Charlotte Corporeau

Interaction of fish oil and a glucocorticoid on metabolic responses to an oral glucose load in healthy human subjects.

Compared with saturated fat, n-3 long-chain PUFA-rich fish oil improves insulin sensitivity in rats. We studied whether n-3 long-chain PUFA could prevent insulin resistance induced by dexamethasone (a glucocorticoid) in healthy human volunteers. A group of eight subjects was studied twice after a 2 d dexamethasone treatment, before and after a 3-week supplementation with fish oil (providing daily doses of 1.1 g 20 : 5n-3 and 0.7 g 22 : 6n-3). The subjects were studied during the basal state and over the 6 h following an oral glucose load (1 g/kg). Plasma glucose fluxes were traced with [6,6-2H2]glucose and [13C]glucose (naturally 13C-enriched corn glucose). Substrate oxidation was obtained from indirect calorimetry. Following fish oil supplementation, plasma glucose fluxes and substrate oxidation were maintained despite a 17 % reduction (P<0.05) in the area under the curve of plasma insulin response, suggesting an insulin-sensitizing effect.

Proteomic identification of quality factors for oocytes in the Pacific oyster Crassostrea gigas

We used a 2-DE proteomic approach to identify abundant proteins linked to oocyte quality in the Pacific oyster Crassostrea gigas, an economically important bivalve. Oocyte quality of 14 females was estimated by recording fertilisation and early developmental success until D-larval stage under controlled conditions. Proteins that were differentially expressed between females showing high or low oocyte quality were identified by nano-liquid chromatography tandem mass spectrometry. Twelve up-accumulated spots associated with low quality oocytes revealed 10 distinct proteins, including vitellogenin - breakdown products and metabolic enzymes. Eight up-accumulated spots from high quality oocytes revealed 6 distinct proteins, including chaperone molecules and cell-cycle control proteins. This is the first proteomic study dedicated to oocytes in C. gigas. Our results improve current knowledge about protein factors associated with oocyte quality in this species, and our understanding of the proteomic processes involved in their developmental competence.

In Vivo RNA Interference of a Gonad-Specific Transforming Growth Factor-β in the Pacific Oyster Crassostrea gigas

We investigated the role of oyster gonadal TGFβ (og-TGFβ) in the reproduction of Crassostrea gigas, using an in vivo RNA interference approach. We designed double-stranded RNA targeting og-TGFβ, which is specifically expressed in the somatic cells surrounding germ cells in the gonad of both male and female oysters. In vivo injection of this og-TGFβ dsRNA into the gonad led to knock-down phenotypes for both sexes, with significant reduction (77.52% relative to controls) of the gonad area, lowered reproductive effort and germ cell under-proliferation. Interestingly, half of the injected females halted their vitellogenesis, since we were only able to observe pre-vitellogenic oocytes. In addition, apoptotic germ cells and haemocytes infiltrated into the gonad, likely as part of the active resorption of degenerating germ cells. Conversely, males showed a normal phenotype at the cellular level, with spermatids and spermatozoids observed in the gonads of control and injected males. As a result, og-TGFβ appears to play an essential role in C. gigas germ cell development by functioning as an activator of germ cell proliferation in both male and female oysters and vitellogenesis in females.

Polyunsaturated fatty acids inhibit Pi3K activity in a yeast-based model system.

The phosphatidylinositol 3-kinase (PI3K) pathway controls the regulation of cell growth, proliferation, migration and apoptosis. In many tumors, the PI3K gene is mutated or overexpressed, and/or the PI3K pathway is hyperactive. PI3K is therefore a potential pharmacological target for the development of anti-tumor drugs. Some polyunsaturated fatty acids (PUFA), when given in the diet, may lead to a decrease in PI3K activity. We used a yeast-based model to reconstitute the PI3K/PTEN/Akt pathway to study the effects of long-chain polyunsaturated n-3 fatty acids on PI3K, and found that various PUFA were able to alleviate toxicity induced by overexpression of PI3K. The various PUFA had no significant effect on the steady-state level of PI3K catalytic subunit proteins (p110alpha) in yeast. However, depletion of phosphatidylinositol 4,5-bisphosphate due to overexpression of the p110alpha subunit was significantly reduced by treating the yeast cells with the various PUFA. The inhibition of mammalian PI3K, expressed in an exogenous cellular context in yeast, is likely to be a direct effect of these PUFA on PI3K rather than on other mammalian endogenous or environmental factors. These results are particularly promising given the abundance of active PUFA in marine foodstuffs and especially fish oils.

