Valérie Amarger

Nutritional programming affects hypothalamic organization and early response to leptin.

Nutritional programming, taking place in utero or early after birth, is closely linked with metabolic and appetite disorders in adulthood. Following the hypothesis that nutritional programming impacts hypothalamic neuronal organization, we report on discrepancies of multiple molecular and cellular early events that take place in the hypothalamus of rats submitted to intrauterine growth restriction (IUGR). Expression screening performed on hypothalami from IUGR rats at birth and at postnatal d 12 identified changes in gene expression of neurodevelopmental process (cell differentiation and cytoskeleton organization). Additionally, a slight reduction of agouti-related protein and a strong reduction of alpha-MSH-immunoreactive efferent fibers were demonstrated in the paraventricular nucleus of IUGR rats. Rapid catch-up growth of IUGR rats, 5 d after birth, had a positive effect on neurodevelopmental factors and on neuronal projections emanating from the arcuate nucleus. The molecular and cellular anomalies detected in IUGR rats can be related to the reduced and delayed plasma leptin surge from d 0-16 when compared with control and IUGR rats with catch-up growth. However, the ability of leptin to activate intracellular signaling in arcuate nucleus neurons was not reduced in IUGR rats. Other mechanism such as epigenetic regulation of the major appetite-regulating neuropeptides genes was analyzed in parallel with their mRNA expression during postnatal development. This study reveals the importance of an early catch-up growth that reduces abnormal organization of hypothalamic pathways involved in energy homeostasis, whereas protein restriction, maintained during postnatal development leads to an important immaturity of the hypothalamus.

Discovery, characterization and validation of single nucleotide polymorphisms within 206 bovine genes that may be considered as candidate genes for beef production and quality

A large number of putative single nucleotide polymorphisms (SNPs) have been identified from the bovine genome-sequencing project. However, few of these have been validated and many will turn out to be sequencing artefacts or have low minor allele frequencies. In addition, there is little information available on SNPs within coding regions, which are likely to be responsible for phenotypic variation. Therefore, additional SNP discovery is necessary to identify and validate polymorphisms both in specific genes and genome-wide. Sequence-tagged sites within 286 genes were resequenced from a panel of animals representing a wide range of European cattle breeds. For 80 genes, no polymorphisms were identified, and 672 putative SNPs were identified within 206 genes. Fifteen European cattle breeds (436 individuals plus available parents) were genotyped with these putative SNPs, and 389 SNPs were confirmed to have minor allele frequencies above 10%. The genes containing SNPs were localized on chromosomes by radiation hybrid mapping and on the bovine genome sequence by Blast. Flanking microsatellite loci were identified, to facilitate the alignment of the genes containing the SNPs in relation to mapped quantitative trait loci. Of the 672 putative SNPs discovered in this work, only 11 were found among the validated SNPs and 100 were found among the approximately 2.3 million putative SNPs currently in dbSNP. The genes studied in this work could be considered as candidates for traits associated with beef production and the SNPs reported will help to assess the role of the genes in the genetic control of muscle development and meat quality. The allele frequency data presented allows the general utility of the SNPs to be assessed.

Characterization of the bovine PRKAG3 gene: structure, polymorphism, and alternative transcripts

The bovine PRKAG3 gene encodes the AMPK γ3 subunit, one isoform of the regulatory γ subunit of the AMP-activated protein kinase (AMPK). The AMPK plays a major role in the regulation of energy metabolism and mutations affecting the genes encoding the γ subunits have been shown to influence AMPK activity. The γ3 subunit is involved in the regulation of AMPK activity in skeletal muscle and strongly inflences glycogen metabolism. Glycogen content in muscle is correlated to meat quality in livestock because it influences postmortem maturation process and ultimate pH. Naturally occurring mutations in the porcine PRKAG3 gene highly affect meat quality by influencing glycogen content before slaughter. We present the characterization of the bovine PRKAG3 gene and a polymorphism analysis in three cattle breeds. Thirty-two SNPs were identified among which 13 are in the coding region, one is in the 3′ UTR, and 18 are in the introns. Five of them change an amino acid in the PRKAG3 protein sequence. Allelic frequencies were determined in the three breeds considered, and mutant alleles affecting the coding sequence are found at a very low frequency. Alternative splicing sites were identified at two positions of the gene, introducing heterogeneity in the population of proteins translated from the gene.

