J Comp Physiol B (1992) 162:351-357

Journal of

Comparative

Systemic, Biochemical, and Environ-

Physiology B Physiology ~"'~'

9 Springer-Verlag 1992

Dietary protein levels affect growth and protein metabolism in trunk muscle of cod, Gadus morhua Alexandra vonder Decken 1. and Einar Lied 2 The Wenner-Gren Institute, Biology F3, University of Stockholm, S-106 91 Stockholm, Sweden 2 Nutrition Institute, N-5024 Bergen, Norway Accepted December 2, 1991

Summary. Cod (Gadus morhua) of 50 g body weight were

kept at 14 ~ The fish were fed ad libitum during 80 days a diet containing protein levels which in terms of total energy corresponded to 25%, 45% or 65%. Growth increased in accordance with protein-energy levels. The protein content per gram of wet weight of white trunk muscle was unchanged, as was the myofibrillar protein myosin heavy chain determined by the antigen-antibody reaction of the enzyme-linked immunosorbent assay. The amount of messenger ribonucleic acid (mRNA) coding for myosin heavy chain was lower at 25% than at 45% or 65 % protein-energy intake, the differences being significant per gram of wet weight of muscle. Acid proteinase activity was highest at the lowest protein-energy intake. Glycogen content in muscle increased with the proteinenergy levels. It is concluded that the metabolic response of white trunk muscle to graded protein-energy intake included a change in the capacity to synthesize myosin heavy chain as judged by its mRNA content. The protein content per gram of wet weight was unaffected by dietary protein-energy levels of 25%, 45% and 65%, but protein accretion and thus growth of the animals increased with the protein intake. Dietary protein-energy restriction caused a rise in acid proteinase activity and a decrease in content of mRNA for myosin heavy chain, resulting in a diminished growth rate at an unchanged protein content per gram of wet weight of muscle. Key words: Protein intake - W h i t e trunk muscle - Myo-

sin heavy chain - Growth - Fish, Gadus morhua

has considerable survival value and muscle is the tissue which is likely to respond to environmental and physiological changes by adaptation at the molecular level. The musculature is anatomically separated into slow-, intermediate- and fast-contracting muscle (Greer-Walker 1970). The white or fast-contracting fibres, form the bulk of the musculature, are larger in size than the red or slow contracting fibres, and their metabolism is anaerobic (Bone 1966). White muscle shows a high efficiency of retention of protein (Houlihan et al. 1988). Protein accretion or growth requires such levels of protein synthesis, which must exceed the need for maintenance metabolism of proteins (Millward et al. 1975). The synthesis of proteins in muscle responds to changes in feeding conditions (Lied et al. 1982; vonder Decken and Lied 1989). A decrease in the daily energy intake diminishes total RNA and protein content and total contractile protein myosin heavy chain ( v o n d e r Decken 1989) as determined by specific antigen-antibody reaction (Lied and von der Decken 1985). The capacity for myosin synthesis is lowered by a decrease in the myosin-specific mRNA relative to total RNA. This contributes to the decline in myosin concentration during restricted food energy supply (von der Decken 1989; von der Decken and Lied 1989). In the present study a graded protein level was fed to growing cod with food available ad libitum. The metabolic response was studied in skeletal muscle by relating the dietary protein content and change in growth rate to the capacity for protein synthesis, especially that of myosin heavy chain, and to the activity of protein degradation and glycogen accumulation in muscle.

Introduction

Muscle tissue contains the major part (about 60%) of the body proteins (Pfeffer 1982). For fish, swimming activity Abbreviations: CTP, cytidine triphosphate; DNA, desoxyribonucleic acid; EDTA, ethylenediaminetetra-acetic acid; mRNA, messenger ribonucleic acid; TRIS, tris(hydroxymethyl)aminomethane

* To whom offprint requests should be sent

Materials and methods Materials. All chemicals were of the highest purity available, sup-

plied by Sigma Chemical Co. (St. Louis, MO, USA) and Serva (Heidelberg, Germany). [3HldCTP (14.8 TBq-mmo1-1) and the nick translation kit to label DNA for the cDNA-mRNA hybridization experiments were obtained from Amersham International (Little Chalfont, England); the restriction enzyme AVAII was obtained

352

A.v.d. Decken and E. Lied: Protein nutrition and protein metabolism of cod

from Boehringer (Mannheim, Germany); the DEAE membrane from Schleicher and Schfill (Dassel, Germany).

