Changes in food intake and glucosensing function of hypothalamus and hindbrain in rainbow trout subjected to hyperglycemic or hypoglycemic conditions

To evaluate the possible role of glucose in the control of food intake (FI) in fish and the involvement of glucosensing system in that role, we have subjected rainbow trout (via intraperitoneal injections) to control, hyperglycemic (500 mg kg(-1) glucose body mass) or hypoglycemic (4 mg kg(-1) bovine insulin) conditions for 10 days. The experimental design was appropriate since hypoglycemia and hyperglycemia were observed the first 5 days after treatment and changes observed in metabolic parameters in liver were similar to those of fish literature. Hyperglycemic conditions elicited small changes in FI accompanied by increased glucose and glycogen levels, glucokinase (GK) activity and glycolytic potential in hypothalamus and hindbrain. In contrast, hypoglycemic conditions elicited a marked increase in FI accompanied by decreased glucose and glycogen levels and GK activity in the same brain regions whereas both regions displayed different responses in glycolytic potential. These results allow us to hypothesize that, despite the relative intolerance to glucose of carnivorous fish, changes in plasma glucose levels in rainbow trout detected by glucosensing areas in brain regions (hypothalamus and hindbrain) are integrated in those or near areas eliciting a response in FI, which was more important under hypoglycemic than under hyperglycemic conditions.     

Altered dietary carbohydrates significantly affect gene expression of the major glucosensing components in Brockmann bodies and hypothalamus of rainbow trout

Carnivorous fish have a limited capacity to utilize dietary carbohydrates even though glucosensing components exist in the hypothalamus and Brockmann bodies. Therefore, we fed trout for 10 days with two experimental diets containing a high level of carbohydrates (20%) or a carbohydrate-free level (<0.3%) to test the capacity of dietary carbohydrates to regulate gene expression of glucosensing components. Fish were fed and killed 1, 6, and 24 h after the meal to analyze plasma glucose levels, glucosensing-related biochemical parameters, and gene expression of the major components of the glucosensing system in the hypothalamus and Brockmann bodies. Glucose facilitative transporter type 2 and glucokinase gene expression were confirmed by real-time PCR data and two new components of the glucosensing mechanism, Kir6.-like and sulfonylurea receptor-like, were detected for the first time in fish in both tissues. In addition, a clear adaptation to dietary carbohydrates was found in trout Brockmann bodies, based on increased gene expression of major components of the system as well as enhanced glucokinase activities and glycogen levels. In contrast, in the hypothalamus, only glucokinase gene expression and activity showed a response to dietary carbohydrates, supporting the key role of that enzyme in glucosensing mechanism. Finally, a differential postprandial profile was found between tissues regarding the glucosensing potential, since the hypothalamus seems to respond to hyperglycemia earlier than the Brockmann bodies, whose response took place later. Altogether, these data describe for the first time in fish a complete response of major glucosensing components to dietary carbohydrates in trout hypothalamus and Brockmann bodies, supporting an efficient adaptation of both tissues to those dietary components.     

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.     

In vitro leptin treatment of rainbow trout hypothalamus and hindbrain affects glucosensing and gene expression of neuropeptides involved in food intake regulation

The aim of the present study was to evaluate in hypothalamus and hindbrain of rainbow trout in vitro the effect of leptin treatment on glucosensing capacity and the expression of orexigenic and anorexigenic peptides involved in the control of food intake. In a first experiment, the response of parameters involved in glucosensing (GK, PK and GSase activities; GK expression and glucose; glycogen and DHAP levels) and the expression of orexigenic (NPY) and anorexigenic (POMC, CART, CRF) peptides was assessed in hypothalami and hindbrain incubated for 1 h with 2, 4 or 8 mM d-glucose alone (controls) or with 10 nM leptin, or with 10 nM leptin plus inhibitors of leptin signaling pathways (50 nM wortmannin and 500 nM AG490). Leptin treatment increased levels in parameters involved in glucosensing. Leptin treatment decreased NPY mRNA levels in hypothalamus without affecting the expression of the other peptides assessed. Leptin effects were reverted in the presence of inhibitors for all parameters assessed suggesting the involvement of JAK/STAT and IRS-PI(3)K pathways. In a second experiment, we observed time-dependent (1-3 h) and dose (10, 20 and 50 nM)- effects of leptin treatment in decreasing NPY mRNA levels without affecting expression of the other peptides assessed. Considering the orexigenic action of NPY in fish, it seems that the anorexic effect of leptin can be mediated by reduced expression of NPY occurring in hypothalamus, and that change can be related to the activation of the glucosensing system occurring simultaneously.     

