Metformin counteracts glucose-dependent lipogenesis and impairs transdeamination in the liver of gilthead sea bream (Sparus aurata)

Metformin is an anti-diabetic drug with a major impact on regulating blood glucose levels by decreasing hepatic gluconeogenesis but also affecting other pathways, including glucose transport and energy/lipid metabolism. Carnivorous fish are considered glucose intolerant, as they exhibit poor ability to using dietary carbohydrates. To increase the current knowledge about the molecular mechanisms by which metformin can improve glucose homeostasis in carnivorous fish, we addressed the effect of intraperitoneal administration of metformin, in the presence or absence of a glucose load, on metabolic rate-limiting enzymes and lipogenic factors in the liver of gilthead sea bream (Sparus aurata). Hyperglycemia markedly up-regulated the expression of glycolytic enzymes (glucokinase and 6-phosphofructo-1-kinase, PFK1) 5 h following glucose administration, while at 24 h post-treatment it increased isocitrate dehydrogenase (IDH) activity, a key enzyme of the tricarboxylic acid cycle, and the expression of lipogenic factors (PGC1b, Lpin1 and SREBP1). Metformin counteracted glucose-dependent effects, and down-regulated glutamate dehydrogenase, alanine aminotransferase and mTOR 5 h post-treatment in the absence of a glucose load, leading to decreased long-term activity of PFK1 and IDH. The results of the present study suggest that hyperglycemia enhances lipogenesis in the liver of S. aurata, and that metformin may exert specific metabolic effects in fish by decreasing hepatic transdeamination and supressing the use of amino acids as gluconeogenic substrates. Our findings highlight the role of amino acid metabolism in the glucose-intolerant carnivorous fish model. KEYWORDS: Glutamate dehydrogenase; Lipogenesis; Liver; Metformin; Sparus aurat

Role of upstream stimulatory factor 2 in glutamate dehydrogenase gene transcription

Glutamate dehydrogenase (Gdh) plays a central role in ammonia detoxification by catalysing reversible oxidative deamination of L-glutamate into α-ketoglutarate using NAD+ or NADP+ as cofactor. To gain insight into transcriptional regulation of glud, the gene that codes for Gdh, we isolated and characterised the 5' flanking region of glud from gilthead sea bream (Sparus aurata). In addition, tissue distribution, the effect of starvation as well as short- and long-term refeeding on Gdh mRNA levels in the liver of S. aurata were also addressed. 5'-deletion analysis of glud promoter in transiently transfected HepG2 cells, electrophoretic mobility shift assays, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis allowed us to identify upstream stimulatory factor 2 (Usf2) as a novel factor involved in the transcriptional regulation of glud. Analysis of tissue distribution of Gdh and Usf2 mRNA levels by reverse transcriptase-coupled quantitative real-time PCR (RT-qPCR) showed that Gdh is mainly expressed in the liver of S. aurata, while Usf2 displayed ubiquitous distribution. RT-qPCR and ChIP assays revealed that long-term starvation down-regulated the hepatic expression of Gdh and Usf2 to similar levels and reduced Usf2 binding to glud promoter, while refeeding resulted in a slow but gradial restoration of both Gdh and Usf2 mRNA abundance. Herein, we demonstrate that Usf2 transactivates S. aurata glud by binding to an E-box located in the proximal region of glud promoter. In addition, our findings provide evidence for a new regulatory mechanism involving Usf2 as a key factor in the nutritional regulation of glud transcription in the fish liver

Chitosan-mediated shRNA knockdown of cytosolic alanine aminotransferase improves hepatic carbohydrate metabolism

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 catabolis

Administration of chitosan-tripolyphosphate-DNA nanoparticles to knockdown glutamate dehydrogenase expression impairs transdeamination and gluconeogenesis in the liver

