Somatotropin increases protein balance by lowering body protein degradation in fed, growing pigs

Rhonda C. Vann1, Hanh V. Nguyen1, Peter J. Reeds1, Douglas G. Burrin1, Marta L. Fiorotto1, Norman C. Steele2, Daniel R. Deaver3, and Teresa A. Davis1

1 United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030; 2 United States Department of Agriculture/Agricultural Research Service Growth Biology Laboratory, Beltsville, Maryland 20705; and 3 Department of Animal and Dairy Science, Pennsylvania State University, University Park, Pennsylvania 16802


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Somatotropin (ST) administration enhances protein deposition in well-nourished, growing animals. To determine whether the anabolic effect is due to an increase in protein synthesis or a decrease in proteolysis, pair-fed, weight-matched (~20 kg) growing swine were treated with porcine ST (150 µg · kg-1 · day-1, n = 6) or diluent (n = 6) for 7 days. Whole body leucine appearance (Ra), nonoxidative leucine disposal (NOLD), urea production, and leucine oxidation, as well as tissue protein synthesis (Ks), were determined in the fed steady state using primed continuous infusions of [13C]leucine, [13C]bicarbonate, and [15N2]urea. ST treatment increased the efficiency with which the diet was used for growth. ST treatment also increased plasma insulin-like growth factor I (+100%) and insulin (+125%) concentrations and decreased plasma urea nitrogen concentrations (-53%). ST-treated pigs had lower leucine Ra (-33%), leucine oxidation (-63%), and urea production (-70%). However, ST treatment altered neither NOLD nor Ks in the longissimus dorsi, semitendinosus, or gastrocnemius muscles, liver, or jejunum. The results suggest that in the fed state, ST treatment of growing swine increases protein deposition primarily through a suppression of protein degradation and amino acid catabolism rather than a stimulation of protein synthesis.

protein synthesis; insulin-like growth factor I; insulin; growth hormone; muscle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE QUEST FOR INCREASED LEAN muscle production has been a focus of many studies involving somatotropin (ST) administration and other anabolic growth promoters. Exogenous administration of ST to domestic animals increases both weight gain and the efficiency with which dietary amino acids are utilized for protein deposition (16, 17). In swine, ST administration increases protein deposition (4, 5) and decreases fat accretion, while simultaneously lowering feed intake (1). The increase in growth that occurs with ST administration includes not only that of skeletal muscle, but also that of skin, intestine, and liver (5, 6).

The increase in protein deposition with ST treatment is accompanied by a reduction in blood urea nitrogen (PUN) concentrations (3, 12, 33, 36), suggesting that ST treatment reduces the catabolism of amino acids. This possibility is supported by direct measurement of amino acid oxidation in adult humans (19, 24). However, the role of amino acid catabolism in the enhanced protein deposition that occurs with ST treatment of domestic animals is not completely understood.

It is generally held that the increase in protein deposition with ST treatment is due to an increase in protein synthesis rather than to a reduction in protein degradation. This premise is based on studies that were performed either in adult humans or in domestic animals approaching maturity (3, 14, 18, 19, 24, 31, 33). On the other hand, our recent study in young pigs (34) suggests that ST treatment has little effect on protein synthesis, despite an enhancement in the gain of tissue mass. Rates of protein degradation were not determined. There is also considerable variability in the protein synthetic responses of different tissues to ST treatment. For example, ST treatment of mature steers increases protein synthesis in some, but not all, skeletal muscles, and it has no effect on protein synthesis in liver and intestine, despite an increase in tissue mass (14). Thus the mechanisms that regulate the enhanced rate of protein deposition in growing animals treated with ST remain unclear.

