Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of Sparus aurata

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish

Alanine aminotransferase: A target to improve utilisation of dietary nutrients in aquaculture

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/67430Alanine aminotransferase (ALT) is a sensitive marker of dietary protein utilisation in fish. Three ALT isoforms (cALT1, cALT2 and mALT) encoded by two genes have been isolated from gilthead sea bream (Sparus aurata). Molecular characterization of ALT isozymes and gene promoters suggest involvement of cALT1 and mALT in postprandial use of dietary amino acids, while cALT2 seems associated to hepatic gluconeogenesis. Inhibition of hepatic cytosolic ALT activity stimulates pyruvate kinase and decreases the renewal rate of alanine in S. aurata. These findings point to ALT as a target to spare protein and improve catabolism of dietary carbohydrates in cultured fish

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

Incorporación de un caso clínico transversal en el Grado de Farmacia. valoración de la experiencia docente en la asignatura de Bioquímica

Introducción. Para implementar la transversalidad docente y evitar así la compartimentación de los conocimientos adquiridos por los estudiantes, se ha iniciado un proyecto docente coordinado y multidisciplinar basado en el desarrollo de un caso clínico sobre el consumo de alcohol. Éste se tratará en diferentes asignaturas del grado. El trabajo presenta las actuaciones realizadas y los resultados obtenidos por profesores que imparten la asignatura de Bioquímica. Material y métodos. Los alumnos de primer curso fueron convocados a una sesión informativa del caso y a un seminario de Bioquímica donde se les presentó un personaje ficticio, Sam, que a los 20 años es un bebedor de riesgo y que a lo largo de su vida va a desarrollar diferentes patologías derivadas de este consumo. También se expusieron los contenidos específicos sobre el metabolismo del alcohol. Los mismos estudiantes, ya en segundo curso, respondieron dos cuestionarios de cinco ítems sobre el seminario, uno de conocimientos y otro de opinión del caso. Resultados. La asistencia al seminario incrementó la puntuación de los ítems de conocimientos 2 y 5 respecto a la no asistencia. Se observó una baja puntuación para el ítem 1 en ambos grupos. Los ítems de opinión 4 y 5 fueron significativamente los más valorados por los alumnos, con puntuaciones medias de 4,23 ± 0,08 y 4,18 ± 0,08 sobre 5, respectivamente. Conclusiones. Los alumnos valoraron positivamente la introducción de casos clínicos en primer curso y su desarrollo de forma transversal. La asistencia al seminario mejoró la nota global de conocimientos de los estudiantes

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

Nutritional regulation of glucose-6-phosphatase gene expression in liver of the gilthead sea bream (Sparus aurata)

To examine the role of glucose-6-phosphatase (G6Pase) in glucose homeostasis in the diabeteslike experimental model of carnivorous fish, we analysed postprandial variations and the effect of starvation, ration size and diet composition on the regulation of G6Pase expression at the enzyme activity and mRNA level in the liver of gilthead sea bream (Sparus aurata ). G6Pase expression increased in long-term starved or energy-restricted fish. In contrast to data reported for other fish species, short-term regulation of G6Pase expression was found in regularly fed S. aurata. G6Pase mRNA levels were lowest between 4 and 15 h after food intake, whereas minimal enzyme activity was observed 10-15 h postprandially. Alterations of plasma glucose levels affect G6Pase in mammals. However, the carbohydrate content of the diet did not affect hepatic expression of G6Pase in S. aurata, suggesting that a different molecular mechanism is involved in the control of G6Pase expression in fish. Although G6Pase was unaffected, highcarbohydrate low-protein diets increased glucokinase (GK) expression and thus allowed a metabolic adaptation favouring glycolysis over gluconeogenesis. Interestingly, only the nutritional conditions that promoted variations in the blood glucose levels resulted in changes in the hepatic expression of G6Pase. These findings indicate a concerted regulation of G6Pase and GK expression and suggest that the direction and rate of the glucose-glucose-6-phosphate substrate cycle flux is finely regulated in the liver of S. aurata, challenging the role attributed to deficient regulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolize glucose

Sterol regulatory element binding protein-1a transactivates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene promoter

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB)catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a)binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription

Sterol regulatory element binding protein-1a transactivates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene promoter

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB)catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a)binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription

Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of Sparus aurata

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish