Somatotropin increases protein balance independent of insulin's effects on protein metabolism in growing pigs

Rhonda C. Vann1, Hanh V. Nguyen1, Peter J. Reeds1, Norman C. Steele2, Daniel R. Deaver3, and Teresa A. Davis1

1 United States Department of Agriculture, Agricultural Research Service (USDA/ARS) Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030; 2 USDA/ARS 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 and elicits profound metabolic responses, including hyperinsulinemia. To determine whether the anabolic effect of ST is due to hyperinsulinemia, pair-fed weight-matched growing swine were treated with porcine ST (150 µg · kg body wt-1 · day-1) or diluent for 7 days (n = 6/group, ~20 kg). Then pancreatic glucose-amino acid clamps were performed after an overnight fast. The objective was to reproduce the insulin levels of 1) fasted control and ST pigs (basal insulin, 5 µU/ml), 2) fed control pigs (low insulin, 20 µU/ml), and 3) fed ST pigs (high insulin, 50 µU/ml). Amino acid and glucose disposal rates were determined from the infusion rates necessary to maintain preclamp blood levels of these substrates. Whole body nonoxidative leucine disposal (NOLD), leucine appearance (Ra), and leucine oxidation were determined with primed, continuous infusions of [13C]leucine and [14C]bicarbonate. ST treatment was associated with higher NOLD and protein balance and lower leucine oxidation and amino acid and glucose disposals. Insulin lowered Ra and increased leucine oxidation, protein balance, and amino acid and glucose disposals. These effects of insulin were suppressed by ST treatment; however, the protein balance remained higher in ST pigs. The results show that ST treatment inhibits insulin's effects on protein metabolism and indicate that the stimulation of protein deposition by ST treatment is not mediated by insulin. Comparison of the protein metabolic responses to ST treatment during the basal fasting period with those in the fully fed state from a previous study suggests that the mechanism by which ST treatment enhances protein deposition is influenced by feeding status.

protein synthesis; insulin-like growth factor I; amino acid catabolism; growth hormone


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EXOGENOUS ADMINISTRATION of somatotropin (ST) to domestic animals increases daily weight gain and the efficiency with which dietary amino acids are utilized for protein deposition (19). This enhanced efficiency in response to ST administration is particularly evident in swine, in which porcine ST administration simultaneously lowers feed intake and increases protein deposition (2, 5, 6).

The regulation of protein deposition occurs through changes in the balance between the rates of protein synthesis and proteolysis. Our recent studies in pigs suggest that ST treatment improves protein balance by reducing protein degradation rather than by stimulating protein synthesis (45, 46). However, other studies primarily suggest that ST treatment does not alter protein degradation but, rather, increases protein synthesis (4, 17, 22, 42). Because nutrient intake profoundly stimulates protein synthesis (9, 11, 25, 41), our studies were performed in animals in the fed steady state, whereas most other studies were performed during postabsorption (17, 22, 23, 42). In addition, our studies were performed in young, rapidly growing animals (45, 46), but those by other investigators were performed in mature animals near market weight or in adult humans (4, 17, 22, 42). Because protein synthesis rates are inherently high in young animals and decrease with development (9, 10, 28), we postulated that the ability of ST administration to further enhance the inherently high rates of protein synthesis in young, rapidly growing, fully fed animals may be minimal. It is possible that ST treatment can enhance protein synthesis during the postabsorptive period, when the rate of protein synthesis is reduced by fasting, and one objective of the present study was to examine this possibility.

The administration of ST is generally accompanied by an increase in both insulin and glucose concentrations (8, 15, 35, 49, 51). This response has traditionally been attributed to the antagonistic effects of ST on insulin's actions (32, 33, 43, 51), and it has been shown that the increase in plasma glucose with ST treatment is due, at least in part, to a reduction in the stimulation of glucose uptake by insulin (1, 20, 38, 48). This appears to be largely reflective of changes in the uptake of glucose by adipocytes (16, 48). Whether there is insulin resistance of protein metabolism during ST treatment has not been determined. Testing whether protein turnover becomes resistant to insulin during ST treatment was a second objective of this study.