Glucose and lipid metabolism in the pancreas of rainbow trout is regulated at the molecular level by nutritional status and carbohydrate intake

Glucose and lipid metabolism in pancreatic islet organs is poorly characterized. In the present study, using as a model the carnivorous rainbow trout, a glucose-intolerant fish, we assessed mRNA expression levels of several genes involved in glucose and lipid metabolism (including ATP-citrate lyase; carnitine palmitoyltransferase-1 isoforms, CPT; the mitochondrial isoform of the phosphoenolpyrutave carboxykinase, mPEPCK and pyruvate kinase, PK) and glucosensing (glucose transporter type 2, Glut2; glucokinase, GK and the potassium channel, K_ATP) in Brockmann bodies. We evaluated the response of these parameters to changes in feeding status (food deprived vs. fed fish) as well as to changes in the amount of carbohydrate (dextrin) in the diet. A general inhibition of the glycolytic (including the glucosensing marker GK) and β-oxidation pathways was found when comparing fed versus food-deprived fish. When comparing fish feeding on either low- or high-carbohydrate diets, we found that some genes related to lipid metabolism were more controlled by the feeding status than by the carbohydrate content (fatty acid synthase, CPTs). Findings are discussed in the context of pancreatic regulation of glucose and lipid metabolism in fish, and show that while trout pancreatic metabolism can partially adapt to a high-carbohydrate diet, some of the molecular actors studied seem to be poorly regulated (K_ATP) and may contribute to the glucose intolerance observed in this species when fed high-carbohydrate diets.     

The role of hepatic, renal and intestinal gluconeogenic enzymes in glucose homeostasis of juvenile rainbow trout

Rainbow trout is unable to utilize high levels of dietary carbohydrates and experiences hyperglycemia after consumption of carbohydrate-rich meals. Carbohydrates stimulate hepatic glycolytic activity, but gene expression of the rate-limiting gluconeogenic enzymes glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) remains high. Although there is significant mRNA expression and activity of gluconeogenic enzymes in trout intestine and kidney, the regulation of these enzymes by diet is not known. We tested the hypothesis that dietary carbohydrate modulates intestinal and renal G6Pase, FBPase and PEPCK. Fish were either fasted or fed isocaloric carbohydrate-free (CF) or high carbohydrate (HC) diets for 14 days. As expected, fish fed HC exhibited postprandial hyperglycemia and enhanced levels of hepatic glucokinase mRNA and activity. Dietary carbohydrates had no significant effect on the expression and activity of PEPCK, FBPase and G6Pase in all three organs. In contrast, fasting enhanced the activity, but not the mRNA expression of both hepatic and intestinal PEPCK, as well as intestinal FBPase. Therefore, the activity of rate-limiting gluconeogenic enzymes in trout can be modified by fasting, but not by the carbohydrate content of the diet, potentially causing hyperglycemia when fed high levels of dietary carbohydrates. In this species consuming low carbohydrate diets at infrequent intervals in the wild, fasting-induced increases in hepatic and intestinal gluconeogenic enzyme activities may be a key adaptation to prevent perturbations in blood glucose during food deprivation. Presented in part at Experimental Biology, April 2006, San Francisco, CA [Kirchner S., Panserat S., Kaushik S. and Ferraris R. FASEB-IUPS-2006 A667.6].     