Glutamate dehydrogenase (GDH) plays a major role in amino acid catabolism. To increase the current knowledge of GDH function, we analysed the effect of GDH silencing on liver intermediary metabolism from gilthead sea bream (Sparus aurata). Sequencing of GDH cDNA from S. aurata revealed high homology with its vertebrate orthologues and allowed us to design short hairpin RNAs (shRNAs) to knockdown GDH expression. Following validation of shRNA-dependent downregulation of S. aurata GDH in vitro, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a selected shRNA (pCpG-sh2GDH) were produced to address the effect of GDH silencing on S. aurata liver metabolism. Seventy-two hours following intraperitoneal administration of chitosan-TPP-pCpG-sh2GDH, GDH mRNA levels and immunodetectable protein decreased in the liver, leading to reduced GDH activity in both oxidative and reductive reactions to about 53-55 % of control values. GDH silencing decreased glutamate, glutamine and aspartate aminotransferase activity, while increased 2-oxoglutarate content, 2-oxoglutarate dehydrogenase activity and 6-phosphofructo-1-kinase/fructose-1,6-bisphosphatase activity ratio. Our findings show for the first time that GDH silencing reduces transdeamination and gluconeogenesis in the liver, hindering the use of amino acids as gluconeogenic substrates and enabling protein sparing and metabolisation of dietary carbohydrates, which would reduce environmental impact and production costs of aquaculture

Gene markers of dietary macronutrient composition and growth in the skeletal muscle of gilthead sea bream (Sparus aurata)

To increase our current knowledge on the nutritional regulation of growth and gene expression pattern in fish skeletal muscle, the effect of dietary macronutrient composition was assessed on digestibility, nutrient retention, growth performance, and the mRNA levels of key genes involved in functionality, growth and development of the skeletal muscle in gilthead sea bream (Sparus aurata). Long-term starvation decreased the expression of myogenic regulatory factors such as Myod2, Myf5, myogenin (Myog) and Myf6 in the skeletal muscle of S. aurata. The supply of high or medium protein, low carbohydrate diets enhanced growth parameters, feed efficiency ratio, feed conversion ratio and significantly upregulated myod2. However, the supply of low protein, high carbohydrate diets restricted growth and stimulated the mRNA levels of myostatin, while downregulated follistatin (fst), igf1, mtor and rps6. Microarray analysis revealed igfals, tnni2, and gadd45a as gene markers upregulated by diets enriched with protein, lipids and carbohydrates, respectively. The results of the present study show that in addition to myod2, fst, igf1, mtor and rps6, the expression levels of igfals, tnni2 and remarkably gadd45a in the skeletal muscle can be used as markers to evaluate the effect of dietary macronutrient changes on fish growth and muscle development in S. aurata

A transcriptomic approach to study the effect of long-term starvation and diet composition on the expression of mitochondrial oxidative phosphorylation genes in gilthead sea bream (Sparus aurata)

Background: The impact of nutritional status and diet composition on mitochondrial oxidative phosphorylation (OXPHOS) in fish remains largely unknown. To identify biomarkers of interest in nutritional studies, herein we obtained a deep-coverage transcriptome by 454 pyrosequencing of liver and skeletal muscle cDNA normalised libraries from long-term starved gilthead sea bream (Sparus aurata) and fish fed different diets. Results: After clean-up of high-throughput deep sequencing reads, 699,991 and 555,031 high-quality reads allowed de novo assembly of liver and skeletal muscle sequences, respectively (average length: 374 and 441 bp; total megabases: 262 and 245 Mbp). An additional incremental assembly was completed by integrating data from both tissues (hybrid assembly). Assembly of hybrid, liver and skeletal muscle transcriptomes yielded, respectively, 19,530, 11,545 and 10,599 isotigs (average length: 1330, 1208 and 1390 bp, respectively) that were grouped into 15,954, 10,033 and 9189 isogroups. Following annotation, hybrid transcriptomic data were used to construct an oligonucleotide microarray to analyse nutritional regulation of the expression of 129 genes involved in OXPHOS in S. aurata. Starvation upregulated cytochrome c oxidase components and other key OXPHOS genes in the liver, which exhibited higher sensitive to food deprivation than the skeletal muscle. However, diet composition affected OXPHOS in the skeletal muscle to a greater extent than in the liver: most of genes upregulated under starvation presented higher expression among fish fed a high carbohydrate/low protein diet. Conclusions: Our findings indicate that the expression of coenzyme Q-binding protein (COQ10), cytochrome c oxidase subunit 6A2 (COX6A2) and ADP/ATP translocase 3 (SLC25A6) in the liver, and cytochrome c oxidase subunit 5B isoform 1 (COX5B1) in the liver and the skeletal muscle, are sensitive markers of the nutritional condition that may be relevant to assess the effect of changes in the feeding regime and diet composition on fish farming

