Liver and white muscle protein turnover rates in the European eel (Anguilla anguilla): effects of dietary protein quality

Aquaculture 179 Ž1999. 203–216

Liver and white muscle protein turnover rates in the European eel žAnguilla anguilla /: effects of dietary protein quality M. de la Higuera a,) , H. Akharbach a , M.C. Hidalgo a , J. Peragon ´ b, a J.A. Lupianez ´˜ c , M. Garcıa-Gallego ´ a

c

Departamento Biologıa ´ Animal y Ecologia, Facultad Ciencias, UniÕersidad de Granada, Campus FuentenueÕa, 18071 Granada, Spain b Departamento Biologıa ´ Experimental y Ciencias de la Salud, UniÕersidad de Jaen, ´ Jaen, ´ Spain Departamento Bioquımica y Biologıa ´ ´ Molecular, Facultad de Ciencias, UniÕersidad de Granada, Granada, Spain

Abstract The influence of the quality of dietary protein source on growth and protein synthesis and degradation rates was studied in the liver and white muscle of the European eel. Fish were fed isonitrogenous diets differing in protein source: one Žcontrol. contained fish meal, three others incorporating meat meal ŽMM. or sunflower meal ŽSFM. as the only protein source, and SFM supplemented with some essential amino acids ŽEAAs. were also tested. Fish fed diets containing unsupplemented MM or SFM exhibited dietary utilization and growth indices poorer than those fed the control, while EAA supplementation greatly improved the performance of the SFM-diet. Liver showed higher rates of protein synthesis Ž k s . and degradation Ž k d . associated with a higher capacity for protein synthesis per unit of DNA but a lower protein deposition efficiency ŽPDE., compared to muscle. Low quality dietary protein increased the protein turnover rate, with a higher protein synthesis rate per unit of DNA and RNA but a decrease of PDE. In white muscle, MM and unsupplemented SFM diets decreased k s without changing k d . The MM diet reduced the efficiency of protein synthesis and deposition. EAA supplementation of the SFM diet raised the protein synthesis rate and capacity as well as protein deposition compared to control values. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Metabolism; Nutrition; Protein; Turnover; Eel

)

Corresponding author. Tel.: q34-58-243240; fax: q34-58-243238; E-mail: [email protected]

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 6 3 - 5

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1. Introduction Protein synthesis in fish has been studied in relation to factors such as growth rate ŽHoulihan et al., 1986, 1988., environmental temperature ŽFauconneau and Arnal, 1985; de la Higuera et al., 1997., starvation-refeeding, ration size ŽHoulihan et al., 1989., feeding frequency ŽPeragon ´ et al., 1992; Espe and Lied, 1994; Langar and Guillaume, 1994. and diet composition in macronutrients and energy ŽLied and Rosenlund, 1984; Rosenlund et al., 1984; Peragon ´ et al., 1994.. Nevertheless, few studies have been devoted to the influence of diet quality or essential nutrient availability on tissue protein turnover rates of growing fish, most studies being dedicated to the influence of protein quality and amino acid ŽAA. availability. In this sense, quality of alternative protein sources is related to the AA pattern they offer for tissue protein synthesis. A correlation between dietary and tissue AA profile has been found in fish ŽWilson et al., 1985; Ogata, 1986. and plasma levels of a given essential amino acid ŽEAA. have been measured to determine requirements ŽWalton et al., 1986; Sierra, 1995.. Fish fed EAA deficient diets undergo reduced growth as a consequence of an altered AA pattern available for protein synthesis; in fact, protein synthesis and deposition rates decrease ŽGarzon, ´ 1995; Sierra, 1995; de la Higuera et al., 1997. in response to being fed AA deficient diets. Muscle-protein synthesis has been shown, in trout ŽFauconneau, 1985. and sea bass ŽLangar et al., 1993; Langar and Guillaume, 1994., to be altered when fed an unbalanced AA diet. Nevertheless, adequate EAA supplementation of diets has been shown to stimulate muscle protein synthesis and deposition ŽGarzon, ´ 1995; Sierra, 1995; de la Higuera et al., 1997.. This sensitivity of muscle protein synthesis has also been used to test in vitro the influence of feeding conditions on fish growth ŽLied et al., 1983; Rosenlund et al., 1983.. Muscle is the most representative tissue of growth when protein synthesis and deposition are considered and, at the same time, it has a low protein turnover in fish ŽMcMillan and Houlihan, 1989; Peragon ´ et al., 1994., as observed in higher vertebrates ŽWaterlow et al., 1978.. Consequently, muscle should show pronounced changes of protein synthesis rate in response to dietary protein quality. On the contrary, protein turnover rates of regulatory tissues such as liver are much higher than of muscle ŽMcMillan and Houlihan, 1989; Peragon ´ et al., 1994. and hence a large proportion of dietary protein must be used to ensure regulatory functions. According to the concept of anabolic drive, diet is required not only to provide substrates for maintenance and growth but to exert a regulatory influence on the organism ŽMillward, 1989.. The present paper investigates the influence of dietary protein quality, using only animal and plant protein, as well as dietary protein supplementation with EAAs, on protein synthesis and degradation rates in the liver and white muscle of the European eel. The phenylalanine free pool-flooding method ŽGarlick et al., 1980. was used to measure protein synthesis rates in vivo. In addition, the relationship between turnover rates and growth rate and tissue RNA and DNA contents were also investigated.

