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In aquaculture, there is high interest in substituting fishmeal protein with carbohydrate (CHO)-based substrates such as vegetable starch. Procurement of fishmeal protein remains highly dependent on overexploited wild fisheries (1); hence any reduction in its consumption by farmed fish would reduce the ecological burden and improve the sustainability of aquaculture (2). Furthermore, to the extent that dietary CHO replaces protein for systemic glucose and energy demands (3), it decreases waste ammonia generation from protein catabolism, thereby reducing nitrogenous effluents. For carnivorous fish such as the European seabass (Dicentrarchus labrax L.), the efficacy of this approach depends on the capacity of the fish to adapt from their natural diet that is high in both protein and fat, but lacking in CHO, to a regime where the proportion of dietary CHO to total caloric content is increased (4). The capacity to digest complex CHO varies widely between different fish species (5). Generally, carnivorous fish are poorly able to digest raw starch (RS). Cooking or gelatinizing the starch significantly improves its digestibility Abstract Farmed seabass have higher adiposity than their wild counterparts and this is often attributed to carbohydrate (CHO) feeding. Whether this reflects a reduction in fat oxidation, increased de novo lipogenesis (DNL), or both, is not known. To study the effects of high CHO diets on hepatic TG biosynthesis, hepatic TG deuterium ( similar hepatic TG levels to CTRL. DS-fed fish showed higher activity for enzymes that can provide NADPH for lipogenesis, relative to CTRL in the case of glucose-6-phosphate dehydrogenase (G6PDH) and relative to RS for both G6PDH and 6-phosphogluconate dehydrogenase. This approach indicated that elevated hepatic adiposity from DS feeding was not attributable to increased DNL.-Viegas, I., I.

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In the present study, the effects of partial substitution of dietary protein by digestible starch on endogenous glucose production were evaluated in European seabass (Dicentrarchus labrax). The fractional contribution of dietary carbohydrates v. gluconeogenesis to blood glucose appearance and hepatic glycogen synthesis was quantified in two groups of seabass fed with a diet containing 30 % digestible starch (DS) or without a carbohydrate supplement as the control (CTRL). Measurements were performed by transferring the fish to a tank containing water enriched with 5 % 2 H 2 O over the last six feeding days, and quantifying the incorporation of 2 H into blood glucose and hepatic glycogen by 2 H NMR. For CTRL fish, gluconeogenesis accounted for the majority of circulating glucose while for the DS fish, this contribution was significantly lower (CTRL 85 (SEM 4) % v. DS 54 (SEM 2) %; P,0·001). Hepatic glycogen synthesis via gluconeogenesis (indirect pathway) was also significantly reduced in the DS fish, in both relative (CTRL 100 (SEM 1) % v. DS 72 (SEM 1) %; P, 0·001) and absolute terms (CTRL 28 (SEM 1) v. DS 17 (SEM 1) mmol/kg per h; P,0·001). A major fraction of the dietary carbohydrates that contributed to blood glucose appearance (33 (SEM 1) % of the total 47 (SEM 2) %) had undergone exchange with hepatic glucose 6-phosphate. This indicated the simultaneous activity of hepatic glucokinase and glucose 6-phosphatase. In conclusion, supplementation of digestible starch resulted in a significant reduction of gluconeogenic contributions to systemic glucose appearance and hepatic glycogen synthesis. Key words:2 H 2 O: Gluconeogenesis: Endogenous glucose production: Glucokinase: Starch utilisationIn aquaculture, substituting costly fishmeal with less expensive plant-derived carbohydrate (CHO) reduces feed input costs. The provision of dietary CHO to carnivorous fish such as the European seabass (Dicentrarchus labrax L.) may also have environmental benefits since CHO utilisation can potentially spare the catabolism of dietary amino acids to glucose and nitrogenous waste (1) . Dietary CHO influences growth, feed utilisation and deposition of nutrients according to species, quantity, origin and pre-treatment for improving digestibility (2 -4) . The effects of partial substitution of dietary protein by plant-derived CHO on food conversion efficiency and nutrient digestibility sources have been tested in seabass (5 -7) . In addition, its effects on energy retention and maintenance requirements (8) as well as the activity of key CHO-metabolising enzymes (6,9) have been studied. These studies have suggested that, on the one hand, carnivorous fish are able to up-regulate their capacity for hepatic glucose utilisation through the increased expression of gateway enzymes such as glucokinase when fed with a high-CHO diet. However, on the other hand, supplementation of fish feed with up to 30 % starch has not been shown to alter the overall protein efficiency ratio or nitrogen retention in seabass (7) . This is despite...

