Nutrient Metabolism

Porcine Skeletal Muscle Myofibrillar Protein Synthesis is Stimulated by Ractopamine1'2 OLAYIWOLA ADEOLA,3 RONALD O. BALL AND LESLIE G. YOUNG Department of Animal and Poultry Science, university of Guelph, Guelph, Ontario NIG 2W1, Canada

ABSTRACT Thirty-two 64-kg individually penned barrows were fed protein at 130 or 170 g/kg diet and ractopamine at 0 or 20 mg/kg in a randomized complete block design for 28 d. Fractional synthesis rates of myofibrillar,sarcoplasmic and connective tissue proteins in longissimus dorsi, biceps femoris and gastrocnemius muscles were examined by primed-continuous infusion of l_-[ring-2,6-3H(N)]phenylalanine over a 4-h period. Fractional synthesis, accretion and breakdown rates of protein in the muscles were not affected by dietary rac topamine, regardless of whether estimated on the basis of plasma or tissue homogenate specific activity. Ab solute rates of protein synthesis (P < 0.05) and breakdown (P < 0.1) in biceps femoris muscle were both elevated by ractopamine feeding at 170 g protein/kg diet. Also in the diet containing 170 g protein/kg, rac topamine increased (P < 0.05) protein contents of lon gissimus dorsi and biceps femoris muscles. Dietary rac topamine increased (P < 0.05) fractional rates of synthesis of myofibrillarbut not sarcoplasmic proteins in longissimus dorsi and biceps femoris muscles of pigs fed the 170 g protein/kg diet. This result demonstrates that ractopamine treatment results in a stimulation of myoflbrillar protein synthesis. J. Nutr. 122: 488-495, 1992. INDEXING KEY WORDS:

•beta-adrenerglc agonists •pigs •myofibrillar protein synthesis

The use of beta-adrenergic agonists to manipulate growth and body composition has received a great deal of attention in recent years (1-3). Several investi gations have demonstrated that the beta-adrenergic agonists ractopamine [in pigs (3, 4)], clenbuterol [in rats (5) and calves (6)] and cimaterol [in pigs (1) and lambs (2)]increase muscle mass and reduce fat deposi tion. Metabolic studies on the action of beta-agonists are few; for protein turnover at least, the evidence concerning their mode of action is contradictory. There are reports of either increases in protein syn thesis in rats injected with clenbuterol (7) or no effect on protein synthesis but a reduction in protein

breakdown in clenbuterol-fed rats and lambs (5, 8). Reports of investigations into the changes in protein turnover that could account for the increase in muscle mass associated with ractopamine feeding in pigs are few. Increased protein synthesis in response to ractopamine in cultured rat myotubes and porcine skeletal muscle alpha-actin were reported recently (9, 10). In an earlier study, Bergen et al. (11) observed an increase in fractional synthesis rate of protein in ractopamine-fed pigs. Furthermore, information on the in vivo synthesis rates of the different protein fractions in muscles of ractopamine-fed pigs is lacking. In a previous study (4), dietary protein concen tration was observed to alter growth and carcass per formance by increasing muscle mass in ractopaminefed pigs. The present study examines fractional syn thesis rates of various muscle protein fractions in ractopamine-fed pigs fed diets containing 130 or 170 g protein/kg diet.

MATERIALS AND METHODS Animals and diets. Thirty-two barrows from the University of Guelph's Arkell Swine Research Station were stratified by weight and within strata randomly

^e thankfully acknowledge the financial support provided by Ontario Ministry of Agriculture and Food, Natural Sciences and Engineering Research Council and ELANCO Division, Eli Lilly Canada Inc., London, ON. Financial support for Olayiwola Adeola's graduate study was generously provided by the Canadian Common wealth Scholarship and Fellowship Committee. ^Presented in part at the 81st Annual Meeting of the American Society of Animal Science, 1989, University of Kentucky, Lex ington, KY. [Adeola, O., Ball, R. O. & Young, L. G. (1989) Rac topamine stimulates porcine myofibrillar protein synthesis. J. Anim. Sci. 67 (suppl. 1): 191 (abs. 475)]. 3Current address: Department of Animal Sciences, Purdue Uni versity, West Lafayette, IN 47907.