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2. Material and methods 2.1. Animals and maintenance The eels Ž Anguilla anguilla. used in this experiment were purchased from a fish farm ŽValenciana de Acuicultura.. In the laboratory, they were divided in 12 groups, each kept in a fibreglass tank with 385 l of dechlorinated tap water. The mean initial weight of the eels was 56 g, and the number of eels in each tank was adjusted to represent a total biomass of 2.5–3 kg. Water was continuously renewed at a rate of 15% hy1 , adequately aerated Žoxygen content never below 7 mg ly1 . and maintained at 25 " 18C. Throughout the experiment the photoperiod used was 12-h lightr12-h dark Žlight: 0800–2000.. The experimental period lasted 12 weeks, preceded by an acclimation period of 1 month. 2.2. Experimental diets Four different experimental diets were tested, each formulated to have similar amounts of gross energy Žabout 18.5 MJ kgy1 , estimated by using the caloric contents proposed by Brafield and Llewellyn Ž1982., for the different sources., carbohydrates, fat and total nitrogen, but different in the nature of the protein component. A diet control ŽCONT. contained fish meal ŽFM. as the only protein source. In diets MM and SFM, FM was fully replaced by meat meal ŽMM. or sunflower meal ŽSFM., respectively. Finally, SFMq AA was formulated the same as SFM but was supplemented with some EAA Žlysine, methionine, histidine, threonine. ŽTable 1.. The amount of EAA added to the SFMq AA diet was calculated from the SFM composition Žprovided by the manufacturer. and the EAA requirements established for the Japanese eel ŽArai, 1986.. Diets were extruded to produce pellets Ž2 mm in diameter= 4 mm long.. Table 1 shows the final composition of the diets and Table 2 presents the AA content. To compare the overall AA profile of the different diets, we calculated the percentage of EAA and the EAA index ŽEAAI.. The EAAI is defined as the geometrical mean of the ratios Ž%. of each EAA in the experimental source and a reference. Two different references were considered: trout egg composition ŽKetola, 1982. and the EAA requirements of the Japanese eel as determined by Arai Ž1986.. Each experimental diet was provided to three groups of eels, twice a day Ž1000 and 1700. to satiation. Fish were offered only a few pellets at a time until they no longer showed any signs of appetite. Uneaten pellets were removed, dried and weighed for a precise measurement of feed intake. 2.3. Measurements Daily food intake was recorded and overall weight increase of the animals was determined by individual weighing Žafter sedation with a solution of 0.1 ml ly1 of ethilenglicol–monophenil–ether. at the beginning and the end of the experimental period. Additionally, every 2 weeks, the entire group contained in a tank was weighed to verify the steadiness of the growth rate throughout the experiment. The following

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Table 1 Composition and biochemical analysis of the experimental diets Diets CONT

