Carbohydrates affect protein-turnover rates, growth, and nucleic acid content in the white muscle of rainbow trout (Oncorhynchus mykiss)1

Aquaculture 179 Ž1999. 425–437

Carbohydrates affect protein-turnover rates, growth, and nucleic acid content in the white muscle of rainbow trout žOncorhynchus mykiss / 1 b Juan Peragon , ´ a, Juan B. Barroso a, Leticia Garcıa-Salguero ´ c b,) Manuel de la Higuera , Jose´ A. Lupianez ´˜ a

Department of Experimental Biology, Area of Biochemistry and Molecular Biology, Centre for Experimental Sciences, UniÕersity of Jaen, ´ Paraje Las Lagunillas sr n, 23071 Jaen, ´ Spain b Department of Biochemistry and Molecular Biology, Centre for Biological Sciences, UniÕersity of Granada, AÕenida FuentenueÕa sr n, 18001 Granada, Spain c Department of Animal Biology and Ecology, Centre for Biological Sciences, UniÕersity of Granada, AÕenida FuentenueÕa sr n, 18001 Granada, Spain

Abstract We have investigated the effect of dietary carbohydrate on different parameters of proteinturnover rate, nature of growth, and nucleic acid content in the muscle of rainbow trout in order to better understand the molecular nature of these growth parameters in the absence of this dietary component. For this, we used a methodology based on the incorporation rate of tritium labelled phenylalanine in muscle protein. Juvenile rainbow trout of an initial body weight of 110 g were fed near to satiety with a control or a non-carbohydrate diet during 7 weeks. The absence of dietary carbohydrate significantly depressed fish growth, as well as daily body weight gain, as a consequence of muscular hypotrophy Žthe cell size diminished by almost 50%. and not by a reduction of number of cells Žhypoplasia.. This nutritional situation also significantly slowed Žby almost 11%. muscle-protein accumulation rate Ž K G . as a result of a significant increase Žeight-fold. in muscle-protein degradation rate Ž K D ., without changing the other protein-turnover rates, protein synthesis rate Ž K S ., protein synthesis capacity Ž CS ., protein synthesis efficiency Ž K RNA ., protein synthesis rate per cell unit Ž K DNA ., or protein retention efficiency ŽPRE.. These results,

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Corresponding author. Tel.: q34-958-243089; fax: q34-958-243089; E-mail: [email protected] Publication no. 194 from ‘Drugs, Environmental Toxics and Cell Metabolism Research Group’, Department of Biochemistry and Molecular Biology, Centre of Biological Sciences, University of Granada, Granada, Spain. 1

0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 1 7 6 - 3

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together with the nucleic acid content, clearly indicate that the absence of carbohydrate significantly exacerbates the muscular-protein degradation without affecting protein synthesis. In conclusion, carbohydrates are needed to prevent amino acids released during protein degradation from being used to synthesize carbohydrates andror to be used for energy and not for growth. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Dietary carbohydrate; Fish; Growth; Nucleic acid; Protein-turnover rate; White muscle

