Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp

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.     

Co-localization of the ketohexokinase and glucokinase regulator genes to a 500-kb region of Chromosome 2p23

The glucokinase regulator (GCKR) is a 65-kDa protein that inhibits glucokinase (hexokinase IV) in liver and pancreatic islet. The role of glucokinase (GCK) as pancreatic β cell glucose sensor and the finding of GCK mutations in maturity onset diabetes of the young (MODY) suggest GCKR as a further candidate gene for type 2 diabetes. The inhibition of GCK by GCKR is relieved by the binding of fructose-1-phosphate (F-1-P) to GCKR. F-1-P is the end product of ketohexokinase (KHK, fructokinase), which, like GCK and GCKR, is present in both liver and pancreatic islet. KHK is the first enzyme of the specialized pathway that catabolizes dietary fructose. We have isolated genomic clones containing the human GCKR and KHK genes. By fluorescent in situ hybridization (FISH), KHK maps to Chromosome (Chr) 2p23.2-23.3, a new assignment corroborated by somatic cell hybrid analysis. The localization of GCKR, originally reported by others as 2p22.3, has been reassessed by high-resolution FISH, indicating that, like KHK, GCKR maps to 2p23.2-23.3. The proximity of GCKR and KHK was further demonstrated both by two-color interphase FISH, which suggests that the two genes lie within 500 kb of each other, and by analysis of overlapping YAC and P1 clones spanning the interval between GCKR and KHK. A new microsatellite polymorphism was used to place the GCKR-KHK locus between D2S305 and D2S165 on the genetic map. The co-localization of these two metabolically connected genes has implications for the interpretation of linkage or allele association studies in type 2 diabetes. It also raises the possibility of coordinate regulation of GCKR and KHK by common cis-acting regulatory elements. Received: 8 December 1995 / Accepted: 27 January 1996     

The level and composition of free fatty acids in the plasma of freshwater fish in a post-absorptive condition

The level and fatty acid composition of plasma free fatty acids (FFA) were determined in four important aquaculture teleost species: goldfish (Carassius auratus), common carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss) and tilapia (Oreochromis mossambicus). Plasma was obtained from animals after a 3 day fast in order to study the mobilization of fatty acids in a post-absorptive condition. Plasma [FFA] amounted to 0.95 in goldfish, 0.77 in both carp and rainbow trout and 0.30 μmol/ml in tilapia. The fatty acid composition was dominated by mono-unsaturates in goldfish and carp, whereas saturates were the main fatty acids in tilapia. The largest amount of PUFA was observed in rainbow trout (41%) which was considerably higher than in the other species (27–29%). The interspecies differences of plasma FFA levels and patterns are discussed in relation to the site of lipid storage, mobility and other factors.     

Proteolytic activity and electrophoretic profiles of proteases from seminal plasma of teleosts

Using gelatin-SDS-PAGE, proteolytic activity was found in the seminal plasma of 10 teleosts: common carp Cyprinus carpio , bream Abramis brama , ide Leuciscus idus , chub Leuciscus cephalus , rainbow trout Oncorhynchus mykiss , grayling Thymallus thymallus , perch Perca fluviatilis , pike Esox lucius , goldfish Carassius carassius and pikeperch Stizostendion lucioperca . This activity was also measured, using azoalbumin as a substrate, in the seminal plasma of these species, with exception of pikeperch and goldfish. When azoalbumin-hydrolysing activity was expressed per volume, it was highest in common carp. Otherwise, as expressed per g of protein, the activity was highest in pike. The lowest proteolytic activity (expressed per g and volume) was observed in perch seminal plasma. Using gelatin containing polyacrylamide gels for detecting gelatinolytic activity, species-specific electrophoretic profiles were found. For all cyprinids two similar bands with a molecular mass of 68 and 74 kDa were found. The seminal plasma of grayling and rainbow trout showed similarities in the 41 kDa band. Perch and pikeperch had one similar main band with a molecular mass of 61 kDa. Proteolytic enzymes of seminal plasma from pike showed high individual variability. These results suggest that multiple forms of proteolytic enzymes exist in seminal plasma of teleosts and they differ among fish families and species.     

