Glycolysis and gluconeogenesis in the liver of catfish fed with different concentrations of proteins, lipids and carbohydrates / Glicólise e gliconeogênese no fígado de jundiá alimentado com diferentes concentrações de proteínas, lipídeos e carboidratos

The activities of enzymes from a number of metabolic pathways have been used as a tool to evaluate the best use of nutrients on fish performance. In the present study the catfish Rhamdia quelen was fed with diets containing crude protein-lipid-carbohydrate (%) as follows: treatment (T) T1: 19-19-44; T2: 26-15-39; T3: 33-12-33; and T4: 40-10-24. The fish were held in tanks of re-circulated, filtered water with controlled temperature and aeration in 2000L experimental units. The feeding experiment lasted 30 days. The following enzymes of the carbohydrate metabolism were determined: Glucokinase (GK), Phosphofructokinase 1 (PFK-1), Pyruvate kinase (PK), Fructose-1,6-biphosphatase 1 (FBP-1). The activities of 6 phosphogluconate dehydrogenase (6PGDH) and glucose 6 phosphate dehydrogenase (G6PDH) were also assayed. The influence of nutrient levels on the enzyme activities is reported. The increase of dietary protein plus reduction of carbohydrates and lipids attenuates the glycolytic activity and induces hepatic gluconeogenesis as a strategy to provide metabolic energy from amino acids. The fish performance was affected by the concentrations of protein, lipid and carbohydrates in the diet. The greatest weight gain was obtained in fish fed diet T4 containing 40.14% of crude protein, 9.70% of lipids, and 24.37% of carbohydrate, respectively.(AU)

As atividades de enzimas das vias metabólicas têm sido utilizadas como uma ferramenta para avaliar a melhor utilização dos nutrientes e o desempenho dos peixes. No presente estudo, o jundiá foi alimentado com rações contendo diferentes concentrações de proteína bruta, lipídeos e carboidratos (%), da seguinte forma: tratamento (T) T1: 19-19-44; T2: 26-15-39; T3: 33-12-33; e T4: 40-10-24. Os peixes foram mantidos em tanques de recirculação, com água filtrada, temperatura controlada e aeração em unidades experimentais de 2.000L. O período experimental foi de 30 dias. Foram aferidas as atividades das enzimas glicoquinase (GK), fosfofrutoquinase 1 (PFK-1), piruvato quinase (PK) e frutose-1,6-difosfatase (FBP-1). Também foram aferidas as atividades da 6-fosfogluconato desidrogenase (6PGDH) e glicose-6-fosfato desidrogenase (G6PDH) da via das pentoses. É relatado que níveis de nutrientes influenciam as atividades enzimáticas das vias metabólicas. No presente estudo, o aumento da proteína da dieta e a redução de hidratos de carbono e lipídeos reduziram a atividade glicolítica e induziram a gliconeogênese hepática como uma estratégia para fornecer energia pelos aminoácidos. O desempenho dos peixes foi afetado pelas concentrações de proteínas, lipídeos e carboidratos na dieta. O maior ganho de peso foi obtido em peixes alimentados com dieta T4 contendo 40,14% de proteína bruta, 9,70% de lipídeos, e 24,37% de carboidratos, respectivamente.(AU)

Sp1 and Sp3 regulate glucokinase gene transcription in the liver of gilthead sea bream (Sparus aurata).

To better understand the transcriptional machinery that governs glucokinase (GCK) expression, we have cloned and characterized the proximal promoter region of GCK from gilthead sea bream (Sparus aurata). The 5'-flanking region of GCK was isolated by chromosome walking. SMART RACE-PCR allowed us to locate the transcription start site 98 bp (bp) upstream from the translational start. Transfection analysis in HepG2 cells revealed the presence of a functional promoter in the 1397 bp 5'-flanking isolated fragment (positions -1321 to +76 relative to the transcription start site). Sequential 5'-deletion analysis indicated a core functional promoter for basal transcription within the 288 bp upstream from the transcription start site. Transient transfection experiments performed in HepG2 cells and electrophoretic mobility shift assays denoted that Sp1 binds and transactivates GCK promoter, whereas Sp3 repressed Sp1-mediated activation of GCK by competing for the same binding site. Mutations in the Sp binding site completely abolished the enhancing effect of Sp1. Treatment with insulin stimulated GCK expression, and increased Sp1 levels in S. aurata liver. We propose a new mechanism that involves Sp1 and Sp3 to mediate insulin activation of GCK transcription.

