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When compared with fish meal and fish oil, plant ingredients differ not only in their protein content and amino acid and fatty acid profiles but are also devoid of cholesterol, the major component of cell membrane and precursor of several bioactive compounds. Based on these nutritional characteristics, plant-based diets can affect fish physiology and cholesterol metabolism. To investigate the mechanisms underlying cholesterol homeostasis, rainbow trout were fed from 1 g body wt for 6 mo with a totally plant-based diet (V), a marine diet (M), and a marine-restricted diet (MR), with feed intake adjusted to that of the V group. The expression of genes involved in cholesterol synthesis, esterification, excretion, bile acid synthesis, and cholesterol efflux was measured in liver. Results showed that genes involved in cholesterol synthesis were upregulated in trout fed the V diet, whereas expression of genes related to bile acid synthesis ( cyp7a1) and cholesterol elimination ( abcg8) were reduced. Feeding trout the V diet also enhanced the expression of srebp-2 while reducing that of lxrα and miR-223. Overall, these data suggested that rainbow trout coped with the altered nutritional characteristics and absence of dietary cholesterol supply by increasing cholesterol synthesis and limiting cholesterol efflux through molecular mechanisms involving at least srebp-2, lxrα, and miR-223. However, plasma and body cholesterol levels in trout fed the V diet were lower than in fish fed the M diet, raising the question of the role of cholesterol in the negative effect of plant-based diet on growth.

Our aim was to investigate the potential role of TOR (target of rapamycin) signaling pathway in the regulation of hepatic glucose metabolism in rainbow trout. Fasted fish were first treated with a single intraperitoneal injection of rapamycin or vehicle and then submitted to a second intraperitoneal administration of glucose 4 h later. Our results revealed that intraperitoneal administration of glucose induced hyperglycemia for both vehicle and rapamycin treatments, which peaked at 2 h. Plasma glucose level in vehicle-treated fish was significantly higher than in rapamycin-treated fish at 8 and 17 h, whereas it remained at the basal level in rapamycin-treated fish. Glucose administration significantly enhanced the phosphorylation of Akt and ribosomal protein S6 kinase (S6K1) in vehicle-treated fish, while rapamycin completely abolished the activation of S6K1 in rapamycin-treated fish, without inhibiting the phosphorylation of Akt on Thr-308 or Ser-473. Despite the lack of significant variation in phosphoenolpyruvate carboxykinase mRNA abundance, mRNA abundance for glucokinase (GK), glucose 6-phosphatase (G6Pase) I and II, and fructose 1,6-bisphosphatase (FBPase) was reduced by rapamycin 17 h after glucose administration. The inhibition effect of rapamycin on GK and FBPase was further substantiated at the activity level. The suppression of GK gene expression and activity by rapamycin provided the first in vivo evidence in fish that glucose regulates hepatic GK gene expression and activity through a TORC1-dependent manner. Unlike in mammals, we observed that acute rapamycin treatment improved glucose tolerance through the inhibition of hepatic gluconeogenesis in rainbow trout., (Copyright © 2014 the American Physiological Society.)

Methionine is a limiting essential amino acid in most plant-based ingredients of fish feed. In the present study, we aimed to determine the effect of dietary methionine concentrations on several main factors involved in the regulation of mRNA translation and the two major proteolytic pathways (ubiquitin-proteasome and autophagy-lysosomal) in the white muscle of rainbow trout (Oncorhynchus mykiss). The fish were fed for 6 weeks one of the three isonitrogenous diets providing three different methionine concentrations (deficient (DEF), adequate (ADQ) and excess (EXC)). At the end of the experiment, the fish fed the DEF diet had a significantly lower body weight and feed efficiency compared with those fed the EXC and ADQ diets. This reduction in the growth of fish fed the DEF diet was accompanied by a decrease in the activation of the translation initiation factors ribosomal protein S6 and eIF2α. The levels of the main autophagy-related markers (LC3-II and beclin 1) as well as the expression of several autophagy genes (atg4b, atg12 l, Uvrag, SQSTM1, Mul1 and Bnip3) were higher in the white muscle of fish fed the DEF diet. Similarly, the mRNA levels of several proteasome-related genes (Fbx32, MuRF2, MuRF3, ZNF216 and Trim32) were significantly up-regulated by methionine limitation. Together, these results extend our understanding of mechanisms regulating the reduction of muscle growth induced by dietary methionine deficiency, providing valuable information on the biomarkers of the effects of low-fishmeal diets.