Comparison of the activity of carp and rat β-actin gene regulatory sequences in tilapia and rainbow trout embryos

A comparative study on the level of expression of lacZ reporter constructs driven by equivalent carp and rat β-actin regulatory sequences was carried out in embryos of tilapia and rainbow trout. DNA was microinjected into fertilised tilapia and rainbow trout eggs and the embryos/fry were assayed at various developmental stages for β-galactosidase expression. We provide evidence to demonstrate that the carp β-actin promoter/lacZ reporter gene is expressed at higher levels than the equivalent rat β-actin construct in both species. © 1996 Wiley-Liss, Inc.     

Human glucokinase regulatory protein (GCKR): cDNA and genomic cloning,complete primary structure,and chromosomal localization

Null mutations in the glucokinase (GCK) gene can cause autosomal dominant type 2 diabetes (maturity onset diabetes of the young, MODY); however, MODY is genetically heterogeneous. In both liver and pancreatic islet, glucokinase is subject to inhibition by a regulatory protein (GCKR). Given the role of GCK in MODY, GCKR is itself a candidate type 2 diabetes susceptibility gene. Here we describe the structure of full-length (2.2 kb) cDNA for human GCKR, from the hepatoblastoma cell line HepG2. The human GCKR translation product has 625 amino acids and a predicted molecular weight of 68,700. It has 88% amino acid identity to rat GCKR. Yeast artificial chromosomes (YAC clones) containing human GCKR were isolated, and the gene was mapped to Chromosome (Chr) 2p23 by fluorescent in situ hybridization and somatic cell hybrid analysis.EMBL database accession numbers: Z48475 and Z48476.     

A comparison of the properties of fructose 1,6-diphosphatase, and the activities of other key enzymes of carbohydrate metabolism, in the livers of embryonic and adult rat, sheep and domestic fowl

1. The activities of some key enzymes of glycolysis and gluconeogenesis were measured in embryonic chick, sheep and rat livers. 2. In chicken the activities of hexokinase, phosphofructokinase and pyruvate kinase are low, but those of glucose 6-phosphatase and fructose diphosphatase are very high; the converse situation exists in the rat (Burch et al. 1963), but in sheep the activities of both phosphofructokinase and fructose diphosphatase are high, and the activities of hexokinase and glucose 6-phosphatase are low. These findings are discussed in relation to carbohydrate metabolism in these embryonic livers. 3. The regulatory properties of fructose diphosphatase from the embryonic livers of these three species were compared with the properties of the enzymes from adult animals. The inhibitions by AMP and fructose diphosphate and the effects of Mg(2+) and pH on the activities of adult and foetal fructose diphosphatase are almost identical. 4. It is concluded that regulatory properties are characteristic of fructose diphosphatase from embryonic and adult tissue, and the importance of this in relation to enzyme development is discussed.     

The in vitro metabolism of Eulan WA New by liver homogenates from freshwater fish

The rate of formation of pentachloro-2-aminodiphenylether (5-PAD) from pentachloro-2-(chloromethylsulphonamido) diphenylether (6-PCSD) by goldfish liver homogenates was studied in a variety of buffers. The highest rate was observed when the homogenate was prepared and assayed in phosphate buffer containing dithiothreitol. The 5-PAD forming activity of liver homogenate preparations from a variety of freshwater fish (goldfish, carp, pike, rainbow trout, perch and eel) showed contrasting species differences. These results could be correlated with information available on the toxicity of the mothproofing agent Eulan WA New (which contains PCSDs) to these fish.     

