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Laboratory of Fish Nutrition, INRA-IFREMER, 64310 St-Pée-sur-Nivelle, France
1To whom correspondence should be addressed. E-mail: panserat{at}st-pee.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • dietary lipids • fish • hepatic glucose metabolism • glucokinase • glucose-6-phosphatase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
). Postprandial hyperglycemia results from the induction of glucose uptake by the liver and inhibition of hepatic endogenous glucose production (3
). Recent studies suggest that the postprandial hyperglycemia after a carbohydrate-rich meal in rainbow trout (Oncorhynchus mykiss) is associated with an expected induction of glycolytic capacity as in mammals but with an unexpected persistent capacity of hepatic endogenous glucose production (4
–8
). On the basis of these observations, it was postulated that retroregulation of relevant hepatic enzymes by blood glucose level may be dysfunctional in this species.
Glucokinase (GK;2
EC 2.7.1.2) and glucose-6-phosphatase (G6Pase; EC 3.1.3.9) are enzymes that catalyze glucose phosphorylation and glucose-6-phosphate hydrolysis, respectively (9
,10
). In this respect, both G6Pase and GK play a key role in glucose homeostasis. In rainbow trout, it has been shown that dietary carbohydrates induce the expression of both GK activity and GK mRNA, whereas they have no effect on G6Pase (7
,8
). Lack of inhibition of G6Pase would contribute to the persistent hepatic glucose production, regardless of the sources of the glucose-6-phosphate, from either glycogenolysis or gluconeogenesis. Such a paradox in the regulation of glucose homeostasis may be causal in the inefficient use of dietary glucose by fish.
Indeed, in mammals, it has been shown that high dietary lipid supplied as fish oil inhibits some of the glycolytic enzymes such as pyruvate kinase and GK (11
,12
), while inducing hepatic gluconeogenesis (13
). Although a few studies have dealt with the effects of dietary macronutrients on the activities of some enzymes involved in carbohydrate metabolism (14
,15
), no data exist currently on the expression of these enzymes at the molecular level. The purpose of this study was to evaluate the effect of dietary lipids in rainbow trout on the expression of two key enzymes, GK and G6Pase, at both the enzyme and mRNA levels. In the context of current practice of increasing dietary digestible energy through supply of high fat diets for salmonids (16
), it is important to examine the role of dietary lipids in dietary glucose utilization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Triplicate groups of juvenile immature rainbow trout (100 fish per tank; initial body weight 95 g) were reared in our experimental fish farm (Donzacq, France) at 18°C under a natural photoperiod in winter. They were hand-fed twice a day a low lipid (LL) diet without fish oil, i.e., 10% of lipids, or a high lipid (HL) diet with fish oil, i.e., 25% of lipids (Table 1
) for 8 wk. Pair-feeding was employed to supply the same quantity of dietary protein and starch to the groups while fat supply was variable; one group (HL) was fed the HL diet at 1.5% of body weight, and the second group (LL) was fed the LL diet at 1.27% of body weight per day. At the end of the growth trial, which lasted 8 wk, and after 2 d of food deprivation, separate groups of fish were given the experimental diets for 2 d. Nine fish from each group were killed by a sharp blow on the head 6 and 24 h after the meal (the food-deprived control corresponds to 2 d of food deprivation). Liver was sampled and frozen in liquid nitrogen and stored at -80°C. Gut contents were checked systematically to ensure that fish had actually consumed the test diets. Blood was sampled from the caudal vein, centrifuged (3000 x g) and kept frozen until analyses for plasma glucose, fatty acids and triglycerides.
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Chemical compositions of the diets and whole fish were analyzed using the following procedures: dry matter after drying at 105°C for 24 h, fat by dichloromethane extraction (Soxhlet), starch by the glucoamylase glucose oxidase method (17
) and gross energy in an adiabatic bomb calorimeter (IKA, Heitersheim Gribheimer, Germany). Protein content (N · 6.25) was determined by the Kjeldahl method after acid digestion. Total liver lipid was determined by the method of Folch et al. (18
) after extraction by dichloromethane rather than chloroform. Total RNA was extracted from each tissue using the method of Chomczynski and Sacchi (19
).