Fish and diets. Cod (Gadus morhua) averaging 30 g were obtained from the Aquaculture Station, Austevoll (Directorate of Fisheries, Bergen, Norway), on the west coast of Norway. The feeding experiments were carried out in sheltered 350-1 aquaria supplied with running sea water maintained at 14 ~ and 35 rag. ml-1 salinity. Photoperiod was automatically regulated to 12 h light and 12 h dark. Fish were acclimated for 4 weeks to the experimental conditions. During the acclimatization period fish were fed daily ad libitum the diet containing 45% protein energy (Table 1). The feeding experiment was carried out with six groups, each containing 16 fish. Duplicate groups were fed to satiety once daily during an experimental period of 80 days (March 14-June 3) with isocaloric diets containing levels of protein corresponding to 25%, 45% and 65% of the total energy (Table 1). Food consumption per day is given in Table 2. The change in protein-energy content was balanced by energy from fat. The level of carbohydrate energy was kept constant at 15 % in all diets. The protein-energy content of the food was controlled by direct analysis. Protein (N x 6.25) was analysed by a modified Kjeldahl procedure (Crooke and Simpson 1971); lipids by gravimetry of the ethylacetate extract of the food; ash by gravimetry after ashing for 24 h at 660 ~ water by weight before and after drying for 24 h at 105 ~ and dry matter by gravimetry after drying. Carbohydrate content was the difference in weight between the sum of the above analytical results and the original weight of the food. At the end of the feeding experiment the fish were killed by a blow to the head and weighed. The epaxial muscle of the white type was dissected and slices of the muscle were wrapped in aluminium foil, immediately frozen between two solid blocks of CO2 and stored at - 8 0 ~ (Lund and von der Decken 1980).

Preparation of muscle homogenates and ribosomes. Muscle (0.5 g wet weight) was thawed and homogenized in 10 volumes of PBS-NaC1 (0.01 M Na-phosphate, 0.45 M NaC1, 0.1% Nonidet, pH 7). Part Table 1. Composition of diets (grams per kilogram wet weight a)

Ingredients

Protein-energy contentb 25%

Squid (Gonatisfabricii) mantlec Cod muscle meaP Capelin (Mallotus vilosus) oile Salmomix basisf Vitamin mixture~ Salt mixtureh Water

105 99 138 140 12 3 502

45% 201 189 88 140 12 3 366

65% 297 279 38 140 12 3 231

a The feed was processed into moist pellets of 5 mm diameter and 4--6 mm length b The values of 18.0, 33.5 and 12.5 kJ 9g-1 were used to calculate the digestible energy of protein, fat and carbohydrates, respecitvely (Brett and Groves 1979) c Lerry, Bergen, Norway d Toro, Bergen, Norway ~ Norsildmel, Bergen, Norway f Skretting, Starvanger, Norway g Composition of the vitamin mixture (mg vitamin per kg vitamin mixture): thiamin H C1 167; riboflavin 333; pyrodoxine 167; Capantothenate 667; niacin 2500; folic acid 83; ascorbic acid 667; retinyl palmitate (Vit A) 35; cholecalciferol (Vit D3) 3; ct-tocopherol acetate (Vit E) 20 000. Dextrin was used as the main constituent of the mixture h Commercial standard mineral mixture, used for poultry and swine, containing (g 9kg-1); phosphorus 60; calcium 240; sodium 60; magnesium 10; iron 2; manganese 2; zinc 2.5; copper 0.4; iodine 0.075; selenium 0.008

of the suspension was saved for determination of myosin heavy chain by ELISA (see below) and for analysis of DNA and protein. The remainder was centrifuged at 4 ~ for 10 min at 1200 x y (max). To the supernatant fraction Triton X-100 was added to a final concentration of 0.1% and ribosomes were recovered by centrifugation for 2 h at 165000 • (av.) as described previously (Lied et al. 1982). The ribosomes were suspended in PBS-NaCI for further analyses.

