Ghrelin is involved in the regulation of growth in vertebrates through controlling different functions, such as feed intake, metabolism, intestinal activity or growth hormone (Gh) secretion. The aim ...of this work was to identify the sequences of preproghrelin and Ghrelin receptors (ghsrs), and to study their responses to different nutritional conditions in gilthead sea bream (Sparus aurata) juveniles. The structure and phylogeny of S. aurata preproghrelin was analyzed, and a tissue screening was performed. The effects of 21 days of fasting and 2, 5, 24 h, and 7 days of refeeding on plasma levels of Ghrelin, Gh and Igf-1, and the gene expression of preproghrelin, ghsrs and members of the Gh/Igf-1 system were determined in key tissues. preproghrelin and the receptors are well conserved, being expressed mainly in stomach, and in the pituitary and brain, respectively. Twenty-one days of fasting resulted in a decrease in growth while Ghrelin plasma levels were elevated to decrease at 5 h post-prandial when pituitary ghsrs expression was minimum. Gh in plasma increased during fasting and slowly felt upon refeeding, while plasma Igf-1 showed an inverse profile. Pituitary gh expression augmented during fasting reaching maximum levels at 1 day post-feeding while liver igf-1 expression and that of its splice variants decreased to lowest levels. Liver Gh receptors expression was down-regulated during fasting and recovered after refeeding. This study demonstrates the important role of Ghrelin during fasting, its acute down-regulation in the post-prandial stage and its interaction with pituitary Ghsrs and Gh/Igf-1 axis.
The tolerance of fish to fasting offers a model to study the regulatory mechanisms and changes produced when feeding is restored. Gilthead sea bream juveniles were exposed to a 21-days fasting period ...followed by 2 h to 7-days refeeding. Fasting provoked a decrease in body weight, somatic indexes, and muscle gene expression of members of the Gh/Igf system, signaling molecules (akt, tor and downstream effectors), proliferation marker pcna, myogenic regulatory factors, myostatin, and proteolytic molecules such as cathepsins or calpains, while most ubiquitin-proteasome system members increased or remained stable. In bone, downregulated expression of Gh/Igf members and osteogenic factors was observed, whereas expression of the osteoclastic marker ctsk was increased. Refeeding recovered the expression of Gh/Igf system, myogenic and osteogenic factors in a sequence similar to that of development. Akt and Tor phosphorylation raised at 2 and 5 h post-refeeding, much faster than its gene expression increased, which occurred at day 7. The expression in bone and muscle of the inhibitor myostatin (mstn2) showed an inverse profile suggesting an inter-organ coordination that needs to be further explored in fish. Overall, this study provides new information on the molecules involved in the musculoskeletal system remodeling during the early stages of refeeding in fish.
World population is expected to increase to approximately 9 thousand million people by 2050 with a consequent food security decline. Besides, climate change is a major challenge that humanity is ...facing, with a predicted rise in mean sea surface temperature of more than 2°C during this century. This study aims to determine whether a rearing temperature of 19, 24, or 28°C may influence musculoskeletal development and muscle lipid metabolism in gilthead sea bream juveniles. The expression of growth hormone (GH)/insulin-like growth factors (IGFs) system-, osteogenic-, myogenic-, and lipid metabolism-related genes in bone and/or white muscle of treated fish, and the
viability, mineralization, and osteogenic genes expression in primary cultured cells derived from bone of the same fish were analyzed. The highest temperature significantly down-regulated
, the receptor
, and the binding proteins
and
in bone, and in muscle,
and
, suggesting impaired musculoskeletal development. Concerning myogenic factors expression, contrary responses were observed, since the increase to 24°C significantly down-regulated
and
, while at 28°C
and
were significantly up-regulated. Moreover, in the muscle tissue, the expression of the fatty acid transporters
and
, and the lipases
and
resulted significantly increased at elevated temperatures, whereas β-oxidation markers
and
were significantly reduced. Regarding the primary cultured bone-derived cells, a significant up-regulation of the extracellular matrix proteins
, and
expression was found with increased temperatures, together with a gradual decrease in mineralization along with fish rearing temperature. Overall, these results suggest that increasing water temperature in this species appears to induce unfavorable growth and development of bone and muscle, through modulating the expression of different members of the GH/IGFs axis, myogenic and osteogenic genes, while accelerating the utilization of lipids as an energy source, although less efficiently than at optimal temperatures.
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.
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.
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.
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.
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.