MM

Ingredients (g d.m.a 100 g diet d.m.y 1) White fish meal 35.90 Meat meal Sunflower meal Essential amino acids b Fish oil 3.79 Corn oil 4.25 Linseed oil 2.37 Manioc meal 37.10 Vitamin pre-mix c 2.00 Mineral pre-mix c 5.00 Chromium oxide 0.50 Sodium alginate 1.00 Cellulose Žmicronized. 7.29 Betaine 0.80

1.00 0.50 2.37 37.10 2.00 5.00 0.50 1.00 0.13 0.80

Component Moisture Ž%. Protein ŽPercent d.m.. Fat ŽPercent d.m.. Ash ŽPercent d.m..

8.94 30.40 12.02 18.59

8.13 29.02 11.80 12.64

SFM

SFMqAA

68.00 7.10 2.73 2.37 10.00 2.00 5.00 0.50 1.00 0.50 0.80

64.00 1.60 7.10 2.80 2.37 11.30 2.00 5.00 0.50 1.00 1.53 0.80

8.56 28.81 13.74 9.97

8.15 28.51 14.41 9.85

49.60

a

Dry matter. 0.65 g Lysine, 0.45 g methionine, 0.3 g histidine, 0.2 g threonine. c According to de la Higuera et al. Ž1989.. b

indices were determined: Specific growth rate ŽSGR.: Žlog e final weight Žg. y log e initial weight Žg. = 100 number of daysy1 .; Feed efficiency ŽFE.: weight increase Žg.rfood intake Žg dry matter.. 2.4. Diet analysis Standard AOAC Ž1984. methods were used to determine moisture, protein, fat and ash content; AA content of the different diets was determined by ionic-exchange chromatography ŽChromakon 500, KONTRON. and by using an AA standard ŽSigma.. 2.5. Protein turnoÕer rate measurement Six animals of each treatment were selected for this measurement. The selected eels had an individual weight close Ž"3 g. to the mean weight of the respective whole lot. Liver and white muscle protein accretion Ž k g . were calculated as the percentage of protein increase per day, using the following equation: k g Ž Percent dayy1 . s 100 Ž log e P2 y log e P1 . Ž t 2 y t 1 .

y1

,

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Table 2 Amino acid composition ŽPercent protein. of the experimental diets. EAA: Essential amino acids; EAAI: Essential amino acids index: Ž1. Reference Japanese eel requirements ŽArai, 1986.; Ž2. Reference trout egg ŽKetola, 1982. Amino acid

Aspartic acid Threonine Serine Glutamic acid Hydroxyproline Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Percent EAA EAAI Ž1. EAAI Ž2. Vs. Control diet Ž1.

Diets CONT

MM

SFM

SFMqAA

10.20 4.88 4.65 17.20 0.00 3.87 6.11 5.96 5.27 1.11 2.60 4.90 8.81 3.11 4.78 8.01 2.53 5.99 47.78 120.41 97.01 1.00

7.63 3.27 3.91 13.29 7.27 10.68 13.80 7.36 4.02 0.91 1.00 2.86 5.74 2.23 3.28 4.71 1.61 6.43 32.92 78.20 63.00 0.65

10.39 4.20 4.83 22.29 0.00 3.60 6.41 4.89 5.39 2.32 1.20 4.46 7.23 2.61 5.11 4.38 2.67 8.02 42.65 103.23 83.17 0.86

9.67 4.66 4.74 22.11 0.00 4.79 5.93 4.27 5.18 2.16 1.78 4.32 6.70 2.42 5.01 5.30 3.23 7.74 43.91 111.25 89.63 0.92