The objectives of this study were 1) to determine whether the increase in protein deposition after ST treatment is due to a suppression in whole body proteolysis and/or a stimulation in whole body protein synthesis, 2) to identify which tissues respond to ST with an increase in protein synthesis, and 3) to determine whether the reduction in PUN with ST administration reflects a lower rate of urea production and amino acid oxidation. Studies were performed in rapidly growing young swine (~20 kg) in which the inherent rate of protein accretion was about threefold higher than that of the mature animals used in most other studies that investigated the effects of ST on protein metabolism (e.g., 50- to 100-kg swine, Ref. 15). In addition, energy and protein intakes were matched in ST and control pigs during the 7 days of treatment and were rigorously controlled during the 8-h isotope infusion study to ensure a fed steady-state status.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and design. Twelve crossbred (Landrace × Yorkshire × Hampshire × Duroc) barrows (castrated males, Agriculture Headquarters, Texas Department of Criminal Justice, Huntsville, TX) weighing 15 kg (~10 wk old) were housed in individual cages. Because relatively high protein intakes are required to obtain the maximum growth-promoting effects of ST (4, 5), pigs were fed a 24% protein diet (Producers Cooperative Association, Bryan, TX). During an initial 7-day adjustment period, the diet was provided at 6% body wt, a level which is ~90% of the ad libitum intake of pigs of this age. Water was provided ad libitum. After the 7-day adjustment period, pigs were assigned randomly to one of two treatment groups, either diluent (saline; control, n = 6) or recombinant porcine ST (n = 6, Southern Cross Biochemicals, Melbourne, Australia) at a rate of 150 µg · kg body wt-1 · day-1 for 7 days. This dose has been demonstrated to be slightly greater than that required to maximally stimulate protein accretion in swine (100 µg · kg body wt-1 · day-1) (15). The dose of saline or ST was divided into two daily injections (75 µg/kg body wt) administered into the neck region in alternating sides. Body weights were measured daily, and the dietary intake and treatment doses were adjusted accordingly. The ST-treated pigs were offered the diet at 6% body wt/day, and the control pigs were pair-fed to the intake level of the ST-treated pigs to minimize confounding effects of differences in feed intake. The daily feed allowance was divided into two meals to coincide with the injection times. Pigs generally consumed all feed presented to them, and if not, any unconsumed or spilled food was accounted for in the estimate of feed intake.

Five days before stable isotope tracer infusions, the pigs were fasted overnight and the jugular vein and carotid artery of each pig were catheterized with use of sterile technique under general anesthesia (Aerrane, Anaquest, Madison, WI) as described previously (7). After surgery, pigs were placed in individual cages, and, after recovery, they resumed their regular treatment regimen. The protocol was approved by the Animal Care and Use Committee of Baylor College of Medicine and was conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

Infusions. Pigs were placed in individual cages and maintained in the fed state throughout the 8-h infusion period by being fed hourly meals. The daily allowance of feed was partitioned into hourly meals based on a rate of 6% body wt/day (i.e., 2% body wt over the 8 h of study). On the day of stable isotope infusions, the pigs were administered their daily dose of ST (150 µg/kg body wt) or diluent and fed their first meal at 60 min before infusion. To measure whole body protein turnover, tissue protein synthesis, and urea production, pigs were infused for 8 h. Pigs were administered a primed (7.5 µmol/kg body wt), continuous (10 µmol · kg body wt-1 · h-1) infusion of [13C]bicarbonate from 0 to 120 min to determine CO2 production rate. A primed (10 µmol/kg body wt), continuous (10 µmol · kg body wt-1 · h-1) infusion of [1-13C]leucine lasting from 120 to 480 min was given to determine whole body leucine flux, leucine disposal into protein, leucine oxidation, and tissue fractional protein synthesis rates. A primed (242 µmol/kg body wt), continuous (24.2 µmol · kg body wt-1 · h-1) infusion of [15N2]urea lasting from 0 to 480 min (Cambridge Isotope Laboratories, Andover, MA) was administered to quantify amino acid catabolism. Blood and breath samples were collected at -10, 0, 90, 100, 110, 120, 420, 440, 460 and 480 min for measurement of 13C and 15N2 enrichment. Additional blood samples were collected every 30 min throughout the 480-min infusion for measurement of glucose and insulin concentrations. Concentrations of insulin-like growth factor I (IGF-I), glucagon, amino acids, ST, and PUN were measured at 0, 240, and 480 min. Pigs were killed at 480 min by exsanguination under anesthesia (pentobarbital sodium). Immediately after exsanguination, tissue samples from the longissimus dorsi, semitendinosus, and gastrocnemius muscles, liver, and jejunum were rapidly removed and frozen in liquid nitrogen.