Northern blot analysis.
Samples of total extracted RNA (20 µg) were submitted to electrophoresis in 1% agarose gels containing 5% formaldehyde and capillary transferred onto nylon membrane (Hybond-N+; Amersham, Buckinghamshire, UK). After transfer, RNA blots were stained with methylene blue to locate 26S and 16S rRNAs and to determine the amount of loaded RNA. Membranes were hybridized with [32P]-DNA probes labeled by random priming (Stratagene, La Jolla, CA), recognizing trout GK and G6Pase mRNAs as described previously (7
,8
). After stringent washing, the membranes were exposed to X-ray film and the autoradiographic images were submitted to quantitative scanning using Visio-Mic II software (Genomic, Lyon, France).
GK activities.
The GK and HK activities were measured using 100 and 0.5 mmol/L of glucose, respectively, as described previously (8
,20
). A frozen sample of liver (500 mg) was homogenized (dilution 1/10) in ice-cold buffer [80 mmol/L Tris, 5 mmol/L EDTA, 2 mmol/L dithiothreitol (DTT), 1 mmol/L benzamidine, 1 mmol/L 4-(2-aminoethyl)benzenesulfonyl fluoride, pH 7.6]. Enzyme activities were measured at 37°C by coupling ribulose-5-phosphate formation from glucose-6-phosphate to the reduction of NADP 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. This assay for measuring GK activity on frozen samples necessitated correction by measuring glucose dehydrogenase activity (EC 1.1.1.47) as described by Tranulis et al. (20
).
G6Pase activities.
To measure G6Pase activity, microsomes were obtained from rainbow trout livers as described previously (7
). The final preparation, which was stored at -80°C, averaged 3–6 g protein/L and was used in the spectrophotometric assays. Microsomes were suspended in the buffer (100 mmol/ LNaH2PO4, 25 mmol/L Na2HPO4, 2 mmol/L EDTA, 1 mmol/L DTT, pH 7), without further treatment. The procedure followed was that of Alegre et al. (21
), monitoring the increase in absorbance (NADH production) using glucose dehydrogenase (Sigma) in excess as the coupling enzyme. One unit of G6Pase activity was defined as the amount of enzyme that catalyzed the hydrolysis of 1 µmol of glucose-6-phosphate/min under the specified conditions (30°C).
Plasma metabolites.
Plasma glucose concentration was determined using the glucose oxidase method in a Beckman glucose analyzer (Beckman II; Fullerton, CA). Plasma triglyceride levels were measured by colorimetric enzymatic assay using hepatic lipase (EC 3.1.1.3), glycerokinase (EC 2.7.1.30), glycerol-3-phosphate oxidase (EC 1.1.3.21) and peroxidase (EC 1.1.11) as enzymes (PAP 150 kit, Biomérieux, Marcy-l’étoile, France). Plasma fatty acid levels were measured by colorimetric enzymatic assay using acyl-CoA synthetase, acyl-CoA oxidase and peroxidase as enzymes (Wako Nefa C kit, Wako Chemicals, Neuss, Germany).
Data analysis.