Muscle insulin binding and plasma levels in relation to liver glucokinase activity,glucose metabolism and dietary carbohydrates in rainbow trout

Rainbow trout were fed for 10 weeks with either a carbohydrate-free diet (C-free) or with four experimental diets containing various levels (20 or 40%) and sources of starch (extruded wheat or peas) in order to examine metabolic utilisation of dietary vegetable carbohydrates and its endocrine control. The study was focused on the parameters described as limiting in glucose metabolism in fish. Feeding trials were conducted at 8 and 18 degrees C to establish whether carbohydrate-rich diets can be used in trout farming irrespective of water temperature. At both temperatures, pea diets (especially the highest level) resulted in a feed efficiency as high as the C-free diet. Fish had similar growth rates except when fed the low wheat content diet. Glycaemia values 6 h after feeding were significantly higher in trout fed carbohydrate diets than those given the C-free diet, whereas plasma insulin levels were similar independently of the levels of dietary starch. This study provides the first evidence that glucokinase (GK) activity and mRNA level in trout liver increase in proportion to the content of dietary starch. Nevertheless, these changes were not correlated with plasma insulin levels. Insulin-like growth factor-I (IGF-I) binding and number of receptors in skeletal muscle were consistently higher than those for insulin but no diet-induced differences were found for any of these parameters. Temperature clearly affected the postprandial profile of glucose and insulin, which both showed lower levels 6 h after feeding at 8 degrees C than at 18 degrees C, which was consistent with a lower feed intake. Glucose and insulin levels decreased markedly 24 h after feeding at 18 degrees C, while they were still high at 8 degrees C, an observation concordant with delayed transit rate. These findings indicate satisfactory adaptation of rainbow trout to diets with a relatively high vegetable starch content, especially when provided as extruded peas, and indicate that diets with increased levels of carbohydrates can be used in this species even when it is reared at low temperature.     

In vitro evidences for glucosensing capacity and mechanisms in hypothalamus, hindbrain, and Brockmann bodies of rainbow trout

We aimed to support in vitro the glucosensing capacity observed in vivo in rainbow trout hypothalamus, hindbrain, and Brockmann bodies (BB) and to obtain preliminary evidence of the mechanisms involved. The response of parameters involved in the glucosensing capacity [hexokinase, hexokinase IV (glucokinase), and pyruvate kinase activities and glucose and glycogen levels] was assessed in these tissues incubated for 1 h with 2, 4, or 8 mM D-glucose alone (control) or with specific agonists/inhibitors of the steps involved in glucosensing capacity in mammals. These agents were a competitor for glucose phosphorylation (15 mM mannose), sulfonylurea receptor-1 effectors (500 microM tolbutamide or diazoxide), glycolytic intermediates (15 mM glycerol, lactate, or pyruvate), and inhibitors of glucose transport (10 microM cytochalasin B), glycolysis [20 mM 2-deoxy-D-glucose (2-DG)], and L-type calcium channel (1 microM nifedipine). Control incubations of the three tissues displayed increased glucose and glycogen levels and glucokinase activities in response to increased medium glucose, thus supporting our previous in vivo studies. Furthermore, critical components of the glucosensing mammalian machinery are apparently functioning in the three tissues. The responses in brain regions to all substances tested (except 2-DG and nifedipine) were similar to those observed in mammals, suggesting a similar glucosensing machinery. In contrast, in BB, only the effects of 2-DG, lactate, pyruvate, diazoxide, and nifedipine were similar to those of mammalian beta-cells, suggesting that some of the components of the piscine glucosensing model are different than those of mammals. Such differences may relate to the importance of amino acids rather than glucose signaling in the trout BB.     