Analysis of tracer enrichment. Tissues were homogenized in 0.2 M perchloric acid (PCA) as previously described (8). Briefly, the homogenate supernatant containing the tissue free amino acid pool was separated from the PCA-insoluble precipitate. The PCA-insoluble precipitate was washed and solubilized. An aliquot of the solubilized pellet was assayed for protein with the use of bicinchoninic acid (Pierce, Rockford, IL) by the method of Smith et al. (32). The remaining solution was reacidified, and the acid-soluble fraction was assayed for RNA by the method of Munro and Fleck (27). The protein pellet was washed (4 times) and hydrolyzed for 24 h with 6 N HCl. The protein and blood supernatants were analyzed for [1-13C]leucine enrichment.

Leucine was isolated by cation exchange chromatography (AG-50W resin, Bio-Rad, Hercules, CA). Leucine was derivatized with a trifluoroacetyl-methyl ester (F3NAM) derivative (26) and prepared for measurement of isotopic leucine enrichment with continuous flow gas chromatography combustion isotope ratio mass spectrometry (GIRMS) (Hewlett-Packard 5890 Series II GC equipped with a Europa Orchid 20/20 stable isotope analyzer; Hewlett-Packard, Palo Alto, CA). In addition, the isotopic enrichment of free [13C]leucine was determined by electron impact ionization (EI) gas chromatography mass spectrometry (GC-MS) by monitoring ions at mass-to-charge ratios (m/z) 153 and 154. The enrichment of 13CO2 from the oxidation of [1-13C]leucine was measured by Europa RoboPrep-G GC-MS analysis (Hewlett-Packard).

Plasma alpha -ketoisocaproic acid (alpha -KICA) was isolated by cation exchange chromatography (AG-50W resin, Bio-Rad). To each eluant, 10 N sodium hydroxide (100 µl) and 0.36 M hydroxylamine HCl (200 µl) were added. Samples were heated at 60°C for 30 min and then cooled at 4°C, and the pH was adjusted to <2. Keto acids were extracted with ethylacetate (5 ml) and dried under nitrogen at room temperature. The alpha -KICA was derivatized by adding 50 µl of N-methyl-N-t-butyl-dimethylsilyl-trifluoroacetamide + 1% t-butyl-dimethylchlorosilane (MTBSTFA + 1% TBDMCS, Regis Chemical). The isotopic enrichment of alpha -[13C]KICA was determined by EI GC-MS (Hewlett-Packard 5970 GC-mass spectrometer equipped with a Hewlett-Packard 5890 Series II GC, Hewlett-Packard) by monitoring ions at 316 m/z and 317 m/z.

Plasma isotopic [15N2]urea concentrations were also determined by EI GC-MS analysis. Plasma proteins were precipitated with cold (0°C) ethanol, and the protein pellet was separated by centrifugation. The supernatant was removed and dried under nitrogen at room temperature. To the dried sample was added a 1:20 dilution of malonaldehyde bis(dimethyl acetal) (Aldrich Chemical, Milwaukee, WI) and concentrated HCl (37 wt%; Fisher Scientific, Fair Lawn, NJ). The sample was incubated at room temperature for 24 h and then evaporated to dryness (Speedvac, Savant Instruments, Forma Scientific, Marietta, OH). The urea was derivatized with MTBSTFA + 1% TBDMCS and the plasma isotopic [15N2]urea enrichments were determined using EI GC-MS analysis by monitoring ions at 153 to 155 m/z.

