Nutritional regulation and tissue specificity of gene expression for proteins involved in hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss)
Laboratory of Fish Nutrition, INRA-IFREMER, 64310 St-Pée-sur-Nivelle, France
*e-mail: panserat{at}st-pee.inra.fr
Accepted May 2, 2001
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Summary |
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Key words: liver metabolism, glucose metabolism, glucose transporter, fructose bisphosphatase, pyruvate kinase, bifunctional enzyme, trout, Oncorhynchus mykiss.
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Introduction |
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The aim of the present study was to analyse two key hepatic glycolytic enzymes: (i) 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, otherwise termed the bifunctional enzyme (6PF-2K/F-2,6BPase, EC2.7.1.105/EC3.1.3.46) (Pilkis and Granner, 1992; Pilkis et al., 1995), which produces fructose 2,6-bisphosphate, a key allosteric regulator of glycolysis/gluconeogenesis in the liver (Pilkis et al., 1995), and (ii) L-type pyruvate kinase (PK, EC2.7.1.40), which catalyses the conversion of phosphoenolpyruvate to pyruvate, the last step in glycolysis (Yamada and Noguchi, 1999). We also studied the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase, EC3.1.3.11); this enzyme catalyses the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate (Marcus et al., 1987). Finally, we looked for a type-2 glucose transporter (Glut2) in the liver of rainbow trout. This transporter maintains the same glucose concentration between the extra and intracellular (hepatocyte) media because of its high Km for glucose (Thorens, 1992).
In mammals, the expression of PK, 6PF-2K/F-2,6BPase and Glut2 proteins is positively controlled by feeding, specifically by dietary carbohydrates (Thorens, 1992; Pilkis et al., 1995; Yamada and Noguchi, 1999); this induction potentiates dietary glucose utilisation in the liver. However, kinase activity of 6PF-2K/F-2,6BPase, which generates the fructose-2,6-bisphosphate, is also positively dependent on protein phosphatase 2A activity. The mechanism of induction of these proteins is mediated largely by induction of their gene expression even though, for the Glut2, the regulation of gene expression in the liver is rather complex (Thorens, 1992; Rencurel et al., 1996; Pilkis et al., 1995; Yamada and Noguchi, 1999). In contrast, feeding and dietary carbohydrates inhibit FBPase gene expression (El-Magrabi et al., 1982; El-Magrabi et al., 1988).
Nutritional regulation of the activities of PK, FBPase and 6PF-2K/F-2,6BPase has been studied previously in fish (Cowey et al., 1977; Cowey et al., 1981; Hilton and Atkinson, 1982; Fideu et al., 1983; Petersen et al., 1987; Lupianez et al., 1989; Garcia de Fructos and Baanante, 1994; Shikata et al., 1994; Shimeno et al., 1995; Meton et al., 1999a; Meton et al., 1999b; Tranulis et al., 1996; Borrebaek and Christophersen, 2000). It seems that there is an increase in the activities of PK and 6PF-2K/F-2,6BPase in response to feeding, although no clear data on the mechanism (transcriptional, post-transcriptional, post-translational) involved in this nutritional regulation are available except for the 6PF-2K/F-2,6BPase in gilthead seabream (Sparus aurata) (Garcia de Fructos and Baanante, 1994; Meton et al., 1999a; Meton et al., 1999b). No specific inhibition of hepatic FBPase activity in response to feeding has been observed in perch (Perca fluviatilis) or salmon (Salmo salar) (Tranulis et al., 1996; Borrebaek and Christophersen, 2000) whereas in European seabass (Dicentrarchus labrax) and gilthead seabream inhibition of FBPase activity in response to feeding has been demonstrated (Garcia-Rejon et al., 1996; Meton et al., 1999b). Finally, the existence of a mammalian-type glucose transporter (Glut) in fish is still a matter of debate; for example, studies using mammalian anti-Glut antibodies suggest, in tilapia (Oreochromis niloticus), a very limited distribution of Glut1 and the complete absence of Glut4 (Wright et al., 1998), whereas recent studies demonstrate the existence of Glut1 and Glut4 cDNAs in rainbow trout and brown trout (Salmo trutta), respectively (Teerijoki et al., 2000; Planas et al., 2000).