Data are presented as means ± SD. When the one-way ANOVA was significant, differences between series of postprandial data from fish fed the same diet were determined using Tukey’s test (Systat 9 software products, SPSS, Chicago, IL). Fish fed the two diets were compared by an unpaired two-tailed Student’s t test (Systat 9 software products, SPSS). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Growth performance of juvenile rainbow trout fed the HL diet was better than that of those fed the LL diet (P < 0.01, t test). Feed efficiency and protein efficiency were better in the HL fish. Whole-body protein and lipid contents varied significantly from 56.2 ± 1.3% (HL diet) to 69.2 ± 1.8% (LL diet) of dry matter and 29.0 ± 1.7% (HL diet) to 13.7 ± 3.0% (LL diet) of dry matter, respectively (initial compositions were 76.8 ± 1.0 and 6.4 ± 0.5% for protein and lipid, respectively). Energy content of fish was 26.2 ± 0.9 and 23.8 ± 0.5 kJ/g of dry matter for HL and LL groups, respectively (P > 0.05; initial value, 21.5 ± 0.5 kJ/g). The liver to body weight ratio was significantly higher in HL fish than in LL fish (P < 0.05, t test) (Table 2)
. Groups did not differ in protein, lipid or energy retentions (as % of intake) nor in protein gain (Table 2)
. In contrast, fat gain was greater in fish fed the HL diet than in those fed the LL diet (P < 0.05, t test) (Table 2)
. As expected, fat intake was greater in fish fed the HL diet than in those fed the LL diet (P < 0.05, t test) (Table 2)
.
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. Both groups exhibited postprandial hyperglycemia (P < 0.05, Tukey’s test). However, the hyperglycemia lasted longer in HL fish than in LL fish (6 h after feeding, HL fish were still hyperglycemic). The plasma free fatty acid (FFA) levels were lower in fed fish than in fish deprived of food for 2 d (P < 0.05, Tukey’s test) and were higher in trout fed the HL diet than in those fed the LL diet (P < 0.05, t test). Plasma triglyceride levels were higher in fed fish at 6 h after feeding than in fed fish at 24 h after feeding (P < 0.05, Tukey’s test) and were higher in those fed the HL diet than in those fed the LL diet (P < 0.05, t test).
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) (P < 0.05, Tukey’s test). Expression of GK mRNA followed a trend similar to the enzyme activity (Fig. 2
A, B). A difference in GK mRNA expression between the two groups of fish was found only at 3 h after feeding (Fig. 2
B); mRNA was higher in fish fed the HL diet than in fish fed the LL diet (P < 0.05, t test).
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) (P > 0.05, Tukey’s test). However, at 6 h after feeding, G6Pase activity was greater in HL fish than in LL fish (P < 0.05, Student’s t test). G6Pase mRNA was always expressed at high levels in both groups (Fig. 3
A). However, at 6, 12 and 24 h after feeding, fish fed the HL diet had higher levels of G6Pase mRNA expression than those fed the LL diet (Fig. 3
B) (P < 0.05, Student’s t test).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The practical diet based on fish meal containing 8% fat provided sufficient amounts of essential fatty acids to meet the requirements of rainbow trout (22
). The increase in dietary lipid level alone led to an overall improvement in growth performance, as evidenced by weight gain and feed and protein efficiencies. Similar observations of a protein-sparing effect of dietary fats have been made earlier in rainbow trout (16
,23
,24
) although this is the first demonstration of such an effect under pair-feeding conditions. Similar trends in protein gain and retention observed in this study confirm the efficiency of the pair-feeding protocol (almost the same protein intakes in the two groups of fish). Fat gain and body lipid composition in fish fed diets with or without additional fish oil revealed better growth performance in the former. However, this improved body weight gain seems due mainly to the higher fat deposition in the perivisceral fat tissue (visual observation) and liver (greater relative weight).
As expected, there were higher levels of plasma lipids (fatty acids and triglycerides) in fish fed additional fish oil than in those fed the LL diet. Although plasma glucose levels did not differ between fish fed the HL and LL diets, there was a tendency (P = 0.08 and P = 0.06 at 3 and 6 h after feeding, respectively, t test) for higher values in fish fed the HL diet. The data suggest that there is possibly a faster decline in glycemia to basal levels in fish fed the LL diet. These results are in accordance with the observations in mammals in which dietary fatty acids negatively affect glucose utilization (13
,25
–27
).