Glucose and lipid metabolism in pancreatic islet organs is poorly characterized. In the present study, using as a model the carnivorous rainbow trout, a glucose-intolerant fish, we assessed mRNA expression levels of several genes involved in glucose and lipid metabolism (including ATP-citrate lyase; carnitine palmitoyltransferase-1 isoforms, CPT; the mitochondrial isoform of the phosphoenolpyrutave carboxykinase, mPEPCK and pyruvate kinase, PK) and glucosensing (glucose transporter type 2, Glut2; glucokinase, GK and the potassium channel, K(ATP)) in Brockmann bodies. We evaluated the response of these parameters to changes in feeding status (food deprived vs. fed fish) as well as to changes in the amount of carbohydrate (dextrin) in the diet. A general inhibition of the glycolytic (including the glucosensing marker GK) and β-oxidation pathways was found when comparing fed versus food-deprived fish. When comparing fish feeding on either low- or high-carbohydrate diets, we found that some genes related to lipid metabolism were more controlled by the feeding status than by the carbohydrate content (fatty acid synthase, CPTs). Findings are discussed in the context of pancreatic regulation of glucose and lipid metabolism in fish, and show that while trout pancreatic metabolism can partially adapt to a high-carbohydrate diet, some of the molecular actors studied seem to be poorly regulated (K(ATP)) and may contribute to the glucose intolerance observed in this species when fed high-carbohydrate diets.

In this study, the effect of acute and chronic insulin treatments on major factors involved in the control of muscle protein turnover were investigated in rainbow trout (Oncorhynchus mykiss). We found that acute but not chronic insulin administration leads to the induction of the phosphorylation of several key factors (IRS1, TOR and 4E-BP1) involved in the control of the protein synthesis and to the concomitant down-regulation of the expression of ubiquitin-proteasome-related genes (atrogin1, C2, C9) and the calpains inhibitor calpastatin. In contrast, no modification of autophagy-related gene (LC3B, gabarpl1, atg4b) expressions was observed suggesting that the mechanisms controlling this proteolytic route have diverged throughout the evolution. Overall, these results provide a possible explanation of the growth-promoting properties of insulin previously described in fish and indicate that this hormone acutely administrated is able to exert a regulatory influence on various factors associated with growth in skeletal muscle., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Most teleost fish are known to require high levels of dietary proteins. Such high-protein intake could have significant effects, particularly on insulin-regulated gene expression. We therefore analyzed the effects of an increase in the ratio of dietary carbohydrates/proteins on the refeeding activation of the Akt-target of rapamycin (TOR) signaling pathways in rainbow trout and the effects on the expression of several genes related to hepatic and muscle metabolism and known to be regulated by insulin, amino acids, and/or glucose. Fish were fed once one of three experimental diets containing high (H), medium (M), or low (L) protein (P) or carbohydrate (C) levels after 48 h of feed deprivation. Activation of the Akt/TOR signaling pathway by refeeding was severely impaired by decreasing the proteins-to-carbohydrates ratio. Similarly, postprandial regulation of several genes related to glucose (Glut4, glucose-6-phosphatase isoform 1), lipid (fatty acid synthase, ATP-citrate lyase, sterol responsive element binding protein, carnitine palmitoyltransferase 1, and 3-hydroxyacyl-CoA dehydrogenase), and amino acid metabolism (serine dehydratase and branched-chain α-keto acid dehydrogenase E2 subunit) only occurred when fish were fed the high-protein diet. On the other hand, diet composition had a low impact on the expression of genes related to muscle protein degradation. Interestingly, glucokinase was the only gene of those monitored whose expression was significantly upregulated by increased carbohydrate intake. In conclusion, this study demonstrated that macro-nutrient composition of the diet strongly affected the insulin/amino acids signaling pathway and expression pattern of genes related to metabolism.

The origin for the poor glucose utilization in carnivorous fish species fed high carbohydrate diets remains under debate. In the present study, we have fed rainbow trout a diet containing 30% carbohydrate for 1 or 5 days. In both cases, fish were implanted with mini-osmotic pumps releasing 0.7 i.u. kg(-1) day(-1) bovine insulin, and mRNA transcripts and the protein phosphorylation status of proteins controlling glycemia and glucose-related metabolism were studied in fish killed 6 h after the last meal. We demonstrate that when the exposure occurs over a short term (30 h), insulin exerts beneficial actions on trout glucose homeostasis, including a lowered glycemia and increased hepatic lipogenic and glycogenic potentials. However, when trout were fed for 5 days, these beneficial actions of insulin infusion were no longer observed. Thus, the increased lipogenic potential observed after one single meal was not present, and this together with the increased glycogenesis and the decreased glucose exported to the blood from the liver explains the lack of hypoglycemic action of insulin. The fact that insulin improved glucose homeostasis when administrated over a short time period implies that endogenous insulin secretion is inadequate in trout to deal with this amount of dietary carbohydrates. Moreover, the fact that a longer exposure to insulin resulted in a reduced response indicates that the rainbow trout is sensitive to insulin, re-enforcing the hypothesis that the hyperglycemia observed following a high carbohydrate meal is an insulin secretion issue rather an insulin action issue.