The administration of chitosan-tripolyphosphate-DNA nanoparticles to express exogenous SREBP1a enhances conversion of dietary carbohydrates into lipids in the liver of Sparus aurata

In addition to being essential for the transcription of genes involved in cellular lipogenesis, increasing evidence associates sterol regulatory element binding proteins (SREBPs) with the transcriptional control of carbohydrate metabolism. The aim of this study was to assess the effect of overexpression SREBP1a, a potent activator of all SREBP-responsive genes, on the intermediary metabolism of Sparus aurata, a glucose-intolerant carnivorous fish. Administration of chitosan-tripolyphosphate nanoparticles complexed with a plasmid driving expression of the N-terminal transactivation domain of SREBP1a significantly increased SREBP1a mRNA and protein in the liver of S. aurata. Overexpression of SREBP1a enhanced the hepatic expression of key genes in glycolysis-gluconeogenesis (glucokinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), fatty acid synthesis (acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2), elongation (elongation of very long chain fatty acids protein 5) and desaturation (fatty acid desaturase 2) as well as reduced nicotinamide adenine dinucleotide phosphate production (glucose-6-phosphate 1-dehydrogenase) and cholesterol synthesis (3-hydroxy-3-methylglutaryl-coenzyme A reductase), leading to increased blood triglycerides and cholesterol levels. Beyond reporting the first study addressing in vivo effects of exogenous SREBP1a in a glucose-intolerant model, our findings support that SREBP1a overexpression caused multigenic effects that favoured hepatic glycolysis and lipogenesis and thus enabled protein sparing by improving dietary carbohydrate conversion into fatty acids and cholesterol

A transcriptomic approach to study the effect of long-term starvation and diet composition on the expression of mitochondrial oxidative phosphorylation genes in gilthead sea bream (Sparus aurata)

Abstract Background The impact of nutritional status and diet composition on mitochondrial oxidative phosphorylation (OXPHOS) in fish remains largely unknown. To identify biomarkers of interest in nutritional studies, herein we obtained a deep-coverage transcriptome by 454 pyrosequencing of liver and skeletal muscle cDNA normalised libraries from long-term starved gilthead sea bream (Sparus aurata) and fish fed different diets. Results After clean-up of high-throughput deep sequencing reads, 699,991 and 555,031 high-quality reads allowed de novo assembly of liver and skeletal muscle sequences, respectively (average length: 374 and 441 bp; total megabases: 262 and 245 Mbp). An additional incremental assembly was completed by integrating data from both tissues (hybrid assembly). Assembly of hybrid, liver and skeletal muscle transcriptomes yielded, respectively, 19,530, 11,545 and 10,599 isotigs (average length: 1330, 1208 and 1390 bp, respectively) that were grouped into 15,954, 10,033 and 9189 isogroups. Following annotation, hybrid transcriptomic data were used to construct an oligonucleotide microarray to analyse nutritional regulation of the expression of 129 genes involved in OXPHOS in S. aurata. Starvation upregulated cytochrome c oxidase components and other key OXPHOS genes in the liver, which exhibited higher sensitive to food deprivation than the skeletal muscle. However, diet composition affected OXPHOS in the skeletal muscle to a greater extent than in the liver: most of genes upregulated under starvation presented higher expression among fish fed a high carbohydrate/low protein diet. Conclusions Our findings indicate that the expression of coenzyme Q-binding protein (COQ10), cytochrome c oxidase subunit 6A2 (COX6A2) and ADP/ATP translocase 3 (SLC25A6) in the liver, and cytochrome c oxidase subunit 5B isoform 1 (COX5B1) in the liver and the skeletal muscle, are sensitive markers of the nutritional condition that may be relevant to assess the effect of changes in the feeding regime and diet composition on fish farming

Análisis transcriptómico para identificar genes biomarcadores del estado nutricional en la dorada (Sparus aurata): efecto de la sobreexpresión hepática de SREBP-1a