1. Introduction Carbohydrates constitute one of the three prime components of the fish diet that are used as an energy source to support animal growth. The biological functions and metabolism of this nutrient in fish are not well understood. Carbohydrate digestibility is better in freshwater and warmwater fish than in marine and coldwater fish ŽShimeno et al., 1977; Hofer and Sturmbauer, 1985., and it is related to the complexity of the carbohydrates ŽBuhler and Halver, 1961; Furuichi and Yone, 1982a; Wilson and Poe, 1987.. A relative inability to metabolise carbohydrates has been found in several studies in different species of fish ŽFuruichi and Yone, 1982b; Wilson and Poe, 1987; Gutierrez ´ et al., 1991.. This inability is reflected in a persistent hyperglycemia ŽPalmer and Ryman, 1972; Furuichi and Yone, 1981; Wilson and Poe, 1987., a lower activity of liver hexokinase ŽFuruichi and Yone, 1982b., a lack of glucokinase ŽNagayama and Ohshima, 1974; Cowey et al., 1977., and a lower number of muscle insulin receptors ŽGutierrez et ´ al., 1991.. Nevertheless, it has been proposed that it is necessary to provide an appropriate level of this nutrient in the diet Žapproximately 20%. to ensure maximum utilization of the other nutrients ŽWilson, 1994.. However, currently, there is a notable lack of knowledge about the influence of dietary carbohydrate on protein-turnover rates and nucleic acid content of different fish tissues. In all fish, and in particular, in rainbow trout, regulation of muscle protein deposition is generally of great importance for control of whole body growth rate. White muscle protein accumulation rate Ž K G . determines whole body growth rate Ž G R . ŽWeatherly and Gill, 1987, 1989. and is the result of the balance between the protein synthesis and protein degradation rates. In the white muscle of trout, the fractional protein synthesis Ž K S . and degradation Ž K D . rates are lower than in other tissues, while the protein retention efficiency ŽPRE. is the highest ŽFauconneau and Arnal, 1985; Houlihan et al., 1988.. Therefore, in this tissue, practically all the protein synthesised in muscle accumulates as growth. In fish, other parameters related to tissue growth and proteinturnover rates are content of nucleic acids, RNA and DNA ŽBuckley, 1984; Bastrop et al., 1992; Foster et al., 1993.. In the present work, we investigate the absence of dietary carbohydrate in relation to the protein-turnover rates and nucleic acid content of white muscle of rainbow trout, both being the main factors responsible for control of white muscle protein accumulation and fish growth rate. A good understanding of the relationship between white muscle protein-turnover rates and the carbohydrate level of fish diets is of great importance to clarify the biological function and metabolism of this nutrient in rainbow trout.

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2. Materials and methods 2.1. Chemicals 3

Hx Phenylalanine Ž2.22 Tbqrmmol. was obtained from Amersham International ŽUK.. L-tyrosine decarboxylase from Streptococcus faecalis, L-phenylalanine, bovine serum albumin, b-phenylethylamine, leucylalanine and pyridoxal 5-phosphate came from Sigma ŽUSA.. All other chemical compounds were bought from Fluka Chemika–Biochemika ŽSwitzerland. and were of analytical grade. L-w2,6-

2.2. Fish and maintenance Rainbow trout Ž Oncorhynchus mykiss . were supplied by a local fish farm ŽRiofrio, Granada, Spain. and kept in 650-l fiber-glass tanks in fresh, temperature-controlled, continuously aerated water Ž1.5 lrmin and 15.0 " 0.58C., under regulated lighting conditions Ž0800 to 2000 h.. The fish were adapted to laboratory conditions for 2 weeks with free access to a standard commercial diet before being divided into two experimental groups: one Žcontrol. was fed a diet composed of 40% protein, 18% fat and 23% carbohydrate and the other Žnon-carbohydrate, NC. was fed a carbohydrate-free diet composed of 40% protein, 18% fat and 0% carbohydrate ŽTable 1.. The total experimental period was 9 weeks including the time for adaptation to laboratory conditions. Each group contained 150 fish selected at random from the original group of 300 fish. The two groups were then separated once more into three different tanks of 50 fish each. The fish were fed by hand to near satiety and the quantity of diet supplied at each feeding recorded in order to calculate feed and protein-conversion efficiency. The composition and formulation of the diets are shown in Table 1. The ingredients were blended with sodium alginate and then thoroughly stirred with distilled water to obtain a homogeneous, moist paste. Pellets were made by passing the diet mixture through an electric meat grinder equipped with a die of 2.5 mm hole size. The diets were dried at 308C and kept in a deep-freeze at y208C. The diets were analysed for crude protein, total lipids and moisture by standard methods ŽAOAC, 1984.. Daily food intake and weekly weight gains were recorded throughout the experiment. The relative daily intake was calculated by dividing the absolute daily diet intake by the mean body weight of fish, calculated from the growth curve. 2.3. Experimental procedure Each fish was killed by a sharp blow to the head and immediately put onto ice, where it was cleaned of everything superfluous to the muscle, except the bones. The entire muscle was weighed, including the bones, and a section was then removed from beneath the skin, above and on either side of the spine, in the region of the neck, discarding any superficial red muscle. The white muscle, thus obtained, was freeze-clamped and stored in liquid nitrogen for protein-turnover assays.

