High or low dietary carbohydrate:protein ratios during first-feeding affect glucose metabolism and intestinal microbiota in juvenile rainbow trout.

Based on the concept of nutritional programming in mammals, we tested whether an acute hyperglucidic-hypoproteic stimulus during first feeding could induce long-term changes in nutrient metabolism in rainbow trout. Trout alevins received during the five first days of exogenous feeding either a hyperglucidic (40% gelatinized starch + 20% glucose) and hypoproteic (20%) diet (VLP diet) or a high-protein (60%) glucose-free diet (HP diet, control). Following a common 105-day period on a commercial diet, both groups were then challenged (65 days) with a carbohydrate-rich diet (28%). Short- and long-term effects of the early stimuli were evaluated in terms of metabolic marker gene expressions and intestinal microbiota as initial gut colonisation is essential for regulating the development of the digestive system. In whole alevins (short term), diet VLP relative to HP rapidly increased gene expressions of glycolytic enzymes, while those involved in gluconeogenesis and amino acid catabolism decreased. However, none of these genes showed persistent molecular adaptation in the liver of challenged juveniles (long term). By contrast, muscle of challenged juveniles subjected previously to the VLP stimulus displayed downregulated expression of markers of glycolysis and glucose transport (not seen in the short term). These fish also had higher plasma glucose (9 h postprandial), suggesting impaired glucose homeostasis induced by the early stimulus. The early stimulus did not modify the expression of the analysed metabolism-related microRNAs, but had short- and long-term effects on intestinal fungi (not bacteria) profiles. In summary, our data show that a short hyperglucidic-hypoproteic stimulus during early life may have a long-term influence on muscle glucose metabolism and intestinal microbiota in trout.

Effects of refeeding with low- or high-carbohydrate diets on intermediary carbohydrate metabolism in juvenile and adult Nile tilapia.

Generally, energy expenditure and compensation according to food deprivation and refeeding often occur along the life cycle of farmed-raised fish. Fasting and refeeding are also hypothesised to modulate carbohydrate metabolism particularly for herbivorous and/or omnivorous. This study aims to investigate the effects of short-term fasting and subsequent refeeding with high or low-carbohydrate diets on the intermediary carbohydrate metabolism of juvenile and adult Nile tilapia (Oreochromis niloticus) which is known to be a good user of carbohydrate as an energy source. Fish were fasted for 4 days and subsequently refed with either a low carbohydrate and high protein (LC/HP) or high carbohydrate and low protein (HC/LP) diet for 4 days. Our results showed that 4 days of refeeding with either one of the diets could compensate for weight loss due to fasting. Thus, we investigated the effects of a 4-day-refeeding strategy and different carbohydrate-refeeding diets on plasma metabolites, nutrient composition, and glucose and its related metabolism in the liver and muscle of adult fish. Refeeding had similar effects in adults and juveniles and induced modulations to the intermediary metabolism: (1) refeeding with the HC/LP diet elevated plasma glucose levels; (2) refeeding with both diets increased triglyceride levels in the plasma, liver, and muscle, but the effect of the HC/LP diet was superior; (3) refeeding elevated plasma cholesterol levels in adults, irrespective of diet; (4) refeeding with both diets increased hepatic lipid levels in juveniles, with stronger effects observed in those fed the HC/LP diet, and refeeding with the HC/LP diet elevated hepatic lipid levels in adults; (5) refeeding with both diets increased the plasma protein content, but the effect of the LC/HP diet was superior; (6) refeeding with the LC/HP diet increased hepatic protein content in adults; and (7) refeeding with both diets increased hepatic glycogen levels, but the effect of the HC/LP diet was superior. Additionally, in juveniles and adults, refeeding with the HC/LP diet upregulated the expression of glycolytic genes in the liver and muscle, lipogenic genes in the liver, and glucose transport genes. Moreover, refeeding with the HC/LP diet downregulated the expression of gluconeogenic and amino acid catabolism genes in the liver and amino acid catabolism genes in the muscle. Collectively, the effect of short-term refeeding with a high carbohydrate diet on intermediary metabolism resembled that of long-term feeding, supporting the hypothesis that Nile tilapia, an omnivorous fish, is highly responsive to dietary carbohydrates.

Glucose injection into the yolk influences intermediary metabolism in adult Nile tilapia fed with high levels of carbohydrates.