It is well established that insulin is a potent anabolic agent (25, 26, 39). Our studies in young swine indicate that insulin mediates the stimulation in skeletal muscle protein synthesis that follows a meal and that this response decreases with development (50). Studies in adult humans suggest that the protein anabolic effect of insulin results from a decrease in the rate of proteolysis rather than a stimulation of protein synthesis (27, 30, 36). Thus the process by which, and the degree to which, insulin stimulates protein deposition likely vary with stage of development.

Because ST administration to growing animals enhances protein deposition in association with increased insulin concentrations (45), the third aim of the present study was to test the hypothesis that the protein anabolic effect of ST treatment is due to the increased circulating insulin concentrations that occur with ST treatment. To achieve these three objectives, pancreatic glucose-amino acid clamps were performed in control and ST-treated young pigs after an overnight fast. Insulin was infused at rates designed to achieve insulin concentrations that are characteristic of 1) the fasting state of control and ST pigs, 2) the fed state of control pigs, and 3) the fed state of ST pigs. Whole body protein turnover and amino acid oxidation rates were determined.


    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 (Agriculture Headquarters, Texas Department of Criminal Justice, Huntsville, TX) weighing 13 kg (~10 wk) were housed in individual cages, fed a 24% protein diet (Producers Cooperative Association, Bryan, TX) at 6% of their body wt, and provided water ad libitum. The pigs were adjusted to the diet for 7 days and then randomly assigned to one of two treatment groups, either diluent (saline) (control, n = 6) or recombinant porcine ST (Southern Cross Biochemicals, Melbourne, Australia; n = 6) at a rate of 150 µg · kg body wt-1 day-1 for 7 days. This dose of ST is slightly greater than the dose that has been demonstrated (see Ref. 18) to maximally stimulate protein accretion in swine (100 µg · kg body wt-1 · day-1). The dose of saline or ST was divided into two daily injections (75 µg/kg body wt) and administered into the neck region (alternating sides for each injection). Body weights were measured daily, and the dietary intake and treatment doses were adjusted accordingly. ST-treated pigs were offered the diet at 6% of their body wt per day, and control pigs were pair-fed to the intake level of the ST-treated pigs to minimize confounding effects of differences in food intake. The daily feed allowance was divided into two meals to coincide with the injection times.

Five days before stable isotope 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 (9). After surgery, pigs were placed in individual cages and, after recovery, 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.

Pancreatic glucose-amino acid clamps. Control (18.2 ± 2.8 kg) and ST-treated (20.2 ± 2.1 kg) pigs were fasted overnight (12 h) before the infusion studies. On the day of the infusions, ST-treated pigs were administered their daily dose of ST (150 µg/kg body wt) or diluent 60 min before infusion. Clamps were performed using techniques similar to those previously described (47, 50), except that an ST infusion was given to suppress endogenous insulin secretion, and replacement doses of glucagon and replacement or elevated doses of insulin were provided (Fig. 1). Over a 30-min period before the clamp procedure was initiated, basal blood glucose (YSI 2300 STAT Plus, Yellow Springs Instruments, Yellow Springs, OH) and branched-chain amino acid (BCAA) concentrations were determined to establish the average fasting concentrations to be used in the glucose-amino acid clamp procedure (50). The clamp was initiated with a primed (4.5 µg/kg), continuous (18 µg · kg-1 · h-1; BACHEM, Torrance, CA) ST infusion. After a 10-min infusion of ST, an infusion of replacement glucagon (72 ng · kg body wt-1 · h-1; Eli Lilly, Indianapolis, IN) was initiated and continued to the end of the clamp period. Insulin was infused at 20, 75, and 200 ng · kg-0.66 · min-1 to reproduce the insulin concentrations normally present in the 1) fasting state of control and ST pigs (5 µU/ml; basal), 2) fed state of control pigs (20 µU/ml; low insulin), and 3) fed state of ST pigs (50 µU/ml; high insulin) (45). Each pig was continuously infused for 8 h on two separate days, with 1 day separating the two infusion days. On the 1st day, the basal insulin (20 ng · kg-0.66 · min-1) dose was infused from 10 to 240 min, and the low insulin (75 ng · kg-0.66 · min-1) dose was primed over a 10-min period and then infused at a constant rate (12 ml/h) from 250 to 480 min. Following the same protocol, on the 2nd day the basal (20 ng · kg-0.66 · min-1) and the high insulin (200 ng · kg-0.66 · min-1) doses were administered. Venous blood samples (0.2 ml) were obtained every 5 min and immediately analyzed for glucose and BCAA concentrations, as previously described (50). A 2.5-min enzymatic kinetic assay was used to determine total BCAA concentrations. Dextrose 70% (Baxter Healthcare, Deerfield, IL) solution and TrophAmine 10% (McGaw, Irvine, CA) were infused to maintain glucose and amino acid values at the preclamp level. Additional blood samples were collected every 30 min throughout the 480-min infusion for measurement of glucose, insulin, glucagon, and C-peptide concentrations. Concentrations of insulin-like growth factor I (IGF-I), amino acids, ST, and blood urea nitrogen (BUN) were measured at 0, 240, and 480 min. On the 2nd day of the insulin infusions, the pigs were killed at 480 min of infusion by exsanguination under anesthesia.


