Fasting/refeeding: an experimental model to study the impact of early thermal manipulation on hepatic metabolism in mule ducks.

An increase in egg incubation temperature was previously shown to enhance the metabolism of mule ducks and increase liver fattening after overfeeding, through a metabolic programming mechanism. Here, we examined whether fasting (F) followed by refeeding (RF) in 11-wk-old mule ducks could become an accelerated model to study the mechanisms of metabolic programming following embryonic thermal manipulation. This study investigated the hepatic response of mule ducks subjected to 23 h of fasting and 1 h of refeeding, in control or thermally programmed animals (with an increase of 1°C, 16 h per day from days 13 to 27 of embryogenesis). Liver weight and energy composition, hepatocyte structure, plasma parameters, and gene expression levels were measured at 1, 2, and 4 h after RF. All these parameters were strongly affected by RF, whereas significant impacts of embryonic programming were measured in cell size (+1 µm on average), lipid composition (+4.2% of saturated fatty acids 4 h after the meal), and relative gene expressions (including HK1, SCD1, ELOVL6, and FASN). In addition to confirming previously identified molecular targets of thermal manipulation, this study revealed new ones, thanks to kinetic sampling after RF. Finally, the detailed description of the impact of the F/RF challenge on the liver structure, composition, and gene expression, but also on plasma parameters allowed us to draw a parallel with these same traits measured during overfeeding. This comparative analysis suggests that this protocol could become a pertinent model to study the mechanisms involved in embryonic liver thermal programming, without overfeeding.

Hepatic Global DNA Hypomethylation Phenotype in Rainbow Trout Fed Diets Varying in Carbohydrate to Protein Ratio.

BACKGROUND: A high carbohydrate-low protein diet can induce hepatic global DNA hypomethylation in trout. The mechanisms remain unclear. OBJECTIVES: We aimed to investigate whether an increase in dietary carbohydrates (dHCs) or a decrease in dietary proteins (dLPs) can cause hepatic global DNA hypomethylation, as well as explore the underlying mechanisms in trout. METHODS: Two feeding trials were conducted on juvenile males, both of which involved a 4-d fasting and 4-d refeeding protocol. In trial 1, trout were fed either a high protein-no carbohydrate [HP-NC, protein 60% dry matter (DM), carbohydrates 0% DM] or a moderate protein-high carbohydrate (MP-HC, protein 40% DM, carbohydrates 30% DM) diet. In trial 2, fish were fed either a moderate protein-no carbohydrate (MP-NC, protein 40% DM, carbohydrates 0% DM), an MP-HC (protein 40% DM, carbohydrates 30% DM), or a low protein-no carbohydrate (LP-NC, protein 20% DM, carbohydrates 0% DM) diet to separate the effects of dHCs and dLPs on the hepatic methylome. Global CmCGG methylation, DNA demethylation derivative concentrations, and mRNA expression of DNA (de)methylation-related genes were measured. Differences were tested by 1-factor ANOVA when data were normally distributed or by Kruskal-Wallis nonparametric test if not. RESULTS: In both trials, global CmCGG methylation concentrations remained unaffected, but the hepatic 5-mdC content decreased after refeeding (1-3%). The MP-HC group had 3.4-fold higher hepatic 5-hmdC and a similar 5-mdC concentration compared with the HP-NC group in trial 1. Both MP-HC and LP-NC diets lowered the hepatic 5-mdC content (1-2%), but only the LP-NC group had a significantly lower 5-hmdC concentration (P < 0.01) compared with MP-NC group in trial 2. CONCLUSIONS: dHC and dLP independently induced hepatic global DNA demethylation in trout. The alterations in other methylation derivative concentrations indicated the demethylation process was achieved through an active demethylation pathway and probably occurred at non-CmCGG sites.

Chaperone-Mediated Autophagy in the Light of Evolution: Insight from Fish.

