Structural and Functional Evolution of Glucose Transporter 4 (GLUT4): A Look at GLUT4 in Fish

The insulin-responsive glucose transporter GLUT4 was first described in 1988 as a result of studies on the regulation of glucose metabolism by insulin [1]. Soon after the discovery of GLUT4, several groups cloned GLUT4 in the human [2], rat [3,4] and mouse [5]. Since its discovery, GLUT4 has received, together with GLUT1, more experimental attention than any other single membrane transport protein. Structurally, GLUT4 follows the predicted model for class I glucose transporters. GLUT4 has a high affinity for glucose, with a Km of approximately 5 mM [6], and also transports mannose, galactose, dehydroascorbic acid and glucosamine [7-10]. In mammals, GLUT4 is mainly expressed in cardiac and skeletal muscle, brown and white adipose tissue, and brain [6,11,12]. GLUT4 plays a pivotal role in whole body glucose homeostasis, mediating the uptake of glucose regulated by insulin [13,14]. GLUT4 is responsible for the reduction in the postprandial rise in plasma glucose levels [6]. Insulin acts by stimulating the translocation of specific GLUT4-containing vesicles from intracellular stores to the plasma membrane (PM) resulting in an immediate increase in glucose transport [6,15]. The disruption of GLUT4 expression has been extensively associated with pathologies of impaired glucose uptake and insulin resistance such as type 2 diabetes and obesity [13,16-18]..

Conseqüències fisiològiques d'una activació del sistema immunitari sobre la reproducció i el creixement en peixos d'interès en aqüicultura

En peixos teleostis, una activació del sistema immunitari comporta la producció de factors immunitaris que ajuden a combatre una infecció i estableixen una comunicació entre la resposta immunitària innata i l'adaptativa. A part d'aquesta funció estrictament immunitària, els factors immunitaris poden afectar el funcionament d'altres cèl·lules i teixits no immunitaris. Dos processos particularment importants, tant per l'estat biològic d'una població de peixos, com per aspectes més relacionats amb la productivitat d'espècies en cultiu, són la reproducció i el creixement. En aquest treball es descriuen estudis encaminats a investigar els efectes d'una activació del sistema immunitari, i dels factors produïts, sobre la reproducció i el creixement en peixos teleostis.In teleost fish, an activation of the immune system entails the production of immune factors that are key to fighting an infeccion and establish a bridge between the innate and D’aquesthe adaptive immune responses. In addition to this strictly immune function, immune factors can also affect the function of other non-immune cells and tissues. Reproduction and growth are two processes that are particularly important for the biological condition of a population of fish as well as for aspects more related to productivity of cultured species. In this paper, we describe studies designed to investigate the effects of an activation of the immune system, as well as of the factors produced, on reproduction and growth in teleost fish

Transcriptional Assessment by Microarray Analysis and Large-Scale Meta-analysis of the Metabolic Capacity of Cardiac and Skeletal Muscle Tissues to Cope With Reduced Nutrient Availability in Gilthead Sea Bream (Sparus aurata L.)

The effects of nutrient availability on the transcriptome of cardiac and skeletal muscle tissues were assessed in juvenile gilthead sea bream fed with a standard diet at two feeding levels: (1) full ration size and (2) 70 % satiation followed by a finishing phase at the maintenance ration. Microarray analysis evidenced a characteristic transcriptomic profile for each muscle tissue following changes in oxidative capacity (heart > red skeletal muscle > white skeletal muscle). The transcriptome of heart and secondly that of red skeletal muscle were highly responsive to nutritional changes, whereas that of glycolytic white skeletal muscle showed less ability to respond. The highly expressed and nutritionally regulated genes of heart were mainly related to signal transduction and transcriptional regulation. In contrast, those of white muscle were enriched in gene ontology (GO) terms related to proteolysis and protein ubiquitination. Microarray meta-analysis using the bioinformatic tool Fish and Chips (http://fishandchips.genouest.org/index.php) showed the close association of a representative cluster of white skeletal muscle with some of cardiac and red skeletal muscle, and many GO terms related to mitochondrial function appeared to be common links between them. A second round of cluster comparisons revealed that mitochondria-related GOs also linked differentially expressed genes of heart with those of liver from cortisol-treated gilthead sea bream. These results show that mitochondria are among the first responders to environmental and nutritional stress stimuli in gilthead sea bream, and functional phenotyping of this cellular organelle is highly promising to obtain reliable markers of growth performance and well-being in this fish species. © 2014 Springer Science+Business Media New York.This work was funded by the EU seventh Framework Programme by the AQUAEXCEL (Aquaculture Infrastructures for Excellence in European Fish Research, FP7/2007-2012; grant agreement no. 262336) project. Additional funding was obtained from the Generalitat Valenciana (research grant PROMETEO 2010/006) and the Spanish Government through AQUAGENOMICS project (Consolider-Ingenio-2010 Programme).Peer Reviewe