Immunoassay. An enzyme-linked immunosorbent assay (ELISA) was used to determine the content of myosin heavy chain in the whole homogenate and the ribosome fraction (Engvall and Perlmann 1972). The competitive inhibition test was applied. Primary antibodies against myosin heavy chain were added (Lied and von der Decken 1985; Persson et al. 1991). Anti-rabbit IgG antibodies conjugated with alkaline phosphatase were used as secondary antibodies, p-Nitrophenyl phosphate was added to develop the colour. Purified myosin heavy chain from cod was used as standard (Lied and von der Decken 1985).

Preparation ofpoly(A) + mRNA. To minimize RNase activity RNA was isolated under sterile conditions. All glassware and media were autoclaved and ribonucleoside vanadyl complex (1 raM) was included into the homogenization medium. Gloves were worn during these procedures. RNA was isolated from the 1200 x g supernatant fraction (vonder Decken and Lied 1989). Poly(A)+mRNA was isolated using the oligo d(T) method (Bantle et al. 1976). cDNA-mRNA dot hybridization. The relative level of the myosin specific mRNA was measured by dot hybridization using ZetaProbe membranes (Bio-Rad, Richmond CA, USA) and following the procedure described by Anderson and Young (1985). Transfer RNA was dotted separately and used for background subtraction. The DNA of the myosin heavy chain of Caenorhabditis elegans was used as the probe (Karn et al. 1983). The bacteriophage L-cloning vector, L 1059, contained the nematode une-54 myosin heavy-chain gene (Karn et al. 1980) and was a gift from L. Barnett. The bacteriophage DNA was digested with the restriction enzyme AVAII and electrophoresed in 0.6% agarose gel (Maniatis et al. 1982). Fragments of the size below 3500 nucleotides were collected electrophoretically directly from the agarose gel as described by Dretzen et al. (1980), using a DEAE membrane and following the supplier's manual (Schleicher and Schiill, Dassel, FRG). The membranes were washed in 0.15 M NaC1, 0.1 mM EDTA, 20 mM TRIS, pH 8. DNA was extracted in high ionic strength buffer (1.0 M NaC1, 0.1 mM EDTA, 20 mM TRIS, pH 8), precipitated with 2.5 volumes of ethanol, and reprecipitated from 0.3 M Na-acetate to remove any NaC1 residue. DNA was solubilized and radioactively labelled using the nick translation kit with [3H]dCTP as a precursor (Arrand 1985). The specific radioactivity was between 4. l06 and 9 ' l06 dpm per microgram of DNA. Hybridization was followed by washing (Meinkoth and Wahl 1984). The dots were punched out and the radioactivity was measured in a scintillation spectrometer. In some experiments a cDNA probe of the beta form of myosin heavy chain was used. It originated from rabbit cardiac ventricle and was a gift from R. Zak (Sinha et al. 1982; Friedman et al. 1984).

Northern blot analysis. Isolated RNA was electrophoresed in 2.2 M formaldehyde/0.8% agarose gels (Maniatis et al. 1982), transferred to membranes and hybridized with the DNA probes.

Analysis. Proteins of the total muscle homogenates and ribosomes were analysed by the Coomassie brilliant blue method (Bradford 1976) with bovine serum albumin used as standard. DNA of the total homogenate was analysed by the fluorescent method with salmon DNA as standard (Setaro and Morley 1976). RNA was analysed by extracting the ribosome fraction with 0.4 M HC104 for 18 min at 70 ~ The absorbance of the soluble fraction was measured at 260 nm and RNA calculated on the basis of 34.2 absorbance units per milligram of RNA.