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Fig. 1.   Pancreatic glucose-amino acid clamp protocol. Somatotropin (ST)-treated and control pigs were fasted overnight and then injected with ST and diluent, respectively, 60 min before infusion. Pigs were infused for 8 h with ST and replacement glucagon on 2 separate days, with 1 day separating the 2 infusion days. On the 1st day, insulin was infused at replacement doses (basal insulin) for 4 h, and then insulin was infused for 4 h to reproduce the insulin level in the fed state of control pigs (low insulin). On the 2nd day of infusion, replacement doses of insulin were infused for 4 h (basal insulin), and then insulin was infused for 4 h to reproduce the insulin level in the fed state of ST pigs (high insulin). Glucose and amino acids were clamped at fasting levels. CO2 production rate and whole body protein turnover were measured using [14C]bicarbonate and [13C]leucine. Blood and breath samples were obtained for whole body protein turnover during the preclamp period and the last hour of each insulin infusion period as indicated. Blood samples were also obtained at 5-min intervals to clamp blood glucose and amino acids and at either 30-min intervals or at the beginning and end of the infusion periods for substrate and hormone concentrations.

Whole body protein turnover was measured during the 2 days of 8-h infusions. Pigs were administered a primed (0.75 µCi/kg body wt), continuous (1.0 µCi · kg body wt-1 · h-1) infusion of [14C]bicarbonate (Amersham Life Sciences, Arlington, Heights, IL) throughout the infusion to determine CO2 production rate. A [14C]bicarbonate prime (5 µmol/kg body wt), and a primed (15 µmol/kg body wt), continuous (15 µmol · kg body wt-1 · h-1) infusion of [1-13C]leucine was given from 0 to 480 min to determine whole body leucine flux, disposal into protein, and oxidation (Cambridge Isotope Laboratories, Andover, MA). Blood and breath samples were collected at -30, -15, 0, 180, 195, 210, 225, 240, 420, 435, 450, 465, and 480 min for measurement of 13C and 14C enrichment and blood gases (Chiron Diagnostics, Halstead, Essex, England). Values between 180 and 240 min were used to measure basal insulin kinetics, and values between 420 and 480 min were used to measure insulin-stimulated kinetics.

Analysis of tracer enrichment. Plasma alpha -ketoisocaproic acid (alpha -KICA) was isolated by cation exchange chromatography (AG-50W resin, Bio-Rad, Hercules, CA). To each eluant, 10 N sodium hydroxide and 0.36 M hydroxylamine hydrochloric acid were added. Samples were heated at 60°C for 30 min and cooled at 4°C, and the pH was adjusted to <2. Keto acids were extracted with ethylacetate and dried under nitrogen at room temperature. The alpha -KICA was derivatized by adding N-methyl-N-t-butyl dimethylsilyl trifluoroacetamide + 1% t-butyl dimethyl-chlorosilane (MTBSTFA + 1% TBDMCS; Regis Chemical, Morton Grove, IL). The isotopic enrichment of alpha -[13C]KICA was determined by electron impact ionization (EI) gas chromatography-mass spectrometry (GC-MS) (Hewlett-Packard 5989 B GC-mass spectrometer equipped with a Hewlett-Packard 5890 Series II GC; Hewlett-Packard, Palo Alto, CA) by monitoring ions at mass-to-charge ratios (m/z) of 316 and 317. The 14C radioactivity of blood supernatants and infusates was measured using ScintiVerse II (Fisher Scientific) in a liquid scintillation counter (TM Analytic, Elk Grove Village, IL).

