title: Chitosan-Mediated shRNA Knockdown of Cytosolic Alanine Aminotransferase Improves Hepatic Carbohydrate Metabolism.
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.
authors.forename: Juan D
authors.initials: JD
authors.lastname: González
authors.forename: Jonás I
authors.initials: JI
authors.lastname: Silva-Marrero
authors.forename: Isidoro
authors.initials: I
authors.lastname: Metón
authors.forename: Albert
authors.initials: A
authors.lastname: Caballero-Solares
authors.forename: Ivan
authors.initials: I
authors.lastname: Viegas
authors.forename: Felipe
authors.initials: F
authors.lastname: Fernández
authors.forename: Montserrat
authors.initials: M
authors.lastname: Miñarro
authors.forename: Anna
authors.initials: A
authors.lastname: Fàbregas
authors.forename: Josep R
authors.initials: JR
authors.lastname: Ticó
authors.forename: John G
authors.initials: JG
authors.lastname: Jones
authors.forename: Isabel V
authors.initials: IV
authors.lastname: Baanante
mesh_terms: D000410:Alanine Transaminase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D050260:Carbohydrate Metabolism / Q000502:physiology*; D048271:Chitosan / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism; D055785:Gene Knockdown Techniques / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism*; D053769:Nanocapsules / Q000737:chemistry; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics*; D021541:Sea Bream / Q000235:genetics / Q000378:metabolism*
chemical_list: D053769:Nanocapsules; D034741:RNA, Small Interfering; D048271:Chitosan; D000410:Alanine Transaminase