Didier Vieau

Sex- and diet-specific changes of imprinted gene expression and DNA methylation in mouse placenta under a high-fat diet.

Background Changes in imprinted gene dosage in the placenta may compromise the prenatal control of nutritional resources. Indeed monoallelic behaviour and sensitivity to changes in regional epigenetic state render imprinted genes both vulnerable and adaptable. Methods and Findings We investigated whether a high-fat diet (HFD) during pregnancy modified the expression of imprinted genes and local and global DNA methylation patterns in the placenta. Pregnant mice were fed a HFD or a control diet (CD) during the first 15 days of gestation. We compared gene expression patterns in total placenta homogenates, for male and female offspring, by the RT-qPCR analysis of 20 imprinted genes. Sexual dimorphism and sensitivity to diet were observed for nine genes from four clusters on chromosomes 6, 7, 12 and 17. As assessed by in situ hybridization, these changes were not due to variation in the proportions of the placental layers. Bisulphite-sequencing analysis of 30 CpGs within the differentially methylated region (DMR) of the chromosome 17 cluster revealed sex- and diet-specific differential methylation of individual CpGs in two conspicuous subregions. Bioinformatic analysis suggested that these differentially methylated CpGs might lie within recognition elements or binding sites for transcription factors or factors involved in chromatin remodelling. Placental global DNA methylation, as assessed by the LUMA technique, was also sexually dimorphic on the CD, with lower methylation levels in male than in female placentae. The HFD led to global DNA hypomethylation only in female placenta. Bisulphite pyrosequencing showed that neither B1 nor LINE repetitive elements could account for these differences in DNA methylation. Conclusions A HFD during gestation triggers sex-specific epigenetic alterations within CpG and throughout the genome, together with the deregulation of clusters of imprinted genes important in the control of many cellular, metabolic and physiological functions potentially involved in adaptation and/or evolution. These findings highlight the importance of studying both sexes in epidemiological protocols and dietary interventions.

Angiotensin-Converting Enzyme 2 (ACE2) and ACE Activities Display Tissue-Specific Sensitivity to Undernutrition-Programmed Hypertension in the Adult Rat

Human epidemiological studies have shown that low birth weight is associated with hypertension in adulthood. Rodent models of intrauterine growth retardation (IUGR) support these findings because offspring from undernourished dams develop hypertension. Angiotensin-converting enzyme 2 (ACE2) is a newly described renin-angiotensin system (RAS) component that competes with ACE for angiotensin peptide hydrolysis and therefore may modulate blood pressure. However, ACE2 potential participation in hypertension programming remains unknown, although RAS alterations were reported in IUGR models. Hence, we first investigated the tissue distribution of ACE2 and ACE in the rat and then whether hypertension programming differentially affects both enzymes. Using multiplex RT-PCR and in situ hybridization, we show that ACE2 mRNA is widely expressed and coregionalized with ACE. Moreover, tissues involved in blood pressure homeostasis (lung, heart, and kidney) express high levels of both enzymes. Enzymatic assays reveal that ACE2 and ACE are coactive in these tissues. Adult (4-month-old) offspring from 70% food-restricted dams throughout gestation (FR30 rats) present mild hypertension, impaired renal morphology, as well as elevated plasma angiotensin II and aldosterone, suggesting alterations of the systemic RAS. In FR30 rats, we show that ACE2 and ACE activities are increased only in the lung, whereas their mRNA expression is not significantly altered, showing that the enzymes display tissue-specific sensitivity to programming. Our results indicate that ACE2 and ACE are coexpressed in numerous rat tissues and that their increased activity in the lung of FR30 rats may participate in hypertension programming.

Crosstalk between nervous and immune systems through the animal kingdom: focus on opioids

During the course of evolution invertebrates and vertebrates have maintained common signaling molecules, such as neuropeptides. For example, complete hormonal-enzymatic systems for the biosynthesis of opioid peptides have been found in both the CNS and immune systems of these animals. These signaling molecules have been found in the blood circulation and act as immunomodulators. In vertebrates, release of the signaling molecules occurs during stress (cognitive or pathogens), which triggers the hypothalamo-hypophysial-adrenal axis. Similarly, these neuropeptides are used as messengers to initiate and stimulate the innate immune response in invertebrates. Thus, the crosstalk between nervous and immune systems has an ancient evolutionary origin and the messengers used have been conserved during the course of evolution reflecting their vital importance.

Maternal prenatal undernutrition alters the response of POMC neurons to energy status variation in adult male rat offspring

Epidemiological studies suggest that maternal undernutrition predisposes the offspring to development of energy balance metabolic pathologies in adulthood. Using a model of a prenatal maternal 70% food-restricted diet (FR30) in rats, we evaluated peripheral parameters involved in nutritional regulation, as well as the hypothalamic appetite-regulatory system, in nonfasted and 48-h-fasted adult offspring. Despite comparable glycemia in both groups, mild glucose intolerance, with a defect in glucose-induced insulin secretion, was observed in FR30 animals. They also exhibited hyperleptinemia, despite similar visible fat deposits. Using semiquantitative RT-PCR, we observed no basal difference of hypothalamic proopiomelanocortin (POMC) and neuropeptide Y (NPY) gene expression, but a decrease of the OB-Rb and an increase of insulin receptor mRNA levels, in FR30 animals. These animals also exhibited basal hypercorticosteronemia and a blunted increase of corticosterone in fasted compared with control animals. After fasting, FR30 animals showed no marked reduction of POMC mRNA levels or intensity of beta-endorphin-immunoreactive fiber projections. By contrast, NPY gene expression and immunoreactive fiber intensity increased. FR30 rats also displayed subtle alterations of food intake: body weight-related food intake was higher and light-dark phase rhythm and refeeding time course were modified after fasting. At rest, in the morning, hyperinsulinemia and a striking increase in the number of c-Fos-containing cells in the arcuate nucleus were observed. About 30% of the c-Fos-expressing cells were POMC neurons. Our data suggest that maternal undernutrition differently programs the long-term appetite-regulatory system of offspring, especially the response of POMC neurons to energy status and food intake rhythm.