Plasma substrates and hormones. Heparinized blood samples were obtained and centrifuged, and the plasma was frozen at -70°C. Plasma glucose concentrations were analyzed using a glucose oxidase reaction (YSI 2300 STAT Plus, Yellow Springs Instruments) during the pancreatic glucose-amino acid clamp. BUN concentrations were measured with the use of an end-point enzyme assay (Roche, Somerville, NJ).

Plasma amino acid concentrations were determined using an HPLC method (11). 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 Chemical, Rockford, IL). Cystine coeluted with a reagent peak on most chromatographs; therefore, mean cystine values are not reported.

Plasma insulin concentrations were measured using a porcine insulin RIA kit (Linco, St. Charles, MO) with porcine insulin antibody and human insulin standards. Plasma glucagon and C-peptide concentrations were measured with the use of glucagon and C-peptide RIA kits (Linco). Plasma total IGF-I and free IGF-I concentrations were measured with a two-site immunoradiometric assay with IGF-coated tubes (Diagnostic System Laboratories, Webster, TX).

Porcine ST was assayed using a previously described method with minor modifications (8). Briefly, purified porcine (p) ST (AFP-6400 pGH-I-1) was iodinated using IODO-GEN [Pierce Chemical (12)]. To improve sensitivity, a nonequilibrium assay format was used. Standard (pST-I-1, range 0.2-20 ng/tube) or sample (100 ml) and baboon anti-porcine ST (a gift from Hoffmann-LaRoche) were incubated for ~18 h. Then ~12,000 counts/min of 125I-labeled pST were added, and tubes were incubated for an additional 24 h. The immune complex was precipitated using goat anti-monkey serum (Organon Teknika) and 1 ml of cold 10% polyethylene glycol. Assay sensitivity was ~3 ng/ml, and the intra-assay and interassay coefficients of variation were <10%.

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

<IT>×</IT>[<SUP><IT>13</IT></SUP>C]leucine IR
where IE is the infusate enrichment, PE is the plasma enrichment at plateau during the leucine infusion, and IR is the leucine infusion rate
CO<SUB><IT>2</IT></SUB> production rate

<IT>=</IT>[(NaH<SUP><IT>14</IT></SUP>CO<SUB><IT>3</IT></SUB>IE<IT>/<SUP>14</SUP></IT>CO<SUB><IT>2</IT></SUB>BE)<IT>-1</IT>]<IT>×</IT>NaH<SUP><IT>14</IT></SUP>CO<SUB><IT>3</IT></SUB> IR
where IE is the infusate enrichment, BE is the enrichment of expired CO2 at plateau during the leucine infusion, and IR is the infusion rate
leucine oxidation

<IT>=</IT>(CO<SUB><IT>2</IT></SUB> PR<IT>×<SUP>13</SUP></IT>CO<SUB><IT>2</IT></SUB> E<SUB>l</SUB>)<IT>/</IT>[<SUP><IT>13</IT></SUP>C]KICA PE
where PR is the production rate, El is the 13C enrichment in the breath at plateau during the leucine infusion, and PE is the enrichment of plasma [13C]KICA during the last 60 min of the leucine infusion. Because flux (Q) equals synthesis (S) plus oxidation (O), which equals breakdown (B) plus intake (I), then leucine disposal into protein (NOLD), an indication of whole body protein synthesis, equals leucine flux (Q) minus leucine oxidation (O) (i.e., Q = B + I = O + S). Leucine appearance from protein (endogenous Ra), an indication of whole body proteolysis, equals leucine flux (Q) minus leucine intake (I). Leucine intake was the rate of leucine infusion from TrophAmine. Protein balance was calculated by protein synthesis minus protein degradation.

