INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

A PRIMARY GOAL OF EXOGENOUS SOMATOTROPIN (ST) treatment is to increase lean body mass. This is, in part, accomplished by the ST-induced increase in the overall efficiency with which dietary amino acids are used for protein deposition (18, 20). ST administration also decreases blood urea nitrogen concentrations (4, 15, 45, 48) and whole body leucine oxidation (45), suggesting a reduction in amino acid catabolism.

Most research in ST-deficient (2, 19, 37), as well as normal, mature animals and humans (1, 7, 18, 24, 25, 35) suggests that ST treatment increases protein deposition by stimulating whole body and muscle protein synthesis. For example, acute ST infusion in adult humans increases limb protein synthesis (24, 25), although differing results have also been reported (11). Chronic ST treatment in cattle increases amino acid uptake by the hindquarter (4) and protein synthesis in skeletal muscle (18). Less is known about its effects on protein degradation, particularly at the tissue level. The equivocal findings of the ST-induced effects on protein synthesis and protein degradation in previous studies may be due to different developmental stages of the subject population (i.e., growing vs. mature), species studied, nutrient status of the subject (i.e., fasted or fed), tissues analyzed, and length and/or mode of ST treatment (4, 5, 17, 24, 25, 30, 37, 38, 42, 45).

Recently, studies in our laboratory have suggested that ST treatment for 7 days in young, growing swine enhances metabolic efficiency by minimizing protein loss during fasting and maximizing protein gain during meal absorption (45, 46). These studies demonstrated that ST treatment improves protein balance by maintaining higher rates of whole body protein synthesis in the postabsorptive condition (45). Feeding increases whole body protein synthesis, but the feeding-induced increase in protein synthesis is not significantly greater in ST-treated than in control pigs (46). Whole body protein degradation rates in the fed condition are reduced with ST treatment, thereby enhancing whole body protein balance. Thus the results suggest that ST treatment attenuates the reduction in whole body protein synthesis that occurs with fasting, further reduces whole body proteolysis that occurs with feeding, and reduces amino acid catabolism in both the postabsorptive and postprandial states. The conservation of amino acids results in an improvement in protein balance and likely contributes to the reduction in circulating amino acid concentrations that we have observed in fully fed ST-treated pigs.

In the current studies, we wished to identify the tissue-specific responses of protein synthesis and protein degradation to 7 days of exogenous ST treatment in fully fed, young, growing swine. Amino acid kinetics were measured in vivo in the hindquarter (HQ) and portal-drained viscera (PDV) with a dual stable isotope tracer/mass transorgan balance technique. Studies were performed in rapidly growing pigs (~25 kg) in which protein intake and ST treatment were rigorously controlled over a 7-day treatment period and during a 6-h isotope infusion study to ensure fully fed and steady-state conditions. The effects of ST treatment on whole body phenylalanine oxidation were reported in a recent paper on ureagenesis (6), but for completeness, oxidation data are presented herein together with data for whole body phenylalanine turnover.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

Animals and dietary intake. The protocol, previously described by Bush et al. (6), 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. Housing and care of the animals conformed to US Department of Agriculture guidelines. Twenty crossbred (Landrace × Yorkshire × Hampshire × Duroc) female pigs were purchased from the Agriculture Headquarters at the Texas Department of Criminal Justice, Huntsville, TX. The pigs were received at the Baylor College of Medicine Animal Facility at 8-10 wk of age, weighing ~10 kg, and were housed in individual cages. During the 2-wk acclimation period, pigs were fed a 24% high-protein dry diet (Producers Cooperative Association, Bryan, TX) at a rate of 6% of body weight per day. Pigs were weighed every other day to calculate feed intake at 6% body weight, thus ensuring that ~90% of ad libitum intake for pigs of this age was consumed. Pigs were offered food in two equal amounts (one-half the total amount of feed, twice daily) each day at 0800 and 1500. Pigs generally consumed all of the food presented to them; any unconsumed food was accounted for when estimating daily food intake and feed efficiency. Water was continuously available. The pigs received ~1,750 kJ · kg¹ · day¹ metabolizable energy in the dry matter diet, which consisted of protein (263.5 g/kg from soybean meal), carbohydrate (470.9 g/kg from crude fiber), fat (69.6 g/kg from soybean oil, rice, and corn), vitamins (0.436 g/kg), minerals (17.264 g/kg), ash (76.010 g/kg), and moisture (89.201 g/kg).