Transcriptional regulation of glucose-6-phosphatase catalytic subunit promoter by insulin and glucose in the carnivorous fish, Sparus aurata.

Increase in glucose-6-phosphatase catalytic subunit (G6Pase, G6pc) transcription enhances hepatic glucose production in non-insulin-dependent diabetes mellitus (NIDDM). The fact that carnivorous fish is an alternative model to study NIDDM led us to clone and characterise the first G6pc promoter region reported for fish and non-mammalian animals. The 5'-flanking region of G6pc from gilthead sea bream (Sparus aurata) was isolated by chromosome walking. With SMART RACE-PCR, the transcription start site was located 106 base pairs (bp) upstream of the translational start. Transfection analysis in HepG2 cells located a functional promoter in the 850 bp 5'-flanking isolated fragment (positions -770 to +80 relative to the transcription start). Sequential 5'-deletion analysis of the promoter fragment revealed that a core functional promoter for basal transcription is comprised within the 190 bp upstream of the transcription start site. In vivo, glucose and insulin reduced G6Pase mRNA levels in the fish liver. Transfection experiments in HepG2 cells showed that insulin repressed S. aurata G6pc under high-glucose conditions. Synergistic activation of piscine G6pc promoter was induced by cotransfection with expression plasmids for hepatocyte nuclear factor-4alpha (HNF-4alpha) and peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1alpha). No direct relationship was found between PGC-1alpha coactivation of HNF-4alpha transactivation and the repressive effect of insulin. Interestingly, insulin hardly affected G6pc promoter activity in the absence of glucose, suggesting that a reduced capacity of insulin-dependent repression of piscine G6pc may lead to insulin resistance in carnivorous fish.

Nutritional regulation of glucose-6-phosphatase gene expression in liver of the gilthead sea bream (Sparus aurata).

To examine the role of glucose-6-phosphatase (G6Pase) in glucose homeostasis in the diabetes-like experimental model of carnivorous fish, we analysed postprandial variations and the effect of starvation, ration size and diet composition on the regulation of G6Pase expression at the enzyme activity and mRNA level in the liver of gilthead sea bream (Sparus aurata). G6Pase expression increased in long-term starved or energy-restricted fish. In contrast to data reported for other fish species, short-term regulation of G6Pase expression was found in regularly fed S. aurata. G6Pase mRNA levels were lowest between 4 and 15 h after food intake, whereas minimal enzyme activity was observed 10-15 h postprandially. Alterations of plasma glucose levels affect G6Pase in mammals. However, the carbohydrate content of the diet did not affect hepatic expression of G6Pase in S. aurata, suggesting that a different molecular mechanism is involved in the control of G6Pase expression in fish. Although G6Pase was unaffected, high-carbohydrate low-protein diets increased glucokinase (GK) expression and thus allowed a metabolic adaptation favouring glycolysis over gluconeogenesis. Interestingly, only the nutritional conditions that promoted variations in the blood glucose levels resulted in changes in the hepatic expression of G6Pase. These findings indicate a concerted regulation of G6Pase and GK expression and suggest that the direction and rate of the glucose-glucose-6-phosphate substrate cycle flux is finely regulated in the liver of S. aurata, challenging the role attributed to deficient regulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolize glucose.

Glucokinase gene expression is nutritionally regulated in liver of gilthead sea bream (Sparus aurata).

Glucose intolerance in carnivorous fish has been attributed to the lack of hepatic glucokinase (GK) activity. Transcription/translation assay and transient transfection of COS-7 cells with a cDNA encoding Sparus aurata liver GK showed the functionality of the enzyme in vitro. The endogenous fish hepatic GK had lower affinity for glucose than the rat enzyme. The GK activity values in fed fish were similar to those reported for starved and diabetic rats. In this study, we also addressed the nutritional regulation of GK gene expression in fish liver. Starvation and energy restriction decreased S. aurata hepatic GK mRNA and activity levels, as previously reported in rats. In contrast, the fish enzyme expression exhibited a delayed onset during the daily feeding rhythm. These findings demonstrate for the first time the presence and the nutritional modulation of a functional GK activity in fish liver and contribute to explain the low ability of carnivorous fish to metabolize carbohydrates.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression is regulated by diet composition and ration size in liver of gilthead sea bream, Sparus aurata.

Modulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P(2)ase) gene expression by diet composition and ration size was studied in the liver of gilthead sea bream, Sparus aurata. From five different types of diet supplied to fish, those with either high carbohydrate/low protein or high carbohydrate/low lipid content stimulated 6PF-2-K/Fru-2,6-P(2)ase expression at the levels of mRNA, immunodetectable protein and kinase activity as well as promoting higher fructose-2,6-bisphosphate (Fru-2,6-P(2)) values. The expression of the bifunctional enzyme and Fru-2,6-P(2) levels showed also direct dependence on the quantity of diet supplied. These findings demonstrate for the first time nutritional regulation of 6PF-2-K/Fru-2,6-P(2)ase at mRNA level by diet composition and ration size and suggest that the carnivorous fish S. aurata can adapt its metabolism, by stimulation of liver glycolysis, to partial substitution of protein by carbohydrate in the diet. In addition, the expression of 6PF-2-K/Fru-2,6-P(2)ase can be used as an indicator of nutritional condition.

Liver insulin-like growth factor-I mRNA is not affected by diet composition or ration size but shows diurnal variations in regularly-fed gilthead sea bream (Sparus aurata).

Nutritional regulation of insulin-like growth factor-I (IGF-I) mRNA was assessed in liver of gilthead sea bream (Sparus aurata). As in mammals, starvation lowered the IGF-I mRNA content, which was recovered by refeeding. However, in contrast to previous observations in rats, neither diet composition nor ration size significantly affected hepatic IGF-I mRNA. Although fish growth depended on the quantity of diet supplied, no relationship was found between growth and liver IGF-I mRNA levels, a fact that challenges the importance, at least in fish, of liver-derived IGF-I on body growth attributed by the classical somatomedin hypothesis. In addition, diurnal modulation of mRNA levels occurred following food intake, suggesting that the intake of food may play a key role in the regulation of the short-term anabolic effects of IGF-I.

Dietary chromic oxide does not affect the utilization of organic compounds but can alter the utilization of mineral salts in gilthead sea bream Sparus aurata.

This study was conducted to determine whether the level of chromic oxide supplemented to diets containing gelatinized starch as the carbohydrate source affects digestibility, body composition, growth performances, and liver enzyme activities in gilthead sea bream, Sparus aurata. Gilthead sea bream fingerlings were fed diets containing gelatinized corn starch as the carbohydrate source and several levels of chromic oxide (0, 5, 10 and 20 g/kg) for 6 wk. No effect of dietary chromium level was detected on carbon, nitrogen, or dry matter digestibility. Calcium and phosphorus digestibility were higher in fish fed the diet supplemented with 5 g/kg chromic oxide than in fish fed the other supplemented diets. Dietary chromium did not affect dry matter, carbon, nitrogen, protein, or lipid concentrations in fish. However, fish fed 5 g/kg chromic oxide generally had higher levels of calcium, phosphorus, and ash than fish fed the other Cr-containing diets. Chromium concentration was significantly higher in fish fed the diets with 0.5 and 1% chromic oxide than in fish fed the control diet. Chromium supplementation of the diets did not affect the specific growth rate, the food efficiency ratio, the protein efficiency ratio, or, protein or nitrogen retention of the fish. Blood glucose and the activity of several liver enzymes involved in carbohydrate metabolism were unaffected by dietary chromic oxide. Alanine aminotransferase was lower in the fish fed the diet with 10 g/kg of chromic oxide than in unsupplemented controls. Our results indicate that chromic oxide can be used as a neutral marker in fish nutrition studies involving organic compounds, but not mineral salts.

Molecular cloning of a cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from liver of Sparus aurata: nutritional regulation of enzyme expression.

A cDNA clone encoding full-length 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) was isolated and sequenced from a Sparus aurata liver cDNA library. The 2527 bp nucleotide sequence of the cDNA contains a 73 bp 5'-untranslated region (5'-UTR), an open reading frame that encodes a 469 amino acid protein and 1041 bp at the 3'-UTR. The deduced amino acid sequence is the first inferred 6PF-2-K/Fru-2, 6-P2ase in fish. The kinase and bisphosphatase domains, where the residues described as crucial for the mechanism of reaction of the bifunctional enzyme are located, present a high degree of homology with other liver isoenzymes. However, within the first 30 amino acids at the N-terminal regulatory domain of the fish enzyme a low homology is found. Nutritional regulation of the 6-phosphofructo-2-kinase activity, together with immunodetectable protein and mRNA levels of 6PF-2-K/Fru-2,6-P2ase, was observed after starvation and refeeding. In contrast to results previously described for rat liver, the decrease in immunodetectable protein and kinase activity caused by starvation was associated in the teleostean fish to a decrease in mRNA levels.

Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, the pentose phosphate pathway and amino acid metabolism in liver of gilthead sea bream (Sparus aurata).

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). High-carbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

The muscle isoform of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase of the teleost Sparus aurata: relationship with the liver isoform.

The liver isoform of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase of the teleost fish Sparaus aurata has several characteristics similar to the skeletal muscle isoform of mammals. In order to ascertain the relation between muscle and liver isoforms in teleost, 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was purified from skeletal muscle of S. aurata. The muscle isozyme is composed of subunits with a molecular weight of 54 kDa, is bifunctional, and has an activity ratio kinase to bisphosphatase of 2.5. Muscle 6-phosphofructo 2-kinase is not sensitive to glycerol 3-phosphate inhibition and has noncooperative KmATP, higher than the liver isozyme. Thus, the kinetic characteristics of the muscle were distinguishable from the liver isozyme. Furthermore, the muscle isozyme is not a substrate of cAMP-dependent protein kinase. Despite those differences, two polyclonal antibodies raised against purified liver and muscle isozymes from S. aurata are not able to distinguish between them. Both antisera recognize with lower affinity recombinant rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase. A third antibody raised against the rat liver isozyme was also able to immunoprecipitate the teleost enzymes. The close immunological properties found suggest that S. aurata isozymes share epitopes in common. Considering the kinetic and immunological data reported, it is likely that the skeletal muscle/liver isozymes in teleost are products of a differentially spliced transcript of the same gene, as it is in rat. As those species are distant in vertebrate evolution, the similitude suggest that a common ancestral gene is involved in the muscle/liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase system in vertebrates.

6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase in liver of the teleost Sparus aurata.

6-Phosphofructo 2-kinase/fructose 2,6-bisphosphatase was purified from the liver of the teleost fish Sparus aurata and the enzymatic activities were characterized kinetically. Both activities copurify, being dimers of relative molecular mass of 98 kDa with subunits of M(r) 54 kDa. Although both specific activities are in the range of mammalian liver isozymes, the Kmfru 6-P of teleost 6-phosphofructo 2-kinase is 3 times that in rat liver. The S. aurata 6-phosphofructo 2-kinase is inhibited by ADP, citrate and phosphoenolpyruvate, and fructose-2,6-bisphosphatase presents inhibition by fru 6-P. Unlike the rat liver enzyme, the kinase reaction is scarcely inhibited by glycerol 3-P. The teleost isozyme is substrate for the cyclic-AMP-dependent protein kinase, as can be followed by the incorporation of 32P from ATP into the enzyme. Phosphorylation of the enzyme changes its kinetic behavior, leading to a form with a lower kinase/bisphosphatase activity ratio. No change is detected in the fru 6-P dependence of 6-phosphofructo 2-kinase, but the phosphorylated form is more sensitive to inhibition by effectors, especially by glycerol 3-phosphate. Phosphorylation enhances the fructose-2,6-bisphosphatase Vmax activity twofold. The implications of all these kinetic characteristics in the control of hepatic fructose-2,6-bisphosphate levels are discussed in the context of the studies in S. aurata in vivo. The results support the hypothesis that differences in the regulation of 6-phosphofructo 2-kinase/fructose-2,6-bisphosphatase are a key point for the specific adaptations of carbohydrate metabolism in this teleost fish.

Fructose 2,6-bisphosphate in liver of Sparus aurata: influence of nutritional state.

1. Fructose 2,6-bisphosphate (fru-2,6-P2) has been measured in liver and muscle of gilthead sea bream fish, Sparus aurata. 2. The fru-2,6-P2 levels in liver depend on the diet given to the fish: in fish fed a high carbohydrate diet, the fru-2,6-P2 levels are higher than any one previously reported. These changes are associated with differences in the phosphofructokinase 2 activity. 3. Fru-2,6-P2 levels has also been measured in liver of Sparus aurata after different fasting periods. In starved fish, fru-2,6-P2 did not decrease as sharply as in rat. The values found in fish starved for 20 days were similar to those reported for rats that had been starved for 24 hr.