Statistics. Analysis of variance for repeated measures (SPSS) was used to assess the effects of ST treatment, insulin, and their interaction. When significant interactions were detected, values at each insulin dose were compared with basal values or, when appropriate, preclamp values, using paired t-tests. Also, values in ST-treated pigs were compared with values in control pigs at each insulin dose. Because values did not differ significantly between the two basal periods in either ST or control pigs, the basal values were averaged and are presented as single basal values. Also, values did not differ significantly between the two preclamp periods; therefore, the preclamp values were averaged and are presented as single preclamp values. Results are presented as means ± SD. Probability values of <0.05 were considered statistically significant and are not presented in the text.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Plasma hormones and substrates. The plasma hormone and substrate concentrations shown in Tables 1 and 2 are mean values obtained during each of the infusion periods. Plasma ST concentrations were elevated in ST-treated pigs compared with the negligible levels in control pigs during the preclamp and the basal, low, and high insulin infusion periods (Table 1). On the day of the infusion, ST-treated pigs were administered one ST injection 1 h before the initiation of the insulin infusions. Therefore, plasma ST values in ST-treated pigs were higher during the preclamp period than during the later clamp periods. Although insulin infusion was associated with a fall in total and free insulin-like growth factor I (IGF-I) concentrations, IGF-I concentrations remained twofold higher in ST-treated than in control pigs throughout the infusion periods.

                              
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Table 1.   Plasma hormone concentrations in ST-treated and control pigs during pancreatic glucose-amino acid clamps


                              
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Table 2.   Plasma amino acid, glucose, and urea nitrogen concentrations in ST-treated and control pigs during pancreatic glucose-amino acid clamps

Using the pancreatic glucose-amino acid clamps in fasted, control, and ST-treated young pigs, we wished to reproduce the insulin concentrations that we have previously shown (45) to be normally present in the 1) fasting state of control and ST pigs (5 µU/ml), 2) fed state of control pigs (20 µU/ml), and 3) fed state of ST pigs (50 µU/ml). The results show that the targeted plasma insulin concentrations were largely achieved (Table 1). However, ST-treated pigs had lower insulin concentrations than control pigs at the high insulin dose, likely due to their higher rate of insulin clearance (1,006 ± 340 vs. 639 ± 176 ml/min). Insulin clearance rates in ST-treated and control pigs were similar during the basal period (566 ± 262 vs. 586 ± 167 ml/min) and the low insulin infusion period (735 ± 225 vs. 664 ± 81 ml/min). C-peptide concentrations were suppressed by the ST infusion during the basal, low, and high insulin infusion periods in both ST and control pigs. Glucagon concentrations were unaffected by ST treatment and insulin infusion. Cortisol concentrations decreased during the clamp periods compared with preclamp values and were unaffected by ST treatment.

Plasma BCAA and essential amino acid (EAA) concentrations were successfully maintained over the course of the insulin infusion periods by use of the amino acid clamp method (Table 2). However, nonessential (NEAA) and, hence, total amino acids (TAA) decreased at the high insulin dose in control pigs and at the low and high insulin doses in ST-treated pigs. The concentrations of TAA, EAA, NEAA, and BCAA did not differ between ST-treated and control pigs. Plasma glucose concentrations were largely maintained within 10% of the fasting, preclamp level throughout the basal, low, and high insulin infusion periods with the glucose clamp method. As expected, plasma glucose was higher in ST-treated pigs than in the control pigs during the basal period. BUN concentrations were ~35% lower in ST-treated pigs than in control pigs during the preclamp, basal, and low insulin infusion periods. Insulin infusion reduced BUN concentrations in control pigs (basal, low, and high insulin doses) and ST-treated pigs (high insulin dose only).

Amino acid and glucose disposal. Because our previous studies demonstrated that the amino acids infused during hyperinsulinemic glucose-amino acid clamps were not catabolized, but likely utilized and deposited, the amino acid infusion rate can be used as an indication of insulin-stimulated whole body net amino acid disposal (47). Amino acid infusion was not required during the basal period (Fig. 2). Insulin stimulated amino acid disposal; however, ST treatment blunted the stimulation of amino acid disposal at the low dose of insulin. On the basis of the changes in dextrose infusion rate during the last hour of each insulin infusion period, glucose disposal was lower in ST-treated pigs than in control pigs during all insulin infusion periods (Fig. 2).