Hepatic glucokinase and glucose-6-phosphatase responses to dietary glucose and starch in gilthead sea bream (Sparus aurata) juveniles reared at two temperatures

The effects of carbohydrate sources/complexity and rearing temperature on hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities and gene expression were studied in gilthead sea bream juveniles. Two isonitrogenous (50% crude protein) and isolipidic (19% crude lipids) diets were formulated to contain 20% waxy maize starch or 20% glucose. Triplicate groups of fish (63.5 g initial body weight) were fed each diet to near satiation during four weeks at 18 degrees C or 25 degrees C. Growth, feed intake, feed efficiency and protein efficiency ratio, were higher at the higher water temperature. At each water temperatures fish growth and feed efficiency were higher with the glucose diet. Plasma glucose levels were not influenced by water temperature but were higher in fish fed the glucose diet. Hepatosomatic index and liver glycogen were higher at the lower water temperature and within each water temperature in fish fed the glucose diet. No effect of water temperature on enzymes activities was observed, except for hexokinase and GK which were higher at 25 degrees C. Hepatic hexokinase and pyruvate kinase activities were not influenced by diet composition, whereas glucose-6-phosphate dehydrogenase activity was higher in fish fed the glucose diet. Higher GK activity was observed in fish fed the glucose diet. GK gene expression was higher at 25 degrees C in fish fed the waxy maize starch diet while in fish fed the glucose diet, no temperature effect on GK gene expression was observed. Hepatic G6Pase activities and gene expression were neither influenced by dietary carbohydrates nor water temperature. Overall, our data suggest that in gilthead sea bream juveniles hepatocytes dietary carbohydrate source and temperature affect more intensively GK, the enzyme responsible for the first step of glucose uptake, than G6Pase the enzyme involved in the last step of glucose hepatic release.     

Hepatic glucokinase and glucose-6-phosphatase responses to dietary glucose and starch in gilthead sea bream (Sparus aurata) juveniles reared at two temperatures.

The effects of carbohydrate sources/complexity and rearing temperature on hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities and gene expression were studied in gilthead sea bream juveniles. Two isonitrogenous (50% crude protein) and isolipidic (19% crude lipids) diets were formulated to contain 20% waxy maize starch or 20% glucose. Triplicate groups of fish (63.5 g initial body weight) were fed each diet to near satiation during four weeks at 18 degrees C or 25 degrees C. Growth, feed intake, feed efficiency and protein efficiency ratio, were higher at the higher water temperature. At each water temperatures fish growth and feed efficiency were higher with the glucose diet. Plasma glucose levels were not influenced by water temperature but were higher in fish fed the glucose diet. Hepatosomatic index and liver glycogen were higher at the lower water temperature and within each water temperature in fish fed the glucose diet. No effect of water temperature on enzymes activities was observed, except for hexokinase and GK which were higher at 25 degrees C. Hepatic hexokinase and pyruvate kinase activities were not influenced by diet composition, whereas glucose-6-phosphate dehydrogenase activity was higher in fish fed the glucose diet. Higher GK activity was observed in fish fed the glucose diet. GK gene expression was higher at 25 degrees C in fish fed the waxy maize starch diet while in fish fed the glucose diet, no temperature effect on GK gene expression was observed. Hepatic G6Pase activities and gene expression were neither influenced by dietary carbohydrates nor water temperature. Overall, our data suggest that in gilthead sea bream juveniles hepatocytes dietary carbohydrate source and temperature affect more intensively GK, the enzyme responsible for the first step of glucose uptake, than G6Pase the enzyme involved in the last step of glucose hepatic release.     

Rearing temperature enhances hepatic glucokinase but not glucose-6-phosphatase activities in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) juveniles fed with the same level of glucose

The aim of this work was to elucidate if the previous results observed in hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) activities in European sea bass and gilthead sea bream are due to temperature per se or to differences in feed intake at different water temperatures. For that purpose triplicate groups of fish (30 g initial body weight) were kept at 18 degrees C or 25 degrees C during two weeks and fed a fixed daily ration of a glucose-free or 20% glucose diet. At the end of the experimental period, plasma glucose levels in both species were not influenced by water temperature but were higher in fish fed the glucose diet. Higher hepatic GK activity was observed in the two fish species fed the glucose diet than the glucose-free diet. In the glucose fed groups, GK activity was higher at 25 degrees C than at 18 degrees C. Glucose-6-phosphatase activities in both species were not influenced by water temperature. In European sea bass and in contrast to gilthead sea bream it was observed an effect of dietary composition on G6Pase activities with surprising higher activities recorded in fish fed the glucose diet than in fish fed the glucose-free diet. Overall, our data strongly suggest that European sea bass and gilthead sea bream are apparently capable to strongly regulate glucose uptake by the liver but not glucose synthesis, which is even enhanced by dietary glucose in European sea bass. Within limits, increasing water temperature enhances liver GK but not G6Pase activities, suggesting that both species are more able to use dietary carbohydrates at higher rearing temperatures.     

