Antarctic Notothenioid Fishes Do Not Display Metabolic Cold Adaptation in Hepatic Gluconeogenesis

Antarctic notothenioid fishes present specializations related to their chronically cold environment, such as high lipid content in tissues (predominantly triacylglycerols, TAG). When TAGs are mobilized, they yield fatty acids and glycerol. Fatty acids are the primary fuel of oxidative muscle tissues. Gluconeogenesis from glycerol has not been studied in Antarctic fishes despite the importance of glycerol as a breakdown product of TAGs. To assess the possible importance of glycerol as a substrate for gluconeogenesis and to determine whether this pathway and Krebs cycle are metabolically cold adapted, key hepatic enzyme activities were measured in Antarctic notothenioid fishes (Notothenia coriiceps, Gobionotothen gibberifrons and Chionodraco rastrospinosus) and Subantarctic notothenioid fishes (Dissostichus eleginoides, Patagonotothen ramsayi and Eleginops maclovinus) . Citrate synthase, fructose 1,6-biphosphatase, glycerol kinase, and phosphoenolpyruvate carboxykinase enzyme activities were measured at lo, 60, 1 l o , and 2 10 C. Levels of specific metabolites in liver (glycerol, glucose and glycogen) and in serum (glycerol and glucose) were measured. My results indicate that gluconeogenesis and aerobic metabolism are not metabolically cold adapted in livers of Antarctic fishes. Levels of glycerol in plasma and liver were generally similar for all fishes studied, but surprisingly lower than the values reported for other teleost. Maximal activities for all enzymes assayed in livers of notothenioids fishes with Antarctic and Subantarctic distribution were similar when measured at the same temperature (loC). In addition, energies of activation for all the enzymes, calculated from the slope of Arrhenius plot, were similar between both groups of fishes. Lack of metabolic cold adaptation in hepatic gluconeogenesis may indicate that this pathway is of low physiological importance in both Antarctic and Subantarctic notothenioids or, more likely, that these two groups are so closely related that insufficient time has elapsed for evolutionary divergence in this tr

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 swim-flume 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 up-regulation 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

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

AMP-Activated Protein Kinase Plays an Important Evolutionary Conserved Role in the Regulation of Glucose Metabolism in Fish Skeletal Muscle Cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP∶ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish

Physiological Adaptations to Swimming in Fish

Swimming is an integral part of the life history of many fish species as is intimately linked with their ability to express feeding and predator avoidance behaviors, habitat selection and environmental preferences, social and reproductive behaviors as well as migratory behaviors. Therefore, swimming is an important determinant factor of fitness in a true Darwinian sense and, not surprisingly, swimming performance has been often used as a measure of physiological fitness in fish. The main aim of this Research Topic is to showcase some of the current studies designed to improve our understanding of the physiological energetic and metabolic requirements of swimming and of the adaptive responses to swimming in fish

In Vivo Molecular Responses of Fast and Slow Muscle Fibers to Lipopolysaccharide in a Teleost Fish, the Rainbow Trout (Oncorhynchus mykiss)

The physiological consequences of the activation of the immune system in skeletal muscle in fish are not completely understood. To study the consequences of the activation of the immune system by bacterial pathogens on skeletal muscle function, we administered lipopolysaccharide (LPS), an active component of Gram-negative bacteria, in rainbow trout and performed transcriptomic and proteomic analyses in skeletal muscle. We examined changes in gene expression in fast and slow skeletal muscle in rainbow trout at 24 and 72 h after LPS treatment (8 mg/kg) by microarray analysis. At the transcriptional level, we observed important changes in metabolic, mitochondrial and structural genes in fast and slow skeletal muscle. In slow skeletal muscle, LPS caused marked changes in the expression of genes related to oxidative phosphorylation, while in fast skeletal muscle LPS administration caused major changes in the expression of genes coding for glycolytic enzymes. We also evaluated the effects of LPS administration on the fast skeletal muscle proteome and identified 14 proteins that were differentially induced in LPS-treated trout, primarily corresponding to glycolytic enzymes. Our results evidence a robust and tissue-specific response of skeletal muscle to an acute inflammatory challenge, affecting energy utilization and possibly growth in rainbow trout

In Vivo Molecular Responses of Fast and Slow Muscle Fibers to Lipopolysaccharide in a Teleost Fish, the Rainbow Trout (Oncorhynchus mykiss)