Plasma hormones and amino acids. Heparinized blood samples were obtained and centrifuged, and the plasma was frozen at -70°C. Plasma insulin concentrations were measured with a porcine insulin RIA kit (Linco, St. Charles, MO) with the use of porcine insulin antibody and human insulin standards. Blood glucose concentrations were rapidly analyzed during the infusion period by means of a glucose oxidase reaction (YSI 2300 STAT Plus, Yellow Springs Instruments, Yellow Springs, OH). Plasma glucagon concentrations were measured with a glucagon RIA kit (Linco). Plasma total IGF-I concentrations were measured by means of a two-site immunoradiometric assay for human IGF-I (Diagnostic System Laboratories, Webster, TX). Porcine ST (pST) was assayed by use of a previously described method with minor modifications (6). Briefly, purified pST was iodinated using IODO-GEN [Pierce Chemical, Rockville, IL (10)]. To improve sensitivity, a nonequilibrium assay format was used. Standard (pST-I-1, range 0.2-20 ng/tube) or sample and baboon antiporcine ST were incubated for ~18 h. Then ~12,000 cpm 125I-labeled pST were added and tubes incubated for an additional 24 h. The immune complex was precipitated with goat anti-monkey serum and cold 10% polyethylene glycol(PEG). Assay sensitivity was ~3 ng/ml, and the intra-assay and interassay coefficients of variation were <10%. PUN concentrations were measured with the use of an end-point enzyme assay (Roche, Somerville, NJ).

Plasma amino acid concentrations were determined with a high-performance liquid chromatography method (9). Plasma spiked with methionine sulfone (internal standard) was filtered through a 10,000-molecular-weight filter. Amino acids were precolumn derivatized with phenyl isothiocyanate and separated on a PICO-TAG reverse-phase column (Waters, Milford, MA). Derivatized amino acids were detected on-line spectrophotometrically, and quantities were calculated with the use of an amino acid standard (Pierce). On most chromatographs, cystine is coeluted with a reagent peak; therefore, mean cystine values are not reported.

Diet was digested with the use of a digestion buffer (protease in phosphate buffer, Sigma-Aldrich, St. Louis, MO) at 37°C for 24 h, and the sample was dried (Speedvac, Savant Instruments, Forma Scientific). The dried residue was hydrolyzed for 24 h at 110°C in 6 N HCl and dried as before, and the amino acids were dissolved in 0.1 M HCl. Amino acid analysis was performed with the same method as that for plasma amino acids.

Calculations. Whole body protein turnover was calculated by means of the following standardized steady-state equations
flux = [([1-<SUP>13</SUP>C]leucine IE/[1-<SUP>13</SUP>C]KICA PE) − 1] 

× [<SUP>13</SUP>C]leucine IR
where IE is the infusate enrichment, PE is the plasma enrichment at plateau during leucine infusion, and IR is the infusion rate.
CO<SUB>2</SUB> production rate = [(NaH<SUP>13</SUP>CO<SUB>3</SUB> IE/<SUP>13</SUP>CO<SUB>2</SUB> BE) − 1] × NaH<SUP>13</SUP>CO<SUB>3</SUB> IR
where IE is the infusate enrichment, BE is the enrichment of expired CO2 at plateau during bicarbonate infusion, and IR is the infusion rate.
leucine oxidation = (CO<SUB>2</SUB> PR ×<SUP> 13</SUP>CO<SUB>2</SUB> E<SUB>l</SUB>)/[1-<SUP>13</SUP>C]KICA PE
where PR is the production rate, El is the 13C enrichment in the breath at plateau during leucine infusion, and PE is the enrichment of plasma [1-13C]KICA during the last 60 min of leucine infusion.
flux = synthesis + oxidation = breakdown + intake
Therefore, leucine disposal into protein (NOLD), an indication of whole body protein synthesis, equals leucine flux minus leucine oxidation. Leucine appearance from protein (endogenous Ra), an indication of whole body proteolysis, equals leucine flux minus leucine intake. To calculate leucine intake, and thus endogenous Ra, the amino acid composition analysis of the 24% protein diet fed during the study was used as a reference for amino acid intake from the diet. This was estimated to be ~124 µmol leucine · kg-1 · h-1. Protein balance equals protein synthesis minus protein degradation.