Surgery. After the 2-wk orientation period, pigs were fasted overnight, and the carotid artery, jugular vein, hepatic portal vein, inferior caudal vena cava (inferior to the renal veins and superior to the common iliac vein), and duodenum were catheterized using sterile techniques and general anesthesia, as previously described (13). The catheters were flushed with 100 U heparin/ml saline and tied off to prevent discharge. Perivascular flow probes (Transonic Systems, Ithaca, NY) were secured around the hepatic portal vein and inferior caudal aorta, adjacent to the vena cava catheter insertion site, for measurement of blood flow. Catheters and flow probes were externalized and enclosed within a pocket of a specialized swine jacket (Lomir Biomedical, Harvard Apparatus, Holliston, MA).

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Experimental design. Pigs were weight-matched and randomly assigned to either the control (saline, n = 10) or recombinant porcine ST group (n = 10) (Southern Cross Biotech, Australia). The ST was administered at a concentration of 150 µg · kg¹ · day¹ for a 7-day period. This dose has been shown to be effective in increasing protein deposition and reducing blood urea nitrogen in domestic animals (4, 9, 26, 45, 46, 48). The dose of ST was divided into two equal daily injections and administered into alternating HQ musculature concurrently with the feeding sessions. Control pigs received equal volume injections of saline. To minimize the confounding effect of differences in feed intake, control pigs were pair-fed to the level of their respective weight-matched ST-treated counterparts during the 7-day treatment period.

Infusions. On the morning of infusions, overnight-fasted pigs were given their final injection of ST (150 µg · kg¹ · day¹) and secured in a swine hammock (Walter Terry Distributor, Houston, TX). To ensure that pigs were in the fully fed state throughout the infusion period, control and ST-treated pigs were infused intraduodenally for 7 h with the nutrient solution (11 ml · kg¹ · h¹; 37.5 kJ · kg¹ · h¹, 0.54 g amino acid · kg¹ · h¹) beginning 1 h before the onset of the tracer infusion. To estimate CO₂ production rate, a primed (7.5 µmol/kg), continuous (10 µmol · kg¹ · h¹) intravenous infusion of NaH¹³CO₃ (Cambridge Isotope Laboratories, Andover, MA) was performed from 0 to 120 min. Arterial whole blood (1.0 ml) and breath samples were obtained at baseline and every 15 min throughout the 2-h NaH¹³CO₃ infusion for analysis of steady-state CO₂ production rate.

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Hormone and substrate concentrations. Heparinized blood (1.0-ml) samples were obtained and centrifuged at 2,500 g for 15 min at 4°C, and the plasma was stored at 80°C until analyzed for IGF-I and PUN concentrations. Plasma IGF-I concentrations were analyzed in duplicate via two-site immunoradiometric assay with prior extraction (Diagnostic Systems Laboratories, Webster, TX). PUN concentrations were analyzed in duplicate via an end-point colorimetric assay in which urease reacts to generate ammonia, which then reacts with bromophenol blue (Vitros Chemistry Products, Johnson & Johnson Clinical Diagnostics, Rochester, NY). Blood glucose concentrations were rapidly analyzed by a glucose oxidase reaction (YSI 2300 STAT Plus, Yellow Springs Instruments, Yellow Springs, OH).

Analysis of tracer enrichment. Blood and breath samples for ¹³CO₂ production were analyzed using isotope ratio mass spectrometry (IRMS; ANCA, RoboPrep-G, Europa Instruments, Crewe, UK). Briefly, to estimate ¹³CO₂ production rate, an aliquot of whole blood (1.0 ml) was placed in a 10-ml vacutainer (Becton Dickinson, Franklin Lakes, NJ) containing 1.0 ml of perchloric acid (10% wt/wt), gently mixed, and placed on ice for 60 s. Room air was filtered via a soda lime filter (Sodasorb, Grace Container Products, Lexington, MA) to obtain air devoid of CO₂. The air (~8 ml) was injected into the 10-ml vacutainer containing 1:1 whole blood-perchloric acid. Eight to ten milliliters of gas were withdrawn and transferred to a sterile 10-ml vacutainer for subsequent analysis of isotopic enrichment of ¹³CO₂ via continuous flow GCIRMS. To analyze expired ¹³CO₂, breath samples were collected from the pig via a two-way valve breath bag for 30 s. The collected expired air was injected into a sterile 10-ml vacutainer for subsequent analysis of isotopic enrichment of ¹³CO₂ via IRMS.