where P1 and P2 represent total tissue protein content at times t 1 and t 2 , respectively. Tissue protein concentration was determined according to Lowry et al. Ž1951. in samples of both tissues taken 2 weeks before and at the end of the experiment Ž n s 6 per dietary treatment.. With the data obtained from periodic weighing during the experiment, we verified the uniformity of growth rate and this, together with the fact that previous studies in our laboratory have revealed that for eels of these ages and weights, the liver weight represents a constant proportion of the total body weight Ž P - 0.001., indicate that the weight increase during the last 2 weeks could be considered as a true indication for the k g calculation. The fractional protein synthesis rate was determined by the method described by Garlick et al. Ž1980. as modified by Peragon ´ et al. Ž1992.. The caudal vein injection solution contained 135 mM L-phenylalanine and L-w2-63 Hxphenylalanine ŽAmersham International. at 37.0 MBq mly1 Ž100 mCi mly1 . and a specific radioactivity of 1640 dpm nmoly1 . The dosage, 50 mCi 100 gy1 body weight at a volume per dosage of 0.5 ml 100 gy1 body weight — taking into account a blood volume of 4 to 10% ŽJones and Randall, 1978. — represents 5 to 12.5% of the blood volume. Eels were killed by a blow on the head at 2 and 45 min after the injection. Immediately afterwards, a median ventral incision was made to remove visceral mass,

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after cutting from the esophagus to anus, and the liver was separated. White muscle was dissected on ice, within 10 min, by removal from one side along the backbone. Both entire fillets were separated and weighed, and their protein content determined. The liver and muscle protein increase was calculated in each fish by substracting the average initial values Žof a reference group. from the final protein content. A sample from the liver and white muscle in the anterior dorsal region were extracted in the cold and then freeze-clamped in liquid nitrogen. Tissues were homogenized Ž1:10 wrv. and proteins precipitated with 2% HClO4 Žvrv. in ice and separated into two equal fractions: one to determine the protein synthesis rate and the other to quantify the DNA and RNA contents. After centrifugation at 2800 = g Ž rav s 8.94 cm. at 48C for 15 min, the insoluble protein fraction was washed twice with 96% ethanol and once with ether, and left overnight in 2 M NaOH at 378C. The protein fraction was then hydrolysed in 6 M HCl for 24 h at 1108C. HCl was removed by evaporating to dryness and the remaining AAs were resuspended in saturated sodium citrate, pH 6.3. The final concentrations of total phenylalanine and L-w2,43 Hxphenylalanine incorporated into soluble Ž SA . and protein Ž SB . fractions were calculated after the conversion of phenylalanine into b-phenylethylamine ŽPEA.. The PEA concentrations were determined by spectrofluorescence vs. a standard curve of 0–15 nmol of PEA ŽSuzuki and Yagi, 1976.. The experimental results were expressed as SA and SB Ždpm nmoly1 .. The fractional protein synthesis rate, k s Žpercent protein synthesis per day. was calculated as: ks s

Ž SB t

2

y SB t 1 . SAy1 100 Ž t 2 y t 1 . Ž t 2 yt 1 .

y1

,

where S B t 1 and S B t 2 are the protein bound specific radioactivity at 2 and 45 min, respectively, of experimental time after injection; SA Ž t 2yt 1 . is the average free pool of specific radioactivity over the period Ž t 2 y t 1 .. The fractional protein degradation rates Ž k d . were calculated as the difference between the protein synthesis Ž k s . and accretion Ž k g . rates. The efficiency of protein deposition ŽPDE. was calculated as being the ratio of the protein synthesized vs. that . retained as growth Ž k g ky1 s = 100 . 2.6. Determination of liÕer and white muscle DNA and RNA concentrations DNA and RNA were separated, purified and quantified by the method recommended by Munro and Fleck Ž1966.. Total DNA and RNA contents were extracted according to the procedure of Schmidt and Thannhauser Ž1945. as modified by Munro and Fleck Ž1966.. The pellet was washed twice with 0.2 M HClO4 to remove low molecular weight contaminants Žnucleotides, coenzymes, inorganic phosphate and phosphorus.. The RNA and DNA fractions were separated by digestion in alkaline medium Ž0.3 M KOH. at 378C for 1 h followed by acidification in 1.2 M HClO4 . The supernatant was diluted to 0.1 M HClO4 and the RNA concentration measured 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 a DNA standard.

