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

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Abstract

Modulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase) 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-P2ase expression at the levels of mRNA, immunodetectable protein and kinase activity as well as promoting higher fructose-2,6-bisphosphate (Fru-2,6-P2) values. The expression of the bifunctional enzyme and Fru-2,6-P2 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-P2ase 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-P2ase can be used as an indicator of nutritional condition.

Introduction

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase, EC 2.7.1.105/EC 3.1.3.46) catalyzes the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P2), a key modulator of glycolysis–gluconeogenesis through its action on 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase [1]. The 6PF-2-K/Fru-2,6-P2ase enzyme activities and gene expression are modulated by nutritional, hormonal and genetic factors in mammals [2], [3], [4], [5], [6]. Carbohydrate metabolism in fish differs from rat and carnivorous mammals. It was reported that carnivorous fish are glucose intolerant and have low ability to utilize dietary carbohydrates [7], [8], [9]. As in other fish species, glycolytic–gluconeogenic enzymes involved in carbohydrate metabolism were found in the liver of gilthead sea bream, Sparus aurata [10], [11], [12], [13]. 6PF-2-K/Fru-2,6-P2ase purified from liver of S. aurata is as in mammals modulated through phosphorylation–dephosphorylation [4], [10], [14]. The fish liver 6PF-2-K activity has a lower affinity for fructose-6-phosphate and a higher affinity for ATP than the rat liver enzyme. Moreover, the dephosphorylated enzyme from liver of S. aurata is scarcely inhibited by glycerol-3-phosphate. Our group recently cloned a cDNA encoding S. aurata liver 6PF-2-K/Fru-2,6-P2ase and reported that the kinase enzyme activity, protein content and mRNA levels decreased in liver of starved fish [15]. In contrast, no modulation of the 6PF-2-K/Fru-2,6-P2ase mRNA abundance was reported to occur in rat by starvation or diabetes, although immunodetectable protein and kinase activity decreased under such conditions [16], [17], [18]. However, a decrease in the 6PF-2-K/Fru-2,6-P2ase mRNA levels has recently been also reported in lean and obese rats during starvation [19]. We hypothesized that the decrease in liver 6PF-2-K enzyme activity observed in starved S. aurata could be caused by two mechanisms. First, specific phosphorylation and allosteric modulation of 6PF-2-K/Fru-2,6-P2ase, simultaneously inhibiting its kinase and activating its bisphosphatase activity, and second, a mechanism that involves a decrease in both mRNA and protein levels. The induction of 6PF-2-K/Fru-2,6-P2ase gene expression during refeeding was similar to that reported in rat [16]. Short-term refeeding promoted a rapid recovery of the active/total 6PF-2-K activity ratio, presumably by dephosphorylation and allosteric modulation of the enzyme, while long-term regulation of the enzyme activity appeared to be modified by an increase in mRNA abundance and protein content [15]. Good progress has recently been made in unravelling the molecular properties of 6PF-2-K/Fru-2,6-P2ase enzyme [20], [21]. Insulin, glucagon, glucocorticoids and growth factors modulate the 6PF-2-K/Fru-2,6-P2ase gene expression. Glucocorticoids stimulate the 6PF-2-K/Fru-2,6-P2ase gene transcription in the rat liver enzyme, while glucagon promotes the opposite effect. The effects of insulin depend on the hormonal context. Insulin treatment results in an in vitro increased rate of transcription, while it inhibits and reverses the glucocorticoid-induced stimulation of transcription of the liver 6PF-2-K/Fru-2,6-P2ase mRNA [3], [6], [22], [23], [24]. A number of studies focused on the interactions between dietary and hormonal conditions and gene expression, are already available for several key enzymes involved in different metabolic pathways [25], [26], [27], [28], [29], [30]. In contrast, knowledge of the effect of the dietary nutrients on 6PF-2-K/Fru-2,6-P2ase gene expression is scarce and it remains unclear how the nutritional conditions modulate in vivo the expression of 6PF-2-K/Fru-2,6-P2ase in mammals and in carnivorous fish. In the present report the regulation of 6PF-2-K/Fru-2,6-P2ase gene expression by diet composition and ration size has been studied at the mRNA, immunodetectable protein and kinase activity levels.

Section snippets

Fish and diets

S. aurata averaging 10 g were maintained in aquaria as previously described [13]. In order to study the effect of the diet composition, the experimental fish were fed five different diets (Table 1) for 18 days at 2% body weight (bw) per day. Analyses of the diets were performed following standard procedures [31]. The composition of the diets was selected to cover a wide range above and below the levels in diets commercially available for fish, presenting increasing amounts of carbohydrate

Results

In liver of 18-day fed fish on five different types of diet, active and total 6PF-2-K enzyme activities and Fru-2,6-P2 levels showed a clear direct dependence on the carbohydrate content of the diet (Fig. 1). Comparison of the groups of fish fed diets with similar protein levels but increasing carbohydrate and decreasing lipid content (diet 2 compared with diet 3 and diet 4 compared with diet 5) indicated that increased Fru-2,6-P2 and 6PF-2-K activity values were observed in fish fed high

Discussion

It is generally accepted that carnivorous fish have a poor capacity to metabolize dietary carbohydrate. Fish tissues have a low ability to utilize glucose and to maintain glycemia. The high protein content of the natural diets of carnivorous fish provides gluconeogenic substrates for de novo glucose and glycogen synthesis. Dietary protein together with the lower energy demand of fish can explain the relatively low need for nutrient carbohydrate as an energy source in carnivorous fish [11], [36]

Acknowledgments

This work was supported by DGICYT (PB93-0757 and PB96-1488) from M.E.C. (Spain) and a grant from Universitat de Barcelona (GRC/96-02643). A.C. is a recipient of a Predoctoral Fellowship from M.E.C. (Spain). We are grateful to Dr. A. Tauler for providing the antibody used in Western blotting analysis.

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