Lucie Marandel

Remodelling of the hepatic epigenetic landscape of glucose-intolerant rainbow trout ( Oncorhynchus mykiss ) by nutritional status and dietary carbohydrates

The rainbow trout, a carnivorous fish, displays a ‘glucose-intolerant’ phenotype revealed by persistent hyperglycaemia when fed a high carbohydrate diet (HighCHO). Epigenetics refers to heritable changes in gene activity and is closely related to environmental changes and thus to metabolism adjustments governed by nutrition. In this study we first assessed in the trout liver whether and how nutritional status affects global epigenome modifications by targeting DNA methylation and histone marks previously reported to be affected in metabolic diseases. We then examined whether dietary carbohydrates could affect the epigenetic landscape of duplicated gluconeogenic genes previously reported to display changes in mRNA levels in trout fed a high carbohydrate diet. We specifically highlighted global hypomethylation of DNA and hypoacetylation of H3K9 in trout fed a HighCHO diet, a well-described phenotype in diabetes. g6pcb2 ohnologs were also hypomethylated at specific CpG sites in these animals according to their up-regulation. Our findings demonstrated that the hepatic epigenetic landscape can be affected by both nutritional status and dietary carbohydrates in trout. The mechanism underlying the setting up of these epigenetic modifications has now to be explored in order to improve understanding of its impact on the glucose intolerant phenotype in carnivorous teleosts.

Evolutionary history of c-myc in teleosts and characterization of the duplicated c-myca genes in goldfish embryos.

c‐Myc plays an important role during embryogenesis in mammals, but little is known about its function during embryonic development in teleosts. In addition, the evolutionary history of c‐myc gene in teleosts remains unclear, and depending on the species, a variable number of gene duplicates exist in teleosts. To gain new insight into c‐myc genes in teleosts, the present study was designed to clarify the evolutionary history of c‐myc gene(s) in teleosts and to subsequently characterize DNA methylation and early embryonic expression patterns in a cyprinid fish. Our results show that a duplication of c‐myc gene occurred before or around the teleost radiation, as a result of the teleost‐specific whole genome duplication giving rise to c‐myca and c‐mycb in teleosts and was followed by a loss of the c‐mycb gene in the Gasterosteiforms and Tetraodontiforms. Our data also demonstrate that both c‐myc genes previously identified in carp and goldfish are co‐orthologs of the zebrafish c‐myca. These results indicate the presence of additional c‐myca duplication in Cyprininae. We were able to identify differences between the expression patterns of the two goldfish c‐myca genes in oocytes and early embryos. These differences suggest a partial sub‐functionalization of c‐myca genes after duplication. Despite differences in transcription patterns, both of the c‐myca genes displayed similar DNA methylation patterns during early development and in gametes. Together, our results clarify the evolutionary history of the c‐myc gene in teleosts and provide new insight into the involvement of c‐myc in early embryonic development in cyprinids. Mol. Reprod. Dev. 79:85–96, 2012.

nanog 5′-upstream sequence, DNA methylation, and expression in gametes and early embryo reveal striking differences between teleosts and mammals

The nanog gene plays a major role in vertebrate development and was only recently discovered in teleosts. In order to gain new insight into its regulation in gametes and early embryo in teleost fish, the present study aimed at characterizing nanog upstream sequence features and DNA methylation, as well as early embryonic expression pattern in a Cyprinid fish, the goldfish. Using an in silico approach, we were able to demonstrate that despite the existence of conserved regulatory motifs in the promoter region of the nanog gene, specific features known to play a major role in the regulation of Nanog in mammals were missing in teleosts. The analysis of CpG sites in the upstream region of the nanog genes in goldfish revealed a significant DNA methylation state in oocytes while a hypomethylated state was observed in sperm. Using both quantitative PCR and whole mount in situ hybridization, we were able to clearly demonstrate the maternal inheritance of the nanog transcript in goldfish. Corresponding mRNA levels subsequently decreased during early gastrulation. Together, our results reveal striking differences in expression and DNA methylation patterns in gametes and during early development and in upstream region features between teleosts and mammals that are consistent with the hypothesis of a rapid evolution of the Nanog gene in vertebrates, at least in some lineages.

