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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION |
* INRA, UR83 Recherches Avicoles, F-37380 Nouzilly, France; and INRA, UE89 Palmipèdes à foie gras, Domaine d’Artiguères, F-40280 Benquet, France
1 Corresponding author. rideau{at}tours.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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600 bp) was obtained from duck livers. It presented 99% identity with chicken partial GK cDNA (gi 44888789) and 82% identity with human GK (gi 15967158). Chicken liver weights represented 1.8 and 3.3% of BW, respectively, for BC and BS (n = 8, P < 0.05). Glucokinase and low-Michaelis constant hexokinase (HK) activity levels were similar in BC (respectively, 0.88 and 1.00 mU/mg of protein). In response to the meal load, GK activity increased significantly (+57%), whereas HK decreased (–46%) in BS. Duck liver weights represented 1.4 and 7.6% of BW, respectively, for DC and DO (n = 8, P < 0.05). In DC livers, GK activity was significantly higher than HK activity (respectively, 1.76 and 0.63 mU/mg of protein). Both activities were significantly increased in DO (2 times, n = 8, P < 0.05). In conclusion, GK is present in ducks as well as chickens, and it is nutritionally regulated in avian species as well as in mammals. Further work will determine whether the higher liver GK activity and GK:HK ratio in DC compared with BC is related to age or BW or linked to the high lipogenic capacity of the duck liver.
Key Words: glucokinase • liver • complementary deoxyribonucleic acid • chicken • duck
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Recent studies in mammals have shown a major role of the hexokinase (HK) D isoform (glucokinase, GK; EC 2.7.1.2) in carbohydrate utilization by the liver (Girard et al., 1997; Kahn, 1998). Glucokinase is a member of the HK protein family, which controls the first step of glucose utilization; it shows some remarkable characteristics that clearly differentiate it from the other mammalian HK. The high Michaelis constant (Km) of GK for glucose, together with its specific expression in the liver and pancreas, allows it to be a sensitive and efficient control step for the maintenance of glucose metabolism. In the liver, GK is sensitive to nutritional conditions, particularly to blood glucose and insulin levels (Matsuda et al., 1990). It regulates the production of glucose-6-phosphate, which will be directed toward energy production through glycolysis and the Krebs cycle, as well as glycogen synthesis and lipogenesis. Hepatic GK is also required for glycolytic and lipogenic gene expression, as recently shown by Dentin et al. (2004). In the pancreas, GK acts as a major sensor of circulating glucose and is a key element regulating insulin secretion (Matschinsky, 1996).
Although found in most vertebrate species, the presence of GK in avian species has long been a matter of debate (Cardenas et al., 1998). This is a relevant problem, because GK plays an important role in mammalian glucose homeostasis. We recently answered this question in the chicken by using available molecular techniques. Using reverse transcription-PCR, we cloned and sequenced a partial cDNA fragment (750 bp) from the chicken liver and pancreas that showed a high degree of identity with a mammalian or fish GK cDNA sequence. Using antibodies directed toward human GK, a 50-kDa band in the chicken liver and pancreas was immunodetected. The molecular mass of the band and its specific interaction with the antibody suggested that this protein corresponds to a chicken homolog of human GK. By spectrophotometry, we also determined a GK-like activity in crude liver homogenates with an apparent half-saturating concentration for glucose of 8.6 mM. We thus provided evidence of the presence of the GK gene and protein in the chicken liver and pancreas and showed that the liver enzyme is active (Berradi et al., 2005). In birds, lipogenesis occurs in the liver (Leveille et al., 1968), so we further questioned the role of GK in avian lipogenesis by using the overfed mule duck, which develops a fatty liver resulting from a dramatic increase in de novo liver lipogenesis. We characterized a GK-like immunoreactive protein and a GK-like activity in the mule duck liver and reported