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Fig. 2.   Amino acid (A) and glucose (B) disposal rates with increasing insulin infusion rates in ST-treated and control pigs. Values are means ± SD; n = 6/treatment group. Amino acid and glucose disposal rates were the average rates of infusion of total amino acids from TrophAmine and dextrose necessary to maintain circulating branched-chain amino acids and glucose near basal fasting level during infusion of insulin. Amino acids were not infused during the basal insulin infusion dose. Repeated-measures ANOVA indicated that amino acid and glucose disposals were increased by insulin (P < 0.001) and reduced by ST treatment (P < 0.01) and that there were interactions of insulin and ST treatment on amino acid and glucose disposals (P < 0.01). Results of paired t-test: * response in ST-treated pigs differed from controls at individual insulin dose (P < 0.05); # response to insulin dose differed from basal in either ST or control pigs (P < 0.01). Values did not differ significantly between the 2 basal periods; therefore, values were averaged and are presented as single basal values.

Whole body protein turnover. Plasma alpha -KICA concentrations were at substrate and isotopic steady state during the last hour of the basal, low, and high insulin infusions when samples were obtained for calculation of protein turnover (data not shown). Leucine flux did not differ significantly between ST-treated and control pigs (Fig. 3). Insulin increased leucine flux during the low and high insulin infusion periods in control pigs, but only during the high insulin infusion period in ST-treated pigs. Leucine oxidation was lower in ST-treated pigs compared with controls during the basal (-47%) and low (-64%) insulin infusion periods. Insulin increased leucine oxidation during the low and high insulin infusion periods in control pigs but only during the high insulin infusion period in ST-treated pigs. NOLD was higher in ST-treated pigs compared with control pigs during the basal (+23%) and low (+52%) insulin infusion periods. NOLD was unaffected by insulin. Leucine appearance from protein (endogenous Ra) did not differ between ST-treated and control pigs. Insulin decreased endogenous Ra during the low and high insulin infusion periods in control pigs but only during the high insulin infusion period in ST-treated pigs.


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Fig. 3.   Whole body leucine kinetics in control and ST-treated pigs during pancreatic glucose-amino acid clamps. Values are means ± SD; n = 6/treatment group. Repeated-measures ANOVA indicated that insulin increased leucine flux (A) and leucine oxidation (B) and reduced endogenous leucine appearance (Ra; D, P < 0.001). ST treatment reduced leucine oxidation (B, P < 0.05) and increased nonoxidative leucine disposal (NOLD, C, P < 0.005). There were interactions of insulin and ST treatment on Ra, NOLD, and leucine oxidation. Results of paired t-test: * response in ST-treated pigs differed from controls at individual insulin dose (P < 0.05); and # response to insulin dose differed from basal in either ST or control pigs (P < 0.05). Values did not differ significantly between the 2 basal periods; therefore, values were averaged and are presented as single basal values.

Leucine kinetic data were expressed in terms of protein kinetics and are presented in Table 3. Whole body protein synthesis was greater in ST-treated than in control pigs and was unaffected by insulin infusion. Whole body protein degradation was unaffected by ST treatment but was reduced by low and high doses of insulin in control pigs and high doses of insulin in ST-treated pigs. Protein balance was less negative in ST-treated than in control pigs during the basal fasting period. Insulin increased protein balance, and in the ST-treated pigs, protein balance became positive during the low and high insulin infusion periods.

                              
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Table 3.   Plasma turnover in ST-treated and control pigs during pancreatic glucose-amino acid clamps


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study suggest that the protein anabolic effect of ST administration is not due to the increased insulin concentrations that occur with ST treatment. Moreover, pancreatic glucose-amino acid clamp studies indicate that ST treatment produces insulin resistance not only for glucose disposal but also for amino acid disposal, leucine oxidation, leucine flux, and protein degradation. This supports the idea that the increase in protein balance with ST treatment is likely a direct effect of ST. Comparison of the results of the current study performed in the fasted state with those of our previous study performed in the fully fed state (45) suggests that the mechanism by which ST improves protein balance is affected by feeding status. The combined results of the two studies indicate that ST treatment blunts the reduction in protein synthesis that occurs with fasting while enhancing the reduction in proteolysis that occurs with feeding. These dual effects thereby maximize the efficiency of dietary protein utilization.

ST induces insulin resistance. Our results demonstrate that 7 days of ST treatment in 20-kg swine produced the characteristic stimulation of the somatotropic axis (6, 15, 46, 51), as indicated by the twofold increase in circulating concentrations of total and free IGF-I. The characteristic diabetogenic effect (8, 15, 49, 51) was also produced by ST treatment in both the current and previous studies (45), as evidenced by the elevation in plasma glucose concentrations in both the fasting and fed states and the increased plasma insulin concentrations in the fed but not the fasting state.