Glucose metabolism ontogenesis in rainbow trout (Oncorhynchus mykiss) in the light of the recently sequenced genome: new tools for intermediary metabolism programming.

ABSTRACT The rainbow trout (Oncorhynchus mykiss), a carnivorous fish species, displays a ‘glucose-intolerant’ phenotype when fed a high-carbohydrate diet. The importance of carbohydrate metabolism during embryogenesis and the timing of establishing this later phenotype are currently unclear. In addition, the mechanisms underlying the poor ability of carnivorous fish to use dietary carbohydrates as a major energy substrate are not well understood. It has recently been shown in trout that duplicated genes involved in glucose metabolism may participate in establishing the glucose-intolerant phenotype. The aim of this study was therefore to provide new understanding of glucose metabolism during ontogenesis and nutritional transition, taking into consideration the complexity of the trout genome. Trout were sampled at several stages of development from fertilization to hatching, and alevins were then fed a non-carbohydrate or a high-carbohydrate diet during first feeding. mRNA levels of all glucose metabolism-related genes increased in embryos during the setting up of the primitive liver. After the first meal, genes rapidly displayed expression patterns equivalent to those observed in the livers of juveniles. g6pcb2.a (a glucose 6-phosphatase-encoding gene) was up-regulated in alevins fed a high-carbohydrate diet, mimicking the expression pattern of gck genes. The g6pcb2.a gene may contribute to the non-inhibition of the last step of gluconeogenesis and thus to establishing the glucose-intolerant phenotype in trout fed a high-carbohydrate diet as early as first feeding. This information is crucial for nutritional programming investigations as it suggests that first feeding would be too late to programme glucose metabolism in the long term. Summary: The g6pcb2.a gluconeogenic gene is atypically up-regulated by dietary carbohydrate as soon as first feeding in rainbow trout and thus may contribute to the glucose-intolerant phenotype in early in life.

Exposure to an acute hypoxic stimulus during early life affects the expression of glucose metabolism-related genes at first-feeding in trout

Rainbow trout (Oncorhynchus mykiss) is considered a “glucose-intolerant” species. With the aim of programming trout to improve their metabolic use of dietary carbohydrates, we hypothesised that a hypoxic stimulus applied during embryogenesis could later affect glucose metabolism at the first-feeding stage. An acute hypoxic stimulus (2.5 or 5.0 mg·L−1 O2) was applied for 24 h to non-hatched embryos or early hatched alevins followed by a challenge test with a high carbohydrate diet at first-feeding. The effectiveness of the early hypoxic stimulus was confirmed by the induction of oxygen-sensitive markers such as egln3. At first-feeding, trout previously subjected to the 2.5 mg·L−1 O2 hypoxia displayed a strong induction of glycolytic and glucose transport genes, whereas these glucose metabolism-related genes were affected much less in trout subjected to the less severe (5.0 mg·L−1 O2) hypoxia. Our results demonstrate that an acute hypoxic stimulus during early development can affect glucose metabolism in trout at first-feeding.

Evolutionary history and epigenetic regulation of the three paralogous pax7 genes in rainbow trout