Distribution of pancreatic elastase and metalloproteinase in vertebrates

Elastase-like enzymes were detected as zymogens in all of the pancreatic extracts from the gummy shark, bullhead shark, angel shark, smooth hammerhead, bestel, rainbow trout, carp, eel, Japanese mackerel, yellowtail, sea bass, parrotfish, bullfrog, chicken, bluewhite dolphin, hog, rat, cat, and dog. The distribution of pancreatic elastase and metalloproteinase was examined on the basis of the effect of specific inhibitors on elastase like-activity in each extract. The results indicate that pancreatic elastases are present in all the species examined and pancreatic metalloproteinases are present only in the teleost fishes.     

Different sensitivity of carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) to the immunomodulatory effects of UVB irradiation

In order to study the sensitivity of two fish species, carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss), to the immunomodulatory effects of ultraviolet B (UVB) radiation, the fish were exposed to a single UVB dose of 50, 250, 500 or 1,000 mJ cm(-2). These species represent different phylogenetic groups of fish, and they differ also in their behaviour inhabitating often dark and turbid (carp) or clear and transparent waters (salmonids). Immune responses were studied on day 1 post-irradiation. Unexposed fish, and fish exposed to radiation depleted of UV wavelengths served as controls. UVB irradiation markedly enhanced the blood respiratory burst and cytotoxic activity in carp, but in the head kidney these parameters were significantly suppressed. Rainbow trout respiratory burst was affected only after exposure with the highest dose of UVB. Lymphopenia and granulophilia were noted in both fish blood after exposure. This study indicates that UVB irradiation modulates immune functions in both fish species studied, and that rainbow trout is more tolerant than carp against UVB. Fish are clearly adapted to the environmental UVB levels prevailing in their usual living habitats, but are also a target of undesired effects of UVB on immune functions whenever exposed to increased radiation levels.     

Association of glucokinase regulatory protein polymorphism with type 2 diabetes and fasting plasma glucose: a meta-analysis

Glucokinase regulatory protein (GCKR) which binds to glucokinase (GCK) in the nucleus and inhibits its activity in the presence of fructose-6-phosphate is critical for glucose metabolism. In the past few years, a number of case–control studies have been carried out to investigate the relationship between the GCKR polymorphism and type 2 diabetes (T2D) since it was first identified to be associated with fasting plasma glucose levels, insulin resistance through genome-wide association approach. After that, a number of studies reported that the rs780094 polymorphism in GCKR has been implicated in T2D risk. However, these studies have yielded contradictory results. To investigate this inconsistency, we performed a meta-analysis of 19 studies involving a total of 298,977 subjects for GCKR rs780094 to evaluate its effect on genetic susceptibility for T2D. In a combined analysis, the summary per-allele odds ratio for T2D of the rs780094 polymorphism was 1.11 (95 % CI: 1.07–1.14, P < 10^?5). Significant results were also observed using dominant (OR = 1.18, 95 % CI: 1.05–1.34, P < 10^?5) or recessive genetic model (OR = 1.20, 95 % CI: 1.12–1.28, P < 10^?5). Significant results were found in Asians and Caucasians when stratified by ethnicity. Besides, the polymorphism was found to be significantly associated with increased fasting plasma glucose level. There was strong evidence of heterogeneity, which largely disappeared after stratification by ethnicity. This meta-analysis suggests that the rs780094 polymorphism in GCKR is associated with elevated T2D risk, but these associations vary in different ethnic populations.     

Anti-Oxidative Defences Are Modulated Differentially in Three Freshwater Teleosts in Response to Ammonia-Induced Oxidative Stress

