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

S. Panserat^*, E. Plagnes-Juan and S. Kaushik

Laboratory of Fish Nutrition, INRA-IFREMER, 64310 St-Pée-sur-Nivelle, France

View larger version (39K): [in a new window]   Fig. 1. Partial cloning of the pyruvate kinase (PK) gene in rainbow trout. (A) Nucleotide and deduced amino acid sequences of the rainbow trout PK clone. Underlined letters correspond to the primer sequences. (B) Alignments of the partial amino acid deduced PK cDNA of the rainbow trout (T_PK) (GenBank accession number: AF246146) with human PK (H_PK) (GenBank accession number: P30613). Underlined letters correspond to the primer sequences. Asterisks mark amino acid residues homologous between PKs. Residues involved in the interaction with the allosteric activator fructose-1,6-bisphosphate are marked by arrows (Jurica et al., 1998).

View larger version (33K): [in a new window]   Fig. 2. Partial cloning of the glucose transporter type 2 (Glut2) gene in rainbow trout. (A) Nucleotide and deduced amino acid sequences of the rainbow trout Glut2 clone. Underlined letters correspond to the primer sequences. (B) Alignments of the partial amino acid sequence deduced from Glut2 cDNA of the rainbow trout (T_Glut2) (GenBank accession number: AF246147) with human Glut2 (H_Glut2) (GenBank accession number: P11168). Underlined letters correspond to the primer sequences. Asterisks mark amino acid residues homologous between Glut2s. Hydrophobic putative transmembrane segments are boxed (Thorens, 1992).

View larger version (34K): [in a new window]   Fig. 3. Partial cloning of the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (6PF-2K/F-2,6BPase) gene in rainbow trout. (A) nucleotide and deduced amino acid sequences of the rainbow trout 6PF-2K/F-2,6BPase clone. Underlined letters correspond to the primer sequences. (B) Alignments of the partial amino acid deduced sequence from 6PF-2K/F-2,6BPase cDNA of the rainbow trout (T_6PF-2K/F-2,6BPase) (GenBank accession number: AF246148) with human 6PF-2K/F-2,6BPase (H_6PF-2K/F-2,6BPase) (GenBank accession number: P16168) and gilthead seabream (Sparus aurata) 6PF-2K/F-2,6BPase (SB_6PF-2K/F-2,6BPase) (GenBank accession number: U84724). Underlined letters correspond to the primer sequences. Asterisks mark amino acid residues homologous between 6PF-2K/F-2,6BPases. Residues marked by an arrow are key residues for the 6PF-2K/F-2,6BPase (Meton et al., 1999a).

View larger version (42K): [in a new window]   Fig. 4. Partial cloning of the FBPase gene in rainbow trout. (A) Nucleotide and deduced amino acid sequences of the rainbow trout FBPase clone. Underlined letters correspond to the primer sequences. (B) Alignments of the partial amino acid deduced sequence from FBPase cDNA of the rainbow trout (T_FBPase) (GenBank accession number: AF333188) with mouse FBPase (M_FBPase) (GenBank accession number: AJ132693). Underlined letters correspond to the primer sequences. Asterisks mark amino acid residues homologous between FBPases. Conserved residues involved in the interaction with fructose-1,6-bisphosphate are marked by arrows (Ke et al., 1989).

View larger version (49K): [in a new window]   Fig. 5. Tissue specificity of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (6PF-2K/F-2,6BPase), pyruvate kinase (PK), fructose-1,6-bisphosphatase (FBPase) and glucose transporter type 2 (Glut2) gene expression in food-deprived rainbow trout and fed rainbow trout with 20% carbohydrates (at 6h after feeding). Analysis by non-quantitative RT–PCR (N=2 fish per treatment). Ma, molecular mass marker phiX174 DNA/HaeIII (Promega, USA); N, negative control, i.e. RT–PCR reactions performed without RNA and with reverse transcriptase (other controls made with RNA and without reverse transcriptase were also performed to determine genomic DNA contamination; data not shown). L, liver; Mu, muscle; H, heart; B, brain; K, kidney; I, intestine. The exact lengths of the 6PF-2K/F-2,6BPase, PK, FBPase and Glut2 fragments (205bp, 300bp, 288bp and 222bp, respectively) were determined from known gene sequences. The quality of the first-strand cDNA used in each of the PCR assays was first confirmed by its ability to support the amplification of FBPase and 6PF-2K/F-2,6BPase cDNAs, and the failure to detect the presence of PK and Glut2 mRNA in certain tissues does not imply poor quality of RNA samples or a low-efficiency reverse transcription reaction.

View larger version (49K): [in a new window]   Fig. 6. Representative northern blots of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (6PF-2K/F-2,6BPase), pyruvate kinase (PK), fructose-1,6-bisphosphatase (FBPase) and glucose transporter type 2 (Glut2) gene expression in the liver of food-deprived fish or fish fed with 20% carbohydrates (+CHO) or without carbohydrates (-CHO). Each band is from a different fish. The 16S rRNA served as an internal control of sample loading. (A) Glut2, (B) PK, (C) 6PF-2K/F-2,6BPase, (D) FBPase.

View larger version (23K): [in a new window]   Fig. 7. Levels of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2K/F-2,6BPase), pyruvate kinase (PK), fructose-1,6-bisphosphatase (FBPase) and glucose transporter type 2 (Glut2) gene expressions in the liver of food-deprived fish and of fish fed with 20% carbohydrates (+CHO) or without carbohydrates (-CHO). (A) Glut2, (B) PK, (C) 6PF-2K/F-2,6BPase, (D) FBPase. For each gene, two different northern blots (loading with fish fed 6h and 24h after feeding) were done; an analysis by densitometry of mRNA levels (arbitrary units) for five fish from each treatment group weighted by 16S rRNA values was performed (Visio-Mic II software). The results are expressed as means ± S.D. (N=5). Significant differences within groups are represented by different letters (Tukey’s test, P<0.05).