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As protein synthesis depends largely on RNA concentrations, protein synthesis capacity Ž Cs . is defined as RNA: protein ratio and expressed as mg gy1 . Similarly, the efficiency of protein synthesis Ž k RN A . is defined as protein synthesized per day Žg. and RNA unit Žg. and is calculated as Ž10 k s Csy1 .. k DNA represents protein synthesis rate: DNA-unit ratio, the two terms of the ratio being expressed in g and calculated: Ž k sr100. multiplied by Žprotein DNAy1 .. 2.7. Statistical methods One-way ANOVA and least significant difference Ž P - 0.05. tests were employed for determining the significance of dietary treatments on variables recorded.

3. Results Results of food intake, growth and feed efficiency are shown in Table 3. Diets formulated with MM or unsupplemented SFM as the only protein source were clearly less well accepted. Food intake of SFM diet supplemented with EAAs ŽSFM q AA. was significantly higher reaching control values. Growth rate and feed efficiency were significantly lower in fish fed the MM diet. AA supplementation of sunflower protein diet increased growth rate by twofold, closely approaching control values. The influence of dietary protein quality on indices of protein turnover of eel liver are shown in Table 4. A significant decrease of liver RNA concentration, as well as a lower RNA:DNA ratio, of fish fed on the MM diet was observed. When liver protein turnover rates and efficiency were considered ŽTable 4., significant differences were detected in response to diets of different protein quality. Liver growth rates changed proportionally to body growth rates and thus the hepatosomatic index was not altered. Lower growth indices were found for diets containing meat or SFM as the only protein source, especially for the MM diet. AAs added to the sunflower protein diet boosted fish growth to control values. Liver protein turnover rates significantly increased in fish fed on diets of a low protein quality Žmeat and unsupplemented sunflower protein diets., increasing both protein synthesis Ž k s . and degradation Ž k d . rates in a similar manner. In all cases, liver k s and k d values were higher than those obtained in the white muscle ŽTable 5., with tissue differences being higher when k d was considered. The capacity of protein synthesis Ž Cs s mg RNArg protein. was the lowest in the liver of fish fed on the MM

Table 3 Main indices of the utilization of the different experimental diets by eels Diets Food intake Žg 100 g fishy1 dayy1 . SGR ŽPercent dayy1 . Feed Efficiency a,b,c

CONT

MM

SFM

SFMqAA

1.47"0.14 a,b 0.39"0.12 a 0.27"0.08 a

1.01"0.07 b 0.04"0.01b 0.06"0.02 b

0.90"0.09 b 0.18"0.04 a,b 0.23"0.04 a

1.45"0.01a 0.35"0.09 a 0.30"0.08 a

Values in the same row with different superscripts are significantly different Ž P - 0.05..

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Table 4 Influence of dietary treatment on liver protein turnover of eels Diets CONT kg ks kd Cs k DN A k RN A PDE wRNAx wDNAx Total DNA ProtrDNA RNArDNA

MM

SFM

a

0.08"0.01 10.16"1.75a 10.08"1.75a 11.68"1.09 b 26.16"6.71a 9.38"2.00 a 1.21"0.21c

3.65"0.95a 0.82"0.16 0.96"0.26 292.05"44.87 4.37"0.42 a

2.34"0.22 b 0.88"0.13 0.76"0.11 262"33.09 2.77"0.21b

0.40"0.13 6.22"1.32 a,b 5.8"1.32 a,b 17.27"1.62 a 13.54"3.32 a 3.81"1.34 b 11.54"6.46 a

c

SFMqAA b

0.35"0.09 a 4.26"0.95 b 3.9"0.95 b 15.28"0.67 a 11.90"3.20 b 2.92"0.73 b 9.56"2.15a

3.57"0.28 a 0.82"0.08 0.68"0.08 303.33"32.73 4.41"0.25a

3.06"0.13 a 0.77"0.05 0.75"0.07 293.2"19.89 3.98"0.11a

0.15" 0.04 9.86"1.97 a 9.69"1.97 a 17.82"1.42 a 27.63"7.79 a 6.17"1.80 a,b 2.05"0.49 b

Abbreviations used: k s , protein synthesis rate; k d , protein degradation rate; k g , protein accumulation rate; C s , protein synthesis capacity: mg RNArg protein; k DN A ,: Ž k s r100.=ŽProt.rDNA., g protein synthesizedrdayrg DNA; k RN A , protein synthesis efficiency: Ž k s r Cs .=10, g protein synthesizedrdayrg RNA; PDE, protein deposition efficiency: Ž k g r k s .=100. Results are mean"S.E.M. of six fish. Significant differences Ž P - 0.05. among diets are indicated by superscripts.