Nutritional programming is a concept proposed to be applied in the field of fish nutrition to improve the use of new diets in aquaculture. This study aimed to investigate for the first time the effects of a glucose injection into the yolk at the alevin stage on intermediary metabolism and growth in adult Nile tilapia (Oreochromis niloticus) at 32-37 weeks later in the life. The early stimulus was performed through direct microinjection of 2 M glucose into yolk sacs of Nile tilapia alevin. Subsequently, in adult tilapia, the long-term effects of glucose stimulus on growth performance, blood metabolites, chemical composition in the liver and muscle, expression of genes involved in glucose transport and metabolism (glycolysis and gluconeogenesis) and related pathways (amino acid catabolism and lipogenesis) were investigated. Our results showed that, even though early glucose injection had no effect on growth performance in adult fish, very few significant effects on glucose metabolism were observed. Furthermore, to evaluate the potential metabolic programming after a dietary challenge, a 2 × 2 factorial design with two early stimuli (0.85% NaCl or 2 M glucose) and two different dietary carbohydrate intakes (medium-carbohydrate diet, CHO-M; high-carbohydrate diet, CHO-H) was performed between weeks 33 and 37. As expected, compared with the CHO-M diet, the CHO-H diet led to decreased growth performance, higher glyceamia and triglyceridemia, higher glycogen and lipid levels in the liver as well as down-regulation of gluconeogenesis and amino acid catabolism gene expressions. More interestingly, although early glucose injection had no significant effect on growth performance, it enhanced the capacities for lipogenesis, glycolysis and gluconeogenesis, particularly in fish that were fed the CHO-H diet. Thus, the nutritional programming of tilapia linked to glucose injection into the yolk of alevins is always visible at the adult stage albeit less intense than what we previously observed in juvenile.

Metformin improves postprandial glucose homeostasis in rainbow trout fed dietary carbohydrates: a link with the induction of hepatic lipogenic capacities?

Carnivorous fish are poor users of dietary carbohydrates and are considered to be glucose intolerant. In this context, we have tested, for the first time in rainbow trout, metformin, a common anti-diabetic drug, known to modify muscle and liver metabolism and to control hyperglycemia in mammals. In the present study, juvenile trout were fed with very high levels of carbohydrates (30% of the diet) for this species during 10 days followed by feeding with pellets supplemented with metformin (0.25% of the diet) for three additional days. Dietary metformin led to a significant reduction in postprandial glycemia in trout, demonstrating unambiguously the hypoglycemic effect of this drug. No effect of metformin was detected on mRNA levels for glucose transporter type 4 (GLUT4), or enzymes involved in glycolysis, mitochondrial energy metabolism, or on glycogen level in the white muscle. Expected inhibition of hepatic gluconeogenic (glucose-6-phosphatase, fructose-1,6-bisphosphatase, and phosphoenolpyruvate carboxykinase) mRNA levels was not found, showing instead paradoxically higher mRNA levels for these genes after drug treatment. Finally, metformin treatment was associated with higher mRNA levels and activities for lipogenic enzymes (fatty acid synthase and glucose-6-phosphate dehydrogenase). Overall, this study strongly supports that the induction of hepatic lipogenesis by dietary glucose may permit a more efficient control of postprandial glycemia in carnivorous fish fed with high carbohydrate diets.

Gluconeogenic enzyme gene expression is decreased by dietary carbohydrates in common carp (Cyprinus carpio) and gilthead seabream (Sparus aurata).

Our objective is to understand the low metabolic utilization of dietary carbohydrates in fish. We compared the regulation of gluconeogenic enzymes at a molecular level in two fish species, the common carp (Cyprinus carpio) and gilthead seabream (Sparus aurata), known to be relatively tolerant to dietary carbohydrates. After cloning of partial cDNA sequences for three key gluconeogenic enzymes (glucose-6-phosphatase (G6Pase), fructose biphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) in the two species, we analyzed gene expressions of these enzymes 6 and 24 h after feeding with (20%) or without carbohydrates. Our data show that there is at least one gluconeogenic enzyme strongly regulated (decreased expression after feeding) in the two fish species, i.e. the PEPCK for common carp and G6Pase/FBPase for gilthead seabream. In these fish species, the regulation seems to be similar to the mammals at least at the molecular level.

Molecular cloning, tissue distribution and sequence analysis of complete glucokinase cDNAs from gilthead seabream (Sparus aurata), rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio).

The enzyme glucokinase (GK) (EC 2.7.1.1) plays an important role in the control of glucose homeostasis. Qualitative and/or quantitative variations in GK enzyme have been postulated by previous studies to explain why dietary carbohydrate utilisation is lower in gilthead seabream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss) than in common carp (Cyprinus carpio). In this study, we report the isolation and characterisation of a full-length cDNA coding for GK in these teleosts. Amino acid sequences derived from these cDNA clones are highly similar to other vertebrate GKs. These findings, including a detailed phylogenetic analysis, reveal that GK gene highly homologous to mammalian GK exists in these fish species with similar tissue specific expression (mainly liver).

Lack of significant long-term effect of dietary carbohydrates on hepatic glucose-6-phosphatase expression in rainbow trout (Oncorhynchus mykiss).