Molecular identification of a glucose transporter from fish muscle11The nucleotide sequence data reported in this paper have been submitted to GenBank under accession number AF247395.

AbstractIn mammals and birds, several isoforms of facilitative glucose transporters have been identified (GLUT1–4), but no information is available regarding the molecules involved in glucose transport in other vertebrates. Here we report the cloning of a GLUT molecule from fish muscle with high sequence homology to GLUT4 and containing features characteristic of a functional GLUT. Fish GLUT is expressed predominantly in skeletal muscle, kidney and gill, which are tissues with known high glucose utilization. These results indicate that fish GLUT is structurally, and perhaps functionally, similar to the other known GLUTs expressed in muscle in mammalian and avian species

Bacterial lipopolysaccharide induces apoptosis in the trout ovary

BACKGROUND: In mammals it is well known that infections can lead to alterations in reproductive function. As part of the innate immune response, a number of cytokines and other immune factors is produced during bacterial infection or after treatment with lipopolysaccharide (LPS) and acts on the reproductive system. In fish, LPS can also induce an innate immune response but little is known about the activation of the immune system by LPS on reproduction in fish. Therefore, we conducted studies to examine the in vivo and in vitro effects of lipopolysaccharide (LPS) on the reproductive function of sexually mature female trout. METHODS: In saline- and LPS -injected brook trout, we measured the concentration of plasma steroids as well as the in vitro steroidogenic response (testosterone and 17alpha-hydroxyprogesterone) of ovarian follicles to luteinizing hormone (LH), the ability of 17alpha,20beta-dihydroxy-4-pregnen-3-one to induce germinal vesicle breakdown (GVBD) in vitro, and that of epinephrine to stimulate follicular contraction in vitro. We also examined the direct effects of LPS in vitro on steroid production, GVBD and contraction in brook trout ovarian follicles. The incidence of apoptosis was evaluated by TUNEL analysis. Furthermore, we examined the gene expression pattern in the ovary of saline- and LPS-injected rainbow trout by microarray analysis. RESULTS: LPS treatment in vivo did not affect plasma testosterone concentration or the basal in vitro production of steroids, although a small but significant potentiation of the effects of LH on testosterone production in vitro was observed in ovarian follicles from LPS-treated fish. In addition, LPS increased the plasma concentration of cortisol. LPS treatment in vitro did not affect the basal or LH-stimulated steroid production in brook trout ovarian follicles. In addition, we did not observe any effects of LPS in vivo or in vitro on GVBD or follicular contraction. Therefore, LPS did not appear to impair ovarian steroid production, oocyte final maturation or follicular contraction under the present experimental conditions. Interestingly, LPS administration in vivo induced apoptosis in follicular cells, an observation that correlated with changes in the expression of genes involved in apoptosis, as evidenced by microarray analysis. CONCLUSION: These results indicate that female trout are particularly resistant to an acute administration of LPS in terms of ovarian hormone responsiveness. However, LPS caused a marked increase in apoptosis in follicular cells, suggesting that the trout ovary could be sensitive to the pro-apoptotic effects of LPS-induced inflammatory cytokines

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout

Background: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). Methods: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses. Results: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha. Conclusions In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction

High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454-Pyrosequencing for the Discovery of Antiviral Immune Genes

Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations

Conseqüències fisiológiques d'una activació del sistema immunitari sobre la reproducción i el creixement en peixos d'interés en aqüicultura

