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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.

Metformin is an antidiabetic drug with a major impact on regulating blood glucose levels by decreasing hepatic gluconeogenesis, but also by affecting other pathways, including glucose transport and energy/lipid metabolism. Carnivorous fish are considered glucose intolerant, as they exhibit poor ability in 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 upregulated the expression of glycolytic enzymes (glucokinase and 6-phosphofructo-1-kinase, PFK1) 5 h following glucose administration, while at 24 h posttreatment, it increased isocitrate dehydrogenase (IDH) activity, a key enzyme of the tricarboxylic acid cycle, and the expression of lipogenic factors (PGC1β, Lpin1, and SREBP1). Metformin counteracted glucose-dependent effects, and downregulated glutamate dehydrogenase, alanine aminotransferase, and mammalian target of rapamycin 5 h posttreatment 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 suppressing the use of amino acids as gluconeogenic substrates. Our findings highlight the role of amino acid metabolism in the glucose-intolerant carnivorous fish model.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In carnivorous fish, conversion of a glucose load to hepatic glycogen is widely used to assess their metabolic flexibility towards carbohydrate utilization, but the activities of direct and indirect pathways in this setting are unclear. We assessed the conversion of an intraperitoneal glucose load (2 g.kg -1 ) enriched with [U- 13 C 6 ]glucose to hepatic glycogen in juvenile seabass and seabream. 13 C-NMR analysis of glycogen was used to determine the contribution of the load to glycogen synthesis via direct and indirect pathways at 48-hr post-injection. For seabass, [U- 13 C 6 ]glucose was accompanied by deuterated water and 2 H-NMR analysis of glycogen 2 H-enrichment, allowing endogenous substrate contributions to be assessed as well. For fasted seabass and seabream, 47 ± 5% and 64 ± 10% of glycogen was synthesized from the load, respectively. Direct and indirect pathways contributed equally (25 ± 3% direct, 21 ± 1% indirect for seabass; 35 ± 7% direct, 29 ± 4% indirect for seabream). In fasted seabass, integration of 2 H- and 13 C-NMR analysis indicated that endogenous glycerol and anaplerotic substrates contributed an additional 7 ± 2% and 7 ± 1%, respectively. In fed seabass, glucose load contributions were residual and endogenous contributions were negligible. Concluding, direct and indirect pathways contributed equally and substantially to fasting hepatic glycogen repletion from a glucose load in juvenile seabream and seabass.

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.

Endocrine factors released from the central nervous system, gastrointestinal tract, adipose tissue and other peripheral organs mediate the regulation of food intake. Although many studies have evaluated the effect of fed-to-starved transition on the expression of appetite-related genes, little is known about how the expression of appetite-regulating peptides is regulated by the macronutrient composition of the diet. The aim of the present study was to examine the effect of diet composition and nutritional status on the expression of four peptides involved in food intake control in gilthead sea bream (Sparus aurata): neuropeptide Y (NPY), ghrelin, cholecystokinin (CCK) and leptin. Quantitative real-time RT-PCR showed that high protein/low carbohydrate diets stimulated the expression of CCK and ghrelin in the intestine and leptin in the adipose tissue, while downregulation of ghrelin and NPY mRNA levels was observed in the brain. Opposite effects were found for the expression of the four genes in fish fed low protein/high carbohydrate diets or after long-term starvation. Our findings indicate that the expression pattern of appetite-regulating peptides, particularly CCK and ghrelin, is modulated by the nutritional status and diet composition in S. aurata., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