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Table 1 Formulation and composition of the experimental diets Ingredients

Formulation, g r k diet Fish meal a Fish solublesb Fish oil Corn oil Pre-cooked starch Vitamin premixture c Mineral premixtured Cellulose Composition (g r100 g dry matter) Protein Lipids Digestible carbohydrates Gross energy ŽkJrg. e PrE ŽmgrkJ. f PErNPE f

Diet Control

Non-carbohydrate

508.7 47.8 54.2 90.0 230.0 20.0 50.0 0.0

508.7 47.8 54.2 90.0 0.0 20.0 50.0 209.3

40.0 18.0 23.0 18.91 21.15 0.71

40.0 18.0 0.0 14.95 26.75 1.10

a

Fish-meal protein was composed of 6.53% fat, 70.77% protein and 8.72% water. Fish soluble mixture was composed of 5.36% lipid, 83.55% protein and 9.65% water. c Vitamin pre-mixture contained Žgrkg pre-mix. thiamin hydrochloride 2, riboflavin 3, pyridoxine hydrochloride 1.5, calcium pantothenate 7.5, nicotinic acid 12.5, folic acid 0.75, myo-inositol 50, choline chloride 250, biotin 0.15, cyanocobalamin 0.33, ascorbic acid 50, vitamin A 0.0075, vitamin D 0.00375, vitamin E 12.5, vitamin K 0.125 and sucrose up to 1000 g mix. d Mineral pre-mixture contained Žgrkg pre-mix.: calcium phosphate monobasic 600, calcium carbonate 130, potassium chloride 50, sodium chloride 80, magnesium sulphate 4, ferric sulphate 30, magnesium chloride 0.0435, aluminium sulphate 0.2 and sucrose up to 1000 mix. e The energy value of protein, lipid and carbohydrate was assumed to be 19.6, 39.5, and 17.2 kJrg, respectively, ŽBrett and Groves, 1979.. f PrE, proteinrenergy, PErNPE, protein-energyrnon-protein energy. b

2.4. Growth rates At the beginning of each experiment, all the fish were weighed individually and separated into different tanks, 50 fish to a tank. An initial sample of 12 fish, different from those used for the experiment, were weighed and the weight and protein content of their white muscle determined. Protein concentration was determined according to Lowry et al. Ž1951.. These initial mean values were used as reference for the determination of whole body growth rate Ž G R . and white muscle protein accumulation rate Ž K G . throughout the experiment. Apart from the fish used to determine the protein synthesis rate, each week, six fish from each group Žtwo per tank. were removed at random, killed by cervical separation, and their white muscle tissue removed and weighed. At the end of the experiment, all the remaining fish were killed to analyse growth, muscle protein and muscle weight curves and to determine the protein synthesis rate.