Pancreatic glucose-amino acid clamps were largely successful in producing the desired hormone and substrate concentrations. C-peptide concentrations were markedly reduced, indicating that the ST infusion effectively suppressed endogenous insulin secretion. The maintenance of circulating glucagon concentrations during the clamp periods indicated that replacement levels of glucagon were achieved. During the basal, low, and high insulin infusion periods, we largely reproduced the circulating insulin concentrations observed in the fasting state of control and ST-treated pigs (5 µU/ml), the fed state in control pigs (20 µU/ml), and the fed state in ST-treated pigs (50 µU/ml) (45). However, at the high insulin dose, plasma insulin levels were slightly lower in ST-treated than in control pigs because of an elevation in insulin clearance. Circulating concentrations of glucose, BCAA, and EAA were largely maintained at the fasting, preclamp level during the pancreatic glucose-amino acid clamps. However, circulating concentrations of NEAA and TAA were not completely maintained, likely because of the use of an amino acid solution (TrophAmine) that is relatively deficient in some NEAA.

The results of the pancreatic glucose-amino acid clamp indicate that ST treatment in young, growing swine induced insulin resistance of glucose disposal. This resistance of ST-treated pigs was present at each insulin dose. Although hyperinsulinemic-euglycemic clamp studies performed in mature swine treated with ST (48) showed resistance of glucose disposal to a low but not a high dose of insulin, the high dose of insulin used in those studies produced supraphysiological insulin concentrations (400 µU/ml) rather than the physiological level of insulin (50 µU/ml) achieved by our highest insulin dose. The reduction in glucose disposal with ST treatment (1, 20, 38) has been attributed to a reduction in glucose uptake in adipose tissue, rather than in skeletal muscle (16, 48).

The results of the current study showed that ST treatment produced insulin resistance not only of glucose metabolism but also of protein metabolism. For all variables that responded to insulin, i.e., whole body amino acid disposal, leucine flux, leucine oxidation, endogenous leucine appearance, and whole body proteolysis, the response to insulin was blunted in ST-treated pigs at the low dose of insulin. These results indicate that ST treatment reduces the sensitivity, but not the responsiveness, of protein metabolism to insulin (33). NOLD was unaffected by insulin, similar to the lack of effect of insulin on whole body and forearm protein synthesis reported in adult humans (7, 13, 27). Although we (50) and others (26) have shown that insulin stimulates protein synthesis in skeletal muscle of much younger growing pigs, the whole body protein synthesis measured in the current study represents the average result of numerous tissues, which may have opposing responses to insulin.

There have been few studies of the effects of ST on insulin-stimulated protein metabolism. However, a report that suggests that insulin's ability to suppress proteolysis and stimulate glucose disposal in the forearm is blunted by ST treatment in adult humans (24) supports the results of the current study. Thus the increase in protein balance with ST treatment is likely a direct effect of ST, rather than an indirect effect of the ST-induced hyperinsulinemia. However, it cannot be ruled out that the response may be an indirect effect of some factor other than insulin, such as IGF-I (21).

Protein anabolic response to ST. The regulation of protein deposition is dependent on the balance between the rates of protein synthesis and proteolysis. The results of the current work suggest that ST treatment of young 20-kg swine improves protein balance by increasing the basal rate of whole body protein synthesis with no change in the rate of proteolysis in the postabsorptive state. These results are consistent with studies in mature swine, steer, and humans (17, 23, 42, 44), also performed in the postabsorptive state, which showed that the protein anabolic effect of ST is generated by an increase in protein synthesis rather than a reduction in proteolysis.

However, comparison of the results of the current study, conducted in the fasting state under basal conditions, with our previous study performed in the fully fed state (45) suggests a more complex mechanism for the enhancement in protein deposition by ST treatment (Fig. 4). In the fully fed 20-kg pigs in the previous study, ST treatment increased protein deposition through a marked suppression of protein degradation rather than by a stimulation of protein synthesis at either the whole body or organ level (45). Because both feeding and immaturity are positive regulators of protein synthesis (9, 10, 28, 39), the results suggest that there may be minimal potential for ST treatment to further enhance the already high rate of protein synthesis that is present in rapidly growing, fully fed animals. By contrast, when protein synthesis is measured in the fasting condition, ST-treated pigs have higher rates of protein synthesis than controls, apparently as a result of the greater reduction in the protein synthesis rates with fasting in controls than in ST-treated animals. Thus ST treatment of young growing swine ameliorates the reduction in protein synthesis that occurs with fasting.