A.v.d. Decken and E. Lied: Protein nutrition and protein metabolism of cod

Acid proteinase (EC 3.4.23.5) activity determination. The original method was used with slight modifications (Anson 1939; Mommsen et al. 1980). Muscle (0.25 g) was homogenized in 1 ml medium (50 m M imidazole, 50 m M KC1, 150 m M sucrose, 1 m M Na-EDTA, 0.2% Triton X-100, final pH 6.8). The homogenate was centrifuged at 4 ~ for 30 min at 15000 x g. The supernatant fraction was used for the enzyme assay. The incubation mixture contained 50 lal Na-acetate buffer (300 m M Na-acetate, 250 m M KC1, final pH 4.05), 50 lal of a 6% hemoglobin solution (previously dialysed against water adjusted to pH 4.05), and 50 lal of the enzyme solution. Final pH was 4.05. Appropriate blanks contained either the haemoglobin solution alone or the enzyme solution alone. A zerotime control was included. Incubation was at 37 ~ for 60 min and for 90 rain. The reaction was terminated by adding 75 lal 0.6 M trichloroacetic acid. The mixture was kept on ice for 30 min. After centrifugation for 5 rain at 5000 x O, 100 gl of the supernatant fraction was transferred to a new tube and diluted with water to a trichloroacetic acid concentration of 60 m m o l . 1-1. The solution was used for analysis of tyrosine as described by Ambrose (1974). Inhibition of acid proteinase activity with pepstatin was studied by adding pepstatin to the incubation mixture at a final concentration of 1 lag" m1-1 (Woessner 1972).

Glycogen determination. Glycogen content was analyzed in muscle as described by Harris et al. (1974). The glycogen content was expressed as glucose after enzymic degradation of glycogen with a-amylase and amyloglucosidase. Glucose was determined using the hexokinase-glucose-6-phosphate dehydrogenase-NADP system (Harris et al. 1974). Polyacrylamide 9el eleetrophoresis. Electrophoresis of proteins was performed in gradient gels of 7%-15% polyacrylamide containing 0.1% sodium dodecylsulphate, using a slab gel apparatus (Haines 1983). Statistical analysis. One-way analysis of variance and NewmanKeul's test for multiple sample comparison were applied for statistical evaluation (Snedecor and Cochran 1980).

Results Growth rates

Growth over 80 days was related to the level of dietary protein-energy supply (Table 2). Growth was expressed as percentage weight gain and daily growth rate. The lowest growth rate was at the 25% protein-energy level, and the 65% protein-energy content gave the highest growth rate. The differences were significant between the three feeding groups. Food intake per day as a percentage of body weight was similar for the groups fed 25 %

353

Table 2. Protein-energy dependent growth of cod

Protein-energy level

25 %

45 %

65 %

Initial body weight ~ (g)

48.7 _+3.2

51.4•

51.0+3.5

Final body weight a (g)

84..9 +_6. I*A

123.5•

*B 168.6• 11.9 *c

Average weight gain (%)

74..5

140.2

Specific growth rate b (% 9day-1) Food intake (dry matter as % of body weight- day- l)

0.70 0.57

230.8

1.10

1.50

0.55

0.49

a Mean values _+SEM, n = 32 b Specific growth rate (% per day)= _ In final body w e i g h t - In initial body weight 100 Experimental days (80) * P

0.5 r'r" :.:.:.:.:.:

:::::::::::-., .:.:.:.:.:...,

:.:.:.:.:.:..,

Protein-energy 1eve1

Tyrosine (l~g) released in 1 h per g of wet weight of muscle

25% 45% 65%

134_+ 8*a 120_ 7 *A,B 105+_ 11*B

a The results are mean values + SEM of seven fish per dietary group and two analyses per fish * P

Dietary protein levels affect growth and protein metabolism in trunk muscle of cod, Gadus morhua.

Cod (Gadus morhua) of 50 g body weight were kept at 14 degrees C. The fish were fed ad libitum during 80 days a diet containing protein levels which i...
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