0022-3166/92 $3.00 ©1992 American Institute of Nutrition. Received 28 February 1991. Accepted 26 August 1991. 488 Downloaded from https://academic.oup.com/jn/article-abstract/122/3/488/4754919 by Washington University, Law School Library user on 12 April 2018

PROTEIN TURNOVER

IN RACTOPAMINE-FED

assigned to four diets (Table 1). The diets were ar ranged factorially with dietary protein at 130 or 170 g/kg and dietary ractopamine at 0 or 20 mg/kg. The barrows were Yorkshire-Landrace crossbreeds with an average initial weight of 64.3 kg. The barrows were individually penned and maintained as described pre viously (12). Infusion procedures. On d 28, the pigs were fitted with polyvinyl chloride catheters (0.86 mm i.d. x 1.27 mm o.d.; Durai Plastics and Engineering; Durai, N.S.W., Australia) in veins of both ears. Catheters were connected to 500-mL sterile bottles containing radioisotopes and driven by Technicon proportioning pump (Technicon Instruments Corporation, Tarrytown, NY). L-[ring-2,6-3H(N)]Phenylalanine (New England Nuclear Products, E. I. Du Pont, Boston, MA) was diluted in physiological saline. Each pig received a primed (88.8 kBq/kg) continuous [88.8 kBq/(kg-h)] infusion of the radioactive phenylalanine for 4 h at 42 mL/h. Total radioactivity infused was 444 kBq/kg body wt. Blood was sampled every 5 min for the first 30 min and every 30 min thereafter from a second catheter to monitor plasma phenylalanine specific ac tivity over the 4-h infusion. Plasma from the blood samples was stored at -18°C until subsequent analysis. Immediately following the 4-h infusion of isotope and blood sampling, pigs were killed with a lethal dose of sodium pentobarbital injected via the sampling catheter. Samples of longissimus dorsi, biceps femoris and gastrocnemius muscles were ex cised, placed in Whirl-Pak bags and immersed in liquid nitrogen. The research protocol was approved by the Animal Care Committee of the University of Guelph. Plasma phenylalanine specific activity. Norleucine (250 nmol) was added to 1 mL of plasma as an in ternal standard and plasma was deproteinized with 4 ml, of acetone. The supernatant was separated from the protein precipitate by centrifugation (1800 x g), and amino acids in the supernatant were separated from other constituents by cation exchange chromatography as described by Adams (13). A slurry of Dowex 50W-8X, H+; 100-200 mesh (Bio-Rad Laborato ries, Mississauga, ON, Canada) was prepared, washed in 1.2 mol/L HC1 followed by distilled water and poured into glass wool-plugged pasteur pipettes to obtain -1 mL of wet resin bed. Supernatant from the deproteinized plasma was passed through the cation exchange resin, and two sequential 2-mL distilled water washes were added to the resin column. Phen ylalanine was eluted from the resin column with two 2-mL washes of 5.5 mol/L ammonium hydroxide. The ammonium hydroxide was reconstituted in 1 mL of distilled water. Radioactivity in a 0.5-mL aliquot was determined by liquid scintillation spectrometry fol lowing l h dark adaptation using Aquasol (New En gland Nuclear Products) as the scintillation cocktail. Counting efficiency was -31%. Phenylthiocarbamyl Downloaded from https://academic.oup.com/jn/article-abstract/122/3/488/4754919 by Washington University, Law School Library user on 12 April 2018