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player of the control of numerous cellular functions, and whose defects have been associated with several human pathologies. To date, this cellular function is presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in nontetrapod species. However, the recent identification of expressed sequences displaying high homology with mammalian LAMP2A in several fish species challenges that view and suggests that CMA likely appeared earlier during evolution than initially thought. In the present study, we provide a comprehensive picture of the evolutionary history of the LAMP2 gene in vertebrates and demonstrate that LAMP2 indeed appeared at the root of the vertebrate lineage. Using a fibroblast cell line from medaka fish (Oryzias latipes), we further show that the splice variant lamp2a controls, upon long-term starvation, the lysosomal accumulation of a fluorescent reporter commonly used to track CMA in mammalian cells. Finally, to address the physiological role of Lamp2a in fish, we generated knockout medaka for that specific splice variant, and found that these deficient fish exhibit severe alterations in carbohydrate and fat metabolisms, in consistency with existing data in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective.

Short-Term Effect of a Low-Protein High-Carbohydrate Diet on Mature Female and Male, and Neomale Rainbow Trout.

Rainbow trout are considered as a poor user of dietary carbohydrates, displaying persistent postprandial hyperglycaemia when fed a diet containing high amounts of carbohydrates. While this phenotype is well-described in juveniles, less attention was given to broodstock. Our objective was to assess for the first time the short-term consequences of feeding mature female and male, and neomale trout with a low-protein high-carbohydrate diet on glucose and lipid metabolism. Fish were fed for two days with a diet containing either no or 32% of carbohydrates. We analysed plasma metabolites, mRNA levels and enzymatic activities of glycolysis, gluconeogenesis, de novo lipogenesis and ß-oxidation in the liver. Results demonstrated that the glucose and lipid metabolism were regulated by the nutritional status in all sexes, irrespective of the carbohydrate intake. These data point out that carbohydrate intake during a short period (5 meals) at 8 °C did not induce specific metabolic changes in broodstock. Finally, we demonstrated, for the first time, sex differences regarding the consequences of two days of feeding on glucose and lipid metabolism.

Early feeding of rainbow trout (Oncorhynchus mykiss) with methionine-deficient diet over a 2 week period: consequences for liver mitochondria in juveniles.

Methionine is a key factor in modulating the cellular availability of the main biological methyl donor S-adenosylmethionine (SAM), which is required for all biological methylation reactions including DNA and histone methylation. As such, it represents a potential critical factor in nutritional programming. Here, we investigated whether early methionine restriction at first feeding could have long-term programmed metabolic consequences in rainbow trout. For this purpose, trout fry were fed with either a control diet (C) or a methionine-deficient diet (MD) for 2 weeks from the first exogenous feeding. Next, fish were subjected to a 5 month growth trial with a standard diet followed by a 2 week challenge (with the MD or C diet) to test the programming effect of the early methionine restriction. The results showed that, whatever the dietary treatment of fry, the 2 week challenge with the MD diet led to a general mitochondrial defect associated with an increase in endoplasmic reticulum stress, mitophagy and apoptosis, highlighting the existence of complex cross-talk between these different functions. Moreover, for the first time, we also observed that fish fed the MD diet at the first meal later exhibited an increase in several critical factors of mitophagy, hinting that the early nutritional stimulus with methionine deficiency resulted in long-term programming of this cell function. Together, these data extend our understanding of the role of dietary methionine and emphasize the potential for this amino acid in the application of new feeding strategies, such as nutritional programming, to optimize the nutrition and health of farmed fish.

DNA methylation of the promoter region of bnip3 and bnip3l genes induced by metabolic programming.

BACKGROUND: Environmental changes of biotic or abiotic nature during critical periods of early development may exert a profound influence on physiological functions later in life. This process, named developmental programming can also be driven through parental nutrition. At molecular level, epigenetic modifications are the most likely candidate for persistent modulation of genes expression in later life. RESULTS: In order to investigate epigenetic modifications induced by programming in rainbow trout, we focused on bnip3 and bnip3l paralogous genes known to be sensitive to environmental changes but also regulated by epigenetic modifications. Two specific stimuli were used: (i) early acute hypoxia applied at embryo stage and (ii) broodstock and fry methionine deficient diet, considering methionine as one of the main methyl-group donor needed for DNA methylation. We observed a programming effect of hypoxia with an increase of bnip3a and the four paralogs of bnip3l expression level in fry. In addition, parental methionine nutrition was correlated to bnip3a and bnip3lb1 expression showing evidence for early fry programming. We highlighted that both stimuli modified DNA methylation levels at some specific loci of bnip3a and bnip3lb1. CONCLUSION: Overall, these data demonstrate that methionine level and hypoxia stimulus can be of critical importance in metabolic programming. Both stimuli affected DNA methylation of specific loci, among them, an interesting CpG site have been identified, namely - 884 bp site of bnip3a, and may be positively related with mRNA levels.