Response of hexokinase enzymes and the insulin system to dietary carbohydrates in the common carp, Cyprinus carpio

The response of the common carp to diets with varying amounts of digestible starch, provided either as pea meal (LP, HP, 30 and 46% peas, respectively) or as cereal (LW, HW, 30 and 46% wheat, respectively), was studied and compared with the response to a carbohydrate-free protein-rich diet (CF). Here we focused on the utilisation of dietary carbohydrates by examining the relationship between dietary starch intake, hepatic hexokinase activities, circulating insulin and muscle insulin receptor system. Plasma glucose concentration and hepatic high Km hexokinase (glucokinase, GK) activity were not affected by the content of digestible starch, but 6 h after feeding enzyme activity was higher in the fish fed carbohydrate diets. Similarly, low Km hexokinase (HK) activity was also higher in the fish 24 h after feeding. Fat gain and protein retention were significantly improved by increased digestible starch intake, especially in the HP group, which in turn, presented the highest plasma insulin levels. Glycogen stores were moderately increased by the ingestion of digestible starch. The number of insulin receptors was greater in the CF group than in fish on carbohydrates, except the HP group. Our results confirmed that the common carp uses dietary carbohydrates efficiently, especially when there are provided by peas. This efficiency might be related to the enhanced response of postprandial insulin observed in the HP group.     

Short-term food restriction and refeeding alter expression of genes likely involved in brain glucosensing

Several genes involved in glucosensing of the endocrine pancreas have been proposed to serve a similar function in the brain. These genes include the glucose transporter-2 (Glut-2) and glucokinase (GK). In addition, the glucagon-like peptide 1 receptor, which serves as a downstream signal modulator in pancreatic glucosensing and centrally alters feeding, is also of interest. We used quantitative real-time RT-PCR to measure changes in hypothalamic and brainstem Glut-2, GK, and Glp-1R expression of these genes induced by food restriction and refeeding. Sprague-Dawley rats were 50% food restricted for 1 day; one-half of the food-restricted rats were refed with chow for 1 hr before sacrifice. In both hypothalamus and brainstem, gene expression of Glut-2, GK, and Glp-1R was significantly lower in refed rats compared with food-restricted rats. The measures of gene expression in two feeding control groups (ad libitum and voluntarily overfed animals) were intermediate between the food-restricted and refed groups, but were not significantly different from each other. The results indicate that putative glucosensing (GK, Glut-2, and Glp-1R) gene expression in the hypothalamus and brainstem is reduced in response to food intake, depending on prior nutritional status.     

Evidence for the Presence of Glucosensor Mechanisms Not Dependent on Glucokinase in Hypothalamus and Hindbrain of Rainbow Trout (Oncorhynchus mykiss)

We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) operate in hypothalamus and hindbrain of the carnivorous fish species rainbow trout and stress affected them. Therefore, we evaluated in these areas changes in parameters which could be related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and sodium/glucose co-transporter 1 (SGLT-1) 6h after intraperitoneal injection of 5 mL.Kg^-1 of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment, 4 mg bovine insulin.Kg^-1 body mass), or D-glucose (hyperglycaemic treatment, 500 mg.Kg^-1 body mass). Half of tanks were kept at a 10 Kg fish mass.m^-3 and denoted as fish under normal stocking density (NSD) whereas the remaining tanks were kept at a stressful high stocking density (70 kg fish mass.m^-3) denoted as HSD. The results obtained in non-stressed rainbow trout provide evidence, for the first time in fish, that manipulation of glucose levels induce changes in parameters which could be related to putative glucosensor systems based on LXR, mitochondrial activity and sweet taste receptor in hypothalamus, and a system based on SGLT-1 in hindbrain. Stress altered the response of parameters related to these systems to changes in glycaemia.     