Sex-Specific Regulation of AMP-Activated Protein Kinase (AMPK) in the Pacific Oyster Crassostrea gigas

ABSTRACT The hermaphrodite Pacific oyster Crassostrea gigas displays a high energy allocation to reproduction. We studied the expression of AMP-activated protein kinase (AMPK) during gametogenesis in the gonad and characterized the mRNA sequences of the AMPK subunits: the AMPK alpha mRNA sequence was previously characterized; we identified AMPK beta, AMPK gamma, and mRNAs of putative AMPK-related targets following bioinformatics mining on existing genomic resources. We analyzed the mRNA expression of the AMPK alpha, beta, and gamma subunits in the gonads of male and female oysters through a reproductive cycle, and we quantified the mRNA expression of genes belonging to fatty acid and glucose metabolism. AMPK alpha mRNA levels were more abundant in males at the first stage of gametogenesis, when mitotic activity and the differentiation of germinal cells occur, and were always more abundant in males than in females. Some targets of fatty acid and glucose metabolism appeared to be correlated with the expression of AMPK subunits at the mRNA level. We then analyzed the sex-specific AMPK activity by measuring the phosphorylation of the catalytic AMPK alpha protein and its expression at the protein level. Both the amount of AMPK alpha protein and threonine 172 phosphorylation appeared to be almost totally inhibited in mature female gonads at stage 3, at the time when accumulation of reserves in oocytes was promoted, while it remained at a high level in mature spermatozoa. Its activation might play a sex-dependent role in the management of energy during gametogenesis in oyster.

Identification of Hypoxia-Regulated Genes in the Liver of Common Sole (Solea solea ) Fed Different Dietary Lipid Contents

Coastal systems could be affected by hypoxic events brought about by global change. These areas are essential nursery habitats for several fish species including the common sole (Solea solea L.). Tolerance of fish to hypoxia depends on species and also on their physiological condition and nutritional status. Indeed, high dietary lipid content has been recently shown to negatively impact the resistance of sole to a severe hypoxic challenge. In order to study the molecular mechanisms involved in the early response to hypoxic stress, the present work examined the hepatic transcriptome in common sole fed diets with low and high lipid content, exposed to severe hypoxia. The activity of AMP-activated protein kinase (AMPK) was also investigated through the quantification of threonine-172 phosphorylation in the alpha subunit. The results show that hypoxia consistently regulates several actors involved in energy metabolism pathways and particularly AMPKα, as well as some involved in cell growth and maintenance or unfolded protein response. Our findings reveal that (1) the expression of genes involved in biological processes with high energy cost or implicated in aerobic ATP synthesis was down-regulated by hypoxia, contrary to genes involved in neoglucogenesis or in angiogenesis, (2) the consumption of high lipid induced regulation of metabolic pathways going against this energy saving, and (3) this control was fine-tuned by the regulation of several transcriptomic factors. These results provide insight into the biological processes involved in the hepatic response to hypoxic stress and underline the negative impact of high lipid consumption on the tolerance of common sole to hypoxia.

A Functional Study of Transforming Growth Factor-Beta from the Gonad of Pacific Oyster Crassostrea gigas

The transforming growth factor (TGF)-β superfamily is a group of important growth factors involved in multiple processes such as differentiation, cell proliferation, apoptosis and cellular growth. In the Pacific oyster Crassostrea gigas, the oyster gonadal (og) TGF-β gene was recently characterized through genome-wide expression profiling of oyster lines selected to be resistant or susceptible to summer mortality. Og TGF-β appeared specifically expressed in the gonad to reach a maximum when gonads are fully mature, which singularly contrasts with the pleiotropic roles commonly ascribed to most TGF-β family members. The function of og TGF-β protein in oysters is unknown, and defining its role remains challenging. In this study, we develop a rapid bacterial production system to obtain recombinant og TGF-β protein, and we demonstrate that og TGF-β is processed by furin to a mature form of the protein. This mature form can be detected in vivo in the gonad. Functional inhibition of mature og TGF-β in the gonad was conducted by inactivation of the protein using injection of antibodies. We show that inhibition of og TGF-β function tends to reduce gonadic area. We conclude that mature og TGF-β probably functions as an activator of germ cells development in oyster.

The Kinome of Pacific Oyster Crassostrea gigas, Its Expression during Development and in Response to Environmental Factors.

