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© 2003 The American Society for Nutritional Sciences J. Nutr. 133:2561-2564, August 2003

Nutrient-Gene Interactions
Research Communication

Low Protein Intake Is Associated with Reduced Hepatic Gluconeogenic Enzyme Expression in Rainbow Trout (Oncorhynchus mykiss)

Séverine Kirchner, Sadasivam Kaushik and Stéphane Panserat2

Laboratory of Fish Nutrition, INRA-IFREMER, 64310 St-Pée-sur-Nivelle, France

2To whom correspondence should be addressed. E-mail: panserat{at}st-pee.inra.fr.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Our objective was to understand the reasons behind the persistent postprandial hyperglycemia in rainbow trout (Oncorhynchus mykiss). We hypothesized that in this species, high levels of dietary protein could increase the hepatic production of glucose, irrespective of the dietary carbohydrate supply. We fed juvenile rainbow trout four diets containing graded levels of protein for 14 d. Pair-feeding was employed to keep lipid and carbohydrate intakes constant. Six hours after feeding, as postulated, activities and mRNA levels of gluconeogenic enzymes (glucose-6-phosphatase, fructose-1,6-bisphosphatase) increased with increasing dietary protein (P < 0.05). However, in fish with a very low protein intake, there was a very strong increase in plasma glucose (18 mmol/L) that was also associated with a high capacity to store excess glucose as indicated by altered pyruvate kinase activity, glucokinase activity, and hepatic glycogen and fat concentrations (P < 0.05). In conclusion, at the same level of carbohydrate intake, a low dietary protein intake was associated with an unexplained increase in glycemia, which was probably responsible for the decrease in hepatic gluconeogenic enzyme expression. The effect of dietary protein on low carbohydrate utilization in this species remains unclear.

KEY WORDS: • protein • glucose • rainbow trout • glycolysis • gluconeogenesis

Rainbow trout (Oncorhynchus mykiss) are recognized for their inefficiency in utilizing high levels of dietary carbohydrates, leading to prolonged postprandial hyperglycemia after oral administration of glucose or a carbohydrate-rich meal (1). In contrast to nondiabetic mammals (2), rainbow trout exhibit a persistent high level of mRNAs of hepatic gluconeogenic enzymes [phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32), fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) and glucose-6-phosphatase (G6Pase; EC 3.1.3.9)] (1,3,4). Dietary lipids or proteins affect the activities of enzymes involved in hepatic carbohydrate metabolism in fish (5–7). Diets of rainbow trout generally contain >40% protein (8); amino acids are utilized as an energy source but also constitute substrates for endogenous glucose synthesis (3). We hypothesized that such high protein intakes could cause a persistent hepatic production of endogenous glucose due to excess availability of gluconeogenic substrates and/or amino acid–based activation (or persistence) of hepatic gluconeogenic enzymes, irrespective of the carbohydrate supply.

To analyze the effects of dietary protein, we evaluated some of the metabolic consequences of graded levels of dietary protein for 14 d using a pair-feeding method. The effects of dietary protein supply on proteins involved in glucose metabolism (glucose transporter, glycolytic and gluconeogenic enzymes) and amino acid metabolism were examined in the liver of rainbow trout.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Experimental protocols.

    Trial A. Juvenile immature rainbow trout (Viviers de France, Sarrance, France) were reared in our experimental fish farm (n = 2 tanks per diet; n = 40 fish per tank) (9). Fish were hand-fed twice a day for 14 d, with one of four experimental diets (P1, P2, P3 and P4), formulated to contain increasing levels of dietary protein (Table 1). A pair-feeding method was employed to supply the same quantity of dietary carbohydrate and fat to the four groups while the protein supply was variable. The control group was fed 1.8 g P4 diet/(100 g body · d). Feeding ratios of P1, P2 and P3 were 1.0, 1.3 and 1.5 g/(100 g body · d), respectively (Table 1). Diet acceptance level was controlled daily and fish were weighed at the end of the trial to calculate daily growth intake, feed and protein efficiencies [Daily Growth Index = 100 · final body wt1/3 – initial body wt1/3; Feed Efficiency = wet wt gain (g)/dry feed intake (g); Protein Efficiency Ratio = wet wt gain (g)/crude protein intake (g)].