Recovery of Exfoliated Cells From the Gastrointestinal Tract of Premature Infants: A New Tool to Perform “Noninvasive Biopsies?”

To gain insight into specific gene expression in the gastrointestinal (GI) tract of preterm infants, we adapted a method to isolate exfoliated epithelial cells. Gastric residual fluid aspirates (n = 89) or stool samples (n = 10) were collected from 96 neonates (gestational age, 24–36 wk). Cells were characterized by microscopic observation, cytokeratin-18 immunodetection, and expression of transcripts. The human origin of cellular DNA was confirmed by amplification of specific X and Y chromosome sequences. Isolation yielded 100–500 cells per sample for gastric aspirates (n = 8) and 10–20 cells for fecal samples (n = 5). Epithelial origin was confirmed by immunodetection of cytokeratin 18. Analyses of reverse transcribed products, using two independent methods, from 15 gastric fluid and two stool samples showed that 18S-rRNA and transcripts of beta-actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), and period1 were in quantities corresponding to at least 10 cells. On 59 aspirates, we found beta-actin transcripts (all but one), cytokeratin 18 (eight positive of eight samples), SLC26-A7-1 (13 positive of 19 samples), period2 (17 positive of 17 samples), and clock (25 positive of 26 samples). Exfoliated cells can be recovered from gastric aspirates and fecal samples and serve as a tool to investigate the impact of therapeutic and nutritional regimens on the maturation of GI functions.

Cotranscription and intergenic splicing of the PPARG and TSEN2 genes in cattle

BackgroundIntergenic splicing resulting in the combination of mRNAs sequences from distinct genes is a newly identified mechanism likely to contribute to protein diversity. Few cases have been described, most of them involving neighboring genes and thus suggesting a cotranscription event presumably due to transcriptional termination bypass.ResultsWe identified bovine chimeric transcripts resulting from cotranscription and intergenic splicing of two neighboring genes, PPARG and TSEN2. These two genes encode the Peroxisome Proliferator Activated Receptors γ1 and γ2 and the tRNA Splicing Endonuclease 2 homolog and are situated in the same orientation about 50 kb apart on bovine chromosome 22q24. Their relative position is conserved in human and mouse. We identified two types of chimeric transcripts containing all but the last exon of the PPARG gene followed by all but the first exon of the TSEN2 gene. The two chimers differ by the presence/absence of an intermediate exon resulting from transcription of a LINE L2 sequence situated between the two genes. Both transcripts use canonical splice sites for all exons coming from both genes, as well as for the LINE L2 sequence. One of these transcripts harbors a premature STOP codon and the other encodes a putative chimeric protein combining most of the PPARγ protein and the entire TSEN2 protein, but we could not establish the existence of this protein.ConclusionBy showing that both individual and chimeric transcripts are transcribed from PPARG and TSEN2, we demonstrated regulation of transcription termination. Further, the existence and functionality of a chimeric protein harboring active motifs that are a priori unrelated is hypothesized.

Protein Content and Methyl Donors in Maternal Diet Interact to Influence the Proliferation Rate and Cell Fate of Neural Stem Cells in Rat Hippocampus

Maternal diet during pregnancy and early postnatal life influences the setting up of normal physiological functions in the offspring. Epigenetic mechanisms regulate cell differentiation during embryonic development and may mediate gene/environment interactions. We showed here that high methyl donors associated with normal protein content in maternal diet increased the in vitro proliferation rate of neural stem/progenitor cells isolated from rat E19 fetuses. Gene expression on whole hippocampi at weaning confirmed this effect as evidenced by the higher expression of the Nestin and Igf2 genes, suggesting a higher amount of undifferentiated precursor cells. Additionally, protein restriction reduced the expression of the insulin receptor gene, which is essential to the action of IGFII. Inhibition of DNA methylation in neural stem/progenitor cells in vitro increased the expression of the astrocyte-specific Gfap gene and decreased the expression of the neuron-specific Dcx gene, suggesting an impact on cell differentiation. Our data suggest a complex interaction between methyl donors and protein content in maternal diet that influence the expression of major growth factors and their receptors and therefore impact the proliferation and differentiation capacities of neural stem cells, either through external hormone signals or internal genomic regulation.