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Whole body growth rate Ž G R . was calculated as the percentage of weight increase per day using the following equation ŽRicker, 1979.: G R Ž %rday. s 100 Ž log e W2 y log e W1 . r Ž t 2 y t 1 . where W1 and W2 are the body weight at times t 1 and t 2 . The white muscle protein accumulation rate Ž K G . was calculated as the percentage of white muscle protein increase per day, using the equation: K G Ž %rday. s 100 Ž log e P2 y log e P1 . r Ž t 2 y t 1 . where P1 and P2 are the total muscle protein contents at time t 1 and t 2 ŽWootton, 1990.. The absolute protein accumulation rate Ž A G . was calculated as the product of K G multiplied by the total protein content of the tissue and expressed as milligram of protein accumulated per day. 2.5. Protein-turnoÕer rates The fractional protein synthesis rate was determined by the method described by Garlick et al. Ž1980., as modified by Peragon ´ et al. Ž1992, 1998.. The caudal vein injection solution contained 135 mM L-Phe and L-w2,6 3 HxPhe at 37.0 MBqrml Ž100 mCirml. and a specific radioactivity of 1640 dpmrnmol. The dosage was 50 mCir100 g body weight at a volume per dosage of 0.5 mlr100 g body weight. Six fish Žtwo per tank. were killed 2 min after injection and 12 Žfour per tank. 45 min after injection. They were immediately put on ice and sections of white muscle were removed and freeze-clamped in liquid nitrogen. The tissues were homogenised Ž1:10 wrv. with cold 0.2 M HClO4 and separated into two equal fractions, one for determining the protein synthesis rate and the other for quantifying DNA and RNA. After centrifuging one of the fractions for 15 min at 2800 = g Ž rav s 8.94 cm. at 48C, saturated tripotassium citrate was added to the supernatant Žacid soluble fraction. and later the KClO4 generated was removed by centrifuging at 2800 = g for 15 min. The pH of the supernatants were adjusted to 6.0. The insoluble protein fraction was washed twice with 96% ethanol and once with ether, whereupon the pellet was hydrolysed in 6 M HCl for 24 h at 1108C. The HCl was removed by evaporation and the amino acids were resuspended in saturated sodium citrate, pH 6.3. The final concentrations of Phe and L-w2,6 3 H xPhe incorporated into the soluble supernatant Ž SA . and insoluble protein Ž SB . fractions were calculated after the conversion of Phe to b-phenylethylamine ŽPEA.. The b-phenylethylamine concentrations were determined by spectrofluorescence using a standard curve based on an analysis of 0 to 15 nmol of PEA ŽSuzuki and Yagi, 1976.. The results were expressed as a function of SA and S B Ždpmrnmol.. The fractional protein synthesis rate, K S Žpercentage protein synthesis per day. was calculated as: KS s

Ž SB t

2

y S B t 1 . rSA Ž t 2yt 1 . 1440r Ž t 2 y t 1 . 100

where SB t 1 and S B t 2 are the protein bound specific radioactivity at 2 min and 45 min after injection, SA Ž t 2yt 1 . is the average free pool of specific radioactivity over the period Ž t 2 y t 1 . and 1440 is the number of minutes in a day ŽGarlick et al., 1980..

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The absolute protein synthesis rate Ž A S . was calculated as the product of K S multiplied by the total protein content of the tissue and expressed as milligram of protein synthesised per day. The fractional protein degradation rates Ž K D . were calculated as being the difference between the protein synthesis Ž K S . and protein accumulation Ž K G . rates calculated for a period of 43 min and expressed as a percentage per day ŽMillward et al., 1975.. Finally, PRE was defined as the ratio between protein retained as growth and total protein synthesised, and was calculated as Ž K G rK S .100 ŽFoster et al., 1991.. 2.6. Determination of DNA and RNA concentrations in the white muscle DNA and RNA were separated and purified from the second fraction of muscle precipitated with 0.2 M HClO4 and the nucleic acids were quantified by the method described by Munro and Fleck Ž1966.. The pellet was washed twice with 0.2 M HClO4 to remove low molecular weight contaminants Žmetabolites, nucleotides, coenzymes, inorganic phosphate and phosphorus.. The RNA and DNA fractions were separated by digestion in an alkaline medium of 0.3 M KOH at 378C for 1 h. Proteins and DNA were precipitated by adding 1.2 M HClO4 . These samples were centrifuged for 15 min at 1000 = g at 48C, RNA was localised in the supernatant and DNA in the pellet. The supernatant was diluted to 0.1 M HClO4 and the RNA concentration determined by measuring the absorbance at 260 nm and 232 nm ŽAshford and Pain, 1986.. The pellet was dissolved in 0.1 M KOH and the DNA concentration was estimated by the Indol test ŽCeriotti, 1952. using herring sperm as the DNA standard. The values of the protein synthesis rate are, to a great extent, proportional to RNA concentration, so the protein synthesis capacity Ž CS . can also be defined as a ratio of RNA:protein and expressed as milligramsrgram Žmgrg. ŽSugden and Fuller, 1991.. Protein synthesis efficiency Ž K RN A . is defined as the amount Žg. of protein synthesized per day and RNA unit Žg. and is calculated as wŽ K SrCS .10x ŽSugden and Fuller, 1991.. Protein synthesis raterDNA unit Ž K DN A . is defined as the amount Žg. or protein synthesized per day and DNA unit Žg. and is calculated as wŽ K Sr100.ŽProteinrDNA.x ŽSugden and Fuller, 1991.. 2.7. Statistical treatment The results are expressed as the means " SEM. To compare the groups of fish, one-way analysis of variance and Student’s unpaired, two-tailed t-test were used. Differences were considered to be significant at a value of P - 0.05. Regression analysis was applied to the results. The linear and logarithmic regressions were determined by a least-square linear-regression analysis.