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Fig. 4.   Comparison of fasted vs. fed values of whole body protein synthesis (A), protein degradation (B), protein balance (C), and leucine oxidation (D) in control and ST-treated pigs. Fed values were obtained from a previous study (45) of ~20 kg control and ST-treated pigs in which whole body protein turnover was measured during the fed steady state. Fasted values are basal values from the current study. ANOVA indicated that protein synthesis was increased by feeding (P < 0.001) and by ST treatment (P < 0.05). Protein degradation was reduced by feeding (P < 0.01) and ST (P 0.01), and the reduction in protein degradation by ST was altered by feeding (P < 0.01). Protein balance was increased by feeding (P < 0.001) and by ST treatment (P < 0.001), and the increase in balance by ST treatment was affected by feeding (P < 0.005). Leucine oxidation was increased by feeding (P < 0.001) and reduced by ST (P < 0.001); the reduction in oxidation by ST was affected by feeding (P 0.005). Results of paired t-test: * response in ST-treated pigs (P < 0.05); # response to feeding (P < 0.05).

The results of the two studies further suggest that ST-treated animals, but not controls, reduce their rate of protein degradation in response to feeding (Fig. 4). Although most studies in adult humans suggest that whole body protein degradation decreases with feeding (31, 37), studies in human neonates suggest that feeding does not alter protein degradation, but rather, feeding stimulates protein synthesis (14), as in the results of the current study in young pigs. Thus the feeding-induced stimulation of protein synthesis, rather than a suppression of protein degradation, may play a more important role in protein deposition in the immature mammal. In the ST-treated young animal, however, feeding both suppresses protein degradation and stimulates protein synthesis, resulting in a marked improvement in protein balance.

The reduction in leucine oxidation associated with ST treatment was maintained during both fasting and feeding conditions in the current and previous (45) studies (Fig. 4). The circulating concentrations of amino acids, an important determinant of amino acid oxidation (40), were unaltered by ST treatment, suggesting that ST treatment directly affects the pathways of amino acid catabolism. This conclusion is supported by recent work in ST-treated rats (3, 29).

Perspectives. The classic metabolic response to ST treatment in domestic animals is an increase in the efficiency with which the diet is used for growth (2, 5, 6, 19). The combined results of our current and previous (45) studies suggest that ST treatment of young growing swine enhances metabolic efficiency by minimizing the loss of protein during fasting and maximizing the protein gained during meal absorption. This is accomplished by blocking the reduction in protein synthesis that occurs with fasting, enhancing the reduction in proteolysis that occurs with feeding, and reducing amino acid catabolism in both the fed and fasting conditions. This conservation of protein results in an improvement in overall protein balance. Whether these responses are unique to the young, growing swine or are also present in older, larger pigs remains to be determined.

Pancreatic glucose-amino acid clamp studies demonstrate that the improvement in protein balance with ST treatment is not due to the ST-induced hyperinsulinemia. ST treatment of young growing swine induces insulin resistance not only for glucose metabolism but also for protein metabolism. Thus the increase in protein balance with ST treatment is likely a direct effect of ST or some factor other than insulin.


    ACKNOWLEDGEMENTS

We thank M. Fiorotto and D. Burrin for helpful discussions. We appreciate the technical assistance of J. Wen, M. Haymond, J. Cunningham, M. Stubblefield, F. Biggs, and J. Vargas. We also thank E. O. Smith for statistical assistance, L. Loddeke for editorial review, A. Gillum for graphics, and M. Alejandro for secretarial assistance. We acknowledge Eli Lilly for the generous donation of porcine insulin, and McGaw for the generous donation of TrophAmine.


    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 (USDA/ARS) under Cooperative Agreement no. 58-6250-6-001, and the 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 USDA, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

Present address of R. C. Vann: Coastal Plains Experiment Station, University of Georgia, Tifton Campus, PO Box 748, Tifton, GA 31793-0748.

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).

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.

Received 12 October 1999; accepted in final form 1 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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