variations during different stages of overfeeding. The intensity of the immunoreactive signal varied significantly between overfed and control ducks, whereas GK-like specific activity (U/mg of protein) was strongly induced by overfeeding (Berradi et al., 2004). These results suggested that a GK-like enzyme may have actively contributed to glucose disposal throughout the period in which mule ducks were overfed a carbohydrate-rich diet. In the current paper, we further characterized a partial cDNA sequence from the mule duck liver and measured GK-like activities from chicken and duck livers under 2 nutritional conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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37% carbohydrates and 22% proteins). Feed was given as a 2-h meal (0900 to 1100 h) from 1 wk of age until sacrifice at 5 wk of age. Water was freely available. At 5 wk of age, chickens were killed by cervical dislocation. Liver samples were obtained from overnight-fasted chickens (control broilers, BC) and 5 h after an oral saccharose load (6 mL/kg of BW of a 50% saccharose solution) given just before the meal (saccharose load broilers, BS). Tissues were quickly removed, frozen, powdered in liquid nitrogen, and stored at –80°C. Male mule ducks [hybrid of male muscovy (Cairina moschata) and female Pekin (Anas plathyrhynchos), cross MMG x PKL, Gourmaud sélection, Saint-André-Treize-Voies, France] were grown under natural conditions of light and temperature at the Experimental Station for Waterfowl Breeding (Institut National de la Recherche Agronomique, Artiguères, France). They were housed collectively from hatching to 13 wk and then in individual cages during the overfeeding period (2 wk). Rearing and overfeeding were conducted as previously described (Rouvier et al., 1994). The preoverfeeding period began at 13 wk of age and continued for 6 d, during which the feed restriction was progressively relaxed to increase the volume of the digestive tract and to initiate metabolic adaptation to overfeeding. At the end of the preoverfeeding period, for 13 d ducks were given 2 carbohydrate-rich meals/d consisting of 35% boiled, salted corn (14.2 MJ, 77 g of protein/kg), 25% corn grain (to improve digestibility), and 40% water. Animals had free access to water at all times. No prophylactic medication was provided before or during overfeeding. Overfed ducks were killed after 13 d (12 h after the last overfeeding meal: overfed ducks, DO). A control group of the same age was fed ad libitum until 15 wk and killed after overnight feed deprivation with water provided (control ducks, DC). Blood was drawn by puncture of the occipital venous sinus and collected on EDTA. Individual plasma samples were separated by centrifugation at 2,680 x g for 10 min. Plasma was frozen at –20°C until analysis. Ducks were then killed in a slaughterhouse by exsanguination while under electronarcosis. The livers were quickly removed and weighed, and a 5-g sample was taken from the ventromedial portion of the main lobe (right lobe) of each liver, frozen in liquid nitrogen, ground into powder, and stored at –80°C until use. The study was carried out according to French legislation on animal experimentation and with the authorization of the French Ministry of Agriculture (Animal Health and Protection Directorate).
Duck Liver cDNA Cloning
Duck liver cDNA cloning was performed as decribed by Berradi et al. (2005). Glucokinase forward (5'-ACT GGA GGA GAT GCA CAA CG-3') and reverse (5'-TCC GAC TGG ATG AAG GTG ATG-3') primers were chosen on the chicken partial cDNA GK sequence (gi 44888789) by using Primer Express software (Applied Biosystems, Courtaboeuf, France). Cloned fragments were sequenced with an ABI automated sequencer (Applied Biosystems). The sequences obtained from liver cDNA samples were compared with GenBank, EMBL, DNA Data Bank of Japan, and PDB databases by using the basic alignment search tool BLAST algorithm. Amino acid sequences of the cloned fragments were also deduced with this algorithm. Sequence alignments and percentages of conservation of amino acids were assessed with the ClustalW multiple-alignment algorithm, which compared the cloned duck sequence with chicken partial GK sequence and sequences of chosen representative species.
Analysis of Enzyme Activity.