3. Results The absence of dietary carbohydrate significantly altered fish growth as reflected by the different nutritional indices ŽTable 2.. The daily body weight gain was 30% less in fish fed the NC diet with respect to the control. In the same way, the growth rate Ž G R .,

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Table 2 Weight gain, nutrient intake, feed and protein conversion efficiencies in fish fed with control and non-carbohydrate diets Control Body weight (g) and growth rate (GR ) Initial 112.12"0.60 Final 211.10"14.2 Daily weight gain Žg. 2.02"0.11 GR 1.12"0.06 RelatiÕe daily intake Diet Žmgrg fish. Energy ŽJrg fish. Nutritional indices FCE a PCE a

13.8"0.2 261.5"3.8

0.96"0.02 2.39"0.04

Non-carbohydrate 106.23"2.00 168.26"6.37 1.27"0.15U 0.94"0.12

13.9"0.1 207.3"2.1U

0.76"0.01U 1.89"0.01U

a

FCEs weight gain Žg.rdiet intake Žg., PCEs weight gain Žg.rprotein intake Žg.. The total number of fish used in this experiment was 150 per treatment separated into three tanks. The results are expressed by the means"SEM. Results were tested with a One-way ANOVA following a Student’s t-test. Probabilities of P - 0.05 or less were considered statistically significant and was represented by U .

expressed as a daily percentage was significantly diminished by 16%. In addition, the nutritional indexes, feed conversion efficiency ŽFCE. and protein conversion efficiency ŽPCE. were also 21% lower in fish fed the NC diet, compared to those fed the control

Fig. 1. Nature of white muscle growth in trout fed the control ŽControl. and NC diet. The results are expressed by the means"SEM of 12 individual fish. Results were tested with a One-way ANOVA following a Student’s t-test using means. Probabilities of P - 0.05 or less were considered statistically significant. For each parameter, a bar with a different superscript is statistically different.

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Table 3 Protein and nucleic acid contents of rainbow trout white muscle fed on control and non-carbohydrate diets Control White muscle concentration, mg r g tissue Protein 173.98"14.70 RNA 1.58"0.27 DNA 0.36"0.04 Total muscle content, g Protein RNA RNArDNA ratio, mgrmg

Non-carbohydrate 148.96"13.96 1.25"0.04 0.56"0.14

21.18"1.27 0.19"0.02

13.78"0.92U 0.12"0.01U

4.39"0.14

2.23"0.32U

The results are expressed by the means"SEM of 12 individual fish. Results were tested with a One-way ANOVA following a Student’s t-test. Probabilities of P - 0.05 or less were considered statistically significant and was represented by U .

Fig. 2. Effects of different carbohydrates dietary levels on white muscle protein-turnover rates. The sample size was 150 fish per treatment, divided into three groups. The results are expressed by the means"SEM of 12 individual fish. Results were tested with a One-way ANOVA following a Student’s t-test using means. Probabilities of P - 0.05 or less were considered statistically significant. For each parameter, a bar with a different superscript is statistically different. K G , protein accumulation rate; K S protein synthesis rate; K D protein degradation rate. K G , K S and K D are expressed as percentage per day Ž%rday.. CS , protein synthesis capacity Žmg RNArg protein.; K RN A , protein synthesis efficiency wŽ K S r CS .10, g protein synthesizedrŽday g RNA.x; K DN A , protein synthesis raterDNA unit wŽ K S r100.ŽproteinrDNA., g protein synthesizedrŽday g DNA.x; PRE, protein retention efficiency wŽ K G r K S .100, percentage of protein synthesized retained as growthx. The values of CS and K DNA are 10-fold higher than that expressed in the figure. The values of PRE are 100-fold higher than that expressed in the figure.