diet. Protein synthesis per cell unit Ž k DN A : g protein synthesized dayy1 rg DNA. and the protein synthesis efficiency Ž k RN A : g protein synthesized dayy1 rg RNA. for diets containing unsupplemented protein sources were at least twice of those obtained for Table 5 Influence of dietary treatment on white muscle protein turnover rates of eels Diets CONT kg ks kd Cs k DN A k RN A PDE wRNAx wDNAx Total DNA ProtrDNA RNArDNA

0.42"0.11a 0.49"0.01a 0.07"0.01 1.21"0.05 c 4.27"2.22 2.92"0.25a 85.8"0.16 a 0.31"0.01 0.1"0.01 6.89"0.01a 2812.85"350.2 a 3.1"0.16

MM 0.08"0.01c 0.14"0.03 c 0.06"0.03 1.38"0.28 b,c 2.63"1.02 1.01"0.22 b 66.05"12.47 b 0.38"0.11 0.11"0.01 4.7"0.23 b 2625.17"194.48 a 3.48"0.13

SFM 0.17"0.04 b 0.30"0.11b,c 0.13"0.11 1.49"0.08 b 8.88"5.58 2.01"0.85a,b 67.10"25.48 a,b 0.4"0.02 0.08"0.01 5.12"0.25 b 2828.54"127.18 a 4.17"0.16

SFMqAA 0.36"0.08 a 0.45"0.06 a,b 0.09"0.06 1.86"0.08 a 3.23"0.49 2.42"0.40 a 80.32"10.73 a,b 0.43"0.02 0.1"0.01 6.87"0.38 a 1933.74"89.97 b 4.81"0.79

Abbreviations used: k s , protein synthesis rate; k d , protein degradation rate; k g , protein accumulation rate; Cs , protein synthesis capacity: mgRNArg protein; k DN A : Ž k s r100.=ŽProt.rDNA., g protein synthesizedrdayrg DNA; k RN A , protein synthesis efficiency: Ž k s r Cs .=10, g protein synthesizedrdayrg RNA; PDE, protein deposition efficiency: Ž k g r k s .=100. Results are mean"S.E.M. of six fish. Significant differences Ž P - 0.05. among diets are indicated by superscripts.

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control and supplemented sunflower protein diets. Furthermore, high protein turnover rates were associated with low efficiency of protein deposition ŽPDE.. In all cases, liver Cs and k DNA were much higher in the liver with respect to white muscle in fish fed any of the experimental diets. k RN A was higher in the liver of fish fed low protein quality diets than in the white muscle of the same fish, while k RN A of fish fed on control and supplemented sunflower protein diets were similar in both tissues. In all cases, PDE was lower in the liver than in the white muscle. Results on protein turnover rates and related parameters measured in the white muscle of fish fed on the experimental diets are presented in Table 5. White muscle RNA and DNA concentrations were not significantly altered by dietary protein quality. The protein synthesis rate Ž k s . was directly related with the growth rate of fish fed on the experimental diets. Muscle k s of fish fed on the MM diet was the lowest, followed by that of fish fed on the unsupplemented sunflower protein diet. The supplementation of sunflower protein diet with EAA significantly increased the white muscle protein synthesis rate up to values obtained for fish fed on the control diet. Protein degradation rates Ž k d . were not affected by dietary regimen. A low efficiency of protein synthesis Ž k RN A . was detected in the muscle of fish fed on the MM diet. The efficiency of protein deposition ŽPDE. was in accordance with growth rates, the highest values being obtained in fish fed on control and sunflower supplemented diets.