PIGS

489

derivative of phenylalanine in 0.2 mL of the recon stituted sample was prepared and separated by HPLC as described previously (12). Plasma phenylalanine specific activity was calculated as the ratio of radioac tivity to concentration of the phenylalanine. Muscle free and protein-bound phenylalanine spe cific activities. Muscle samples (2 g) were homoge nized in 5 mL of trifluoroacetic acid (50 mL/L methanol) in a Polytron homogenizer (Brinkmann Instruments, Rexdale, ON, Canada) and centrifuged at 13,200 x g for 10 min at 4°Cto separate the super natant from protein precipitate. The supernatant was applied to a cation exchange column as described above. The homogenization-centrifugation process was repeated twice, and the homogenate free phenyla lanine was eluted from the column and analyzed for specific activity as described above. The protein pre cipitate was dried under vacuum, and a 20-mg sample was hydrolyzed in 4 mol/L methanesulfonic acid (14) at IIO'C for 24 h. Protein hydrolysates were neu tralized with 3.5 mol/L NaOH and analyzed for phen ylalanine by HPLC. A portion (200 mg) of the dried protein precipitate was dissolved in NCS tissue solubilizer (Amersham Corp., Oakville, ON, Canada). Radioactivity was determined in the solubilized tissue following neutralization with acetic acid and bleaching in H2Û2 (300 mL/L). Specific activity of phenylalanine in the protein-bound portion of the muscle was calculated as described above. Preparation of muscle protein fractions. The scheme for the separation of muscle proteins into different protein fractions followed modifications of methods described by Goldberg (15) and Rikimaru et al. (16). The accuracy of this protein separation pro cedure was previously established (16). Following de velopment and standardization of the procedure, myofibrillar and sarcoplasmic proteins accounted for 55-60% and 30-35%, respectively, of the total pro tein. Muscles were homogenized in 15 mL of cold 0.01 mol/L phosphate buffer (pH 7.4) in a Polytron homogenizer. The homogenate was centrifuged (13,200 x g) for 20 min at 4°Cto separate the soluble fraction (sarcoplasmic and intracellular proteins) from the residue (myofibrillar and connective tissue pro teins). The homogenization-centrifugation procedure was repeated twice. The soluble fraction was mixed with 3 ml, of trichloroacetic acid (200 g/L) and centri fuged as described. The supernatant was analyzed for phenylalanine specific activity as described above and 4 mol/L methanesulfonic acid hydrolysate of the residue was used in determining sarcoplasmic protein-bound phenylalanine specific activity. The phosphate buffer precipitable protein was mixed with 15 mL of 0.3 mol/L NaOH, allowed to stand overnight and centrifuged for separation into connective tissue and myofibrillar proteins. The con nective tissue pellet was mixed with NaOH and reextracted. These protein fractions were hydrolyzed in 4 mol/L methanesulfonic acid, neutralized with 3.5

490

ADEOLA ET AL.

TABLE 1 Ingredient

and nutrient

composition

of diets

Dietary protein 130 g/kg

170 g/kg Dietary ractopamine

0 mg/kg

20 mg/kg

0 mg/kg

20 mg/kg

g/kg

IngredientsCornSoybean mealCalcium phosphate(18.5% P)Limestone Ca, 20.5% Ca)Salt1Vitamin-mineral (38% premix^Ractopamine3Nutrients4Dry

matterProteinEther

extractAshCalciumPhosphorus89075.5131056.5—870.6127.827.537.97.95.888975.5131056.51866.2126.831.837.875.9788.5177131056.5868.317127.442.27

Cobalt-iodized salt with 40 mg cobalt/kg, 70 mg iodine/kg and 990 g sodium chloride/kg. 2Vitamin-mineral premix supplied the following (per kg of diet): 1.14 mg vitamin A, 17.5 (ig vitamin 03, 13.5 mg vitamin E (d-otocopherol), 2 mg menadione sodium bisulfite, 4 mg riboflavin, 20 mg niacin, 10 mg D-calcium pantothenate, 20 pigvitamin B12, 100 mg choline chloride, 60 mg manganese, 70 mg iron, 10 mg copper, 100 mg zinc, 100 |ig selenium. 3Ractopamine, generously donated by BLANCO Division, Eli Lilly Canada Inc. (London, ON), contained 20 g ractopamine/kg premix. 4Nutrient analysis on as fed basis.

mol/L NaOH and analyzed by HPLC and liquid scin tillation spectrometry to determine the specific ac tivity of phenylalanine in connective tissue and myofibrillar proteins. Calculations. The specific activity in the four plasma samples taken at 30-min intervals during the last 2 h of infusion was averaged and used as the phenylalanine specific activity at plateau, because the slope of a line calculated from these points was not different from zero. Phenylalanine accounted for 93 and 99% of total radioactivity present in plasma and muscle, respectively (21). Phenylalanine flux (mmol/ d) was calculated as the ratio of the radioisotope infusion rate to the phenylalanine specific activity at plateau. Whole body protein synthesis rate (g/d) was estimated as flux x 165.19/34, where flux is in mmol/ d, 165.19 is the molecular weight of phenylalanine, and phenylalanine content of whole pig carcass protein was assumed to be 34 mg/g protein (18). Frac tional synthesis rate (%/d) was calculated according to Smith et al. (19) as follows: fractional synthesis rate = (Sß/Sp) x (2400/t), where 5Bis the protein-bound phenylalanine specific activity, Sp is the specific ac tivity of free phenylalanine in plasma or tissue homogenate and t is 4 h. Daily accretion rate of protein in muscles was determined as described in Downloaded from https://academic.oup.com/jn/article-abstract/122/3/488/4754919 by Washington University, Law School Library user on 12 April 2018