The rate of urea appearance was calculated as
urea flux = [([<SUP>15</SUP>N<SUB>2</SUB>]urea IE/[<SUP>15</SUP>N<SUB>2</SUB>]urea PE) − 1] × [<SUP>15</SUP>N<SUB>2</SUB>]urea IR
where IE is the infusate enrichment, PE is the plasma enrichment at plateau during urea infusion, and IR is the infusion rate.

The fractional rate of tissue protein synthesis (Ks, percent protein mass synthesized in a day) was calculated as
K<SUB>s</SUB> (%/day) = [(E<SUB>b</SUB>/E<SUB>a</SUB>) × (1,440/<IT>t</IT>)] × 100
where Eb is the enrichment of protein-bound leucine, Ea is the enrichment in the precursor pool, and t is the time of labeling in minutes. Enrichment in the protein-bound and precursor pool was corrected for baseline enrichment. The RNA-to-protein ratio (mg RNA/g protein) was used as an estimate of protein synthetic capacity (Cs) (7).

Statistics. ANOVA for repeated measures (SPSS) was used to test for changes with treatment during intensive sampling during the 8-h infusion for plasma insulin, glucose, IGF-I, ST, PUN, glucagon concentrations, and [1-13C]leucine and [1-13C]KICA enrichment. Two-way ANOVA (SPSS) was used to test for changes with treatment for body weight, daily weight gain, gain-to-feed ratio, fractional rate of protein synthesis, whole body protein turnover, and urea synthesis. Results are presented as means ± SD. Probability values of <0.05 were considered statistically significant and are not reported in the text.


    RESULTS
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INTRODUCTION
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RESULTS
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Animal weights. Body weight did not differ significantly between control and ST-treated pigs at the initiation of the treatment period (18.6 ± 0.74 vs. 17.7 ± 1.68 kg, respectively) and at the end of the 7 days of treatment (24.0 ± 1.82 vs. 24.2 ± 1.91 kg, respectively). However, the daily gain in body weight for the treatment period tended (P < 0.07) to be higher in the ST-treated pigs compared with controls (0.91 ± 0.14 vs. 0.78 ± 0.23 kg, respectively). The efficiency with which feed was used for growth was greater in ST-treated pigs than in controls, as reflected by their higher gain-to-feed ratios (0.75 ± 0.11 vs. 0.61 ± 0.15 kg gain/kg feed intake).

Plasma hormone and substrate concentrations. The plasma hormone and substrate concentrations shown in Table 1 are mean values over the 8-h infusion period. Plasma ST concentrations were elevated in ST-treated pigs compared with negligible levels in controls. Plasma IGF-I and insulin concentrations were twofold higher in ST-treated pigs than in controls, and glucose concentrations were also elevated (+22%) in ST-treated pigs compared with controls. Glucagon concentrations were lower (-25%) in ST-treated than in control pigs; PUN concentrations were decreased by >50% in ST-treated pigs compared with controls.

                              
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Table 1.   Plasma hormone and substrate concentrations in ST-treated and control pigs

Plasma total amino acid (TAA) concentrations were lower (-23%) in pigs treated with ST compared with control pigs (Fig. 1). Correspondingly, the plasma amino acid concentrations for the essential (EAA) and branched-chain amino acids (BCAA) were lower, and the nonessential amino acid (NEAA) concentrations tended (P < 0.07) to be lower in ST-treated pigs compared with control pigs.


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Fig. 1.   Circulating amino acid concentrations in control and somatotropin (ST)-treated pigs in the fed state. Values are means ± 1 SD; n = 6/treatment group. Total amino acid (TAA), essential amino acid (EAA), and branched chain amino acid (BCAA) concentrations were lower (P < 0.01) in ST-treated compared with control pigs. Nonessential amino acid (NEAA) concentrations tended (P < 0.07) to be lower in ST-treated pigs than in control pigs. * P < 0.05, ST vs. control animals.