Oxidative stress and the antioxidant response induced by high environmental ammonia (HEA) were investigated in the liver and gills of three freshwater teleosts differing in their sensitivities to ammonia. The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed to 1 mM ammonia (as NH₄HCO₃) for 0 h (control), 3 h, 12 h, 24 h, 48 h, 84 h and 180 h. Results show that HEA exposure increased ammonia accumulation significantly in the liver of all the three fish species from 24 h–48 h onwards which was associated with an increment in oxidative stress, evidenced by elevation of xanthine oxidase activity and levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). Unlike in trout, H₂O₂ and MDA accumulation in carp and goldfish liver was restored to control levels (84 h–180 h); which was accompanied by a concomitant increase in superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase activity and reduced ascorbate content. Many of these defence parameters remained unaffected in trout liver, while components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase) enhanced to a greater extent. The present findings suggest that trout rely mainly on glutathione dependent defensive mechanism while carp utilize SOD, CAT and ascorbate as anti-oxidative sentinels. Hepatic cells of goldfish appear to utilize each of these protective systems, and showed more effective anti-oxidative compensatory responses towards HEA than carp, while trout were least effective. The present work also indicates that HEA exposure resulted in a relatively mild oxidative stress in the gills of all three species. This probably explains the almost complete lack of anti-oxidative responses in branchial tissue. This research suggests that oxidative stress, as well as the antioxidant potential clearly differ between salmonid and cyprinid species.     

Glucokinase is highly induced and glucose-6-phosphatase poorly repressed in liver of rainbow trout (Oncorhynchus mykiss) by a single meal with glucose

The low dietary starch utilisation by rainbow trout (Oncorhynchus mykiss) may be attributed to a dysfunction of the nutritional regulation of the hepatic glucose/glucose-6-phosphate cycle. The present study was initiated to analyse the regulation of activity and gene expression of hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) by dietary carbohydrates in this species. We found that even a single meal containing 24% of glucose is sufficient to induce the GK expression (mRNA and activity) as in mammals. In contrast, although the inhibitory effect of dietary glucose on G6Pase expression is observed at the molecular level, the G6Pase activity is not significantly inhibited by dietary glucose. Thus, in contrast to the gluconeogenic G6Pase enzyme, a rapid adaptation of the hepatic glycolytic GK enzyme to dietary glucose seems effective in rainbow trout. These results suggest that in carnivorous rainbow trout, the liver is capable to strongly regulate the utilisation of glucose but not the synthesis of glucose.     

Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss

Scanning electron microscopic studies were conducted on rainbow trout Oncorhynchus mykiss in the first 60 min after their exposure to the triactinomyxon spores of Myxobolus cerebralis. The results demonstrated that as early as 1 min post exposure the whole process, from the attachment of the triactinomyxon spores to the complete penetration of their sporoplasm germs, had occurred. The triactinomyxon spores sought out the secretory openings of mucous cells of the epidermis, the respiratory epithelium and the buccal cavity of trout and used them as portals of entry. Exposure experiments of the triactinomyxon spores of M. cerebralis to non-salmonid fish, such as goldfish Carassius auratus, carp Cyprinus carpio, nose Chondrostoma nasus, medaka Oryzias latipes, guppy Poecilia reticulata and also the amphibian tadpole Rana pipiens as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucus prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results suggest that the simultaneous presence of both mechano- and chemotactic stimuli was required for finding the salmonid fish host.     

金鱼同工酶的发生遗传学研究——Ⅲ.金鱼同工酶基因座位的加倍与多倍性

以鲫鱼和金鱼为材料,用葡萄糖-6-磷酸脱氢酶(G6PD)、乳酸脱氢酶(LDH)和苹果酸脱氢酶(MDH)同工酶体系作为基因标志,从检测同工酶的多重组合形式来研究基因的加倍与演化。对彩鲫与金鱼G6PD和LDH同工酶的分析结果表明,它们均具有与四倍体鱼类相应的谱带。因而说明了金鱼的G6PD和LDH同工酶基因座位的加倍与染色体的多倍性有关,为金鱼是四倍体的假说提供了证据。而对MDH同工酶的分析却得到了与二倍体鱼类相同的谱带数。这可能与加倍基因发生突变而不表达有关。     

Dietary fructose does not specifically induce hepatic glucokinase expression in rainbow trout

Glucokinase (GK) catalyses the first step of hepatic glycolysis to store the excess dietary glucose. After rainbow trout Oncorhynchus mykiss were fed a single meal with dietary glucose and fructose, there was induction of GK expression, although no specific effect of dietary fructose was observed. This suggested that dietary fructose is not essential to improve dietary glucose Utilization.     