The extraordinary muscle growth potential of teleost fish, particular those of the Salmoninae clade, elicits questions about the regulation of the relatively highly conserved transcription factors of the myogenic program. The pseudotetraploid nature of the salmonid genome adds another layer of regulatory complexity that must be reconciled with epigenetic data to improve our understanding of the achievement of lifelong muscle growth in these fish. We identify three paralogous pax7 genes (pax7a1, pax7a2 and pax7b) in the rainbow trout genome. During in vitro myogenesis, pax7a1 transcripts remain stable, whereas pax7a2 and pax7b mRNAs increase in abundance, similarly to myogenin mRNAs but in contrast to the expression pattern of the mammalian ortholog. We also profile the distribution of repressive H3K27me3 and H3K9me3 and permissive H3K4me3 marks during in vitro myogenesis across these loci and find that pax7a2 expression is associated with decreased H3K27 trimethylation, whereas pax7b expression is correlated with decreased H3K9me3 and H3K27me3. These data link the unique differential expression of pax7 paralogs with epigenetic histone modifications in a vertebrate species displaying growth divergent from that of mammals and highlight an important divergence in the regulatory mechanisms of pax7 expression among vertebrates. The system described here provides a more comprehensive picture of the combinatorial control mechanisms orchestrating skeletal muscle growth in a salmonid, leading to a better understanding of myogenesis in this species and across Vertebrata more generally.

Trout coelomic fluid suitability as Goldfish oocyte extender can be determined by a simple turbidity test

Regeneration technologies such as androgenesis, intracytoplasmic sperm injection, and nuclear transfer require that handling conditions do not alter oocyte ability to sustain embryo development. One important parameter in the maintenance of oocyte quality in fish is the possibility to prevent oocytes activation during manipulation. In Cyprinid, such activation is known to be delayed when Salmonid coelomic fluid is used as incubation medium. Coelomic fluid however is a biological fluid whose ability to sustain oocyte quality during in vitro incubation may be variable. The purpose of the present work was to explore this variability using Rainbow Trout (Oncorhynchus mykiss) coelomic fluid (TCF) and Goldfish (Carassius auratus) oocytes, and to set up a test which would reflect TCF suitability for Goldfish oocyte incubation. We showed that different TCF induced very different development rates after oocyte incubation for 30 min at 20 °C: at 24h post fertilization (pf) and at hatching, rates ranged between 35% and 110% of the non-incubated controls. When TCF (1 volume) was mixed with tap water (9 volumes), a precipitate developed whose extent was measured by spectrophotometry. This turbidity test proved to be highly correlated to development rates after Goldfish oocyte incubation in TCF (r(2) = 0.83 at hatching, n = 150): TCF with the highest turbidity (> 1.5 absorbance unit at 400 nm) were the ones which altered the most the development rates after incubation (less than 50 % at hatching). This easy and rapid turbidity test can therefore be used as a reliable estimator of TCF suitability for Goldfish oocyte incubation and manipulation.

Long-term programming effect of embryonic hypoxia exposure and high-carbohydrate diet at first feeding on glucose metabolism in juvenile rainbow trout

ABSTRACT Environmental conditions experienced during early life play an important role in the long-term metabolic status of individuals. The present study investigated whether hypoxia exposure [for 24 h: 2.5 mg O2 l−1 (20% dissolved O2)] during the embryonic stage alone (hypoxic history) or combined with a 5-day high-carbohydrate (60%) diet stimulus at first feeding (HC dietary history) can affect glucose metabolism later in life, i.e. in juvenile fish. After 19 weeks of growth, we observed a decrease in final body mass in fish with an HC dietary history. Feed efficiency was significantly affected by both hypoxic and HC dietary histories. After a short challenge test (5 days) performed with a 30% carbohydrate diet in juvenile trout, our results also showed that, in trout that experienced hypoxic history, mRNA levels of gluconeogenic genes in liver and glucose transport genes in both liver and muscle were significantly increased at the juvenile stage. Besides, mRNA levels of glycolytic genes were decreased in fish with an HC dietary history. Both hypoxic and dietary histories barely affected plasma metabolites or global epigenetic modifications in juvenile fish after the challenge test. In conclusion, our results demonstrated that an acute hypoxic stimulus during early development alone or combined with a hyperglucidic stimulus at first feeding can modify growth performance and glucose metabolism at the molecular level in juvenile trout. Summary: Embryonic hypoxia and dietary stimuli can affect growth performance and the mRNA levels of glucose-metabolism-related genes in juvenile rainbow trout.