The spectrophotometric method described by Panserat et al. (2000) was used. Briefly, a sample of liver (500 mg) was homogenized (dilution 1:10) in ice-cold buffer [in mM: 80 Tris, 5 EDTA, 2 1,4-dithiothreitol, 1 benzamidine, 1 4-(2-aminoethyl)benzenesulfonyl fluoride, pH 7.6]. The homogenate was centrifuged for 5 min at 900 x g. Enzyme activities were measured at 37°C by coupling ribulose-5-phosphate formation from glucose-6-phosphate to the reduction of nicotinamide adenine dinucleotide phosphate by using purified glucose-6-phosphate dehydrogenase (Sigma, St. Louis, MO) and 6-phosphogluconate dehydrogenase (Sigma) as coupling enzymes. One unit of enzyme activity was defined as the amount that phosphorylates 1 µmol of glucose/min. The GK activity of the crude homogenate was estimated by the standard method by subtracting the rate of reduced nicotinamide adenine dinucleotide phosphate formation (at 340 nm; Uvikon 933, Kontron Instruments, UK) in the presence of 0.5 mM glucose (scoring low-Km HK activities) from that at 100 mM glucose (scoring total HK activities) as proposed for mammals and Atlantic salmon (Tranulis et al., 1991). In fish liver, glucose dehydrogenase (EC 1.1.1.47
[EC]
), a moderately active microsomal enzyme, can introduce significant bias into GK measurements on frozen tissue samples, which necessitates correction when measuring GK activity (Tranulis et al., 1991). This was not necessary with chicken and duck samples because we detected no glucose dehydrogenase activity in frozen liver samples (data not shown). N-Acetyl-glucosamine, a known inhibitor of GK activity in mammals, significantly inhibited chicken GK activity by increasing the apparent Km for glucose (data not shown), which confirmed the specificity of the GK activity measurement. Protein concentrations were determined by using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).
Determination of Plasma Glucose and Insulin Levels.
Plasma glucose levels were measured by the glucose oxidase method with an automated analyzer (Beckman Glucose Analyzer 2, Beckman, Palo Alto, CA), and plasma insulin levels were determined by RIA with a guinea pig antiporcine insulin antibody (Ab 27-6, generously provided by G. Rosselin, Hôpital Saint-Antoine, Paris, France), with chicken insulin as the standard (Ruffier et al. 1998).
Statistical Analysis.
Statistical analyses were performed by using the Mann-Whitney test. Data are expressed as means, and P < 0.05 was considered statistically significant.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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600 bp was obtained, purified, cloned, and sequenced. The sequence analyzed with the BLAST algorithm showed 99% identity with the partial GK cDNA sequence from chicken liver (gi 44888789) and 82% similarity with the human GK cDNA sequence (gi 15967158). The corresponding amino acid sequence was deduced from the cDNA sequence and compared with protein sequences from databases by using the BLAST-P algorithm. It presented 75% identity with the human GK isoform (amino acids 232 to 448).
With regard to chicken partial GK cDNA, the duck partial amino acid sequence showed 50% identities with cloned avian HKI and II sequences available in the databases in the region corresponding to putative sites for adenosine triphosphate and glucose binding (from amino acids 646 to 893 of the human HKI protein sequence), and no significant similarity was found in the other part of the sequence. Structure prediction of the translated cDNA with Geno3D (Combet et al., 2002) showed that the most probable structure was the folding of 
-helices and ß-strands into 2 domains (data not shown), typical of HK family clefts, within which glucose and adenosine triphosphate binding sites lie (Mahalingam et al., 1999). Thus, although partial, the duck cDNA sequence may correspond to those of functional mRNA encoding functional GK protein.
GK-Like Activity in Chicken and Duck Livers
Table 1
presents liver relative weights, plasma glucose and insulin levels, and high-Km (GK) and low-Km (HK) HK activities. At 5 wk of age, livers of fasted chickens represented 1.8% of BW. Five hours after the meal with a saccharose load, liver weights increased significantly and represented 3.3% of BW (n = 8, P < 0.05). At 15 wk of age, livers of fasted ducks represented 1.4% of BW; liver weights increased 8-fold after 13 d of overfeeding and represented 7.6% of BW (n = 8, P < 0.05).