Oysters play an important role in estuarine and coastal marine habitats, where the majority of humans live. In these ecosystems, environmental degradation is substantial, and oysters must cope with highly dynamic and stressful environmental constraints during their lives in the intertidal zone. The availability of the genome sequence of the Pacific oyster Crassostrea gigas represents a unique opportunity for a comprehensive assessment of the signal transduction pathways that the species has developed to deal with this unique habitat. We performed an in silico analysis to identify, annotate and classify protein kinases in C. gigas, according to their kinase domain taxonomy classification, and compared with kinome already described in other animal species. The C. gigas kinome consists of 371 protein kinases, making it closely related to the sea urchin kinome, which has 353 protein kinases. The absence of gene redundancy in some groups of the C. gigas kinome may simplify functional studies of protein kinases. Through data mining of transcriptomes in C. gigas, we identified part of the kinome which may be central during development and may play a role in response to various environmental factors. Overall, this work contributes to a better understanding of key sensing pathways that may be central for adaptation to a highly dynamic marine environment.

Energy and antioxidant responses of pacific oyster exposed to trace levels of pesticides.

Here, we assess the physiological effects induced by environmental concentrations of pesticides in Pacific oyster Crassostrea gigas. Oysters were exposed for 14 d to trace levels of metconazole (0.2 and 2 μg/L), isoproturon (0.1 and 1 μg/L), or both in a mixture (0.2 and 0.1 μg/L, respectively). Exposure to trace levels of pesticides had no effect on the filtration rate, growth, and energy reserves of oysters. However, oysters exposed to metconazole and isoproturon showed an overactivation of the sensing-kinase AMP-activated protein kinase α (AMPKα), a key enzyme involved in energy metabolism and more particularly glycolysis. In the meantime, these exposed oysters showed a decrease in hexokinase and pyruvate kinase activities, whereas 2-DE proteomic revealed that fructose-1,6-bisphosphatase (F-1,6-BP), a key enzyme of gluconeogenesis, was up-regulated. Activities of antioxidant enzymes were higher in oysters exposed to the highest pesticide concentrations. Both pesticides enhanced the superoxide dismutase activity of oysters. Isoproturon enhanced catalase activity, and metconazole enhanced peroxiredoxin activity. Overall, our results show that environmental concentrations of metconazole or isoproturon induced subtle changes in the energy and antioxidant metabolisms of oysters.

Non-additive effects of ocean acidification in combination with warming on the larval proteome of the Pacific oyster, Crassostrea gigas

UNLABELLED Increasing atmospheric carbon dioxide results in ocean acidification and warming, significantly impacting marine invertebrate larvae development. We investigated how ocean acidification in combination with warming affected D-veliger larvae of the Pacific oyster Crassostrea gigas. Larvae were reared for 40h under either control (pH8.1, 20 °C), acidified (pH7.9, 20 °C), warm (pH8.1, 22 °C) or warm acidified (pH7.9, 22 °C) conditions. Larvae in acidified conditions were significantly smaller than in the control, but warm acidified conditions mitigated negative effects on size, and increased calcification. A proteomic approach employing two-dimensional electrophoresis (2-DE) was used to quantify proteins and relate their abundance to phenotypic traits. In total 12 differentially abundant spots were identified by nano-liquid chromatography-tandem mass spectrometry. These proteins had roles in metabolism, intra- and extra-cellular matrix formations, stress response, and as molecular chaperones. Seven spots responded to reduced pH, four to increased temperature, and six to acidification and warming. Reduced abundance of proteins such as ATP synthase and GAPDH, and increased abundance of superoxide dismutase, occurred when both pH and temperature changes were imposed, suggesting altered metabolism and enhanced oxidative stress. These results identify key proteins that may be involved in the acclimation of C. gigas larvae to ocean acidification and warming. SIGNIFICANCE Increasing atmospheric CO2 raises sea surface temperatures and results in ocean acidification, two climatic variables known to impact marine organisms. Larvae of calcifying species may be particularly at risk to such changing environmental conditions. The Pacific oyster Crassostrea gigas is ecologically and commercially important, and understanding its ability to acclimate to climate change will help to predict how aquaculture of this species is likely to be impacted. Modest, yet realistic changes in pH and/or temperature may be more informative of how populations will respond to contemporary climate change. We showed that concurrent acidification and warming mitigates the negative effects of pH alone on size of larvae, but proteomic analysis reveals altered patterns of metabolism and an increase in oxidative stress suggesting non-additive effects of the interaction between pH and temperature on protein abundance. Thus, even small changes in climate may influence development, with potential consequences later in life.