Hepatic glucose-6-phosphatase (G6Pase) plays an important role in glucose metabolism because it catalyzes the release of glucose to the circulatory system in the processes of glycogenolysis and gluconeogenesis. The present study was initiated to analyze the regulation of hepatic G6Pase expression by dietary carbohydrates in rainbow trout. The first step in our study was the identification of a partial G6Pase cDNA in rainbow trout that was highly homologous to that of mammals. Hepatic G6Pase activities and mRNA levels were measured in trout fed one of the experimental diets, with or without carbohydrates. We found no significant effect of intake of dietary carbohydrates on G6Pase expression (mRNA and activity) 6 hours and 24 hours after feeding. These results suggest that there is no control of G6Pase synthesis by dietary carbohydrates in rainbow trout and that the lack of regulation of gluconeogenesis by dietary carbohydrates could at least partially explain the postprandial hyperglycemia and the low dietary glucose utilization observed in this species.

Glucokinase is highly induced and glucose-6-phosphatase poorly repressed in liver of rainbow trout (Oncorhynchus mykiss) by a single meal with glucose.

The low dietary starch utilisation by rainbow trout (Oncorhynchus mykiss) may be attributed to a dysfunction of the nutritional regulation of the hepatic glucose/glucose-6-phosphate cycle. The present study was initiated to analyse the regulation of activity and gene expression of hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) by dietary carbohydrates in this species. We found that even a single meal containing 24% of glucose is sufficient to induce the GK expression (mRNA and activity) as in mammals. In contrast, although the inhibitory effect of dietary glucose on G6Pase expression is observed at the molecular level, the G6Pase activity is not significantly inhibited by dietary glucose. Thus, in contrast to the gluconeogenic G6Pase enzyme, a rapid adaptation of the hepatic glycolytic GK enzyme to dietary glucose seems effective in rainbow trout. These results suggest that in carnivorous rainbow trout, the liver is capable to strongly regulate the utilisation of glucose but not the synthesis of glucose.

Nutritional regulation and tissue specificity of gene expression for proteins involved in hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) are known to use dietary carbohydrates poorly. One of the hypotheses to explain the poor utilisation of dietary glucose by these fish is a dysfunction in nutritional regulation of hepatic glucose metabolism. In this study, we obtained partial clones of rainbow trout cDNAs coding for a glucose transporter (Glut2), and for the enzymes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2K/F-2,6BPase), fructose-1,6-bisphosphatase (FBPase) and pyruvate kinase (PK). Their deduced amino acid sequences were highly similar to those of mammals (up to 80% similarity). In a study of nutritional regulation, the Glut2 gene was highly expressed in the liver irrespective of the nutritional status of the trout, in agreement with the role of this transporter in the input (during refeeding) and output (during fasting) of glucose from the liver. Moreover, whereas PK and FBPase gene expression was high irrespective of the nutritional status, levels of hepatic 6PF-2K/F-2,6BPase mRNA were higher in fish fed with carbohydrates than in fish deprived of food. The high levels of hepatic PK, Glut2 and 6PF-2K/F-2,6BPase gene expression observed in this study suggest a high potential for tissue carbohydrate utilisation in rainbow trout. The persistence of a high level of FBPase gene expression suggests an absence of regulation of the gluconeogenic pathway by dietary carbohydrates.

Hepatic phosphoenolpyruvate carboxykinase gene expression is not repressed by dietary carbohydrates in rainbow trout (Oncorhynchus mykiss).

Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-limiting enzyme in hepatic gluconeogenesis and therefore plays a central role in glucose homeostasis. The aim of this study was to analyse the nutritional regulation of PEPCK gene expression in rainbow trout (Oncorhynchus mykiss), which are known to use dietary carbohydrates poorly. A full-length hepatic PEPCK cDNA (2637 base pairs with one open reading frame putatively encoding a 635-residue protein) was cloned and found to be highly homologous to mammalian PEPCKs. The presence of a putative peptide signal specific to a mitochondrial-type PEPCK in the deduced amino acid sequence suggests that this PEPCK gene codes for a mitochondrial form. In gluconeogenic tissues such as liver, kidney and intestine, this PEPCK gene was expressed at high levels and, in the liver we found no regulation of PEPCK gene expression by dietary carbohydrates. These results suggest that the first step of the hepatic gluconeogenic pathway in rainbow trout is functional and highly active irrespective of the dietary carbohydrate supply.

Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp.

Glucokinase (GK) plays a central role in glucose homeostasis in mammals. The absence of an inducible GK has been suggested to explain the poor utilization of dietary carbohydrates in rainbow trout. In this context, we analyzed GK expression in three fish species (rainbow trout, gilthead seabream, and common carp) known to differ in regard to their dietary carbohydrate tolerance. Fish were fed for 10 wk with either a diet containing a high level of digestible starch (>20%) or a diet totally deprived of starch. Our data demonstrate an induction of GK gene expression and GK activity by dietary carbohydrates in all three species. These studies strongly suggest that low dietary carbohydrate utilization in rainbow trout is not due to the absence of inducible hepatic GK as previously suggested. Interestingly, we also observed a significantly lower GK expression in common carp (a glucose-tolerant fish) than in rainbow trout and gilthead seabream, which are generally considered as glucose intolerant. These data suggest that other biochemical mechanisms are implicated in the inability of rainbow trout and gilthead seabream to control blood glucose closely.