Kinetic characterization of glycogen phosphorylase B from skeletal muscle of the mullet Liza ramada.

1. Glycogen phosphorylase purified from muscle of mullet (Liza ramada) has been kinetically characterized. 2. Kinetic analysis for the substrates glucose-1-P and glycogen showed no homotropic co-operativity. AMP exhibited only a slight homotropic co-operative behaviour, although it caused a decrease in the Km for glucose-1-P. 3. Glucose, ATP and glucose-6-P behaved as phosphorylase b inhibitors. Kinetic analysis of the inhibition showed the characteristic heterotropic effect both for the substrate glucose-1-P and the activator AMP. 4. However, glucose-6-P, which enhances the co-operativity between AMP molecules, lost its heterotropic effect on the glucose-1-P saturation curve.

Purification and characterization of glycogen phosphorylase B from skeletal muscle of the mullet Liza ramada: amino acid sequence of the phosphorylation site.

1. Skeletal muscle glycogen phosphorylase b has been purified from Liza ramada (mullet). 2. The Mr of the purified enzyme subunit was found to be 97,000. By gel filtration a relative Mr of 190,000 was found. 3. Proteolytic digestion of 32P-phosphorylated mullet phosphorylase gave a [32P]-labelled peptide which is observed to contain Ser, its sequence being -Gln-Ile-Ser-Val-Pro-. 4. During 'in vitro' phosphorylation of mullet phosphorylase, 32P was incorporated in different protein bands resolved by isoelectric focusing. The degree of radioactivity associated with each one changed with the incubation time.

Hormonal effects on the phosphorylation of glycogen synthase in rat hepatocytes.

The effects of epinephrine and vasopressin on the phosphorylation state of glycogen synthase were studied using rat hepatocytes incubated with 32P. After the incubation with hormones, 32P-labeled glycogen synthase was isolated using antibodies against rat liver enzyme. The immunoprecipitate showed a single radioactive band ( Mapp 88 kDa) when subjected to SDS-gel electrophoresis. Both epinephrine and vasopressin inactivated the enzyme and increased the 32P content of glycogen synthase. Cleavage of the immunoprecipitate with CNBr yielded two major 32P-labeled fragments of Mapp approximately 27 and 12 kDa. Both hormones increased the 32P content of both fragments. These results prove that epinephrine and vasopressin increase the phosphate content of the enzyme promoting its phosphorylation at multiple sites.

Comparison of the mechanism of isoproterenol-stimulated glycogenolysis in skeletal muscle of normal and phosphorylase kinase-deficient mice (I strain).

Diaphragm extracts from mice with the phosphorylase kinase deficiency mutation (I strain) have only 3.7% of the phosphorylase kinase activity of muscle extracts of the control strain (C57BL). Nevertheless, previous studies have shown that isoproterenol-stimulated glycogenolysis in I strain diaphragm muscle at a rate 57% (average relative response at 10 isoproterenol concentrations) of that of C57BL (GROSS, S.R., MAYER, S.E. and LONGSHORE, M.A.: J. Pharmacol. Exp. Ther. 198: 523-538, 1976). The present studies were initiated to compare the mechanism of isoproterenol-stimulated glycogenolysis in I and C57BL diaphragms. Isoproterenol was found to stimulate phosphorylase b to a conversion with an EC50 of 8 nM in muscles from mice of either strain, and the maximum increase in phosphorylase alpha activity in I diaphragms was 23% of that in C57BL diaphragms. Moreover, the initial rate of increase in phosphorylase alpha activity in I diaphragms incubated with 40 nM isoproterenol was 24% of that in C57BL muscles. The isoproterenol-stimulated increases in cyclic AMP content in diaphragms of the two strains were the same. Incubation of I diaphragms with isoproterenol did not significantly increase the concentrations of AMP, IMP or inorganic phosphate, activators of phosphorylase beta activity, nor was there a decrease in ATP and glucose 6-phosphate content, allosteric inhibitors of phosphorylase beta activity. Thus, phosphorylase alpha formation is the principal, if not only, catalyst of isoproterenol-stimulated glycogenolysis in skeletal muscle of phosphorylase kinase-deficient mice, and no evidence was obtained indicating that allosteric regulation of phosphorylase beta activity is part of the mechanism.