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Glucose phosphorylating activity was assayed spectrophotometrically in crude liver homogenates at low (0.5 mM) and high (100 mM) glucose concentrations. Glucokinase activity was obtained by subtracting the activity at low glucose concentrations from that measured at high concentrations. In the basal state, GK specific activity was twice as low in the livers of chickens (BC) than in the livers of mule ducks (DC). Glucokinase and HK specific activities were similar in fasted chickens (BC; respectively, 0.88 ± 0.18 and 1.00 ± 0.18 mU/mg of protein, n =8, P > 0.05), with a GK:HK ratio of 1. In fasted mule ducks (DC), GK activity was significantly higher than HK activity (respectively, 1.76 ± 0.17 and 0.63 ± 0.09 mU/mg of protein for GK and HK, n = 8, P < 0.05), with a GK:HK ratio of 3. Five hours after the meal, GK activity increased significantly in BS (+57%), whereas HK activity decreased (–46%) as compared with the fasted state (BC); the resulting GK:HK ratio increased 3-fold. In DO, both GK and HK activities doubled (n = 8, P < 0.05) as compared with DC; the GK:HK ratio did not differ between DO and DC. According to the nutritional state, GK activities varied between 0.9 and 1.4 mU/mg of protein (or 0.15 to 0.22 U/g of liver; data not shown) in chicken livers and 1.76 and 3.56 mU/mg of protein (or 0.25 to 0.32 U/g of liver; data not shown) in mule duck livers. These values are in the same range as the liver GK activities measured by Wals and Katz (1981) and Klandorf et al. (1986) in chickens, and they are 10-fold lower than the activities measured by Wals and Katz (1981) in rats. Using a radio-chemical assay, Stanley et al. (1984) also noted a 10-fold lower GK activity in the livers of some avian species (chickens, pigeons, and ducks) compared with mammals (rats, mice, and humans). When compared with fish species, chicken liver GK activities were similar to those of common carp and gilthead seabream but were lower than those of rainbow trout (Panserat et al., 2000). At the moment, there is no hypothesis for the low GK activities in the livers of chickens and ducks as well as in those of some fish.
In this study, liver GK activities were higher in DC than in BC. This difference, which may be related to the age or to the BW of the birds (because the chickens were younger than the ducks), needs to be examined further. However, a species-related difference is possible and may be a characteristic of avian species susceptible to hepatic steatosis, such as Muscovy ducks or Landes geese (Anser anser; Mourot et al., 2000). Bedu et al. (2002) noted a 2-fold higher incorporation rate of 14C from glucose into fatty acids of duckling hepatocytes than into those of quail hepatocytes. These authors further observed that the high rate of glucose incorporation into duckling hepatocytes occurred in parallel with high activities of key limiting enzymes of lipogenesis. Moreover, in the absence of overfeeding, Hsu et al. (1992) showed that the fatty acid synthase activity of duckling livers was 10-fold higher than that of chicken livers. The molecular basis for the high incorporation rate of glucose into fatty acids and the high level of lipogenic enzymes may involve GK, as suggested by the high level of GK and by the GK:HK ratio observed in fasted ducks that were not overfed.
In response to the food supply, GK activity increased in parallel with plasma insulin level in broiler chickens (BS) and mule ducks (DO). This observation suggests that liver GK activity is regulated by insulin in avian species as well as in mammals (Goodridge, 1987).
In conclusion, GK is present in duck livers as well as in chicken livers, and it is nutritionally regulated in avian species as well as in mammals. Further work will determine whether the higher liver GK activity and the GK:HK ratio in DC, as compared with BC, is related to age or BW or is linked to the high lipogenic capacity of the duck liver.
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ACKNOWLEDGMENTS |
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Received for publication April 20, 2007. Accepted for publication June 25, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Berradi, H., G. Guy, and N. Rideau. 2004. A glucokinase-like enzyme induced in Mule duck livers by overfeeding. Poult. Sci. 83:161–168.
Berradi, H., M. Taouis, S. Cassy, and N. Rideau. 2005. Glucokinase in chicken (Gallus gallus). Partial cDNA cloning, immunodetection and activity determination. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 141:129–139.[Medline]
Cardenas, M. L., A. Cornish-Bowden, and T. Ureta. 1998. Evolution and regulatory role of the hexokinases. Biochim. Biophys. Acta 1401:242–264.[Medline]
Combet, C., M. Jambon, G. Deleage, and C. Geourjon. 2002. Geno3D: Automatic comparative molecular modelling of protein. Bioinformatics 18:213–214.
Dentin, R., J. P. Pegorier, F. Benhamed, F. Foufelle, P. Ferre, V. Fauveau, M. A. Magnuson, J. Girard, and C. Postic. 2004. Hepatic glucokinase is required for the synergistic action of ChREBP and SREBP-1c on glycolytic and lipogenic gene expression. J. Biol. Chem. 279:20314–20326.