Whole body protein turnover. Plasma leucine, alpha -KICA, urea, and breath carbon dioxide had reached substrate and isotopic steady state during the last 3 h of infusion (data not shown). ST treatment decreased leucine flux (-18%), endogenous Ra (-34%), and leucine oxidation (-63%) in comparison with controls (Fig. 2). NOLD was virtually identical in the two treatment groups.


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Fig. 2.   Whole body leucine kinetics in control and ST-treated pigs in the fed state. Values are means ± 1 SD; n = 6/treatment group. Leucine flux, endogenous Ra, and leucine oxidation were lower in ST-treated pigs than in controls (P < 0.05). Nonoxidative leucine disposal into protein (NOLD) was similar in ST-treated and control pigs (P > 0.10). * P < 0.05, ST vs. control animals.

In terms of protein kinetics, whole body protein synthesis was similar in ST-treated and control pigs (Table 2); however, whole body protein degradation was lower in ST-treated than in control pigs. The reduction in protein degradation in ST-treated pigs resulted in a marked increase in protein balance in the ST-treated group compared with the control group (+177%). The rate of urea synthesis was lower (-70%) in ST-treated pigs compared with control pigs and was proportional to the reduction in leucine oxidation.

                              
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Table 2.   Whole body protein kinetics and urea synthesis in ST-treated and control pigs

Tissue protein synthesis. The fractional rates of protein synthesis (Ks) were determined with several precursor pool models (intracellular free leucine pool, plasma leucine, and plasma alpha -KICA). Also, two different methods (GIRMS and GC-MS) were used to analyze isotopic enrichment values. Each precursor pool model and analytical method gave similar results (data not shown); therefore, the alpha -KICA precursor pool model was used to calculate Ks for all tissues. The Ks in the longissimus dorsi, semitendinosus, and gastrocnemius muscles, and in the liver and jejunum were similar in ST-treated and control pigs (Table 3).

                              
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Table 3.   Tissue fractional rates of protein synthesis and protein synthetic capacity in control and ST-treated pigs in the fed state

Because most of the RNA in tissues is ribosomal RNA (23), the RNA-to-protein ratio was used as an estimate of Cs. The Cs was similar in the longissimus dorsi and semitendinosus muscles and in the jejunum in ST-treated compared with control pigs (Table 3). However, the Cs was higher in gastrocnemius muscle (+13%) and in liver (+26%) of ST-treated pigs than in control pigs.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study demonstrate that a week of ST treatment in rapidly growing swine increases protein balance and the efficiency with which feed is used for growth. This improvement in protein anabolism can be attributed to a suppression in both whole body proteolysis and amino acid catabolism in the fed, steady state. This is evident from the reduction in total leucine flux and leucine appearance from protein (endogenous Ra), an indicator of whole body protein proteolysis, in ST-treated pigs. In addition, leucine oxidation and the rate of urea synthesis were markedly lower in ST-treated pigs. However, neither the rates of whole body protein synthesis nor the individual tissue fractional rates of protein synthesis were altered by ST treatment. It is critical to emphasize that the amino acid and energy intakes both before and during the isotope infusions were the same for both treatment groups. Despite the carefully maintained pair-feeding design, plasma amino acid concentrations were lower in ST-treated pigs, likely reflecting the altered balance between protein synthesis and proteolysis.

