Dietary cobalt supplementation improves growth and body composition and induces the expression of growth and stress response genes in Tor putitora

A 90-day randomized feeding experiment was performed to assess the effects of dietary cobalt (Co) supplementation on the growth performance, muscle composition, status of iron and manganese in the muscle as well as the expression of growth-related genes in the muscle (myoblast determination protein 1 homolog (MyoD) and myogenin) and the stress-related gene heat shock protein 70 KDa (Hsp-70) in the liver of mahseer (Tor putitora). Feeding trial was conducted in triplicate under controlled semi-static conditions, and graded levels of dietary cobalt (0.5-3 mg/kg) were fed to six groups of advanced fry of T. putitora. The results obtained indicated a curvilinear relationship of dietary Co levels with body crude protein content and weight gain (%). A positive correlation was observed with up to 2 mg Co/kg diet. However, a decreasing trend was found with values over 2 mg Co/kg diet. The expression of muscle growth biomarkers MyoD and myogenin showed a similar response, upregulation up to 2 mg Co/kg diet and decreased expression at 3 mg Co/kg diet. Indeed, the highest dietary Co supplementation increased the expression of Hsp-70, a key gene expressed in response to stress. Moreover, the muscle content of iron and manganese showed an inverse relationship with the dietary Co supplementation. Our findings suggest that 2 mg/kg Co dietary supplementation stimulates myogenesis and optimize muscle growth and body composition, while higher levels enhanced the expression of stress response genes and impaired growth of T. putitora

Chitosan-Based Drug Delivery System: Applications in Fish Biotechnology

Chitosan is increasingly used for safe nucleic acid delivery in gene therapy studies, due to well-known properties such as bioadhesion, low toxicity, biodegradability and biocompatibility. Furthermore, chitosan derivatization can be easily performed to improve the solubility and stability of chitosan-nucleic acid polyplexes, and enhance e cient target cell drug delivery, cell uptake, intracellular endosomal escape, unpacking and nuclear import of expression plasmids. As in other fields, chitosan is a promising drug delivery vector with great potential for the fish farming industry. This review highlights state-of-the-art assays using chitosan-based methodologies for delivering nucleic acids into cells, and focuses attention on recent advances in chitosan-mediated gene delivery for fish biotechnology applications. The e ciency of chitosan for gene therapy studies in fish biotechnology is discussed in fields such as fish vaccination against bacterial and viral infection, control of gonadal development and gene overexpression and silencing for overcoming metabolic limitations, such as dependence on protein-rich diets and the low glucose tolerance of farmed fish. Finally, challenges and perspectives on the future developments of chitosan-based gene delivery in fish are also discussed

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 cholesterolPeer ReviewedPostprint (published version

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

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

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

Chitosan-Based Sustained Expression of Sterol Regulatory Element-Binding Protein 1a Stimulates Hepatic Glucose Oxidation and Growth in <i>Sparus aurata</i>

The administration of a single dose of chitosan nanoparticles driving the expression of sterol regulatory element-binding protein 1a (SREBP1a) was recently associated with the enhanced conversion of carbohydrates into lipids. To address the effects of the long-lasting expression of SREBP1a on the growth and liver intermediary metabolism of carnivorous fish, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid expressing the N terminal active domain of hamster SREBP1a (pSG5-SREBP1a) were injected intraperitoneally every 4 weeks (three doses in total) to gilthead sea bream (Sparus aurata) fed high-protein–low-carbohydrate and low-protein–high-carbohydrate diets. Following 70 days of treatment, chitosan-TPP-pSG5-SREBP1a nanoparticles led to the sustained upregulation of SREBP1a in the liver of S. aurata. Independently of the diet, SREBP1a overexpression significantly increased their weight gain, specific growth rate, and protein efficiency ratio but decreased their feed conversion ratio. In agreement with an improved conversion of dietary carbohydrates into lipids, SREBP1a expression increased serum triglycerides and cholesterol as well as hepatic glucose oxidation via glycolysis and the pentose phosphate pathway, while not affecting gluconeogenesis and transamination. Our findings support that the periodical administration of chitosan-TPP-DNA nanoparticles to overexpress SREBP1a in the liver enhanced the growth performance of S. aurata through a mechanism that enabled protein sparing by enhancing dietary carbohydrate metabolisation

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 cholesterolPeer Reviewe

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

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