Girard, J., P. Ferre, and F. Foufelle. 1997. Mechanisms by which carbohydrates regulate expression of genes for glycolytic and lipogenic enzymes. Annu. Rev. Nutr. 17:325–352.[Web of Science][Medline]
Goodridge, A. G. 1987. Dietary regulation of gene expression: Enzymes involved in carbohydrate and lipid metabolism. Annu. Rev. Nutr. 7:157–185.[Web of Science][Medline]
Gracia, M. I., M. J. Aranibar, R. Lazaro, P. Medel, and G. G. Mateos. 2003. Alpha-amylase supplementation of broiler diets based on corn. Poult. Sci. 82:436–442.
Hsu, J. C., K. Tanaka, I. Inayama, and S. Ohtani. 1992. Effects of pantethine on lipogenesis and CO2 production in the isolated hepatocytes of the chick (Gallus domesticus). Comp. Biochem. Physiol. Comp. Physiol. 102:569–572.[Medline]
Kahn, A. 1998. From the glycogenic function of the liver to gene regulation by glucose. C. R. Seances Soc. Biol. Fil. 192:813–827.[Medline]
Klandorf, H., B. L. Clarke, A. C. Scheck, and J. Brown. 1986. Regulation of glucokinase activity in the domestic fowl. Biochem. Biophys. Res. Commun. 139:1086–1093.[Web of Science][Medline]
Leveille, G. A., E. K. O’Hea, and K. Chakbabarty. 1968. In vivo lipogenesis in the domestic chicken. Proc. Soc. Exp. Biol. Med. 128:398–401.[Medline]
Mahalingam, B., A. Cuestamunoz, E. A. Davis, F. M. Matschinsky, R. W. Harrison, and I. T. Weber. 1999. Structural model of human glucokinase in complex with glucose and ATP: Implications for the mutants that cause hypo- and hyperglycemia. Diabetes 48:1698–1705.[Abstract]
Matschinsky, F. 1996. Banting Lecture 1995. A lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm. Diabetes 45:223–241.[Abstract]
Matsuda, T., T. Noguchi, K. Yamada, M. Takenaka, and T. Tanaka. 1990. Regulation of the gene expression of glucokinase and L-type pyruvate kinase in primary cultures of rat hepatocytes by hormones and carbohydrates. J. Biochem. (Tokyo) 108:778–784.
Mignon-Grasteau, S., N. Muley, D. Bastianelli, J. Gomez, A. Peron, N. Sellier, N. Millet, J. Besnard, J. M. Hallouis, and B. Carre. 2004. Heritability of digestibilities and divergent selection for digestion ability in growing chicks fed a wheat diet. Poult. Sci. 83:860–867.
Mourot, J., G. Guy, S. Lagarrigue, P. Peiniau, and D. Hermier. 2000. Role of hepatic lipogenesis in the susceptibility to fatty liver in the goose (Anser anser). Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 126:81–87.[Medline]
Panserat, S., F. Medale, C. Blin, J. Breque, C. Vachot, E. Plagnes-Juan, E. Gomes, R. Krishnamoorthy, and S. Kaushik. 2000. Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278:R1164–R1170.
Rouvier, R., G. Guy, D. Rousselot-Paillet, and B. Poujardieu. 1994. Genetic parameters from factorial cross breeding in two duck strains (Anas platyrhynchos) Brown Tsaiya and Pekin, for growth and fatty liver traits. Br. Poult. Sci. 35:509–517.[Web of Science][Medline]
Ruffier, L., J. Simon, and N. Rideau. 1998. Isolation of functional glucagon islets of Langerhans from the chicken pancreas. Gen. Comp. Endocrinol. 112:153–162.[Web of Science][Medline]
Stanley, J. C., G. L. Dohm, B. S. McManus, and E. A. Newsholme. 1984. Activities of glucokinase and hexokinase in mammalian and avian livers. Biochem. J. 224:667–671.[Web of Science][Medline]
Tranulis, M. A., B. Christophersen, A. K. Blom, and B. Borrebaek. 1991. Glucose dehydrogenase, glucose-6-phosphate dehydrogenase and hexokinase in liver of rainbow trout (Salmogairdneri). Effects of starvation and temperature variations. Comp. Biochem. Physiol. B 99:687–691.[Medline]
Wals, P. A., and J. Katz. 1981. Glucokinase in bird liver: A membrane bound enzyme. Biochem. Biophys. Res. Commun. 100:1543–1548.[Web of Science][Medline]
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