4. Discussion Regardless of the dietary regimen, liver RNA concentrations were about 10 times higher than those in the white muscle. In this study, the direct relationship observed between tissue RNA, as well as RNA:protein ratio Ž Cs s capacity for synthesis., and mean protein synthesis rate, in the liver and white muscle of eel, is attributed to the fact that eukaryotic cell protein synthesis is a function of total ribosome number and activity ŽPain and Clemens, 1980.. In most cells, the majority of RNA is found in the ribosomal fraction, and in fish ŽLeipoldt et al., 1984. as in mammals ŽReeds, 1989., total tissue RNA concentrations have been used as a measure of protein synthesis capacity ŽMillward et al., 1981; Preedy et al., 1988.. Correlation between Cs and k s have also been observed in other fish species ŽMcMillan and Houlihan, 1988; Houlihan et al., 1989; Garzon, ´ 1995; Sierra, 1995.. In the present study, dietary protein quality did not have a clear influence on tissue nucleic acid contents. Body growth rate was related to the quality of the dietary protein source, being lower for fish fed diets containing meat or SFM as the only protein source. The sunflower diet supplemented with EAAs significantly improved growth, raising values almost to the control. The protein synthesis rate showed opposite behaviour in liver and muscle. Liver protein turnover rates Ž k s and k d . increased in response to a lower quality of dietary protein, while muscle protein turnover rates showed a direct relationship with dietary protein quality and growth. Different mechanisms of controlling protein metabolism seem to operate in liver and muscle tissue. With respect to the liver, the diets of lower protein quality stimulated protein degradation in a higher proportion than protein

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synthesis. From studies in mammals, it has been deduced that the stimulation of liver growth by means of feeding ŽBotbol and Scornik, 1985. and protein intake ŽBur and Conde, 1982. is achieved mainly by decreasing protein degradation, which was the main change observed in this study. On the other hand, white muscle response to a decreasing quality of dietary protein lowered the rates of protein synthesis Ž k s . and degradation Ž k d ., the decrease in k s being proportionally higher. In this sense, response to feeding in mammals ŽGarlick et al., 1983. is characterized by a k s stimulation, generally associated with a k d decrease ŽReeds and Palmer, 1986.. In the present study, k s of eel muscle increased with diets of better protein quality, although k d values remained unchanged. White muscle is the tissue most representative of growth, from the standpoint of protein turnover ŽHoulihan et al., 1988; Peragon, ´ 1993., being sensitive to dietary AA availability. Consequently, muscle is the tissue that shows the highest efficiency of protein deposition for growth ŽFauconneau and Arnal, 1985; Houlihan et al., 1988; Peragon, ´ 1993, Peragon ´ et al., 1994.. A decrease in white muscle k s has been reported in trout ŽGarzon, ´ 1995., gilthead sea bream ŽSierra, 1995. in response to diets deficient in EAA. The limitation of protein synthesis by EAA deficiency is well known, probably by inhibition of 43s initiating complex formation, as demonstrated in the rat by Everson et al. Ž1989., and is reversible by the addition of absent AAŽs.. In the present study, protein quality correlated with growth indices, muscle k s and efficiency of protein . in the white muscle. On the contrary, synthesis Ž k RN A . and deposition ŽPDE: 100k g ky1 s liver showed an increased k s associated to a low protein deposition in response to meat and sunflower protein diets, emphasizing the regulating role of this tissue ŽReeds, 1989. in response to diets of low quality protein. The utilization of dietary protein for growth ultimately depends on the availability of an adequate pattern of AAs at the sites of protein synthesis. In this sense, dietary protein quality was found to be directly related with k s . Furthermore, the improvement of the protein quality of sunflower protein diet by supplementation with EAAs Žhistidine, lysine, methionine and threonine. increased the white muscle protein synthesis rate, over that obtained with unsupplemented SFM diet, attaining control values. Similarly, liver k s registered for the supplemented sunflower protein diet diminished to control values. In fish, an improvement of the lysine:arginine ratio in the diet was shown to stimulate white muscle k s in the rainbow trout ŽFauconneau, 1985.. In warmwater fish such as carp ŽGarzon, ´ 1995; de la Higuera et al., 1997. and gilthead sea bream ŽSierra, 1995., supplementation of a single AA deficient diet, respectively with lysine and methionine, significantly improved white muscle k s when the supplemented AA was coated with egg albumin and not in free form. Stimulation of muscle protein synthesis by supplementing the diet with lysine has also been demonstrated in mammals ŽSalter et al., 1990. The European eel proved to be able to utilize free dietary AA quite efficiently. In fact, most liver and muscle protein turnover parameters improved after the diet supplementation with free AAs. Liver and muscle showed, respectively, a regulating and growth adaptive response when improving the available dietary AA pattern. Studies have reported low growth rates in certain fish species when fed on diets containing free AAs ŽMurai et al., 1984; Walton et al., 1986; Coloso et al., 1988; Moon and Gatlin, 1989., the results being attributed to differences in AA absorption rates ŽCowey and Walton, 1988; Murai et al., 1987; Garzon, ´ 1995; Sierra, 1995.. In fact, dietary protein