detail elsewhere (4). The entire longissimus dorsi, biceps femoris and gastrocnemius muscles from an initial slaughter group of six pigs and pigs from each of the four treatments (six pigs/treatment) were ex cised, weighed and analyzed for protein as described previously (4). Daily protein accretion rate (g/d) was calculated as follows: (total muscle protein at d 29 total muscle protein of an initial slaughter group)/29. Fractional accretion rate of muscle protein (%/d) was calculated as daily accretion rate divided by total muscle protein at d 29. Fractional breakdown rate of protein (%/d) was determined as the difference be tween fractional synthesis (based on homogenate spe cific activity) and fractional accretion rates (20). Statistical methods. Data were analyzed as a 2 x 2 factorial experiment in a randomized complete block design. Linear regression was used to validate plateau values during individual infusions. Treatment means were separated using protected least significant dif ference (21).

RESULTS Plasma specific activity of phenylalanine reached a plateau within 30 min of priming dose administration

PROTEIN TURNOVER

ESTRACTOPAMINE-FED

and the start of infusion, and this plateau was main tained throughout the 4-h continuous infusion for the four dietary treatments. In all three muscles, there was a tendency for depression of fractional synthesis rate (based on homogenate specific activity) when pigs were fed the 130 g protein/kg diet with 20 mg ractopamine/kg. In pigs fed the 170 g protein/kg diets, there was a tendency for ractopamine to increase the fractional synthesis rate of protein in longissimus dorsi, biceps femoris and gastrocnemius muscles (Table 2). Similar relationships among treatments were observed when fractional synthesis rates of protein in longissimus dorsi, biceps femoris and gas trocnemius muscles were calculated from plasma spe cific activity. The fractional rates of protein synthesis calculated on the basis of plasma free phenylalanine specific activity (data not shown), which represent the minimum estimate of fractional synthesis rate, were 72 to 98% of the corresponding estimates of fractional synthesis rates based on tissue homogenate free phen ylalanine specific activity. Fractional accretion and breakdown rates (%/d) of proteins in longissimus dorsi, biceps femoris and gastrocnemius muscles showed no detectable differences in either of the vari ables in any muscle among treatments (Table 2). Rac topamine feeding resulted in a higher (P < 0.05) protein content in longissimus dorsi and biceps femoris muscles in pigs that received the 170 g protein/kg diets (Table 2). Phenylalanine flux tended to be increased by rac topamine (but not significantly at P < 0.05) in pigs fed the 170 g protein/kg diets (Table 3). Phenylalanine flux is its flow out of the free amino acid pool of the animal and represents the total amount entering the pathways of protein synthesis, oxidation and con version to other metabolites. If metabolic pathways of phenylalanine other than protein synthesis are dis regarded, the flux is an estimate of the amount of phenylalanine entering protein at a given time (22). From the average phenylalanine content of pig body proteins, it was possible to calculate the amount of protein synthesized in the whole body,- this was in excess of 700 g/d (Table 3). Whole body protein syn thesis (g/d) across treatments followed a pattern similar to that of phenylalanine flux. Absolute rates of protein synthesis and breakdown (Table 3) were obtained from the product of fractional synthesis rate and muscle protein content. Protein synthesis rate was higher (P < 0.05) in the biceps femoris muscle in ractopamine-fed than control pigs fed the 170 g protein/kg diet. Absolute rates of protein breakdown tended to be elevated (P < 0.1) in pigs fed the 170 g protein/kg diet with ractopamine. Figure 1 depicts fractional synthesis rate of myofibrillar, sarcoplasmic and connective tissue proteins in longissimus dorsi muscle of pigs that received the four experimental diets. The fractional rates of myofibrillar protein synthesis were higher (P < 0.05) in Downloaded from https://academic.oup.com/jn/article-abstract/122/3/488/4754919 longissimus dorsi muscles of ractopamine-fed pigs fed by Washington University, Law School Library user on 12 April 2018