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

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.

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

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.

Long-term feeding a plant-based diet devoid of marine ingredients strongly affects certain key metabolic enzymes in the rainbow trout liver.

Incorporation of a plant blend in the diet can affect growth parameters and metabolism in carnivorous fish. We studied for the first time the long-term (1 year) metabolic response of rainbow trout fed from first feeding with a plant-based diet totally devoid of marine ingredients. Hepatic enzymes were analyzed at enzymatic and molecular levels, at 3, 8 and 24 h after the last meal to study both the short-term effects of the last meal and long-term effects of the diet. The results were compared with those of fish fed a control diet of fish meal and fish oil. Growth, feed intake, feed efficiency and protein retention were lower in the group fed the plant-based diet. Glucokinase and pyruvate kinase activity were lower in the livers of trout fed the plant-based diet which the proportion of starch was lower than in the control diet. Glutamate dehydrogenase was induced by the plant-based diet, suggesting an imbalance of amino acids and a possible link with the lower protein retention observed. Gene expression of delta 6 desaturase was higher in fish fed the plant-based diet, probably linked to a high dietary level of linolenic acid and the absence of long-chain polyunsaturated fatty acids in vegetable oils. Hydroxymethylglutaryl-CoA synthase expression was also induced by plant-based diet because of the low rate of cholesterol in the diet. Changes in regulation mechanisms already identified through short-term nutritional experiments (<12 weeks) suggest that metabolic responses are implemented at short term and remain in the long term.

New insights into the nutritional regulation of gluconeogenesis in carnivorous rainbow trout (Oncorhynchus mykiss): a gene duplication trail.

The rainbow trout (Oncorhynchus mykiss) is considered to be a strictly carnivorous fish species that is metabolically adapted for high catabolism of proteins and low utilization of dietary carbohydrates. This species consequently has a "glucose-intolerant" phenotype manifested by persistent hyperglycemia when fed a high-carbohydrate diet. Gluconeogenesis in adult fish is also poorly, if ever, regulated by carbohydrates, suggesting that this metabolic pathway is involved in this specific phenotype. In this study, we hypothesized that the fate of duplicated genes after the salmonid-specific 4th whole genome duplication (Ss4R) may have led to adaptive innovation and that their study might provide new elements to enhance our understanding of gluconeogenesis and poor dietary carbohydrate use in this species. Our evolutionary analysis of gluconeogenic genes revealed that pck1, pck2, fbp1a, and g6pca were retained as singletons after Ss4r, while g6pcb1, g6pcb2, and fbp1b ohnolog pairs were maintained. For all genes, duplication may have led to sub- or neofunctionalization. Expression profiles suggest that the gluconeogenesis pathway remained active in trout fed a no-carbohydrate diet. When trout were fed a high-carbohydrate diet (30%), most of the gluconeogenic genes were non- or downregulated, except for g6pbc2 ohnologs, whose RNA levels were surprisingly increased. This study demonstrates that Ss4R in trout involved adaptive innovation via gene duplication and via the outcome of the resulting ohnologs. Indeed, maintenance of ohnologous g6pcb2 pair may contribute in a significant way to the glucose-intolerant phenotype of trout and may partially explain its poor use of dietary carbohydrates.

Frequency and distribution of mixed Plasmodium falciparum-vivax infections in French Guiana between 2000 and 2008.

BACKGROUND: The two main plasmodial species in French Guiana are Plasmodium vivax and Plasmodium falciparum whose respective prevalence influences the frequency of mixed plasmodial infections. The accuracy of their diagnosis is influenced by the sensitivity of the method used, whereas neither microscopy nor rapid diagnostic tests allow a satisfactory evaluation of mixed plasmodial infections. METHODS: In the present study, the frequency of mixed infections in different part of French Guiana was determined using real time PCR, a sensitive and specific technique. RESULTS: From 400 cases of malaria initially diagnosed by microscopy, real time PCR showed that 10.75 % of the cases were mixed infections. Their prevalence varied considerably between geographical areas. The presence, in equivalent proportions, of the two plasmodial species in eastern French Guiana was associated with a much higher prevalence of mixed plasmodial infections than in western French Guiana, where the majority of the population was Duffy negative and thus resistant to vivax malaria. CONCLUSION: Clinicians must be more vigilant regarding mixed infections in co-endemic P. falciparum/P. vivax areas, in order to deliver optimal care for patients suffering from malaria. This may involve the use of rapid diagnostic tests capable of detecting mixed infections or low density single infections. This is important as French Guiana moves towards malaria elimination.