In liver, through the reaction catalysed by alanine aminotransferase (ALT), alanine becomes an effective precursor for gluconeogenesis. In the present study amino-oxyacetate (AOA) was used to evaluate its effect on liver ALT activity of the carnivorous fish Sparus aurata. Moreover, the derived metabolic effects on metabolites and other key enzymes of glycolysis, gluconeogenesis and the pentose phosphate pathway were also studied. A dose-effect-dependent inhibition of AOA on hepatic cytosolic and mitochondrial ALT activity was observed in vitro. In vivo, AOA behaved as an inhibitor of hepatic cytosolic ALT activity. A long-term exposure to AOA increased pyruvate kinase activity in the liver irrespective of the composition of the diet supplied to fish. 1H NMR studies showed that inclusion of AOA to the diet decreased the hepatic levels of alanine, glutamate and glycogen. Moreover, 2H NMR analysis indicated a higher renewal rate for alanine in the liver of fish fed with a high-carbohydrate/low-protein diet, while AOA decreased alanine 2H-enrichment irrespective of the diet. The present study indicates that AOA-dependent inhibition of the cytosolic ALT activity could help to increase the use of dietary carbohydrate nutrients.

Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in the kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5'-Deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to the downregulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver, and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L: -alanine to pyruvate than the reverse reaction. From these results, we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.

Alanine aminotransferase (Alt) provides a molecular link between carbohydrate and amino acid metabolism. In the cell context, the predominant Alt isozyme is located in the cytosol. To gain insight into the transcriptional regulation of the cytosolic alt gene (calt), we cloned and characterized the calt promoter from gilthead sea bream (Sparus aurata). Transient transfection of sea bass larvae cells with deleted calt promoter constructs and electrophoretic mobility shift assays allowed us to identify p300 and c-Myb as new factors in the transcriptional regulation of calt expression. Transfection studies carried out with an acetylase-deficient mutant p300 (p300DY) revealed that the acetyltransferase activity of p300 is essential for the p300-mediated transcriptional activation of S. aurata calt. We had previously found up-regulation of liver cAlt2, an alternatively spliced isoform of calt, under gluconeogenic conditions and in streptozotocin (STZ)-treated S. aurata. Quantitative RT-PCR assays showed that increased p300 and c-Myb mRNA levels in the liver of starved S. aurata contribute to enhancing the transcription of cAlt2. Consistently, the administration of insulin decreased both p300 and c-Myb expression. The mRNA levels of p300 and c-Myb were also analyzed in the liver of STZ-induced diabetic S. aurata. Treatment with STZ increased the expression of p300, whereas it decreased c-Myb. Our findings suggest an involvement of p300 and c-Myb in up-regulation of cAlt2 in the liver of S. aurata under starvation. In addition, these results provide evidence for a role of p300 in diabetes.

6-phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P2) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P2 counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P2, whereas combination of activation by fru-2,6-P2 and increase in protein content may determine the long-term recovery of the enzyme activity.

Insulin induction of glucokinase (GCK) transcription in the liver is essential for maintaining glucose homeostasis. To study the molecular mechanism underlying the regulation of hepatic GCK expression in the carnivorous fish gilthead sea bream (Sparus aurata), we analysed the role of sterol regulatory element binding protein-1a (SREBP-1a) and specificity protein (Sp) 1 in insulin-dependent GCK transcription. Transient transfection experiments performed in HepG2 cells and electrophoretic mobility shift assays allowed us to identify a cis-element in the proximal region of GCK promoter implicated in transactivation by SREBP-1a. Consistently, mutations in the SRE binding site completely abolished the enhancing effect of SREBP-1a. These results and previous findings suggest that SREBP-1a plays a role in the transcriptional regulation of key enzymes in glycolysis-gluconeogenesis. Since SREBP-1a and Sp1 may mediate insulin action on S. aurata GCK transcription, we analysed the effect of insulin on HepG2 cells transfected with GCK promoter reporter constructs carrying intact or mutated SRE or Sp boxes. Insulin transactivated GCK irrespective of the presence of an intact or mutated SRE box. However, insulin failed to induce GCK transcription when using reporter constructs that had either a mutated Sp site or no Sp site. Our findings indicate that Sp1, rather than SREBP-1a, mediates the insulin-dependent induction of S. aurata GCK.