PIGS

ffî CONNECTIVE

DIETARY PROTEIN: HACTOPAMINE :

N:

TISSUE PROTEINS

[g]

SARCOPLASMIC

[""]

MYOFIBRILLAR

13%

491

PROTEINS PROTEINS

13% 20 ppm 565

17% 0 ppm

17% 20 ppm

FIGURE 1 Fractional synthesis rates of myofibrillar (SD 0.49), sarcoplasmic (SD - 1.36) and connective tissue (SD 0.22) proteins in longissimus dorsi muscle of ractopaminefed pigs. Values are least squares means from two-way ANOVA; SD are represented by the vertical bars.

the 170 g protein/kg diet than in longissimus dorsi muscles of pigs not receiving ractopamine (4.7 vs. 3.19 %/d). Ractopamine did not alter fractional rates of myofibrillar protein synthesis in pigs fed the 130 g protein/kg diets (3.06 vs. 2.73 %/d). Sarcoplasmic protein synthesis tended to be reduced with rac topamine feeding regardless of the level of dietary protein (Fig. 1). Ractopamine reduced fractional rates of sarcoplasmic protein synthesis by 36% (2.27 vs. 1.45 %/d) and 37% (3.01 vs. 1.89 %/d) in pigs fed the 130 and 170 g protein/kg diets, respectively. Con nective tissue protein synthesis rate was similar at 130 (0.93 vs. 0.91 %/d) and 170 (0.90 vs. 0.96 %/d) g protein/kg levels in pigs fed ractopamine at 20 vs. 0 mg/kg (Fig. 1). Fractional rates of myofibrillar protein synthesis in biceps femoris muscle (Fig. 2) followed a trend similar to that of longissimus dorsi muscle. Ractopamine (20 vs. 0 mg/kg) increased the fractional rates of myofi brillar protein synthesis in pigs fed the 170 g protein/ kg diets (3.27 vs. 1.94 %/d) but had no effect in pigs fed the 130 g protein/kg diets (2.19 vs. 2.02 %/d). Harmonious with the observation in longissimus dorsi muscle, the fractional rates of sarcoplasmic protein synthesis in biceps femoris muscle tended to be depressed with ractopamine feeding (20 vs. 0 mg/ kg) at 130 g protein/kg (1.04 vs. 2.36 %/d) and 170 g protein/kg (2.01 vs. 3.22 %/d). hi pigs fed the 130 g protein/kg diets, fractional rates of connective tissue protein synthesis were 0.53 and 0.66 %/d in biceps femoris muscle of pigs that consumed 20 and 0 mg ractopamine kg/diet, respectively; corresponding figures for pigs fed the 170 g protein/kg diets were 0.82 and 0.55 %/d (Fig. 2). The fractional rates of synthesis of protein frac tions in gastrocnemius muscle were not significantly affected by dietary treatments (Fig. 3). In pigs fed the 130 g protein/kg diets, the fractional rates of myofi brillar protein synthesis were 3.72 and 3.79 %/d for

492

ADEOLA ET AL.

TABLE 2 Interaction

between

dietary protein level and ractopamine on fractional rates of protein synthesis, and protein content in pig muscle

breakdown

and accretion (%/d);

Dietary protein 130 g/kg

170 g/kg Dietary ractopamine

0 mg/kg

20 mg/kg

20 mg/kg

0 mg/kg

SD

Fractional %/dLongissimus synthesis rate, dorsiBiceps femorisGastrocncmiusFractional

%/dLongissimus Accretion Rate, dorsiBiceps femorisGastrocnemiusaFractional

%/dLongissimus Breakdown Rate, dorsiBiceps femorisGastrocncmiusMuscle

gLongissimus protein, dorsiBiceps femorisGastrocnemiusn5.214.89X76.361.011.250.8964.213.635.47387"241»8564.973.93X5.671.411.361.0853.552.574.58416ab247*9165.794.71*76.521.0

Values are least squares means from two-way ANOVA. a

Porcine skeletal muscle myofibrillar protein synthesis is stimulated by ractopamine.

Thirty-two 64-kg individually penned barrows were fed protein at 130 or 170 g/kg diet and ractopamine at 0 or 20 mg/kg in a randomized complete block ...
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