[spa] La acuicultura es una importante actividad económica vinculada a la producción de alimentos que se encuentra en etapa de expansión. Sin embargo, la producción de especies carnívoras como la dorada (Sparus aurata) presenta limitaciones, tales como la necesidad de incluir en los piensos altas concentraciones de proteína (incorporada en la forma de harina de pescado). Es por ello que debido a la importancia comercial que representa la dorada en la Unión Europea y particularmente en España, se han hecho grandes esfuerzos en investigación con el fin de entender los mecanismos moleculares encargados de regular el metabolismo intermediario de estos peces. En este sentido, son relevantes y cada vez más abundantes los estudios concernientes a los mecanismos de regulación génica como consecuencia del estado nutricional en peces. Gracias a las tecnologías de secuenciación de nueva generación (que permiten obtener resultados en un período corto de tiempo y a un costo significativamente inferior a los métodos tradicionales), en la última década son diversos los proyectos que han permitido el incremento de la información transcriptómica de dorada. A pesar de ello, ninguno de estos estudios se ha llevado a cabo sobre muestras de hígado, así como tampoco se han estudiado las variaciones consecuencia del estado nutricional y la composición de la dieta. El objetivo principal de esta tesis doctoral fue desarrollar un transcriptoma con muestras de hígado y músculo esquelético de cinco grupos de doradas alimentadas durante un período de 23 días, con piensos de distinta composición de macronutrientes (carbohidratos, proteínas o lípidos), así como de un sexto grupo de peces sometidos a ayuno por ese mismo período de tiempo. Este enfoque metodológico permitió caracterizar el transcriptoma de ambos tejidos, así como la obtención de una base de datos transcriptómicos de amplia cobertura y de interés para el desarrollo de estudios nutricionales. Se ensamblaron 21.093 secuencias únicas a partir de 660.166 y 547.544 lecturas de alta calidad de muestras de hígado y músculo esquelético respectivamente, en un transcriptoma que denominamos híbrido. Estas secuencias fueron utilizadas para el diseño de microarrays de oligonucleótidos que nos permitieron analizar cambios en el patrón de expresión génica como consecuencia del ayuno y la composición de la dieta, con el fin de identificar rutas metabólicas y genes biomarcadores relevantes del estado nutricional y la asimilación de nutrientes por parte de la dorada. Este análisis permitió establecer la importancia de la cadena respiratoria y la fosforilación oxidativa, así como del metabolismo lípidico, como consecuencia del estado nutricional y la composición de la dieta en estos peces. Mediante RT-qPCR se validaron los resultados obtenidos y se identificaron algunos genes biomarcadores, tales como algunos miembros del complejo IV de la cadena respiratoria, que incrementan su expresión en peces sometidos a un ayuno prolongado; o genes relacionados con la síntesis de ácidos grasos y colesterol que varían su expresión en relación con la composición de la dieta. Debido a la importancia que presenta el metabolismo de los lípidos en dorada y a la relación existente del factor de transcripción SREBP-1a tanto con el metabolismo de lípidos como con el metabolismo de carbohidratos, se estudió el efecto metabólico de la sobreexpresión de SREBP-1a en el hígado de doradas alimentadas con dietas de diferente composición. Para ello se administraron nanopartículas de quitosán-tripolifosfato acomplejadas con un plásmido de expresión del fragmento nuclear de SREBP-1a de hámster. El incremento de la expresión hepática de SREBP-1a promovió el aprovechamiento de los carbohidratos de la dieta mediante un incremento en la expresión de genes glucolíticos, así como la de genes relacionados con la síntesis de lípidos. Estos datos sugieren un incremento en la producción de lípidos aparejado a un mayor aprovechamiento de carbohidratos, lo que a su vez permitiría una disminución en la utilización de proteínas de la dieta en la acuicultura.[eng] Aquaculture is an economic activity linked to food production, which is currently an expanding activity. However, the production of carnivorous species such as Gilthead Sea Bream (Sparus aurata) is limited by inclusion of high levels of dietary proteins (fish meal) in feedstuffs. Due to the economic impact of S. aurata farming in the European Union and, in particular, in Spain, remarkable efforts are nowadays devoted to understand the molecular mechanisms that govern the intermediary metabolism of S. aurata. In this regard, studies addressing genetic regulation by the nutritional status in fish are of increasing relevance. The new generation sequencing technologies allow to obtain transcriptomic data in a shorter period of time and at a lower cost than traditional methods. Therefore, during the last decade a number of scientific projects increased transcriptomic data available for S. aurata. In spite of these studies, there are no data concerning the pattern of gene expression in the liver due to changes in the nutritional status and diet composition. The main goal of the present doctoral thesis was to obtain a transcriptome from liver and skeletal muscle samples from five groups of fish fed during 23 days with diets differing in macronutrient composition (carbohydrates, proteins or lipids) and a sixth group of fish subjected to fasting for that same period of time. This approach allowed us to obtain the liver and skeletal muscle transcriptomes of S. aurata and a deep-coverage database of nutritional interest. A total of 21,093 unique sequences were assembled from 660,166 and 547,544 high quality reads of liver and skeletal muscle samples, respectively, to obtain the herein named hybrid transcriptome. Unique sequences were used to design an oligonucleotide microarray that was subsequently used to analyze the pattern of gene expression in fish submitted to starvation and changes in diet composition in order to identify metabolic pathways and biomarker genes relevant for the nutritional status and nutrient utilization in S. aurata. We concluded that nutritional status and diet composition highly affect the respiratory chain, oxidative phosphorylation and lipid metabolism in S. aurata. Microarray data were validated by RT-qPCR, leading to the proposal of several key biomarkers genes, such as some members of complex IV of the respiratory chain, whose gene expression increases in fish subjected to prolonged fasting, and dependence on dietary macronutrient composition of the expression of genes involved in fatty acid and cholesterol biosynthesis. Given the relevance of lipid metabolism in S. aurata and the role exerted by the transcription factor SREBP-1a in the control of both lipid and carbohydrate metabolism, the metabolic effect of SREBP-1a overexpression was studied in the liver of S. aurata fed with different diets. To this end, chitosan-tripolyphosphate nanoparticles complexed with a plasmid expressing the nuclear fragment of hamster SREBP-1a were administered to S. aurata. Overexpression of SREBP-1a stimulated the expression of genes involved in glycolysis and lipid synthesis, suggesting the use of dietary carbohydrates coupled to lipid production, a mechanism that would enable a protein sparing effect in fish farming

