Dietary starch promotes hepatic lipogenesis in barramundi (Lates calcarifer).

Barramundi (Lates calcarifer) are a highly valued aquaculture species, and, as obligate carnivores, they have a demonstrated preference for dietary protein over lipid or starch to fuel energetic growth demands. In order to investigate how carnivorous fish regulate nutritional cues, we examined the metabolic effects of feeding two isoenergetic diets that contained different proportions of digestible protein or starch energy. Fish fed a high proportion of dietary starch energy had a higher proportion of liver SFA, but showed no change in plasma glucose levels, and few changes in the expression of genes regulating key hepatic metabolic pathways. Decreased activation of the mammalian target of rapamycin growth signalling cascade was consistent with decreased growth performance values. The fractional synthetic rate (lipogenesis), measured by TAG 2H-enrichment using 2H NMR, was significantly higher in barramundi fed with the starch diet compared with the protein diet (0·6 (se 0·1) v. 0·4 (se 0·1) % per d, respectively). Hepatic TAG-bound glycerol synthetic rates were much higher than other closely related fish such as sea bass, but were not significantly different (starch, 2·8 (se 0·3) v. protein, 3·4 (se 0·3) % per d), highlighting the role of glycerol as a metabolic intermediary and high TAG-FA cycling in barramundi. Overall, dietary starch significantly increased hepatic TAG through increased lipogenesis. Compared with other fish, barramundi possess a unique mechanism to metabolise dietary carbohydrates and this knowledge may define ways to improve performance of advanced formulated feeds.

Regulation of de novo hepatic lipogenesis by insulin infusion in rainbow trout fed a high-carbohydrate diet.

Carbohydrate energy intake in excess of total energy expenditure is converted to fat. In fish, the liver is considered to be the main lipogenic tissue. Its regulation by insulin is not fully understood, and some of the available in vivo findings are contradictory. In this study, bovine insulin was infused for 5 d into rainbow trout fed a high-carbohydrate diet, and variables of de novo hepatic lipogenesis were measured. We found that hepatic lipogenesis in trout is stimulated by insulin, reflected in enhanced mRNA and protein abundance and enzyme activity of ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase. These results were further supported by parallel changes in enzymes acting as NAD phosphate donors, especially those participating in the pentose phosphate pathway. This is the first time that the main enzymes involved in de novo hepatic lipogenesis have been studied at the molecular, protein, and activity levels in fish. We hypothesize that some of the delayed changes found in the different levels of regulation were probably related to the insulin resistance achieved by the trout liver after 5 d of insulin infusion. We assessed enzyme activity and mRNA abundance of lipid oxidation-related enzymes in the livers of insulin-infused fish in which paradoxically increased ß-oxidation potential was found. The insulin-stimulated de novo hepatic lipogenesis in carbohydrate-fed trout reinforces the hypothesis that this pathway may act as an important sink for excess glucose, which could ultimately contribute to improved glucose homeostasis in this carnivorous and glucose-intolerant species when fed high-carbohydrate diets.

Insulin stimulates lipogenesis and attenuates Beta-oxidation in white adipose tissue of fed rainbow trout.

As lipid deposition tissue in fish, the white adipose tissue (WAT) has important functions related to reproduction and the challenges of long-term fasting. In the study reported here, we infused fish fed a high-carbohydrate diet with two doses of insulin for 5 days in order to explore the effects of this hormone on lipogenesis and beta-oxidation-related enzymes. We demonstrated the presence of some of the main lipogenic enzymes at molecular, protein and activity levels (ATP-citrate lyase and fatty acid synthase). However, while ATP-citrate lyase was unexpectedly down-regulated, fatty acid synthase was up-regulated (at protein and activity levels) in an insulin dose-dependent manner. The main enzymes acting as NADPH donors for lipogenesis were also characterized at biochemical and molecular levels, although there was no evidence of their regulation by insulin. On the other hand, lipid oxidation potential was found in this tissue through the measurement of gene expression of enzymes involved in ß-oxidation, highlighting two carnitine palmitoyltransferase isoforms, both down-regulated by insulin infusion. We found that insulin acts as an important regulator of trout WAT lipid metabolism, inducing the final stage of lipogenesis at molecular, protein and enzyme activity levels and suppressing ß-oxidation at least at a molecular level. These results suggest that WAT in fish may have a role that is important not only as a lipid deposition tissue but also as a lipogenic organ (with possible involvement in glucose homeostasis) that could also be able to utilize the lipids stored as a local energy source.

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.

Liver and muscle metabolic changes induced by dietary energy content and genetic selection in rainbow trout (Oncorhynchus mykiss).