Perinatal Hypercholesterolemia Exacerbates Atherosclerosis Lesions in Offspring by Altering Metabolism of Trimethylamine-N-Oxide and Bile Acids

Objective— Experimental studies suggest that maternal hypercholesterolemia may be relevant for the early onset of cardiovascular disease in offspring. We investigated the effect of perinatal hypercholesterolemia on the atherosclerosis development in the offspring of apolipoprotein E–deficient mice and the underlying mechanism. Approach and Results— Atherosclerosis and related parameters were studied in adult male or female apolipoprotein E–deficient mice offspring from either normocholesterolemic or hypercholesterolemic mothers and normocholesterolemic fathers. Female born to hypercholesterolemic mothers had more aortic root lesions than female born to normocholesterolemic mothers. Lesions in whole aorta did not differ between groups. Higher trimethylamine-N-oxide levels and Fmo3 hepatic gene expression were higher in female born to hypercholesterolemic mothers offspring compared with female born to normocholesterolemic mothers and male. Trimethylamine-N-oxide levels were correlated with the size of atherosclerotic root lesions. Levels of hepatic cholesterol and gallbladder bile acid were greater in male born to hypercholesterolemic mothers compared with male born to normocholesterolemic mothers. At 18 weeks of age, female born to hypercholesterolemic mothers showed lower hepatic Scarb1 and Cyp7a1 but higher Nr1h4 gene expression compared with female born to normocholesterolemic mothers. Male born to hypercholesterolemic mothers showed an increase in Scarb1 and Ldlr gene expression compared with male born to normocholesterolemic mothers. At 25 weeks of age, female born to hypercholesterolemic mothers had lower Cyp7a1 gene expression compared with female born to normocholesterolemic mothers. DNA methylation of Fmo3, Scarb1, and Ldlr promoter regions was slightly modified and may explain the mRNA expression modulation. Conclusions— Our findings suggest that maternal hypercholesterolemia may exacerbate the development of atherosclerosis in female offspring by affecting metabolism of trimethylamine-N-oxide and bile acids. These data could be explained by epigenetic alterations.

Perinatal high methyl donor alters gene expression in IGF system in male offspring without altering DNA methylation

Aim: To investigate the effect of a protein restriction and a supplementation with methyl donor nutrients during fetal and early postnatal life on the expression and epigenetic state of imprinted genes from the IGF system. Materials & methods: Pregnant female rats were fed a protein-restricted diet supplemented or not with methyl donor. Results: Gene expression of the Igf2, H19, Igf1, Igf2r and Plagl1 genes in the liver of male offspring at birth and weaning was strongly influenced by maternal diet. Whereas the methylation profiles of the Igf2, H19 and Igf2r genes were remarkably stable, DNA methylation of Plagl1 promoter was slightly modified. Conclusion: DNA methylation of most, but not all, imprinted gene regulatory regions was resistant to methyl group nutritional supply.

Haplotype association analysis of meat quality traits at the bovine PRKAG3 locus

Abstract The current study presents the results of a preliminary haplotype association analysis at the bovine PRKAG3 locus with meat quality traits in the Chianina breed. No significant association was shown between haploid haplotypes (or diplotypes) and phenotypical traits after applying a Bonferroni correction for multiple comparison. Nonetheless, data from Longissimus dorsi muscle suggest the presence of a statistically non-significant trend toward an influence of the PRKAG3 haploid haplotypes on meat colour (a*) and water holding capacity (M/T) traits, as confirmed also by diplotype-based association analysis. A less clear set of results was observed for the Triceps brachii and Semitendinosus muscles.