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supplementation with coated AAs improved growth, by stimulating protein synthesis and deposition ŽGarzon, ´ 1995; Sierra, 1995; de la Higuera et al., 1997.. On the contrary, some fish species, such as salmonids, achieve good utilization for growth of the dietary free AAs ŽEspe and Lied, 1994.. Few studies have examined the influence of dietary protein source on protein synthesis and degradation rates in fish. Data obtained in our laboratory with trout and carp ŽGarzon, ´ 1995. and gilthead sea bream ŽSierra, 1995. showed increased k s and unchanged or decreased k d , respectively, in the muscle of these species on increasing dietary protein quality. Trout and gilthead sea bream liver k s and k d did not significantly change in response to dietary protein quality, whereas carp liver increased k s and k d at 188C and decreased k s and k d at 258C. Eel muscle k s and k d Žprovided that dietary protein source was supplemented with the limiting EAAs. showed the same behaviour as the forementioned species but eel liver showed a different response, increasing k s and k d for diets of a low protein quality as in the carp when maintained at 188C ŽGarzon, ´ 1995.. An increase of body protein synthesis and degradation has been reported in the European sea bass fed diets with low quality protein sources of unbalanced AA content ŽLangar et al., 1993; Langar and Guillaume, 1994., results that coincide with those found in the liver but not in the muscle of the European eel. An explanation might be that muscle contribution to body protein synthesis is 30–40% ŽFauconneau and Arnal, 1985; Houlihan et al., 1988.. Furthermore, protein turnover of different tissues is much higher than muscle; for example, in the present study, liver presents a mean of 53-fold higher k s and about 120-fold higher k d than those of muscle for meat and sunflower protein diets. McMillan and Houlihan Ž1988. observed differences of 50- to 100-fold between liver and muscle of trout. Consequently, we can reasonably deduce that body turnover response of sea bass to low quality protein diets ŽLangar et al., 1993; Langar and Guillaume, 1994. is the mean of tissues with markedly different turnover rates, so that body k s and k d would reflect the higher turnover rate of certain tissues such as the liver and the digestive tract. With that assumption, the results obtained for European eel liver would be comparable to those obtained for the whole body of the sea bass. The lower intake of fish fed on MM and sunflower protein diets could have also contributed to the results obtained. In that sense, a direct relationship between ration size and body k s and k d has been observed in the cod ŽHoulihan et al., 1989.. Similarly, a decreased protein and energy intake has been found to decrease the RNA content as well as the protein synthesis rate and efficiency in the muscle of cod Žvon der Decken and Lied, 1989., saithe ŽRosenlund et al., 1984. and rainbow trout ŽPeragon ´ et al., 1994.. White muscle protein synthesis rate Ž k s ., efficiency Ž k RNA . and deposition ŽPDE. showed a clear relationship to growth, indicating a higher sensitivity than liver to the influence of dietary protein quality on growth. White muscle appears to be the tissue with the highest proportion of synthesized protein that is retained as growth ŽHoulihan, 1991.. At the same time, liver showed a response related to its regulatory role associated with high protein turnover requirements Žincreased k s and k d . during periods of nutritional unbalance. The results of the present study emphasize the need for further research on fish protein metabolism, especially muscle protein turnover and growth, as a function of dietary protein quality and subsequent pattern of the EAAs available at the sites of protein synthesis.

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Acknowledgements This work was supported by a Grant of the Spanish Government ŽCICYT, MAR890420..

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