Epigenetics in teleost fish: From molecular mechanisms to physiological phenotypes

While the field of epigenetics is increasingly recognized to contribute to the emergence of phenotypes in mammalian research models across different developmental and generational timescales, the comparative biology of epigenetics in the large and physiologically diverse vertebrate infraclass of teleost fish remains comparatively understudied. The cypriniform zebrafish and the salmoniform rainbow trout and Atlantic salmon represent two especially important teleost orders, because they offer the unique possibility to comparatively investigate the role of epigenetic regulation in 3R and 4R duplicated genomes. In addition to their sequenced genomes, these teleost species are well-characterized model species for development and physiology, and therefore allow for an investigation of the role of epigenetic modifications in the emergence of physiological phenotypes during an organisms lifespan and in subsequent generations. This review aims firstly to describe the evolution of the repertoire of genes involved in key molecular epigenetic pathways including histone modifications, DNA methylation and microRNAs in zebrafish, rainbow trout, and Atlantic salmon, and secondly, to discuss recent advances in research highlighting a role for molecular epigenetics in shaping physiological phenotypes in these and other teleost models. Finally, by discussing themes and current limitations of the emerging field of teleost epigenetics from both theoretical and technical points of view, we will highlight future research needs and discuss how epigenetics will not only help address basic research questions in comparative teleost physiology, but also inform translational research including aquaculture, aquatic toxicology, and human disease.

Hepatic glucose metabolic responses to digestible dietary carbohydrates in two isogenic lines of rainbow trout

ABSTRACT Rainbow trout (Oncorhynchus mykiss) was recognized as a typical ‘glucose-intolerant’ fish and poor dietary carbohydrate user. Our first objective was to test the effect of dietary carbohydrates themselves (without modification of dietary protein intake) on hepatic glucose gene expression (taking into account the paralogs). The second aim was to research if two isogenic trout lines had different responses to carbohydrate intake, showing one with a better use dietary carbohydrates. Thus, we used two isogenic lines of rainbow trout (named A32h and AB1h) fed with either a high carbohydrate diet or a low carbohydrate diet for 12 weeks. We analysed the zootechnical parameters, the plasma metabolites, the hepatic glucose metabolism at the molecular level and the hormonal-nutrient sensing pathway. Globally, dietary carbohydrate intake was associated with hyperglycaemia and down regulation of the energy sensor Ampk, but also with atypical regulation of glycolysis and gluconeogenesis in the liver. Indeed, the first steps of glycolysis and gluconeogenesis catalysed by the glucokinase and the phospenolpyruvate carboxykinase are regulated at the molecular level by dietary carbohydrates as expected (i.e. induction of the glycolytic gck and repression of the gluconeogenic pck); by contrast, and surprisingly, for two other key glycolytic enzymes (phosphofructokinase enzyme – pfkl and pyruvate kinase – pk) some of the paralogs (pfklb and pklr) are inhibited by carbohydrates whereas some of the genes coding gluconeogenic enzymes (the glucose-6-phosphatase enzyme g6pcb1b and g6pcb2a gene and the fructose1-6 biphosphatase paralog fbp1a) are induced. On the other hand, some differences for the zootechnical parameters and metabolic genes were also found between the two isogenic lines, confirming the existence of genetic polymorphisms for nutritional regulation of intermediary metabolism in rainbow trout. In conclusion, our study determines some new and unexpected molecular regulations of the glucose metabolism in rainbow trout which may partly lead to the poor utilization of dietary carbohydrates and it underlines the existence of differences in molecular regulation of glucose metabolism between two isogenic lines which provides arguments for future selection of rainbow trout. Summary: Using isogenic lines, this study determines some new, unexpected molecular regulation of the glucose metabolism in rainbow trout, which may partly lead to the poor utilization of dietary carbohydrates.