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Chitosan-Mediated shRNA Knockdown of Cytosolic Alanine Aminotransferase Improves Hepatic Carbohydrate Metabolism

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Abstract

Alanine aminotransferase (ALT) catalyses a transamination reaction that links carbohydrate and amino acid metabolism. In this study, we examined the effect of silencing cytosolic ALT (cALT) expression on the hepatic metabolism in Sparus aurata. A number of siRNA and shRNA designed to down-regulate cALT expression were validated in HEK-293 cells transfected with plasmids expressing S. aurata cALT or mitochondrial ALT (mALT) isoforms: ALT silencing significantly decreased the expression levels of S. aurata mRNA cALT1 to 62 % (siRNA) and 48 % (shRNA) of the values observed in control cells. The effect of cALT silencing was analysed in the liver of S. aurata 72 h after intraperitoneal injection of chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a shRNA to down-regulate cALT expression (pCpG-si1sh1). In fish fed diets with different ratio of protein to carbohydrate and treated with chitosan-TPP-pCpG-si1sh1, cALT1 and cALT2 mRNA levels significantly decreased irrespective of the diet. Consistently, ALT activity decreased in liver of treated animals. In the liver of S. aurata treated with chitosan-TPP-pCpG-si1sh1 nanoparticles, down-regulation of cALT expression increased the activity of key enzymes in glycolysis (6-phosphofructo-1-kinase and pyruvate kinase) and protein metabolism (glutamate dehydrogenase). Besides showing for the first time that administration of chitosan-TPP-pCpG-si1sh1 nanoparticles silences hepatic cALT expression in vivo, our data support that down-regulation of cALT could improve the use of dietary carbohydrates to obtain energy and spare protein catabolism.

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Acknowledgments

This work was supported by the BIO2009-07589 (MCI, Spain) and AGL2012-33305 (MEC, Spain, co-funded by the European Regional Development Fund, ERDF, EC) grants. The authors thank Piscimar (Burriana, Spain) for providing the fish, the Aquarium of Barcelona for providing filtered seawater, Eurocoyal (Sant Cugat del Valles, Barcelona, Spain) for the provision of the fishmeal and Dr. José C. Perales (Departament de Ciències Fisiològiques II, Universitat de Barcelona, Spain) for providing the pCpG-siRNA and pCpG-siRNA-Scramble plasmids.

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Authors and Affiliations

  1. Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Joan XXIII 27, 08028, Barcelona, Spain

    Juan D. González, Jonás I. Silva-Marrero, Isidoro Metón & Isabel V. Baanante

  2. Departament d’Ecologia, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028, Barcelona, Spain

    Albert Caballero-Solares & Felipe Fernández

  3. Departament de Farmàcia i Tecnologia Farmacèutica, Facultat de Farmàcia, Universitat de Barcelona, Joan XXIII 27, 08028, Barcelona, Spain

    Montserrat Miñarro, Anna Fàbregas & Josep R. Ticó

  4. CNC—Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517, Coimbra, Portugal

    Ivan Viegas & John G. Jones

  5. CFE—Center for Functional Ecology, Department Life Sciences, University of Coimbra, Calçada Martins de Freitas, 3000-456, Coimbra, Portugal

    Ivan Viegas

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  1. Juan D. González
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  2. Jonás I. Silva-Marrero
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  3. Isidoro Metón
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  6. Felipe Fernández
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  7. Montserrat Miñarro
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  8. Anna Fàbregas
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  10. John G. Jones
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  11. Isabel V. Baanante
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Correspondence to Isabel V. Baanante.

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González, J.D., Silva-Marrero, J.I., Metón, I. et al. Chitosan-Mediated shRNA Knockdown of Cytosolic Alanine Aminotransferase Improves Hepatic Carbohydrate Metabolism. Mar Biotechnol 18, 85–97 (2016). https://doi.org/10.1007/s10126-015-9670-8

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