We combined genetic selection and dietary treatment to produce a model to study metabolic pathways involved in genetic and nutritional control of fat deposition in fish muscle. Two experimental lines of rainbow trout, selected for a lean (L) or fat (F) muscle, were fed with diets containing either 10 or 23% lipids from the first feeding, up to 6 mo. At the end of the feeding trial, trout were distinguished by very different muscle fat content (from 4.2 to 10% wet weight), and line x diet interactions were observed for parameters related to fat storage. We analyzed the activity and gene expression of key enzymes involved in lipid metabolism (fatty acid synthase, hydroxyacyl-CoA dehydrogenase, carnitine palmitoyltransferase 1 isoforms, and peroxisome proliferator-activated receptor alpha) and glycolysis (hexokinase 1 and pyruvate kinase) as well as energy production (isocitrate dehydrogenase, citrate synthase, and cytochrome oxidase) in the liver and the white muscle of rainbow trout. The lipid-rich diet repressed the activity of the lipogenic enzymes and stimulated enzymes involved in fatty acid oxidation and glycolysis in liver but had little effect on muscle enzymes assessed in this study. Regarding the selection effect, enzyme activity and expression suggest that compared with the L line, the F line presented reduced hepatic fatty acid oxidation as well as reduced mitochondrial oxidative capacities and enhanced glucose utilization in both liver and muscle. Very few line x diet interactions were found, suggesting that the two factors (i.e., dietary energy content and selection) used in this study to modify muscle lipid content exerted some additive but mostly independent effects on these metabolic actors.

Contribution of dietary arginine to nitrogen utilisation and excretion in juvenile sea bass (Dicentrarchus labrax) fed diets differing in protein source.

The role of dietary arginine in affecting nitrogen utilisation and excretion was studied in juvenile European sea bass (Dicentrarchus labrax) fed for 72 days with diets differing in protein sources (plant protein-based (PM) and fish-meal-based (FM)). Fish growth performance and nitrogen utilisation revealed that dietary Arg surplus was beneficial only in PM diets. Dietary Arg level significantly affected postprandial plasma urea concentrations. Hepatic arginase activity increased (P<0.05) in response to dietary Arg surplus in fish fed plant protein diets; conversely ornithine transcarbamylase activity was very low and inversely related to arginine intake. No hepatic carbamoyl phosphate synthetase III activity was detected. Dietary arginine levels did not affect glutamate dehydrogenase activity. A strong linear relationship was found between liver arginase activity and daily urea-N excretion. Dietary Arg excess reduced the proportion of total ammonia nitrogen excreted and increased the contribution of urea-N over the total N excretion irrespective of dietary protein source. Plasma and excretion data combined with enzyme activities suggest that dietary Arg degradation via hepatic arginase is a major pathway for ureagenesis and that ornithine-urea cycle is not completely functional in juvenile sea bass liver.

Dietary arginine degradation is a major pathway in ureagenesis in juvenile turbot (Psetta maxima).

Recent studies indicate that urea excretion is responsive to protein intake and that turbot, Psetta maxima, appear to differ from other species by their urea excretion pattern and levels. This study was undertaken to evaluate the influence of dietary nitrogen and arginine on ureagenesis and excretion in turbot. Juvenile turbot (29 g) were fed semi-purified diets containing graded levels of nitrogen (0-8% dry matter) and arginine (0-3% dry matter) for 6 weeks. Growth data showed that turbot have high dietary nitrogen (123 mg/kg metabolic body weight/day) and very low dietary arginine (9.3 mg/kg metabolic body weight/day) requirements for maintenance. Requirements for unit body protein accretion were 0.31 g and 0.15 g for nitrogen and arginine respectively. Post-prandial plasma urea levels and urea excretion rates showed that urea production was significantly (P<0.05) influenced by dietary arginine levels. While hepatic arginase (EC 3.5.3.1) activity increased significantly (P<0.05) with increasing dietary arginine levels, activities of other enzymes of the ornithine urea cycle were very low. Our data strongly suggest that the ornithine urea cycle is not active in the turbot liver and that dietary arginine degradation is a major pathway of ureagenesis in turbot.

Protein and arginine requirements for maintenance and nitrogen gain in four teleosts.

Besides being an indispensable amino acid for protein synthesis, arginine (Arg) is also involved in a number of other physiological functions. Available data on the quantitative requirement for Arg in different teleosts appear to show much variability. So far, there are very limited data on the maintenance requirements of indispensable amino acids (IAA) in fish. In the present study, we compared N and Arg requirements for maintenance and growth of four finfish species: rainbow trout (Oncorhynchus mykiss), turbot (Psetta maxima), gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax). Groups of fish having an initial body weight close to 5-7 g were fed semi-purified diets containing graded levels of N (0 to 8 % DM) and Arg (0 to 3 % DM) over 4 to 6 weeks. For each species, N and Arg requirements for maintenance and for growth were calculated regressing daily N gain against daily N or Arg intakes. N requirement for maintenance was estimated to be 37.8, 127.3, 84.7 and 45.1 mg/kg metabolic body weight per d and 2.3, 2.2, 2.6 and 2.5 g for 1 g N accretion, in rainbow trout, turbot, gilthead seabream and European seabass respectively. The four species studied appear to have very low or no dietary Arg requirements for maintenance. Arg requirement for g N accretion was calculated to be 0.86 g in rainbow trout and between 1.04-1.11 g in the three marine species. Turbot required more N for maintenance than the other three species, possibly explaining its reputedly high overall dietary protein requirement. Data suggest a small but sufficient endogenous Arg synthesis to maintain whole body N balance and differences between freshwater and marine species as regards Arg requirement. It is worth verifying this tendency with other IAA.

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.

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.