Multiple independent introductions of Plasmodium falciparum in South America.

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Patterns of selection on Plasmodium falciparum erythrocyte-binding antigens after the colonization of the New World.

Pathogens, which have recently colonized a new host species or new populations of the same host, are interesting models for understanding how populations may evolve in response to novel environments. During its colonization of South America from Africa, Plasmodium falciparum, the main agent of malaria, has been exposed to new conditions in distinctive new human populations (Amerindian and populations of mixed origins) that likely exerted new selective pressures on the parasite's genome. Among the genes that might have experienced strong selective pressures in response to these environmental changes, the eba genes (erythrocyte-binding antigens genes), which are involved in the invasion of the human red blood cells, constitute good candidates. In this study, we analysed, in South America, the polymorphism of three eba genes (eba-140, eba-175, eba-181) and compared it to the polymorphism observed in African populations. The aim was to determine whether these genes faced selective pressures in South America distinct from what they experienced in Africa. Patterns of genetic variability of these genes were compared to the patterns observed at two housekeeping genes (adsl and serca) and 272 SNPs to separate adaptive effects from demographic effects. We show that, conversely to Africa, eba-140 seemed to be under stronger diversifying selection in South America than eba-175. In contrast, eba-181 did not show any sign of departure from neutrality. These changes in the patterns of selection on the eba genes could be the consequence of changes in the host immune response, the host receptor polymorphisms and/or the ability of the parasite to silence or express differentially its invasion proteins.

Insulin regulates lipid and glucose metabolism similarly in two lines of rainbow trout divergently selected for muscle fat content.

Two experimental rainbow trout lines were developed through divergent selection for low (Lean 'L' line) or high (Fat 'F' line) muscle fat content. Previous nutritional studies suggested that these lines differed in their regulation of lipid and glucose metabolism. Since insulin acts as an anabolic hormone by regulating lipid and glucose metabolism, we put forward the hypothesis that F line might have a stronger sensitivity to insulin than L line. In order to test this hypothesis, bovine insulin was injected into rainbow trout of the two lines fasted for 48 h. As expected, insulin induced hypoglycemia and activated Akt-TOR signaling both in the liver and muscle of the two lines. We demonstrate that this was coupled with increased expression of insulin dependent glucose transporter (GLUT4) and transcription factors of fatty acid anabolism (LXR and SREBP1c) in the muscle and liver, respectively, and lower mRNA levels of fatty acid oxidation enzymes (CPT1a, CPT1b and HOAD) in the white muscle of both lines. Regarding the genotype effect, TOR signaling response to insulin was stronger in F line as reflected by the higher phosphorylation of S6 protein and elevated mRNA levels of lipogenic enzyme (FAS) in the liver of F line. This observation was concordant with the higher plasma concentrations of free fatty acids and triglycerides in F line. Moreover, mRNA levels of hepatic gluconeogenic enzymes (G6Pase2, FBPase and PEPCK) and muscle fatty acid oxidation enzymes (CPT1a, CPT1b, HOAD and ACO) were higher in the F line. However, very few insulin-genotype interactions were detected, indicating that insulin induced similar changes in lipid and glucose metabolism in both lines.

Exploring the effects of dietary inulin in rainbow trout fed a high-starch, 100% plant-based diet.