Increased alanine aminotransferase (ALT) activity is associated with insulin resistance and the development of type 2 diabetes. The aim of this study was to characterize the modulation of cytosolic ALT expression in liver of gilthead sea bream (Sparus aurata) under conditions associated with increased gluconeogenesis and in streptozotocin (STZ)-treated fish. RT- and RACE-PCR assays allowed us to isolate a novel ALT isozyme (cALT2) generated from alternative splicing of cALT gene in S. aurata. HEK293 cells transfected with constructs expressing cALT2 as a C-terminal fusion with the enhanced green fluorescent protein allowed us to demonstrate that cALT2 is cytosolic. To unravel the molecular functions of cALT1 and cALT2 in liver of S. aurata, we examined tissue distribution, kinetic characterization of piscine cALT isozymes expressed in Saccharomyces cerevisiae, and regulation of hepatic cALT1 and cALT2 expression in various metabolic conditions. Kinetic analysis indicates that cALT2 is more efficient in catalysing the conversion of l-alanine to pyruvate than cALT1. Starvation increased cALT2 expression and decreased cALT1 mRNA in liver. Opposite effects were found in regularly fed fish at postprandial time 4-8h, and 6h after treatment with glucose or insulin. From these results we conclude that increased cALT2 expression occurred in liver under gluconeogenic conditions, while cALT1 was predominant during postprandial utilization of dietary nutrients. Since up-regulation of hepatic cALT2 expression occurred in STZ-induced diabetic S. aurata, increased hepatic cALT2 expression may be a promising marker in the prognosis of diabetes.

The kinetic properties of 6-phosphofructo-1-kinase (PFK) from skeletal muscle (PFKM) of gilthead sea bream (Sparus aurata) were studied, after 10,900-fold purification to homogeneity. The native enzyme had an apparent molecular mass of 662 kDa and is composed of 81 kDa subunits, suggesting a homooctameric structure. At physiological pH, S. aurata PFKM exhibited sigmoidal kinetics for the substrates, fructose-6-phosphate (fru-6-P) and ATP. Fructose-2,6-bisphosphate (fru-2,6-P(2)) converted the saturation curves for fru-6-P to hyperbolic, activated PFKM synergistically with other positive effectors of the enzyme such as AMP and ADP, and counteracted ATP and citrate inhibition. The fish enzyme showed differences regarding other animal PFKs: it is active as a homooctamer, and fru-2,6-P(2) and pH affected affinity for ATP. By monitoring incorporation of (32)P from ATP, we show that fish PFKM is a substrate for the cAMP-dependent protein kinase. The mechanism involved in PFKM activation by phosphorylation contrasts with previous observations in other species: it increased V(max) and did not affect affinity for fru-6-P. Unlike the mammalian muscle enzyme, our findings support that phosphorylation of PFKM may exert a major role during starvation in fish muscle.

To examine the relationship between structure and function of glucose-6-phosphatase (G6Pase) in fish, we undertook molecular cloning and modulation of G6Pase expression by starvation and refeeding on diets with different nutrient composition in the liver of the carnivorous fish, Sparus aurata. A cDNA encoding the full-length G6Pase catalytic subunit from the liver of S. aurata was isolated. This cDNA encodes a 350-amino acid protein, with low homology to the mammalian G6Pase, although it contains most of the key residues required for catalysis. Based on hydrophobicity and membrane structure prediction, we propose a model containing nine-transmembrane regions for S. aurata G6Pase. Northern blots showed that refeeding after a prolonged starvation rapidly reverses the glucose/glucose-6-phosphate substrate cycle flux in the fish liver through decreased G6Pase expression and strong glucokinase (GK) induction. The effect of refeeding different diets on G6Pase and GK expression, indicated that hepatic intermediary metabolism of fish fed diets with low protein/high carbohydrate diets is impelled towards utilization of dietary carbohydrates, by means of modulation of GK mRNA levels rather than G6Pase expression. These findings challenge the role attributed to dysregulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolise glucose.