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  		TABLE 1 Composition of the experimental diets

 
    Trial B. Juvenile immature rainbow trout (body weight ~180 g) were reared (10 fish per tank) in our experimental facilities. Fish were anesthetized (phenoxyethanol 0.2 mL/L) and force-fed the two extreme diets, P1 or P4, following the same pair-feeding procedure.

Fish from each group (n = 9, trial A and n = 6, trial B) were killed by a sharp blow on the head 6 h after the meal. Liver and blood were sampled as previously described (9). The study was performed according to the current legislation on animal experiments in France with the recommendations of the Nantes Animal Care and Use committee (France).

Analytical methods.

We verified the chemical composition of the diets using previously described procedures (Table 1) (9). Liver glycogen and total lipid concentrations were determined according to the methods of Murat and Serfaty (10) and Folch et al. (11), respectively. Plasma glucose, triglyceride, and fatty acid levels were determined by colorimetric enzymatic methods using commercial kits (Glucose RTU kit; PAP 150 kit, Biomerieux, Marcy-l’étoile, France; Wako NEFA C kit; Wako Chemicals, Neuss, Germany). Total plasma free amino acid levels were determined by the ninhydrin reaction according to Moore (12) with glycine as a standard.

Enzyme analysis: activities and gene expressions.

Activities of glutamate dehydrogenase (GDH; EC 1.4.1.2), alanine aminotransferase (ALAT; EC 2.6.1.2), aspartate aminotransferase (ASAT; EC 2.6.1.1), glucokinase (GK; EC 2.7.1.11), L-type pyruvate kinase (PK; EC 2.7.1.40), PEPCK, FBPase and G6Pase were measured as previously described (9,13) except no distinction was made between mitochondrial and cytosolic PEPCK activities. Analysis of GK, PK, G6Pase, FBPase, PEPCK and type 2 glucose transporter (GluT2) gene expression was performed by Northern blotting procedure (9).

Data analysis.

Data are presented as mean ± SD. For trial A, data were analyzed by one-way ANOVA and Tukey’s test (Systat 9 software products, SPSS, Chicago, IL). Before analysis, daily growth index data were arc sin transformed, and feed efficiency and protein feed efficiency data were log transformed. For trial B, the two groups were compared using an unpaired two-tailed Student’s t test (Systat 9 software products, SPSS). In both cases, differences were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The daily growth index and feed efficiency were lower in fish fed P1 than in the other three groups, whereas the protein efficiency was only lower than in fish fed the P2 diet (P < 0.05) (Table 2). Although protein intakes varied (P < 0.01), daily fat intakes did not differ among the groups; daily carbohydrate intakes were greater in fish fed P1 than in those fed P3 or P4 with an intermediate intake in those fed P2 (P = 0.018) (Table 2). Liver to body weight ratios were higher in fish fed P1 or P2 than in those fed P3 and P4 (P << 0.01). Hepatic glycogen and fat concentrations generally were higher in fish fed P1 than in other groups (Table 2). Plasma free amino acid levels of fish fed the different diets increased concomitantly with protein intake levels (P < 0.01) (Table 2). Plasma glucose concentrations were higher in the P1 group than in all others (P << 0.01) (Table 2). Plasma free fatty acid levels were higher in fish fed the P1 diet than in those fed the P3 diet (P < 0.05), whereas plasma triglyceride levels were lower in fish fed P1 than in those fed the P4 and P2 diets (P << 0.01) (Table 2).


