult male rat offspring under high-fat diet.

Several studies have shown that maternal undernutrition leading to low birth weight predisposes offspring to the development of metabolic pathologies such as obesity. Using a model of prenatal maternal 70% food restriction diet (FR30) in rat, we evaluated whether postweaning high-fat (HF) diet would amplify the phenotype observed under standard diet. We investigated biological parameters as well as gene expression profile focusing on white adipose tissues (WAT) of adult offspring. FR30 procedure does not worsen the metabolic syndrome features induced by HF diet. However, FR30HF rats displayed catch-up growth to match the body weight of adult control HF animals, suggesting an increase of adiposity while showing hyperleptinemia and a blunted increase of corticosterone. Using quantitative RT-PCR array, we demonstrated that FR30HF rats exhibited leptin and Ob-Rb as well as many peptide precursor and receptor gene expression variations in WAT. We also showed that the expression of genes involved in adipogenesis was modified in FR30HF animals in a depot-specific manner. We observed an opposite variation of STAT3 phosphorylation levels, suggesting that leptin sensitivity is modified in WAT adult FR30 offspring. We demonstrated that 11ß-HSD1, 11ß-HSD2, GR, and MR genes are coexpressed in WAT and that FR30 procedure modifies gene expression levels, especially under HF diet. In particular, level variation of 11ß-HSD2, whose protein expression was detected by Western blotting, may represent a novel mechanism that may affect WAT glucocorticoid sensitivity. Data suggest that maternal undernutrition differently programs the adult offspring WAT gene expression profile that may predispose for altered fat deposition.

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.

Maternal perinatal undernutrition programs a "brown-like" phenotype of gonadal white fat in male rat at weaning.

Several studies indicate that maternal undernutrition sensitizes the offspring to the development of metabolic disorders, such as obesity. Using a model of perinatal maternal 50% food-restricted diet (FR50), we recently reported that rat neonates from undernourished mothers exhibit decreased leptin plasma levels associated with alterations of hypothalamic proopiomelanocortin system. The present study aimed at examining the consequences of FR50 on the brain-adipose axis in male rat neonates. Using quantitative RT-PCR array containing 84 obesity-related genes, we demonstrated that most of the genes involved in energy metabolism regulation are expressed in rat gonadal white adipose tissue (WAT) and are sensitive to maternal perinatal undernutrition (MPU). In contrast, hypothalamic gene expression was not substantially affected by MPU. Gene expression of uncoupling protein 1 (UCP1), a marker of brown adipocytes, showed an almost 400-fold stimulation in postnatal day 21 (PND21) FR50 animals, suggesting that their gonadal WAT possesses a brown-like phenotype. This was confirmed by histological and immunoshistochemical procedures, which demonstrated that PND21 FR50 gonadal adipocytes are multilocular, resembling those present in interscapular brown adipose tissue, and exhibit an overexpression of UCP1 and neuropeptide Y (NPY) at the protein level. Control animals contained almost exclusively "classical" unilocular white adipocytes that did not show high UCP1 and NPY labeling. After weaning, FR50 animals exhibited a transient hyperphagia that was associated with the disappearance of brown-like fat pads in PND30 WAT. Our results demonstrate that MPU delays the maturation of gonadal WAT during critical developmental time windows, suggesting that it could have long-term consequences on body weight regulation in the offspring.

Effects of a single footshock followed by situational reminders on HPA axis and behaviour in the aversive context in male and female rats.

Gender is an important factor in the vulnerability to develop psychopathologies. At the biological level, stress-related pathologies such as depression or post-traumatic stress disorder (PTSD) are associated with profound disturbances of the hypothalamo-pituitary-adrenal (HPA) axis. The aim of the present study was to assess sex-differences in the long-term effect of an intense stressful procedure on HPA function and behaviour in the aversive context in rats. Female and male rats experienced an aversive procedure consisting in an electric footshock (2mA, 10s) in a dark chamber followed by 3 weekly situational reminders (SR, 2min in the white chamber close to the footshock chamber). Our results indicate that 41 days after the end of the aversive procedure, female rats showed an increase of the corticosterone negative feedback in response to restraint stress, whereas such effect was not observed in males. Despite this change in the hormonal response, glucocorticoid receptors mRNA expression in the hippocampus was not affected in shocked females. In contrast, a significant increase of the mineralocorticoid receptors mRNA was observed in the CA2 of the hippocampus in shocked males. Finally, CRH mRNA levels in the paraventricular nucleus of the hypothalamus (PVN) were decreased in both female and male animals exposed to the aversive procedure. Behavioural observation revealed that shocked males and shocked females showed a high level of avoidance. However, the latency to visit the shock box was lower in females, which spent also more time in this area than males. In conclusion, our results suggest that gender might be a key factor impacting the direction of the effects induced by an intense stress. Interestingly, only females exhibited an increased negative feedback of the HPA axis response to stress, which could parallel endocrine changes of PTSD.