BACKGROUND: High dietary carbohydrates can spare protein in rainbow trout (Oncorhynchus mykiss) but may affect growth and health. Inulin, a prebiotic, could have nutritional and metabolic effects, along with anti-inflammatory properties in teleosts, improving growth and welfare. We tested this hypothesis in rainbow trout by feeding them a 100% plant-based diet, which is a viable alternative to fishmeal and fish oil in aquaculture feeds. In a two-factor design, we examined the impact of inulin (2%) as well as the variation in the carbohydrates (CHO)/plant protein ratio on rainbow trout. We assessed the influence of these factors on zootechnical parameters, plasma metabolites, gut microbiota, production of short-chain fatty acids and lactic acid, as well as the expression of free-fatty acid receptor genes in the mid-intestine, intermediary liver metabolism, and immune markers in a 12-week feeding trial. RESULTS: The use of 2% inulin did not significantly change the fish intestinal microbiota, but interestingly, the high CHO/protein ratio group showed a change in intestinal microbiota and in particular the beta diversity, with 21 bacterial genera affected, including Ralstonia, Bacillus, and 11 lactic-acid producing bacteria. There were higher levels of butyric, and valeric acid in groups fed with high CHO/protein diet but not with inulin. The high CHO/protein group showed a decrease in the expression of pro-inflammatory cytokines (il1b, il8, and tnfa) in liver and a lower expression of the genes coding for tight-junction proteins in mid-intestine (tjp1a and tjp3). However, the 2% inulin did not modify the expression of plasma immune markers. Finally, inulin induced a negative effect on rainbow trout growth performance irrespective of the dietary carbohydrates. CONCLUSIONS: With a 100% plant-based diet, inclusion of high levels of carbohydrates could be a promising way for fish nutrition in aquaculture through a protein sparing effect whereas the supplementation of 2% inulin does not appear to improve the use of CHO when combined with a 100% plant-based diet.

Lipid digestion, absorption and uptake in Solea senegalensis.

Dietary lipids are the major energy source for metabolic purposes in most fish species, and improve dietary protein utilization for growth. In a previous study we have reported a low tolerance of Senegalese sole juveniles to dietary lipid levels and suggested a maximal dietary inclusion level of 8% lipids for both optimal growth and nutrient utilization. The mechanisms behind this apparent poor utilization of the dietary lipids are still to be elucidated. The primary aim of the present study was to investigate the overall process of digestion and lipid absorption in relation to dietary lipid levels. Triplicate groups of twenty fish (mean initial mass 29g) were fed two isonitrogenous diets (54% of protein dry matter basis) with different lipid levels (L4 and L17, 4 and 17% lipids dry matter basis), for 88days. Protein and lipid apparent digestibility coefficients as well as lipase activity were similar in both groups suggesting that Solea senegalensis has the ability to digest equally well a low fat or a high fat diet. Plasma triglyceride concentrations were significantly higher 5 and 16h after feeding in fish fed the L17 compared to those fed L4, following dietary lipid supply, demonstrating effective lipid absorption. Expression of proteins related to lipid transport (microsomal triglyceride transfer protein), trafficking (Fatty acid binding protein 11) and fatty acid uptake (VLDL-r) was significantly higher in liver of fish fed the high fat diet 16h after the meal, but remained unchanged in muscle. In conclusion, it seems that high fat diets do not impair lipid digestion and absorption.

More Than an Antioxidant: Role of Dietary Astaxanthin on Lipid and Glucose Metabolism in the Liver of Rainbow Trout (Oncorhynchus mykiss).

This study investigated the influence of dietary astaxanthin (AX) on glucose and lipid metabolism in rainbow trout liver. Two iso-nitrogenous and iso-lipidic diets were tested for 12 weeks in rainbow trout with an initial mean weight of 309 g. The S-ASTA diet was supplemented with 100 mg of synthetic AX per kg of feed, whereas the control diet (CTRL) had no AX. Fish fed the S-ASTA diet displayed lower neutral and higher polar lipids in the liver, associated with smaller hepatocytes and lower cytoplasm vacuolization. Dietary AX upregulated adipose triglyceride lipase (atgl), hormone-sensitive lipase (hsl2) and 1,2-diacylglycerol choline phosphotransferase (chpt), and downregulated diacylglycerol acyltransferase (dgat2), suggesting the AX's role in triacylglycerol (TAG) turnover and phospholipid (PL) synthesis. Dietary AX may also affect beta-oxidation with the upregulation of carnitine palmitoyltransferase 1 (cpt1α2). Although hepatic cholesterol levels were not affected, dietary AX increased gene expression of sterol regulatory element-binding protein 2 (srebp2). Dietary AX upregulated the expression of 6-phosphogluconate dehydrogenase (6pgdh) and downregulated pyruvate kinase (pkl). Overall, results suggest that dietary AX modulates the oxidative phase of the pentose phosphate pathway and the last step of glycolysis, affecting TAG turnover, ß-oxidation, PL and cholesterol synthesis in rainbow trout liver.