H 2 O Gluconeogenesis Glycogenolysis Glucose 6-phosphatase Glucokinase NMR Sources of blood glucose in European seabass (initial weight 218.0 ± 43.0 g; mean ± S.D., n = 18) were quantified by supplementing seawater with deuterated water (5%-2 H 2 O) for 72 h and analyzing blood glucose 2 H-enrichments by 2 H NMR. Three different nutritional states were studied: continuously fed, 21-day of fast and 21-day fast followed by 3 days of refeeding. Plasma glucose levels (mM) were 10.7 ± 6.3 (fed), 4.8 ± 1.2 (fasted), and 9.3 ± 1.4 (refed) (means ± S.D., n = 6), showing poor glycemic control. For all conditions, 2 H-enrichment of glucose position 5 was equivalent to that of position 2 indicating that blood glucose appearance from endogenous glucose 6-phosphate (G6P) was derived by gluconeogenesis. G6P-derived glucose accounted for 65 ± 7% and 44 ± 10% of blood glucose appearance in fed and refed fish, respectively, with the unlabeled fraction assumed to be derived from dietary carbohydrate (35 ± 7% and 56 ± 10%, respectively). For 21-day fasted fish, blood glucose appearance also had significant contributions from unlabeled glucose (52 ± 16%) despite the unavailability of dietary carbohydrates. To assess the role of hepatic enzymes in glycemic control, activity and mRNA levels of hepatic glucokinase (GK) and glucose 6-phosphatase (G6Pase) were assessed. Both G6Pase activity and expression declined with fasting indicating the absence of a classical counter-regulatory stimulation of hepatic glucose production in response to declining glucose levels. GK activities were basal during fed and fasted conditions, but were strongly stimulated by refeeding. Overall, hepatic G6Pase and GK showed limited capacity in regulating glucose levels between feeding and fasting states.

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In carnivorous fish, conversion of a glucose load to hepatic glycogen is widely used to assess their metabolic flexibility towards carbohydrate utilization, but the activities of direct and indirect pathways in this setting are unclear. We assessed the conversion of an intraperitoneal glucose load (2 g.kg−1) enriched with [U-13C6]glucose to hepatic glycogen in juvenile seabass and seabream. 13C-NMR analysis of glycogen was used to determine the contribution of the load to glycogen synthesis via direct and indirect pathways at 48-hr post-injection. For seabass, [U-13C6]glucose was accompanied by deuterated water and 2H-NMR analysis of glycogen 2H-enrichment, allowing endogenous substrate contributions to be assessed as well. For fasted seabass and seabream, 47 ± 5% and 64 ± 10% of glycogen was synthesized from the load, respectively. Direct and indirect pathways contributed equally (25 ± 3% direct, 21 ± 1% indirect for seabass; 35 ± 7% direct, 29 ± 4% indirect for seabream). In fasted seabass, integration of 2H- and 13C-NMR analysis indicated that endogenous glycerol and anaplerotic substrates contributed an additional 7 ± 2% and 7 ± 1%, respectively. In fed seabass, glucose load contributions were residual and endogenous contributions were negligible. Concluding, direct and indirect pathways contributed equally and substantially to fasting hepatic glycogen repletion from a glucose load in juvenile seabream and seabass.

The stimulation of hepatic glycogenesis is a ubiquitous response to a glucose challenge and quantifying its contribution to glucose uptake informs its role in restoring euglycemia. Glycogenesis can be quantified with labeled water provided that exchange of glucose-6-phosphate hydrogen 2 (G6P-H2) and body water via glucose-6-phosphate isomerase, and exchange of positions 4, 5 and 6 hydrogens (G6P-H456) via transaldolase, are known. These exchanges were quantified in 24-h fasted rats (Rattus norvegicus; n=6) and 21-day fasted seabass (Dicentrarchus labrax; n=8) by administration of a glucose load (2000mg·kg(-1)) enriched with [U-(2)H7]glucose and by quantifying hepatic glycogen (2)H-enrichments after 2h (rats) and 48h (seabass). Direct pathway contributions of the glucose load to glycogenesis were also estimated. G6P-H2 and body water exchange was 61±1% for rat and 47±3% for seabass. Transaldolase-mediated exchange of G6P-H456 was 5±1% for rat and 10±1% for seabass. Conversion of the glucose load to hepatic glycogen was significant in seabass (249±54mg·kg(-1)) but negligible in rats (12±1mg·kg(-1)). Preload plasma glucose levels were similar for seabass and rats (3.3±0.7 and 4.4±0.1mmol·L(-1), respectively) but post-load plasma glucose was significantly higher in seabass compared to rats (14.6±1.8 versus 5.8±0.3mmol·L(-1), p<0.01). In conclusion, G6P-H2 and body water exchange is incomplete for both species and has to be accounted for in estimating hepatic glycogen synthesis and direct pathway activities with labeled water tracers. Transaldolase-mediated exchange is insignificant. Hepatic direct pathway glycogenesis plays a prominent role in seabass glucose load disposal, but a negligible role in the rat.