To explore further the mechanism of action of dietary lipids, we analyzed the two key hepatic enzymes of glucose metabolism, GK and G6Pase. Although a modest increase in GK mRNA expression is attributable to an increase in fat intake, no such increase in GK enzyme activity was noted. This is in direct contrast with results obtained in mammals in which feeding of fish oil reduces the expression of GK at both the enzyme and mRNA levels (11
). Indeed, long-chain polyunsaturated fatty acids (PUFA) in mammals inhibit hepatic PK and GK gene expression (11
,12
). One potential explanation for such discrepant data between mammals and trout could be the difference in the lipid levels, i.e., the lowest lipid content of the fish meal–based diet is already 10%; such a level may be well above the threshold and thus too high to allow observation of any difference in GK expression. Undertaking studies with fat-free purified diets supplemented with only the minimum levels of essential fatty acids [(n-3) PUFA] would clarify these issues.
For G6Pase expression, there was a strong effect of dietary lipid levels, i.e., higher G6Pase mRNA levels and higher G6Pase activity; however, these occurred only at 6 h after the meal in fish fed diets with additional fish oil vs. those fed no additional fish oil. Expressed per unit body weight, there were small differences in total protein and carbohydrate intakes, but these were minimal compared with the big change in fat intake. These minor differences cannot explain the effects on G6Pase enzyme activities and G6Pase gene expression because in a previous study in which fish were fed either a high protein:low carbohydrate (55%:0%) diet or a low protein:high carbohydrate (40%:20%) diet, we did not find any differences in G6Pase expression (7
). Our data suggest that dietary lipids modulate the level of expression of G6Pase in vivo via plasma lipid concentrations (FFA and triglycerides). In mammals, the effects of dietary lipids on G6Pase mRNA and enzyme expressions are not clear (28
). Nevertheless, studies with rats have suggested that prolonged hyperlipidemia (high plasma FFA concentrations) may contribute to increased production of glucose via increased expression of G6Pase by an undefined mechanism (29
).
Our data suggest that high dietary lipids may negatively affect dietary glucose utilization, mainly by up-regulating hepatic glucose production in rainbow trout. Increased hepatic glucose output due to dietary lipids has been observed in mammals (13
). Analyses at the molecular level of other gluconeogenic enzymes (2
), phosphoenolpyruvate carboxykinase (EC 4.1.1.32) and fructose-1,6-bisphosphatase ( EC 3.1.3.11), as well as other glycolytic enzymes such as pyruvate kinase (EC 2.7.1.40) (2
), were also performed on some samples of this study (preliminary results, data not shown). No effects of dietary fish oil were observed 6 and 24 h after feeding on the mRNA expressions of these enzymes. Thus, only G6Pase, an enzyme at the junction of gluconeogenesis and glycogenolysis that catalyzes hepatic glucose production, seems to be induced by high levels of fish oil.
In conclusion, although the ability of specific fatty acids to modulate hepatic glucose metabolism gains some support from our study, the underlying mechanisms are far from being understood either in mammals or in fish. Finally, to improve dietary carbohydrate utilization by fish nutrition, it is important to gain further insight into the effect of other dietary macronutrients. Such studies are all the more relevant given the current search in aquaculture for alternate sources to fish meal and fish oil.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 23 March 2001. Initial review completed 25 October 2001. Revision accepted 13 November 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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27. Bizeau, M. E. & Hazel, J. R. (1999) Dietary fat type alters glucose metabolism in isolated rat hepatocytes. J. Nutr. Biochem. 10:709-715.
28. Mithieux, G., Bordet, J. C., Minassian, C., Ajzannay, A., Mercier, I. & Riou, J. P. (1993) Characteristics and specificity of the inhibition of liver glucose-6-phosphatase by arachidonic acid. Lesser inhibitability of the enzyme of diabetic rats. Eur. J. Biochem. 213:461-466.
29. Massillon, D., Barzilai, N., Hawkins, M., Prus-Wertheimer, D. & Rossetti, L. (1997) Induction of hepatic glucose-6-phosphatase gene expression by lipid infusion. Diabetes 46:153-157.[Abstract]
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