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Fig. 3. Absolute protein accumulation Ž A G ., synthesis Ž A S . and degradation Ž A D . rates in fish fed with control and NC diets. The values of absolute rates were expressed as milligrams of protein per day. Results were tested with a One-way ANOVA and Student’s t-test. For each parameter, a bar with a different superscript are significantly different Ž P - 0.05..

diet. Fish fed the NC diet showed a significantly lower white muscle, weight gain of roughly 18%. Growth of fish fed the NC diet was also measured in terms of total and relative concentrations of white muscle protein, RNA and DNA ŽFig. 1 and Table 3.. Feeding of a diet without digestible carbohydrate did not alter the number of cell units Žindicator of hyperplasia. measured as total DNA, whereas a significant reduction Žalmost 50%. in cell size Žindicator of hypertrophy. was reflected by the ratio between protein and DNA. Furthermore, total protein, RNA and the RNA:DNA ratio in fish fed the NC diet were lower by a 35, 37 and 49%, respectively, than in fish fed the control diet. The behaviour of white muscle protein accumulation and protein-turnover rates in fish fed the NC diet are shown in Fig. 2. Under this nutritional regimen, the white muscle protein accumulation rate Ž K G ., expressed as percentage per day Ž%rday., significantly decreased by 11%, whereas the protein degradation rate Ž K D . increased significantly, some eight-fold over the control. On the other hand, the rest of the parameters related to the protein-turnover rate, such as protein synthesis rate Ž K S ., protein synthesis capacity Ž CS ., protein synthesis efficiency Ž K RNA ., protein synthesis rate per cell unit Ž K DN A . and PRE showed no significant changes. Given the significant reduction in growth and in total content of muscle protein, the absolute values of protein accumulation Ž A G ., protein synthesis Ž A S . and protein degradation Ž A D . underwent significant changes ŽFig. 3.. Both A G and A S diminished significantly Žby 42 and 36%, respectively., whereas the absolute protein degradation Ž A D . increased significantly Žalmost four-fold., as might be expected. 4. Discussion The present work explored the effect of dietary carbohydrate on growth and proteinturnover rates in the white muscle of rainbow trout. This tissue is the best for studying

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the relationship between changes in rates of protein-turnover and tissue growth, because muscle protein accumulation is a direct reflection of animal growth. Muscle protein growth proved to be the consequence of significant alterations in protein-turnover rates in this tissue, this being positive when the rate of protein synthesis rate exceeded the rate of protein degradation, as previously demonstrated ŽHoulihan et al., 1988; Peragon, ´ 1993; Peragon ´ et al., 1994, 1998.. In addition, it is well-documented that there is a close relationship between food ration and diet composition with cellular growth. Several reports have examined the relationship between ration size and growth dynamism ŽHoulihan et al., 1989; Jurss ¨ and Bittorf, 1990; Kiessling et al., 1991; Bastrop et al., 1992.. However, very few works relate the variation in diet composition with protein-turnover rates and growth, to determine how different nutrients affect protein synthesis and degradation rates ŽPeragon, ´ 1993; Peragon ´ et al., 1994.. Under our experimental conditions, fish fed a carbohydrate-free diet showed a significant decrease in the nutritional indices measured ŽFCE and PCE., as well as in both growth rate Ž G R . of the whole body and in protein accumulation rate in the white muscle Ž K G .. The values of total protein and DNA indicate that both changes were due to a clear case of muscular hypotrophy and not to a change in the level of muscular hyperplasia. This behaviour in the nature of growth is indicated by significant changes in protein:DNA ratio without variations in total DNA content. Our results agree with those of Stickland et al. Ž1988. in Atlantic salmon, and of Kiessling et al. Ž1991. in trout. These authors observed an increase in hypertrophy in muscle fibres of fish that presented rapid growth. Kiessling et al. Ž1991. have demonstrated that in trout of different age submitted to feeding with rations of different size, the size of muscle fibres diminished in accordance with the reduction in amount of food ingested. In the same way, our results indicated that fish fed a deficient diet composition showed a lower growth rate and reduced size of muscle cells. The RNA:DNA ratio has commonly been used as one of the best biochemical indexes of specific growth rate because of a positive relationship between RNA:DNA values and growth rates has been described under different physiological and nutritional conditions ŽBastrop et al., 1992; Foster et al., 1993.. The lower values in amount of RNA per DNA unit ŽRNA:DNA ratio. found in fish fed the NC diet, confirms the lower growth rates in fish fed this diet. In addition, the significant increase in relative DNA concentration in fish fed the NC diet is a consequence of both the constancy in values of cell DNA and the significant decrease in total protein content and thus in cell size. The absence of an exogenous carbohydrate supply significantly altered both protein accumulation and protein-turnover rates in the white muscle of trout. Under this nutritional situation, a high and significant increase in protein degradation rate Ž K D . was found without changes in the protein synthesis rate Ž K S . and so the values of K D exceeded the values of K S , accounting for the decrease in growth rate of the whole fish Ž G R . and in the protein accumulation rate of white muscle Ž K G . in fish fed the NC diet. On the other hand, the rest of the parameters related with protein-turnover, CS , K RNA , K DN A and PRE did not change with respect to control values. These latter parameters are closely related to the processes of protein synthesis, and the capacity of protein synthesis Ž CS . is often used as a marker of transcription efficiency ŽRNA synthesis. or of total content of ribosomes, whereas the efficiency of protein synthesis rate Ž K RN A . is