The physiological consequences of the activation of the immune system in skeletal muscle in fish are not completely understood. To study the consequences of the activation of the immune system by bacterial pathogens on skeletal muscle function, we administered lipopolysaccharide (LPS), an active component of Gram-negative bacteria, in rainbow trout and performed transcriptomic and proteomic analyses in skeletal muscle. We examined changes in gene expression in fast and slow skeletal muscle in rainbow trout at 24 and 72 h after LPS treatment (8 mg/kg) by microarray analysis. At the transcriptional level, we observed important changes in metabolic, mitochondrial and structural genes in fast and slow skeletal muscle. In slow skeletal muscle, LPS caused marked changes in the expression of genes related to oxidative phosphorylation, while in fast skeletal muscle LPS administration caused major changes in the expression of genes coding for glycolytic enzymes. We also evaluated the effects of LPS administration on the fast skeletal muscle proteome and identified 14 proteins that were differentially induced in LPS-treated trout, primarily corresponding to glycolytic enzymes. Our results evidence a robust and tissue-specific response of skeletal muscle to an acute inflammatory challenge, affecting energy utilization and possibly growth in rainbow trout

Hepatic Glycerol Metabolism-Related Genes in Carnivorous Rainbow Trout (Oncorhynchus mykiss): Insights Into Molecular Characteristics, Ontogenesis, and Nutritional Regulation

Glycerol metabolism in rainbow trout is poorly studied even though it is at the interface between lipid and glucose metabolism. Moreover, glycerol can be an important ingredient in new aquafeed formulation to decrease the catabolism of dietary amino acids. Thus, the present study aimed to characterize for the first time the different genes coding for key enzymes and proteins involved in hepatic glycerol metabolism. From the trout genomes, all the paralogous genes coding for glycerol transport (aqp9b), glycerol kinase (gk2a and gk5), glycerol-3-phosphate phosphatase (pgp), and glycerol-3-phosphate dehydrogenase (gpd1a, gpd1b, and gpd1c) were identified. The ontogenesis determined that the capacity to metabolize glycerol begins with the apparition of the liver during the development (stage 22) and are more expressed at the endogenous-exogenous feeding period (stage 35). The postprandial regulation of the expression of these genes in juvenile trout showed that the postprandial peak of expression is between 4 and 24 h after the last meal for many of the genes, demonstrating that glycerol metabolism could be nutritionally regulated at a molecular level. However, surprisingly, no regulation of the mRNA abundance for the glycerol metabolism-related genes by different levels of dietary glycerol (0, 2.5, and 5%) have been detected, showing that hepatic glycerol metabolism is poorly regulated at a molecular level by dietary glycerol in rainbow trout juveniles.This work was supported by Fundação para a Ciência e Tecnologia (FCT; Portugal) through national funds with cofunding from ERDF/FEDER, within the PT2020 Partnership Agreement, and COMPETE 2020: research grant to IV (POCI-01-0145-FEDER-016828 - PTDC/CVT-NUT/2851/2014); individual grant to MP through Centro2020 (ReNATURE; Centro-01-0145-FEDER-000007); and structural funds to Center for Neuroscience and Cell Biology (UID/NEU/04539/2013) and Centre for Functional Ecology (UID/BIA/04004/2019) and Interdisciplinary Centre of Marine and Environmental Research (UID/Multi/04423/2019). The research about gene ontogenesis was internally funded by the European Commission (European project FP7-KBBE-2011-5, ARRAINA project no. 288925, Advanced Research Initiatives for Nutrition 1241 and Aquaculture

Proposed model for the role of AMPK in fish skeletal muscle cells.

<p>We propose that activation of endogenous AMPK by pharmacological activators (i.e. AICAR or metformin) results in increased glucose uptake by fish skeletal muscle cells through two mechanisms involving GLUT4: 1) increased mRNA levels of GLUT4 (possibly through the increased expression of peroxisome proliferator-activated receptor γ coactivator 1α or PGC-1α) and 2) stimulation of the translocation of GLUT4 to the plasma membrane. Furthermore, we propose that pharmacological activation of AMPK may also increase glucose utilization by stimulating expression of hexokinase (HK), 6-phosphofuctokinase (6-PFK), pyruvate kinase (PK) and citrate synthase (CS) mRNA levels. Future studies should be devoted to examine the possibility that AMPK may be activated as a result of the contraction of skeletal muscle fibers in swimming fish.</p