In rainbow trout, no molecular data for 6PF-2K/F-2,6BPase, PK, FBPase and Glut2 proteins are yet available. The aim of this study was the partial cloning of PK, GLUT2, FBPase and 6PF-2K/F-2,6BPase cDNAs expressed in the liver, followed by the analysis of their expression in different tissues and in fish under different nutritional conditions.
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Materials and methods |
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RNA isolation and reverse transcription
Total RNA was extracted from rainbow trout tissues as described by Chomczinski and Sacchi (Chomczinski and Sacchi, 1987). cDNA was obtained by annealing 2µg of total RNA with 1µg of random primers and incubating with AMV reverse transcriptase (Boehringer, Roche Molecular Biochemicals, Germany) for 1h at 42°C.
Cloning of partial 6PF-2K/F-2,6BPase, PK, FBPase and Glut2 cDNAs
6PF-2K/F-2,6BPase, PK, FBPase and Glut2 sequences from different species (see Table1) were compared using the Clustal-W multiple alignment algorithm (Higgins and Sharp, 1989). The sequences of the upstream and downstream (degenerate) primers are presented in Table2. cDNA (1µl) was amplified by polymerase chain reaction (PCR) using 100pmol of the degenerate primers in a reaction mixture containing 2 mmoll-1MgCl2, 50 mmoll-1KCl, 20 mmoll-1Tris-HCl, 0.25 mmoll-1dNTP and 2.5 units of Taq polymerase (Boehringer, Roche Molecular Biochemicals, Germany). 35 cycles of denaturation for 1min at 94°C, annealing at 56°C for 30s, and extension at 72°C for 30s were performed. PCR products were subjected to electrophoresis in 1% agarose gels and fragments of the expected size range were purified (Micropure System, Amicon, USA). The purified DNA fragments were inserted into the pCRTMII plasmid and used for transformation of One ShotTMcompetent cells (Invitrogen, Carlsbad, CA, USA). Inserts were detected by EcoRI digestion of the extracted plasmid DNA. Two clones with inserts were sequenced (Cybergène, Evry, France).
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Analysis of gene expression
Northern analysis
Extracted total RNA (20µg) samples were electrophoresed in 1% agarose gels containing 5% formaldehyde and transferred by capillary onto nylon membrane (Hybond-N+, Amersham, England). After transfer, RNA blots were stained with Methylene Blue to locate 26S and 16S rRNAs and to determine the amount of loaded RNA. Membranes were hybridized with fish 32P-labelled DNA probes labelled by random priming (Stratagene, USA) and recognizing partial rainbow trout 6PF-2K/F-2,6BPase, PK, FBPase and Glut2 cDNAs. After stringent washing (2x SSC, 0.1% SDS for 20min; 1x SSC, 0.1% SDS for 20min; 0.2x SSC, 0.1% SDS for 15min), the membranes were exposed to X-ray film and the resulting images were quantified using Visio-Mic II software (Genomic, France).
RTPCR analysis
cDNAs were amplified by PCR using specific primers chosen from the partial sequences for rainbow trout 6PF-2K/F-2,6BPase, PK, FBPase and Glut2 cDNAs (Table2). PCR reactions were carried out in a final volume of 25µl containing 1.5 mmoll-1 MgCl2 and 4pmol of each primer, 2µl cDNA and 1 unit of Taq polymerase (Boehringer, Roche Molecular Biochemicals, Germany). 35 cycles of 20s for hybridization (at 57°C), 20s for elongation (at 72°C) and 20s for denaturation (at 94°C) were performed. The PCR products were characterized by sequencing (Cybergène, Evry, France).
Statistical analyses
The results are expressed as means ± S.D. When there were significant differences in variances (one-way analysis of variance, ANOVA), statistical differences between series of data were determined using Tuckeys post-hoc test (Systat 9 software products, SPSS Inc.). Differences were considered significant at 5%.