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  		TABLE 2 Growth performance, feed efficiencies and plasma metabolite concentrations in juvenile rainbow trout fed different levels of protein for 14 d (trial A)1

 
The activities of amino acid–degrading enzymes (GDH, ALAT and ASAT) did not differ among the groups (Table 3). In contrast, fish fed P1 had lower activities of the two gluconeogenic enzymes than did those fed other diets (Table 3). G6Pase activity was lower in fish fed P1 than in those fed P4 (P = 0.028) and FBPase activity was lower in fish fed the P1 diet than in those fed the P4 and P2 diets (P<<0.01). FBPase and G6Pase gene expression were also lower in fish fed the P1 diet (and P2 for FBPase) than in those fed the P3 and P4 diets (P < 0.01). PEPCK activity and gene expression did not differ among the groups. Glycolytic enzymatic activities (GK and PK) were greater in fish fed P1 than in the other groups (P < 0.04) (Table 3). In contrast, GK and PK gene expressions (Table 3), as well as GluT2 gene expression (data not shown), did not differ among the groups.


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  		TABLE 3 Hepatic enzyme expression in rainbow trout fed different levels of protein for 14 d (trial A) or in a single meal (trial B)1,2

 
After a single meal, there were no differences in plasma glucose or metabolite concentrations between the P1 and P4 groups (data not shown). PEPCK activity was lower in fish fed diet P1 than in those fed diet P4 (P = 0.038) (Table 3). FBPase and G6Pase activities did not differ between the two groups. PEPCK, FBPase and G6Pase gene expression did not differ between the two groups (Table 3) nor did activities or gene expressions of GK, PK and GluT2 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Our initial hypothesis was that in rainbow trout, high levels of dietary protein would lead to a persistent production of endogenous glucose by the liver, interfering with dietary glucose utilization. Because data from previous studies on the effect of dietary protein on the regulation of hepatic glucose metabolism are contradictory and because the effects of dietary protein alone could not be determined in these studies (14,15), we explored the effect of increased protein intakes using similar intakes of lipids and carbohydrates. The success of the pair-feeding protocol is reflected by the plasma free amino acids levels. The semipurified diet containing the highest protein level (P4) met the nutrient requirements of rainbow trout (8) and led to very good growth performance. Because the other diets had lower crude protein levels and because we did not want large differences in growth rates, we restricted our trial to 2 wk. This period was sufficient to yield significant differences in growth performance. However, catabolism of body tissues, which could have confounded our interpretations, was avoided because of the positive nitrogen balance status in all groups including P1.

The absence of any significant differences in GDH, ALAT or ASAT activities among the groups confirmed the lack of adaptation of these enzymes to changes in dietary protein content already reported in most fish (16). Because the capacity of fish to catabolize amino acids in the liver is the same irrespective of the dietary protein level, this metabolic pathway cannot be a priori a limiting factor for hepatic glucose production from (dietary) amino acids.

This is the first demonstration of a significant inhibition of gluconeogenic capacity (FBPase and G6Pase) in rainbow trout. Activities and gene expression of FBPase and G6Pase were lower in fish with low protein intake than in those with adequate protein intakes; regulation of G6Pase and FBPase activities is linked to a molecular mechanism previously described in mammals (17,18). In contrast, there was no inhibition of PEPCK gene expression and activity in fish fed diet P1. However, no distinction between cytosolic and mitochondrial PEPCK forms was made in our study. Thus, nutritional regulation of the expression of the cytosolic PEPCK could have been masked because PEPCK activity in rainbow trout is mainly mitochondrial (19), and only the cytosolic form is acutely regulated by diet (20).