Does the natural diet influence the intestine's ability to regulate glucose absorption?

Summary Two fish species (rainbow trout and common carp) that differ in natural diet also exhibit differences in the adaptive flexibility of their intestinal nutrient transport mechanisms in response to changes in dietary nutrient composition. When carp ingested a feed that was 24% glucose by weight, there was an increase in both the intestinal length and rates of nutrient absorption, particularly for glucose, when compared to carp fed an isonitrogenous diet devoid of digestible carbohydrate. As a result, the intestine's uptake capacity (nmol of glucose and proline absorbed per min per g body weight) was higher in carp fed the 24% glucose feed. Due to the greater increase in glucose uptake, the ratio of glucose uptake relative to proline (G/P ratio) was higher in carp fed the 24% glucose. Thus, carp are able to adapt to the quantity, and apparently also to the type, of digestible carbohydrate in the diet. In contrast, glucose uptake by trout was unresponsive to the quantity of dietary carbohydrate. Insted, the elevated glucose paradoxically resulted in a greater uptake capacity for proline and a lower G/P ratio. Hence, the adaptive capabilities of the intestinal nutrient transport processes are matched to the potential variation in the carbohydrate content of the natural diet.Abbreviation PEG polyethylene glycol     

Limited species differences in estrogen receptor alpha-medicated reporter gene transactivation by xenoestrogens

Estrogen receptors (ERs) play an important role in estrogen function. However, it is well known that there are species differences in amino acid sequences of the ligand binding domains. Here, we report on the analysis of species differences in ER-dependent transactivation with some chemicals using reporter gene assays. Full-length ER cDNAs from human, rat, chicken, alligator (Caiman), whiptail lizard, African clawed frog and rainbow trout were prepared from hepatic mRNA by the RT-PCR method and inserted into expression plasmids. Both expression and reporter plasmids were transiently transfected into HeLa cells, and then the estrogenic effects of chemicals were analyzed in terms of induction of luciferase activity. No species differences in transactivation were found among human, rat, chicken, alligator, whiptail lizard and African clawed frog ERs. However, thermo-dependent alteration in susceptibility to 17-beta-estradiol was observed with the rainbow trout ER because of thermo-dependence of estrogen binding.     

Identification of keratins and analysis of their expression in carp and goldfish: comparison with the zebrafish and trout keratin catalog

With more than 50 genes in human, keratins make up a large gene family, but the evolutionary pressure leading to their diversity remains largely unclear. Nevertheless, this diversity offers a means to examine the evolutionary relationships among organisms that express keratins. Here, we report the analysis of keratins expressed in two cyprinid fishes, goldfish and carp, by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot binding assay, and immunoblotting. We further explore the expression of keratins by immunofluorescence microscopy. Comparison is made with the keratin expression and catalogs of zebrafish and rainbow trout. The keratins among these fishes exhibit a similar range of molecular weights and isoelectric points, with a similar overall pattern on two-dimensional gels. In addition, immunofluorescence microscopy studies of goldfish and carp tissues have revealed the expression of keratins in both epithelial and mesenchymally derived tissues, as reported previously for zebrafish and trout. We conclude that keratin expression is qualitatively similar among these fishes, with goldfish and carp patterns being more similar to each other than to zebrafish, and the cyprinid fishes being more similar to each other than to the salmonid trout. Because of the detected similarity of keratin expression among the cyprinid fishes, we propose that, for certain experiments, they are interchangeable. Although the zebrafish distinguishes itself as being a developmental and genetic/genomic model organism, we have found that the goldfish, in particular, is a more suitable model for both biochemical and histological studies of the cytoskeleton, especially since goldfish cytoskeletal preparations seem to be more resistant to degradation than those from carp or zebrafish. This work was supported by grants to J.M. from the Stiftung Rheinland Pfalz für Innovation (836-386261/138) and the Deutsche Forschungsgemeinschaft (Ma 843/5-1) and a grant to D.G. from the National Science Foundation (INT-0078261).