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.

In mammals, the ubiquitin-proteasome proteolytic pathway is a major route of protein degradation and has been shown to be regulated by the feeding status via the protein kinase B (PKB)-Forkehead box-O transcription factor signaling pathway-mediated transcription regulation of atrophy-related ubiquitin ligases, atrogin1 and muscle RING finger 1. In contrast, in rainbow trout (Oncorhynchus mykiss), the activity of the proteasome in muscle was not affected during starvation-induced muscle degradation. The aim of this study was therefore to explore the molecular basis for this lack of induction of this proteolytic route during starvation. In this study, rainbow trout were food deprived for 7 and 14 d, refed ad libitum, and the effect of the nutritional status was assessed on the different steps involved in the regulation of the ubiquitin-proteasome system in muscle. We observed that starvation reduced the phosphorylation of PKB and enhanced the expression of atrogin1 in muscle, whereas refeeding led to the opposite effects. The level of polyubiquitinated proteins in muscle increased to over 2 times the initial value on d 0 after 14 d of starvation and decreased significantly at 12 h after refeeding, but there were no major changes in the activity of the main proteasomal peptidases (chymotrypsin-like and trypsin-like). Altogether, these results indicate that in rainbow trout muscle, the polyubiquitination step of the ubiquitin-proteasome route is regulated by the feeding status similarly to what is observed in mammals.

Our objective was to understand the reasons behind the persistent postprandial hyperglycemia in rainbow trout (Oncorhynchus mykiss). We hypothesized that in this species, high levels of dietary protein could increase the hepatic production of glucose, irrespective of the dietary carbohydrate supply. We fed juvenile rainbow trout four diets containing graded levels of protein for 14 d. Pair-feeding was employed to keep lipid and carbohydrate intakes constant. Six hours after feeding, as postulated, activities and mRNA levels of gluconeogenic enzymes (glucose-6-phosphatase, fructose-1,6-bisphosphatase) increased with increasing dietary protein (P < 0.05). However, in fish with a very low protein intake, there was a very strong increase in plasma glucose (18 mmol/L) that was also associated with a high capacity to store excess glucose as indicated by altered pyruvate kinase activity, glucokinase activity, and hepatic glycogen and fat concentrations (P < 0.05). In conclusion, at the same level of carbohydrate intake, a low dietary protein intake was associated with an unexplained increase in glycemia, which was probably responsible for the decrease in hepatic gluconeogenic enzyme expression. The effect of dietary protein on low carbohydrate utilization in this species remains unclear.

To contribute to the understanding of the mechanisms involved in poor metabolic utilization of dietary carbohydrates by rainbow trout (Oncorhynchus mykiss), we explored in this study the effects of dietary lipids on the regulation of two hepatic key enzymes, i.e., glucokinase (GK, first enzyme of the glycolytic pathway) and glucose-6-phosphatase (G6Pase, last enzyme of the gluconeogenesis). Two groups of juvenile trout were pair-fed for 8 wk either a low (10%) or a high (25%) level of dietary lipids supplied as fish oil; the pair-feeding technique was adopted to vary fat intake while keeping the protein and carbohydrate intakes more or less constant. Fish fed the high level of dietary lipids had inefficient control of glycemia compared with fish fed the low level of lipids. Levels of dietary lipids did not affect GK activity even though there was a small increase of GK mRNA level at 3 h after feeding high levels of lipids. By contrast, the high level of dietary lipids significantly increased G6Pase mRNA expression at 3, 6 and 12 h and enzyme activity at 6 h after food consumption. Thus, these data suggest that poor dietary carbohydrate utilization in rainbow trout may be related at least in part to increased hepatic glucose production under conditions of high dietary fat intake.

No abstract

1. The study of the CYP2D genotype and phenotype of a Caucasian family revealed that a XbaI‐9 kb allele was associated with the poor metabolizer phenotype. 2. A Polymerase Chain Reaction (PCR)‐based assay showed that the previously described mutations D6A and D6B are not associated with the XbaI‐9 kb allele. 3. To explore the molecular basis of the poor metabolizer phenotype associated with the XbaI‐9 kb allele, complete sequencing of the nine exons and intron‐exon boundaries of the CYP2D6 gene was undertaken after amplification by PCR. 4. All the exons were successfully amplified using CYP2D6 gene‐specific primers except exon 1 which required a combination of CYP2D7 gene‐specific 5' primer and a CYP2D6 gene‐specific 3' primer. 5. Sequence data derived from this amplified product revealed that the XbaI‐9 kb allele corresponds to a novel rearrangement of the locus. This involved a deletion of an approximately 20 kilobase (kb) DNA segment generating a hybrid 5' CYP2D7/CYP2D6 3' gene. 6. The chimeric gene is non‐functional presumably due to an insertion in exon 1 (characteristic of the exon 1 of the CYP2D7 gene) which causes a shift in the reading frame with premature termination of translation.

No abstract

SUMMARY 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.