Growth and metabolic effects of ST. The results of the present study indicate that 7 days of administration of ST to rapidly growing swine successfully elicited the classic metabolic responses that are characteristic of ST treatment in domestic animals. Two of the fundamental anabolic effects of ST treatment in domestic animals are increased body weight gain and an increase in the efficiency with which the diet is used for growth (16, 17, 31, 34). In the present study, administration of ST to rapidly growing swine improved feed efficiency, and after only 7 days of treatment, differences in weight gain were already emerging. Well established metabolic effects of ST administration include the characteristic stimulation of the somatotropic axis, indicated by an increase in circulating IGF-I concentrations (5, 12, 34, 36), and the diabetogenic effect, indicated by an increase in circulating concentrations of both insulin and glucose (6, 12, 35, 36). In the present study, a 22-fold elevation in plasma ST concentrations resulted in a twofold increase in plasma IGF-I and insulin concentrations and a 22% increase in plasma glucose concentrations. Plasma glucagon levels were lower in ST-treated pigs compared with controls, a response which has been reported previously in the fed but not the fasting state (25, 35).

Another characteristic metabolic effect of ST treatment is a reduction in PUN concentrations (3, 12, 28, 36). In this study, PUN concentrations were decreased by 53% in ST-treated pigs. These results, in combination with the observed improvement in total feed efficiency, suggest that ST treatment of young growing animals results in a more efficient use of dietary amino acids for growth. Reports of the effect of ST treatment on plasma amino acid concentrations are limited and variable (3, 11, 24). In this study, total plasma amino acid concentrations, as well as essential and branched-chain amino acid concentrations, were lower in ST-treated pigs compared with controls, despite the similar intakes of both protein and energy in the two groups of animals. This reduction in plasma amino acid concentrations could reflect either an increase in removal from, and/or a decrease in entry to, the plasma amino acid pool in response to ST treatment.

Amino acid catabolism. Our results indicate that ST treatment reduces amino acid catabolism. This reduction in amino acid catabolism by ST treatment was demonstrated by the reduction in leucine oxidation, urea synthesis, and plasma urea nitrogen concentrations. Moreover, the percentages of reduction by ST treatment in all three measures of amino acid catabolism were similar, i.e., 63, 70, and 53%, respectively.

Two possible mechanisms can explain the ST-induced decrease in leucine oxidation and urea production. First, ST treatment lowered circulating amino acid concentrations. Because the rate of catabolism of amino acids is a direct function of the circulating level of those amino acids (29), the lower circulating leucine concentrations may well be responsible for the lower rate of amino acid catabolism. Second, lower rates of amino acid oxidation and urea production may reflect a direct effect of ST on the pathways of amino acid catabolism. This hypothesis is supported by the reduction in leucine oxidation in ST-treated heifers (13), the reduction in lysine, methionine, and valine oxidation in livers of ST-treated rats (2), and the reduced urea nitrogen synthesis capacity and mRNA levels of urea cycle enzymes in ST-treated rats (22).

Proteolysis. The regulation of protein deposition occurs through changes in the balance between the rates of protein synthesis and proteolysis. Based on previous reports in the literature (3, 14, 33), we had hypothesized that ST treatment would increase protein deposition through an increase in protein synthesis rather than by a reduction in protein degradation. For example, studies in mature 65-kg pigs (33) and 240-kg cattle (3) found either an increase (33) or no change (3) in proteolysis with ST treatment. However, a recent study by Dawson et al. (11) demonstrated that ST treatment of 168-kg steers reduced leucine flux in cattle that were fed an energy- and protein-rich diet, but not in those fed at a lower plane of nutrition.

The results of the present study demonstrate that in the fed steady state, ST increases protein deposition in rapidly growing swine through a suppression of protein degradation rather than by a stimulation of protein synthesis. This is evident from the reduction in total leucine flux and leucine appearance from protein (endogenous Ra), an indication of whole body proteolysis, in ST-treated pigs compared with controls. The reduction of protein degradation in ST-treated pigs resulted in a dramatic increase in protein balance (177%), which suggests an increase in the overall metabolic efficiency of ST-treated pigs. However, the current study does not indicate in which tissues the reductions in proteolysis occurred.