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.

Trophic and steroidogenic effects of water deprivation on the adrenal gland of the adult female rat.

The effects of a 3-day water deprivation were studied in adult female rats in order to know what are the different zones of the adrenal gland and the hormonal factors involved in the growth and the activity of the adrenal gland. Water deprivation significantly increased plasma renin activity (PRA), plasma Angiotensin II (AII), vasopressin (AVP), epinephrine, aldosterone and corticosterone concentrations but did not modify the plasma adrenocorticotropin hormone (ACTH) level. Water deprivation significantly increased the absolute weight of the adrenal capsule containing the zona glomerulosa without modification of the density of cells per area unit suggesting that the growth of the adrenal capsule was due to a cell hyperplasia of the zona glomerulosa. Water deprivation significantly increased the density of AII type 1 (AT(1)) receptors in the adrenal capsule but did not modify the density of AII type 2 (AT(2)) receptors in the adrenal capsule and core containing the zona fasciculata, the zona reticularis and the medulla. The treatment of dehydrated female rats with captopril, which inhibits the angiotensin converting enzyme (ACE) in order to block the production of AII, significantly decreased the absolute weight of the adrenal capsule, plasma aldosterone and the density of AT(1) receptors in the adrenal capsule. The concentration of corticosterone in the plasma, the density of AT(2) receptors and the density of cells per unit area in the zona glomerulosa of the adrenal capsule were not affected by captopril-treatment. In conclusion, these results suggest that AII seems to be the main factor involved in the stimulation of the growth and the secretion of aldosterone by the adrenal capsule containing the zona glomerulosa during water deprivation. The low level of plasma ACTH is not involved in the growth of the adrenal gland but is probably responsible for the secretion of corticosterone by the zona fasciculata.

Perinatal maternal food restriction induces alterations in hypothalamo-pituitary-adrenal axis activity and in plasma corticosterone-binding globulin capacity of weaning rat pups.

We investigated the effects of perinatal maternal malnutrition on the hypothalamo-pituitary-adrenal (HPA) axis activity in both basal and stressful conditions in newborn rats at weaning. Mothers from the control group were fed ad libitum. Mothers exposed to food restriction received 50% (FR50) of the daily intake of pregnant dams during the last week of gestation (Pre group), lactation (Post group) or both periods (PP group) in order to compare the long-term effects of gestational and/or lactational restriction. FR50 reduced the body growth of pups from the Post and PP groups as soon as day 11 until day 21 after birth. At weaning, pups of the Post and PP groups showed reduced adrenal, thymus and liver weights. Although the plasma adrenocorticotropic hormone (ACTH) level was reduced in pups, FR50 affected neither corticotropin-releasing hormone expression and peptide synthesis in the hypothalamus nor proopiomelanocortin expression in the adenohypophysis. Basal circulating levels of corticosterone were not markedly affected by FR50, but free corticosterone concentration was increased in the PP group. Plasma corticosterone-binding globulin (CBG) was decreased in newborns from both the Post and PP groups. Mineralocorticoid receptor gene expression was significantly increased in both CA1 and CA3 hippocampal areas in the PP group. Glucocorticoid receptor gene expression was increased in CA1, CA2 and dentate gyrus hippocampal areas in the Pre group, as well as in CA1, CA3 and DG areas in the Post group. The ether inhalation-induced plasma ACTH increase was weaker in pups from the Post and PP groups. Similarly, the ether inhalation-induced plasma corticosterone increase returned to basal levels in the Post group, or to weaker values than baseline in the PP group 90 min after this stressful procedure. The present work suggests that maternal food restriction during the perinatal period (gestation and lactation) or during lactation only reduces the postnatal somatic growth of pups and disturbs the activity of the HPA axis at weaning under both resting and stress conditions. A reduction in the plasma CBG-binding capacity, associated with a probable increase in hippocampal corticosteroid receptors, could reinforce glucocorticoid-mediated negative feedback and shorten stress-induced activation of the HPA axis in pups at weaning.