En peixos teleostis, una activació del sistema immunitari comporta la producció de factors immunitaris que ajuden a combatre una infecció i estableixen una comunicació entre la resposta immunitària innata i l'adaptativa. A part d'aquesta funció estrictament immunitària, els factors immunitaris poden afectar el funcionament d'altres cèl·lules i teixits no immunitaris. Dos processos particularment importants, tant per l'estat biològic d'una població de peixos, com per aspectes més relacionats amb la productivitat d'espècies en cultiu, són la reproducció i el creixement. En aquest treball es descriuen estudis encaminats a investigar els efectes d'una activació del sistema immunitari, i dels factors produïts, sobre la reproducció i el creixement en peixos teleostis.In teleost fish, an activation of the immune system entails the production of immune factors that are key to fighting an infeccion and establish a bridge between the innate and the adaptive immune responses. In addition to this strictly immune function, immune factors can also affect the function of other non-immune cells and tissues. Reproduction and growth are two processes that are particularly important for the biological condition of a population of fish as well as for aspects more related to productivity of cultured species. In this paper, we describe studies designed to investigate the effects of an activation of the immune system, as well as of the factors produced, on reproduction and growth in teleost fish

Extending immunological profiling in the gilthead sea bream, sparus aurata, by enriched cDNA library analysis, microarray design and initial studies upon the inflammatory response to PAMPs

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response

Deep RNA Sequencing of the Skeletal Muscle Transcriptome in Swimming Fish

Deep RNA sequencing (RNA-seq) was performed to provide an in-depth view of the transcriptome of red and white skeletal muscle of exercised and non-exercised rainbow trout (Oncorhynchus mykiss) with the specific objective to identify expressed genes and quantify the transcriptomic effects of swimming-induced exercise. Pubertal autumn-spawning seawater-raised female rainbow trout were rested (n = 10) or swum (n = 10) for 1176 km at 0.75 body-lengths per second in a 6,000-L swimflume under reproductive conditions for 40 days. Red and white muscle RNA of exercised and non-exercised fish (4 lanes) was sequenced and resulted in 15–17 million reads per lane that, after de novo assembly, yielded 149,159 red and 118,572 white muscle contigs. Most contigs were annotated using an iterative homology search strategy against salmonid ESTs, the zebrafish Danio rerio genome and general Metazoan genes. When selecting for large contigs (.500 nucleotides), a number of novel rainbow trout gene sequences were identified in this study: 1,085 and 1,228 novel gene sequences for red and white muscle, respectively, which included a number of important molecules for skeletal muscle function. Transcriptomic analysis revealed that sustained swimming increased transcriptional activity in skeletal muscle and specifically an upregulation of genes involved in muscle growth and developmental processes in white muscle. The unique collection of transcripts will contribute to our understanding of red and white muscle physiology, specifically during the long-term reproductive migration of salmonids.Fil: Palstra, Arjan P.. Universidad de Barcelona. Facultad de Biología; España;Fil: Beltran, Sergi. Universitat de Barcelona. Centres Cientifics i Tecnològics. Unitat de Bioinformàtica; España;Fil: Burgerhout, Erik. Leiden University. Institute of Biology. Molecular Cell Biology; Países Bajos; ZF-screens; Países Bajos;Fil: Brittijn, Sebastiaan A.. Leiden University. Institute of Biology. Molecular Cell Biology; Países Bajos; ZF-screens; Países Bajos;Fil: Magnoni, Leonardo Julián. Universidad de Barcelona. Facultad de Biología; España; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús. Instituto de Investigaciones Biotecnológicas (sede Chascomús); Argentina;Fil: Henkel, Christiaan V.. ZF-screens; Países Bajos;Fil: Jansen, Hans J.. ZF-screens; Países Bajos;Fil: Van Den Thillart, Guido E. E. J. M.. Leiden University. Institute of Biology. Molecular Cell Biology; Países Bajos; ZF-screens; Países Bajos;Fil: Spaink, Herman P.. Leiden University. Institute of Biology. Molecular Cell Biology; Países Bajos; ZF-screens; Países Bajos;Fil: Planas, Josep V.. Universidad de Barcelona. Facultad de Biologia; España