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usually used as a marker of the process of translation, which is essentially a measure of mean protein synthesis rate per ribosome ŽSugden and Fuller, 1991.. The behaviour of the protein-turnover rate in the white muscle of fish fed the NC diet indicates that the essentiality of carbohydrate may centre on the processes of protein degradation, changing its rate instead of that of protein synthesis. This contrasts with what happens in the same tissue when levels of dietary protein fall. In this sense, we previously showed ŽPeragon ´ et al., 1994. that in fish fed a low-protein diet, both growth rate and K G decreased significantly, although in this case, it was due to a significant decrease in all parameters related to protein synthesis rate Ž K S , CS , K RNA , and K DNA . without changes in values of protein degradation rates Ž K D .. As can be seen, the contrary happened with the absence of carbohydrates. It is evident that with a NC diet, amino acids come from the higher rate of protein degradation and are probably used to obtain energy andror, on the other hand, its carbon skeleton is utilized for biosynthesis processes, especially as muscle glycogen. In this case, under our nutritional condition, the activities of some gluconeogenic enzymes from trout white muscle, such as phosphoenolpyruvate carboxykinase ŽPEPCK. and fructose bisphosphatase ŽFBPase. increased significantly Žresult not shown.. Finally, to confirm that carbohydrate directly affects protein degradation, it is necessary to study the kinetic behaviour of the different protease activities in the white muscle of this fish, which is our aim in the near future.

5. Conclusions Fish fed a carbohydrate-free ŽNC. diet showed a significant decrease of growth, due to a phenomenon of muscular hypotrophy and not to a change in the level of muscular hyperplasia. This behaviour in the nature of growth is indicated by significant changes in the protein:DNA ratio without variations in total DNA content. In addition, the absence of carbohydrate increased protein degradation and decreased the absolute protein synthesis rate in the white muscle of rainbow trout. This behaviour in the protein-turnover would also explain the significant decrease in the growth rate and white muscle weight gain. In short, the absence of dietary carbohydrate would induce an important fraction of the amino acids, released from dietary protein digestion and muscle protein breakdown, to be used for gluconeogenic purposes and not for protein synthesis and for growth.

Acknowledgements This work was supported by a grant ŽNo. MAR-92-0412. from the Comision ´ Interministerial de Ciencia y Tecnologıa ´ ŽCICYT., Ministerio de Educacion ´ y Ciencia, Spain, and research grants ŽNs CVI 157 y RNM 156. from the Plan Andaluz de Investigacion ´ ŽPAI., Consolidacion ´ de Grupos de Investigacion, ´ Consejerıa ´ de Educacion ´ y Ciencia, Junta de Andalucıa, ´ Spain. The authors thank David Nesbitt for revising the English text.

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