During the last decades, adipogenesis has become an emerging field of study in aquaculture due to the relevance of the adipose tissue in many physiological processes and its connection with the ...endocrine system. In this sense, recent studies have translated into the establishment of preadipocyte culture models from several fish species, sometimes lacking information on the mRNA levels of adipogenic genes. Thus, the aim of this study was to determine the gene expression profile of gilthead sea bream (Sparus aurata) primary cultured mesenchymal stem cells (MSCs) from different origin (adipose tissue and vertebra bone) during adipogenesis. Both cell types differentiated into adipocyte-like cells, accumulating lipids inside their cytoplasm. Adipocyte differentiation of MSCs from adipose tissue resulted in downregulation of several adipocyte-related genes (such as lpl, hsl, pparα, pparγ and gapdh2) at day 4, gapdh1 at day 8, and fas and pparβ at day 12. In contrast, differences in lxrα mRNA expression were not observed, while g6pdh levels increased during adipocyte maturation. Gapdh and Pparγ protein levels were also detected in preadipocyte cultures; however, only the former increased its expression during adipogenesis. Moreover, differentiation of bone-derived cells into adipocytes also resulted in the downregulation of several adipocyte gene markers, such as fas and g6pdh at day 10 and hsl, pparβ, and lxrα at day 15. On the other hand, the osteogenic genes fib1a, mgp, and op remained stable, but an increase in runx2 expression at day 20 was observed. In summary, the present study demonstrates that gilthead sea bream MSCs, from both adipose tissue and bone, differentiate into adipocyte-like cells, although revealed some kind of species- and cell lineage-specific regulation with regards to gene expression. Present data also provide novel insights into some of the potential key genes controlling adipogenesis in gilthead sea bream that can help to better understand the regulation of lipid storage in fish.
Calpains are non-lysosomal calcium-activated neutral proteases involved in a wide range of cellular processes including muscle proteolysis linked to post-mortem flesh softening. The aims of this ...study were (a) to characterise several members of the calpain system in gilthead sea bream and (b) to examine their expression in relation to nutritional status and muscle tenderisation. We identified the complete open reading frame of gilthead sea bream calpains1-3, sacapn1, sacapn2, sacapn3, and two paralogs of the calpain small subunit1, sacapns1a and sacapns1b. Proteins showed 63-90% sequence identity compared with sequences from mammals and other teleost fishes, and the characteristic domain structure of vertebrate calpains. Transcripts of sacapn1, sacapn2, sacapns1a and sacapns1b had a wide tissue distribution, whereas sacapn3 was almost exclusively detected in skeletal muscle. Next, we assessed transcript expression in skeletal muscle following alteration of nutritional status by (a) fasting and re-feeding or (b) feeding four experimental diets with different carbohydrate-to-protein ratios. Fasting significantly reduced plasma glucose and increased free fatty acids and triglycerides, together with a significant increase in sacapns1b expression. Following 7 days of re-feeding, plasma parameters returned to fed values and sacapn1, sacapn2, sacapns1a and sacapns1b expression was significantly reduced. Furthermore, an increase in dietary carbohydrate content (11 to 39%) diminished growth but increased muscle texture, which showed a significant correlation with decreased sacapn1 and sacapns1a expression, whilst the other calpains remained unaffected. This study has demonstrated that calpain expression is modulated by nutritional status and diet composition in gilthead sea bream, and that the expression of several calpain members is correlated with muscle texture, indicating their potential use as molecular markers for flesh quality in aquaculture production.
Ghrelin is involved in the regulation of growth in vertebrates through controlling different functions, such as feed intake, metabolism, intestinal activity or growth hormone (Gh) secretion. The aim ...of this work was to identify the sequences of
and Ghrelin receptors (
), and to study their responses to different nutritional conditions in gilthead sea bream (
) juveniles. The structure and phylogeny of
was analyzed, and a tissue screening was performed. The effects of 21 days of fasting and 2, 5, 24 h, and 7 days of refeeding on plasma levels of Ghrelin, Gh and Igf-1, and the gene expression of
and members of the Gh/Igf-1 system were determined in key tissues.
and the receptors are well conserved, being expressed mainly in stomach, and in the pituitary and brain, respectively. Twenty-one days of fasting resulted in a decrease in growth while Ghrelin plasma levels were elevated to decrease at 5 h post-prandial when pituitary
expression was minimum. Gh in plasma increased during fasting and slowly felt upon refeeding, while plasma Igf-1 showed an inverse profile. Pituitary
expression augmented during fasting reaching maximum levels at 1 day post-feeding while liver
expression and that of its splice variants decreased to lowest levels. Liver Gh receptors expression was down-regulated during fasting and recovered after refeeding. This study demonstrates the important role of Ghrelin during fasting, its acute down-regulation in the post-prandial stage and its interaction with pituitary Ghsrs and Gh/Igf-1 axis.
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.
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