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.     

Influence of dietary composition on gluconeogenesis from L-(U-14C)glutamate in rainbow trout (Salmo gairdneri)

The effect of dietary composition (high-protein, high-carbohydrate and high-fat diets) and starvation on in totum gluconeogenesis from L-(U-14C)glutamate was studied in the rainbow trout. High-fat and high-carbohydrate diets produced a significant hyperglycaemia. Lower blood glucose values were obtained in trout fed on a high-protein diet. Liver glycogen levels were significantly lower in trout fed on carbohydrate-free diets (high-protein and high-fat diets) and in starved fish. Gluconeogenesis from L-(U-14C)glutamate was markedly reduced in fish given the high-carbohydrate diet and significantly enhanced in starved fish. Radioactive liver glycogen was higher in starved fish, although the amount of radioactivity incorporated into glycogen was very low.     

Dietary carbohydrate-to-protein ratio affects TOR signaling and metabolism-related gene expression in the liver and muscle of rainbow trout after a single meal

Most teleost fish are known to require high levels of dietary proteins. Such high-protein intake could have significant effects, particularly on insulin-regulated gene expression. We therefore analyzed the effects of an increase in the ratio of dietary carbohydrates/proteins on the refeeding activation of the Akt-target of rapamycin (TOR) signaling pathways in rainbow trout and the effects on the expression of several genes related to hepatic and muscle metabolism and known to be regulated by insulin, amino acids, and/or glucose. Fish were fed once one of three experimental diets containing high (H), medium (M), or low (L) protein (P) or carbohydrate (C) levels after 48 h of feed deprivation. Activation of the Akt/TOR signaling pathway by refeeding was severely impaired by decreasing the proteins-to-carbohydrates ratio. Similarly, postprandial regulation of several genes related to glucose (Glut4, glucose-6-phosphatase isoform 1), lipid (fatty acid synthase, ATP-citrate lyase, sterol responsive element binding protein, carnitine palmitoyltransferase 1, and 3-hydroxyacyl-CoA dehydrogenase), and amino acid metabolism (serine dehydratase and branched-chain α-keto acid dehydrogenase E2 subunit) only occurred when fish were fed the high-protein diet. On the other hand, diet composition had a low impact on the expression of genes related to muscle protein degradation. Interestingly, glucokinase was the only gene of those monitored whose expression was significantly upregulated by increased carbohydrate intake. In conclusion, this study demonstrated that macro-nutrient composition of the diet strongly affected the insulin/amino acids signaling pathway and expression pattern of genes related to metabolism.     

Molecular cloning of hepatic glucose-6-phosphatase catalytic subunit from gilthead sea bream (Sparus aurata): response of its mRNA levels and glucokinase expression to refeeding and diet composition

To examine the relationship between structure and function of glucose-6-phosphatase (G6Pase) in fish, we undertook molecular cloning and modulation of G6Pase expression by starvation and refeeding on diets with different nutrient composition in the liver of the carnivorous fish, Sparus aurata. A cDNA encoding the full-length G6Pase catalytic subunit from the liver of S. aurata was isolated. This cDNA encodes a 350-amino acid protein, with low homology to the mammalian G6Pase, although it contains most of the key residues required for catalysis. Based on hydrophobicity and membrane structure prediction, we propose a model containing nine-transmembrane regions for S. aurata G6Pase. Northern blots showed that refeeding after a prolonged starvation rapidly reverses the glucose/glucose-6-phosphate substrate cycle flux in the fish liver through decreased G6Pase expression and strong glucokinase (GK) induction. The effect of refeeding different diets on G6Pase and GK expression, indicated that hepatic intermediary metabolism of fish fed diets with low protein/high carbohydrate diets is impelled towards utilization of dietary carbohydrates, by means of modulation of GK mRNA levels rather than G6Pase expression. These findings challenge the role attributed to dysregulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolise glucose.