Protein synthesis. We observed no effect of seven days of ST treatment on protein synthesis in rapidly growing swine when this was measured under fed steady-state conditions. This lack of effect of ST treatment on protein synthesis in individual tissues was consistent with our finding on a whole body basis, as indicated by the lack of change in the rate of total leucine disposal into protein. In addition, the fractional rates of protein synthesis in three muscles, the longissimus dorsi, semitendinosus, and gastrocnemius, and in two visceral tissues, liver and jejunum, were unaltered by ST treatment. This lack of effect of ST treatment on the fractional rates of tissue protein synthesis contrasts with that of other studies in mature 52-kg swine (31), 289-kg steers (14), and adult humans (19). On the other hand, the protein synthetic capacity (RNA-to-protein ratio) of one of the three muscles and the liver was increased by ST treatment in the present study, suggesting that there is the potential for an elevation in protein synthesis in growing pigs treated with ST.

We hypothesize that protein synthesis was not stimulated by ST treatment in the present experiment because the inherent rate of protein synthesis in these rapidly growing, fully fed pigs was already elevated and, theoretically, near maximum. There are several lines of evidence to support this hypothesis. First, fractional rates of protein synthesis, particularly in skeletal muscle, are quite elevated in young, growing animals, and they decrease with age (7, 8, 21). In this study, muscle protein synthesis rates were almost as high as those of neonatal pigs (7, 34) and were substantially higher than those in mature swine (31), cattle (14), and humans (19). In our previous study with neonatal pigs, ST treatment had no effect on tissue protein synthesis, despite an increase in body weight and tissue protein mass (34). This suggests that an enhancement of protein synthesis rates in young, rapidly growing animals, which have inherently high rates of protein synthesis, may be difficult to attain.

Second, one of the most potent stimulators of protein synthesis is eating (7, 9, 20, 30). In the current study, feed intake was rigorously controlled during the 8-h infusion to ensure the fed steady-state status; thus it is likely that protein synthesis rates were fully stimulated. However, most previous studies that have examined the effects of ST on protein turnover were not performed in the fully fed state (14, 18, 19, 31, 33). This suggests that in fully fed animals with elevated rates of protein synthesis, the potential for ST administration to further stimulate the already high rate of protein synthesis may be minimal. On the other hand, the study does not exclude the possibility that ST administration can enhance protein synthesis in the postabsorptive state in young swine or even in the postprandial state in older animals. Moreover, the elevation in protein synthetic capacity in muscle and liver of ST-treated pigs suggests that the potential for stimulation of protein synthesis by ST can be realized if protein synthesis rates are reduced by fasting or when the inherent rate of protein synthesis declines as the animal matures.

Perspectives. The results of this study suggest that, during meal absorption, ST treatment can enhance protein deposition in rapidly growing swine by suppressing protein degradation and amino acid catabolism rather than by stimulating protein synthesis. This inability of ST to stimulate protein synthesis may be due to the inherently high rate of protein synthesis, which is characteristic of the fully fed pig during early stages of development. Thus the more efficient use of amino acids for protein deposition, which is characteristic of ST treatment, is attained by the suppression of proteolysis, rather than by a stimulation of protein synthesis, during meal absorption in growing pigs administered ST. Whether ST treatment of young pigs can enhance protein synthesis in conditions when protein synthesis is submaximal, e.g., postabsorption, remains to be established.


    ACKNOWLEDGEMENTS

The authors thank M. Haymond and F. Jahoor for their helpful suggestions. We also express appreciation for the technical assistance of J. Wen, R. Parsons, J. Cunningham, M. Stubblefield, and F. Biggs. We thank E. O. Smith for statistical assistance, L. Loddeke for editorial review, A. Gillum for graphics, and M. Alejandro for secretarial assistance.


    FOOTNOTES

This project has been funded by the US Department of Agriculture, National Research Initiative Grant 96-35206-3657, the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement no. 58-6250-6-001, and National Institute of Child Health and Human Development Training Grant T32-HD-07445.

This work is a publication of the USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. A. Davis, USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030 (E-mail: tdavis{at}bcm.tmc.edu).

Received 16 July 1999; accepted in final form 27 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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