Metformin counteracts glucose-dependent lipogenesis and impairs transdeamination in the liver of gilthead sea bream (Sparus aurata)

Metformin is an anti-diabetic drug with a major impact on regulating blood glucose levels by decreasing hepatic gluconeogenesis but also affecting other pathways, including glucose transport and energy/lipid metabolism. Carnivorous fish are considered glucose intolerant, as they exhibit poor ability to using dietary carbohydrates. To increase the current knowledge about the molecular mechanisms by which metformin can improve glucose homeostasis in carnivorous fish, we addressed the effect of intraperitoneal administration of metformin, in the presence or absence of a glucose load, on metabolic rate-limiting enzymes and lipogenic factors in the liver of gilthead sea bream (Sparus aurata). Hyperglycemia markedly up-regulated the expression of glycolytic enzymes (glucokinase and 6-phosphofructo-1-kinase, PFK1) 5 h following glucose administration, while at 24 h post-treatment it increased isocitrate dehydrogenase (IDH) activity, a key enzyme of the tricarboxylic acid cycle, and the expression of lipogenic factors (PGC1b, Lpin1 and SREBP1). Metformin counteracted glucose-dependent effects, and down-regulated glutamate dehydrogenase, alanine aminotransferase and mTOR 5 h post-treatment in the absence of a glucose load, leading to decreased long-term activity of PFK1 and IDH. The results of the present study suggest that hyperglycemia enhances lipogenesis in the liver of S. aurata, and that metformin may exert specific metabolic effects in fish by decreasing hepatic transdeamination and supressing the use of amino acids as gluconeogenic substrates. Our findings highlight the role of amino acid metabolism in the glucose-intolerant carnivorous fish model. KEYWORDS: Glutamate dehydrogenase; Lipogenesis; Liver; Metformin; Sparus aurat