Surprisingly, the decrease in hepatic gluconeogenic enzyme expression in fish fed a low level of protein was not sufficient to decrease the postprandial glycemia. Indeed, we observed a very high hyperglycemia in fish fed the P1 diet, comparable to values in rainbow trout administered oral or intravenous glucose tolerance tests, or fed a starch-rich diet (1). The decrease in gluconeogenic capacities may have been due to this atypical hyperglycemia. High glycemia leads to a strong induction of the capacities to store excess glucose in the liver. Indeed, the glycolytic capacity (GK and PK activities), as well as fat and glycogen concentrations of the liver, and liver size were far higher in fish fed the P1 diet than in those fed adequate or high protein. However, we cannot eliminate the possibility that dietary protein per se is not the regulator of lipogenesis. Indeed, previous studies showed that malic enzyme gene expression and activity were enhanced in chicks fed a low protein diet (21). A similar repression was also suggested in fish (22).

The hyperglycemia in fish fed diet P1 was unexpected and cannot be explained. It was not linked to the meal per se. Indeed, the test meal was not capable of modifying glycemia and glycolysis/gluconeogenesis in liver. The slightly higher carbohydrate intake of the P1 group also cannot explain this hyperglycemia because carbohydrate intake did not differ between the P1 and P2 groups. In mammals, the association between fatty liver and hyperglycemia is due to increased hepatic gluconeogenesis (23), whereas we observed reduced hepatic gluconeogenic capacity in these fish. Hyperglycemia in fish fed a low dietary protein more likely suggests a lack of dietary glucose utilization due to a disruption of its muscular transport or phosphorylation capacity (24) and/or high endogenous production due to other gluconeogenic tissues such as gut or kidney (25,26). Further studies are necessary to clarify these issues.

In conclusion, a low protein intake is associated with hyperglycemia in rainbow trout. The high level of plasma glucose seems to be sufficient to activate glucose storage and inhibit gluconeogenic capacity as in mammals (2). The association of hyperglycemia with the low level of dietary protein, despite a decrease in gluconeogenic capacity, is surprising. In humans, dietary protein does not affect plasma glucose levels despite an increase in gluconeogenesis (27). Thus, our results in fish are even more intriguing and warrant further exploration.


   FOOTNOTES
 
1 Supported by the European Commission (FAIR- N°QLK5–2000-30068, "Perspectives of Plant Protein Use in Aquaculture"). The French Minister of Research and Technology provided a fellowship to S.K. Back

3 Abbreviations used: ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; FBPase, fructose-1,6-bisphosphatase; GDH, glutamate dehydrogenase; GK, glucokinase; GluT2, type 2 glucose transporter; G6Pase, glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase; PK, L-type pyruvate kinase. Back

Manuscript received 6 February 2003. Initial review completed 18 March 2003. Revision accepted 12 May 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Moon, T. W. (2001) Glucose intolerance in fish: fact or fiction?. Comp. Biochem. Physiol. 129B:243-249.

2. Pilkis, S. J. & Granner, D. K. (1992) Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu. Rev. Physiol. 54:885-909.[Medline]

3. Dabrowski, K. & Guderley, H. (2002) Intermediary metabolism. Halver, J. E. Hardy, R. W. eds. Fish Nutrition 2002:309-365 Academic Press New York, NY. .

4. Panserat, S., Plagnes-Juan, E. & Kaushik, S. (2001) Nutritional regulation and tissue specificity of gene expression for key proteins involved in hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 204:2351-2360.[Abstract/Free Full Text]

5. Shimeno, S., Kheyyali, D. & Shikata, T. (1995) Metabolic response to dietary carbohydrate to protein ratios in carp. Fish. Sci. 61:277-281.

6. Shimeno, S., Hosokawa, H. & Takeda, M. (1996) Metabolic response of juvenile yellowtail to dietary carbohydrate to lipid ratios. Fish. Sci. 62:945-949.

7. Panserat, S., Perrin, A. & Kaushik, S. (2002) High dietary lipids induce glucose-6-phosphatase expression in rainbow trout (Oncorhynchus mykiss). J. Nutr. 132:137-141.[Abstract/Free Full Text]

8. National Research Council (1993) Nutrient Requirements of Fish 1993 National Academy Press Washington, DC.

9. Kirchner, S., Kaushik, S. & Panserat, S. (2003) Effect of partial substitution of dietary protein by a single dispensable amino acid on hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 134A:337-347.