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 11β-HSD1, 11β-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 11β-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.

The angiotensin system elements in invertebrates

In this review, the different components of the renin-angiotensin system (RAS) in invertebrates are discussed. This system is implicated in osmoregulation, reproduction, memory processes and immune system regulation. As the elements of this hormone-enzymatic system also exist in invertebrates, it appears that the RAS originated very early in evolution.

Serpins: an evolutionarily conserved survival strategy

Abstract Serpins regulate the activity of serine proteases derived from inflammatory cells and other tissues involved in enzymatic processes. The presence of serpins in invertebrates demonstrates that this important immune regulatory mechanism evolved much earlier than had previously been thought.

Up-regulation of Neurohemerythrin Expression in the Central Nervous System of the Medicinal Leech, Hirudo medicinalis, following Septic Injury

We report here some results of a proteomic analysis of changes in protein expression in the leech Hirudo medicinalis in response to septic injury. Comparison of two-dimensional protein gels revealed several significant differences between normal and experimental tissues. One protein found to be up-regulated after septic shock was identified, through a combination of Edman degradation, mass spectrometry, and molecular cloning, as a novel member of the hemerythrin family, a group of non-heme-iron oxygen transport proteins found in four invertebrate phyla: sipunculids, priapulids, brachiopods, and annelids. We found by in situ hybridization and immunocytochemistry that the new leech protein, which we have called neurohemerythrin, is indeed expressed in the leech central nervous system. Both message and protein were detected in the pair of large glia within the ganglionic neuropile, in the six packet glia that surround neuronal somata in each central ganglion, and in the bilateral pair of glia that separate axonal fascicles in the interganglionic connective nerves. No expression was detected in central neurons or in central nervous system microglia. Expression was also observed in many other, non-neuronal tissues in the body wall. Real-time PCR experiments suggest that neurohemerythrin is up-regulated posttranscriptionaly. We consider potential roles of neurohemerythrin, associated with its ability to bind oxygen and iron, in the innate immune response of the leech nervous system to bacterial invasion.

Hypothalamic Apelin/Reactive Oxygen Species Signaling Controls Hepatic Glucose Metabolism in the Onset of Diabetes

AIMS We have previously demonstrated that central apelin is implicated in the control of peripheral glycemia, and its action depends on nutritional (fast versus fed) and physiological (normal versus diabetic) states. An intracerebroventricular (icv) injection of a high dose of apelin, similar to that observed in obese/diabetic mice, increase fasted glycemia, suggesting (i) that apelin contributes to the establishment of a diabetic state, and (ii) the existence of a hypothalamic to liver axis. Using pharmacological, genetic, and nutritional approaches, we aim at unraveling this system of regulation by identifying the hypothalamic molecular actors that trigger the apelin effect on liver glucose metabolism and glycemia. RESULTS We show that icv apelin injection stimulates liver glycogenolysis and gluconeogenesis via an over-activation of the sympathetic nervous system (SNS), leading to fasted hyperglycemia. The effect of central apelin on liver function is dependent of an increased production of hypothalamic reactive oxygen species (ROS). These data are strengthened by experiments using lentiviral vector-mediated over-expression of apelin in hypothalamus of mice that present over-activation of SNS associated to an increase in hepatic glucose production. Finally, we report that mice fed a high-fat diet present major alterations of hypothalamic apelin/ROS signaling, leading to activation of glycogenolysis. INNOVATION/CONCLUSION: These data bring compelling evidence that hypothalamic apelin is one master switch that participates in the onset of diabetes by directly acting on liver function. Our data support the idea that hypothalamic apelin is a new potential therapeutic target to treat diabetes.

Characterization of the first non-insect invertebrate functional angiotensin-converting enzyme (ACE): leech TtACE resembles the N-domain of mammalian ACE

Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood homoeostasis and reproduction in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two homologous active sites, have been characterized. So far, several ACEs from invertebrates have been cloned, but only in insects. They are soluble and display a single active site. Using biochemical procedures, an ACE-like activity was detected in our model, the leech, Theromyzon tessulatum. Annelida is the most distant phylum in which an ACE activity has been observed. To gain more insight into the leech enzyme, we have developed a PCR approach to characterize its mRNA. The approx. 2 kb cDNA has been predicted to encode a 616-amino-acid soluble enzyme containing a single active site, named TtACE (T. tessulatum ACE). Surprisingly, its primary sequence shows greater similarity to vertebrates than to invertebrates. Stable in vitro expression of TtACE in transfected Chinese-hamster ovary cells revealed that the leech enzyme is a functional metalloprotease. As in mammals, this 79 kDa glycosylated enzyme functions as a dipeptidyl carboxypeptidase capable of hydrolysing angiotensin I to angiotensin II. However, a weak chloride inhibitory effect and acetylated N-acetyl-SDKP (Ac SDAcKP) hydrolysis reveal that TtACE activity resembles that of the N-domain of mammalian ACE. In situ hybridization shows that its cellular distribution is restricted to epithelial midgut cells. Although the precise roles and endogenous substrates of TtACE remain to be identified, characterization of this ancestral peptidase will help to clarify its physiological roles in non-insect invertebrate species.