Chaperone-mediated autophagy protects against hyperglycemic stress.

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homeostasis and metabolism, and whose defects have been associated with several human pathologies. While CMA has been well described in mammals, functional evidence has only recently been documented in fish, opening up new perspectives to tackle this function under a novel angle. Now we propose to explore CMA functions in the rainbow trout (RT, Oncorhynchus mykiss), a fish species recognized as a model organism of glucose intolerance and characterized by the presence of two paralogs of the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A). To this end, we validated a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track functional CMA in mammalian cells, in an RT hepatoma-derived cell line (RTH-149). We found that incubation of cells with high-glucose levels (HG, 25 mM) induced translocation of the CMA reporter to lysosomes and/or late endosomes in a KFERQ- and Lamp2A-dependent manner, as well as reduced its half-life compared to the control (5 mM), thus demonstrating increased CMA flux. Furthermore, we observed that activation of CMA upon HG exposure was mediated by generation of mitochondrial reactive oxygen species, and involving the antioxidant transcription factor Nfe2l2/Nrf2 (nfe2 like bZIP transcription factor 2). Finally, we demonstrated that CMA plays an important protective role against HG-induced stress, primarily mediated by one of the two RT Lamp2As. Together, our results provide unequivocal evidence for CMA activity existence in RT and highlight both the role and regulation of CMA during glucose-related metabolic disorders.Abbreviations: AREs: antioxidant response elements; CHC: α-cyano -4-hydroxycinnamic acid; Chr: chromosome; CMA: chaperone-mediated autophagy; CT: control; DMF: dimethyl fumarate; Emi: endosomal microautophagy; HG: high-glucose; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; KFERQ: lysine-phenylalanine-glutamate-arginine-glutamine; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MCC: Manders' correlation coefficient; Manders' correlation coefficient Mo: morpholino oligonucleotide; NAC: N-acetyl cysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; PA-mCherry: photoactivable mCherry; PCC: Pearson's correlation coefficient; ROS: reactive oxygen species; RT: rainbow trout; siRNAs: small interfering RNAs; SOD: superoxide dismutase; Tsg101: tumor susceptibility 101; TTFA: 2-thenoyltrifluoroacetone; WGD: whole-genome duplication.

Histoplasma capsulatum in Cayenne, French Guiana.

We carried out a soil sampling survey in September 2008 in central Cayenne, French Guiana, using molecular methods to assess the presence of the dimorphic fungus Histoplasma capsulatum. Four of the 31 samples collected (12.9 %) tested positive by PCR, with confirmation of the result by DNA sequencing. H. capsulatum is therefore present in urban environments in French Guiana. These results provide additional support for the primary prophylaxis of AIDS-related histoplasmosis in French Guiana.

Characteristics and specificities of Cryptococcus infections in French Guiana, 1998-2008.

Cryptococcosis is a common opportunistic systemic mycosis caused by members of the encapsulated yeast genus Cryptococcus. The aetiological agents of the disease are classified into two species, i.e., Cryptococcus neoformans (serotypes A, D, and AD) affecting mainly immunocompromised patients and Cryptococcus gattii (serotypes B and C) usually found in immunocompetent individuals. Limited data exist on Cryptococcus infections in French Guiana, the department with the highest HIV incidence in the French territories. A retrospective study between January 1998 and December 2008 was performed on all patients with cryptococcosis, admitted to hospitals in French Guiana. Epidemiological data, clinical forms and diagnosis methods were studied. Of the 43 patients with cryptococcosis, 29 (67.4%) had HIV infections. Among the HIV-negative patients, two (4.7%) presented identified risk factors for cryptococcosis. This study has shown a high frequency of patients with HTLV-1 positive serology (12.1%). C. neoformans var. grubii was recovered with a frequency of 77.3% and was mainly isolated from patients with AIDS (13/17), whereas C. gattii (22.7%) was strictly isolated from HIV-negative patients with no apparent risk factors. Despite a socio-economic environment closer to western countries, the overall epidemiology of cryptococcosis in French Guiana is more similar to that found in South America, particularly with reference to the serotypes of the causative yeasts. Indeed, French Guiana presents an especially high proportion of cases caused by C. gattii, which may explain the significant incidence of this disease, particularly in HIV-negative and immunocompetent patients.