10. Murat, J. C. & Serfaty, A. (1974) Simple enzymatic determination of polysaccharide (glycogen) content of animal tissues. Clin. Chem. 20:1576-1577.[Abstract]

11. Folch, J., Lees, M. & Sloane-Stanley, G. H. S. (1957) A simple method for the isolation and purification of total lipids from animals tissues. J. Biol. Chem. 226:497-509.[Free Full Text]

12. Moore, S. (1968) Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J. Biol. Chem. 1243:6281-6283.

13. Mambrini, M., Vachot, C. & Kaushik, S. (1998) The effect in including soy protein concentrates in diets fed to rainbow trout on the activities of transdeaminating enzymes. Rep. Nutr. Dev. 38:199.

14. Cowey, C. B., Cooke, D. J., Matty, A. J. & Adron, J. W. (1981) Effects of quantity and quality of dietary protein on certain enzyme activities in rainbow trout. J. Nutr. 111:336-345.

15. Walton, M. J. (1986) Metabolic effects of feeding a high protein/low carbohydrate diet as compared to a low protein/high carbohydrate diet to rainbow trout (Salmo gairdneri). Fish Physiol. Biochem. 1:7-15.

16. Cowey, C. B. & Walton, M. (1989) Intermediary metabolism. Cowey, C. B. Walton, M. eds. Intermediary Metabolism 1989:259-329 Academic Press New York, NY. .

17. Marcus, F., Rittenhouse, J., Gontero, B. & Harrsch, P. (1987) Function, structure and evolution of fructose-1, 6-bisphosphatase. Arch. Biol. Med. Exp. 20:371-378.

18. Van de Verve, G., Lange, A., Newgard, C., Méchin, M., Li, Y. & Berteloot, A. (2000) New lessons on regulation of glucose metabolism taught by the glucose-6-phosphatase system. Eur. J. Biochem. 267:1533-1549.[Medline]

19. Mommsen, T. P. & Suarez, R. K. (1984) Control of neoglucogenesis in rainbow trout hepatocytes: role of pyruvate branchpoint and phosphoenol pyruvate-pyruvate cycle. Mol. Physiol. 6:9-18.

20. Hanson, R. W. & Reshelf, L. (1997) Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression. Annu. Rev. Biochem. 17:325-352.

21. Adams, K. A. & Davis, A. J. (2001) Dietary protein concentration regulates the mRNA expression of chicken hepatic malic enzyme. J. Nutr. 131:2269-2274.[Abstract/Free Full Text]

22. Dias, J., Alvarez, M. J., Diez, A., Arzel, J., Corraze, G., Bautista, J. M. & Kaushik, S. J. (1998) Regulation of hepatic lipogenesis by dietary protein/energy in juvenile European seabass (Dicentrarchus labrax). Aquaculture 161:169-186.

23. Song, S. (2002) The role of increased liver triglyceride content: a culprit of diabetic hyperglycaemia?. Diabetes Metab. Res. Rev. 18:5-12.[Medline]

24. Legate, N. J., Bonen, A. & Moon, T. W. (2001) Glucose tolerance and peripheral glucose utilization in rainbow tout (Oncorhynchus mykiss), American eel (Anguilla rostrata), and black bullhead catfish (Ameiurus melas). Gen. Comp. Endocrinol. 122:48-59.[Medline]

25. Rajas, F., Croset, M., Zitoun, C., Montano, S. & Mithieux, G. (2000) Induction of PEPCK gene expression in insulinopenia in rat small intestine. Diabetes. 49:1165-1168.[Abstract]

26. Cano, N. (2002) Bench-to-bedside review: glucose production from the kidney. Crit. Care. 6:317-321.[Medline]

27. Franz, M. J. (1997) Protein: metabolism and effect on blood glucose levels. Diabetes Educ. 23:643-651.




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