In mammals, new evidence has demonstrated the important role of the autophagic/lysosomal pathway in regulating muscle mass and identified the transcription factor FoxO3 as a key factor of the control of this proteolytic system by inducing several autophagy-related genes. In contrast, the mechanisms responsible for the regulation of autophagy have not been investigated in teleosts, known to exhibit different muscle growth dynamics. The present work aimed to characterize both in vivo and in vitro the transcriptional regulation of several major genes involved in autophagy (LC3B, gabarapl1, atg12l, atg4b) in the white skeletal muscle of rainbow trout. We found that fasting fish for 14days or serum depletion of trout myocytes strongly induces the expression of all studied genes. Our in vitro study on trout myocytes indicated that IGF1 induces FoxO3 phosphorylation but has a low or no effect on autophagy-related gene expression, suggesting a moderate role for this transcription factor on the autophagic/lysosomal pathway in this species. Data reported here show for the first time in a lower vertebrate, the existence and the regulation of several major genes involved in the autophagy, opening a new area of research on the molecular bases of muscle protein degradation in teleosts., (Copyright 2010 Elsevier Inc. All rights reserved.)

Amino acids are considered to be regulators of metabolism in several species, and increasing importance has been accorded to the role of amino acids as signalling molecules regulating protein synthesis through the activation of the TOR transduction pathway. Using rainbow trout hepatocytes, we examined the ability of amino acids to regulate hepatic metabolism-related gene expression either alone or together with insulin, and the possible involvement of TOR. We demonstrated that amino acids alone regulate expression of several genes, including glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, pyruvate kinase, 6-phospho-fructo-1-kinase and serine dehydratase, through an unknown molecular pathway that is independent of TOR activation. When insulin and amino acids were added together, a different pattern of regulation was observed that depended upon activation of the TOR pathway. This pattern included a dramatic up-regulation of lipogenic (fatty acid synthase, ATP-citrate lyase and sterol responsive element binding protein 1) and glycolytic (glucokinase, 6-phospho-fructo-1-kinase and pyruvate kinase) genes in a TOR-dependent manner. Regarding gluconeogenesis genes, only glucose-6-phosphatase was inhibited in a TOR-dependent manner by combination of insulin and amino acids and not by amino acids alone. This study is the first to demonstrate an important role of amino acids in combination with insulin in the molecular regulation of hepatic metabolism.

The aim of the present study was to analyse the effects of partial or total replacement of fish meal (FM) and fish oil (FO) by a mixture of plant protein (PP) and a mixture of vegetable oils (VO) on the hepatic insulin-nutrient-signalling pathway and intermediary metabolism-related gene expression in rainbow trout (Oncorhynchus mykiss). Triplicate groups of fish were fed four practical diets containing graded levels of replacement of FM and FO by PP and VO for 12 weeks: diet 0/0 (100 % FM, 100 % FO); diet 50/50 (50 % FM and 50 % PP, 50 % FO and 50 % VO); diet 50/100 (50 % FM and 50 % PP, 100 % VO); diet 100/100 (100 % PP, 100 % VO). Samplings were performed on trout starved for 5 d then refed with their allocated diet. In contrast to partial substitution (diet 50/50), total substitution of FM and FO (diet 100/100) led to significantly lower growth compared with diet 0/0. The insulin-nutrient-signalling pathway (protein kinase B (Akt), target of rapamycin (TOR), S6 protein kinase 1 (S6K1) and S6) was characterised in trout liver and found to be activated by refeeding. However, changes in diet compositions did not differentially affect the Akt-TOR-signalling pathway. Moreover, expression of genes encoding fructose-1,6-biphosphatase, mitochondrial phosphoenolpyruvate carboxykinase, glucokinase, pyruvate kinase and carnitine palmitoyl transferase 1 were not affected by refeeding or by dietary changes. Refeeding down- and up-regulated the expression of gluconeogenic glucose-6-phosphatase isoform 1 and lipogenic fatty acid synthase genes, respectively. Expression of both genes was also increased with partial replacement of FM and total replacement of FO (diet 50/100). These findings indicate that plant-based diets barely affect glucose and lipid metabolism in trout.