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Results |
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Discussion |
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As in mammals (Marcus et al., 1987; Thorens, 1992; Pilkis et al., 1995; Yamada and Noguchi, 1999), PK, 6PF-2K/F-2,6BPase, FBPase and Glut2 genes are expressed in the liver, intestine and kidney. Moreover, there is no apparent nutritional effect on the regulation of PK, Glut2, FBPase and 6PF-2K/F-2,6BPase gene expression in these different tissues, except for the FBPase gene, which is expressed in non-gluconeogenic tissues such as the muscle and heart in fed fish only. To our knowledge, the biochemical consequences of FBPase gene expression in muscle (white muscle and heart) have not been studied.
PK and 6PF-2K/F-2,6BPase genes code for hepatic glucose metabolism proteins that are induced by feeding in mammals (Pilkis et al., 1995; Yamada and Noguchi, 1999). Induction of hepatic trout 6PF-2K/F-2,6BPase gene expression by feeding (and specifically by dietary carbohydrates) in rainbow trout confirms what has been already shown in mammals and gilthead seabream (Garcia de Fructos and Baanante, 1994; Meton et al., 1999a; Meton et al., 1999b; Meton et al., 2000). However, a significant change in 6PF-2K/F-2,6BPase gene expression may either increase or decrease concentrations of fructose-2,6-bisphosphate; more data on the phosphorylation/dephosphorylation status of the enzyme (induced by protein kinase A or protein phosphatase 2A activities) (Pilkis et al., 1995) would be needed to determine in which direction 6PF-2K/F-2,6BPase affects fructose-2,6-bisphophate concentrations. The trout hepatic PK gene is always highly expressed, irrespective of the nutritional status. Using enzymatic methods, some authors (Cowey et al., 1977; Cowey et al., 1981; Fideu et al., 1983) found that PK activity was induced by feeding in rainbow trout, while others (Hilton and Atkinson, 1982; Shikata et al., 1994) found no such induction. Our data suggest that this induction is not (mainly) due to a transcriptional mechanism, but is more probably linked either to post-transcriptional regulation or to qualitative alterations such as phosphorylation/dephosphorylation of PK, as in mammals (Yamada and Noguchi, 1999). Moreover, we were unable to demonstrate a significant decrease in FBPase gene expression in response to dietary carbohydrates in rainbow trout liver (at least for the major FBPase mRNA species), in accordance with the absence of inhibition of FBPase activity by dietary carbohydrates in other fish species such as perch (Perca fluviatilis) or Atlantic salmon (Salmo salar) (Tranulis et al., 1996; Borrebaek and Christophersen, 2000). It is also worth noting that some individuals (irrespective of the nutritional status) show very low levels of FBPase gene expression; the explanation for this result is unknown.
Our data demonstrate the existence of a glucose transporter very similar to the mammalian Glut2 (cloning of a full-length Glut2 cDNA is in progress in collaboration with Dr A. Krasnov and confirms the existence of a Glut2 protein in trout; our unpublished data). Glut2 seems to be expressed at high levels in rainbow trout, irrespective of nutritional status. This result is expected since Glut2 is important for the entry of glucose into hepatocytes but to allow glucose (produced by gluconeogenesis and glycogenolysis) out of the hepatocytes during fasting (Thorens, 1992).
In conclusion, our data demonstrate the existence of mammalian-type induction of 6PF-2K/F-2,6BPase gene expression by feeding, as has been shown previously for trout glucokinase (Panserat et al., 2000a; Panserat et al., 2000b) in association with high levels of PK gene expression. Taken together, these data suggest the existence of an efficient enzymatic mechanism for storing excess glucose. The existence of a type-2 glucose transporter further supports the possibility that hepatocytes store excess dietary glucose. In contrast, FBPase is not strongly inhibited by dietary carbohydrates. Such a lack of inhibition has been observed previously for two other key hepatic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (Panserat et al., 2000a; Panserat et al., 2000b; Panserat et al., 2001), suggesting an absence of nutritional control of hepatic glucose production in trout. Further analysis of hepatic glucose production in vivo and ex vivo is necessary.
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Acknowledgments |
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References |
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