Calculations. A schematic model of phenylalanine kinetics across the HQ and PDV is shown in Fig. 1. Calculations for whole body phenylalanine turnover and phenylalanine kinetics across the HQ and PDV are provided in the APPENDIX.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the metabolic fate of systemic phenylalanine in the hindquarters (A) and systemic and enteral phenylalanine in the portal-drained viscera (B). A, arterial flux of phenylalanine; A', dietary intake of phenylalanine; B, venous outflow of phenylalanine; C, tissue utilization of phenylalanine; D, oxidation of phenylalanine; E, phenylalanine used for protein synthesis; F, phenylalanine released from protein degradation; G, nonmetabolized systemic phenylalanine; G', nonmetabolized systemic and dietary phenylalanine; and H, recycled phenylalanine in the systemic circulation derived from dietary phenylalanine intake.

Statistics. Individual t-tests were performed on the data to detect significant differences among treatment groups for phenylalanine kinetics across the HQ and PDV. ANOVA with repeated measures was used to detect changes with treatment during sampling over the 7-h infusion for hormone and substrate concentrations and isotopic enrichments. Three control pigs and one ST-treated pig died during the treatment period because of complications following surgery; therefore, the resulting sample size was seven for the control group and nine for the ST-treated group. Results are presented as means ± SD. Probability values of P  0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

Animal growth. Body weight did not differ significantly (P = 0.2) between ST-treated and control pigs before the treatment period began (17.1 ± 1.6 vs. 15.6 ± 1.7 kg, respectively). At the end of the 7-day treatment period, the body weight of the ST-treated pigs was significantly (P < 0.05) greater than that of the control pigs (22.1 ± 2.2 vs. 19.5 ± 2.3 kg, respectively). The weight-scaled average daily gain tended to be higher (P = 0.1) in the ST-treated than in the control pigs during the 7-day treatment period (33.4 ± 6.2 vs. 27.2 ± 11.5 g · kg¹ · day¹, respectively). Feed efficiency, as reflected by the gain-to-feed ratio, during the 7-day treatment period was not statistically different (P = 0.2) between ST-treated (0.56 ± 0.10 g gain/g intake) and control pigs (0.48 ± 0.18 g gain/g intake).

Hormone and substrate concentrations. To verify the effectiveness of ST treatment in growing pigs, circulating concentrations of IGF-I and PUN were determined. As expected, IGF-I concentration was significantly (P < 0.001) higher (+464%) in the ST-treated group than in controls (352.0 ± 126.2 vs. 62.3 ± 24.1 ng/ml, respectively). There was a significant (P < 0.001) decrease (46%) in PUN concentrations after 7 days of ST treatment compared with controls (9.7 ± 2.3 vs. 18.2 ± 2.7 mg/dl, respectively). Blood glucose concentrations increased (+25%) significantly (P < 0.04) in the ST-treated group compared with controls (151.4 ± 31.3 vs. 120.2 ± 9.2 mg/dl, respectively).

Phenylalanine and tyrosine concentrations. Phenylalanine concentrations were lower in ST-treated than in control pigs in the arterial (8%), portal venous (9%), and vena cava (11%) circulation (Table 1). Tyrosine concentrations were also lower in ST-treated than in control pigs in the arterial (150%), portal venous (170%), and vena cava (176%) circulation.

Whole body phenylalanine turnover. Isotope-labeled tracers (i.e., [1-¹³C]- and [²H₅]phenylalanine and NaH¹³CO₃) were utilized to estimate whole body phenylalanine turnover under steady-state conditions. There was no significant difference in whole body phenylalanine flux when calculated with either the [1-¹³C]- or the [²H₅]phenylalanine tracers. Therefore, arterial [1-¹³C]phenylalanine was used to estimate whole body phenylalanine flux. There was no significant difference (P > 0.1) between the isotopic enrichment of ¹³CO₂ in expired breath and that of ¹³CO₂ in arterial blood in either treatment group during metabolic steady state of NaH¹³CO₃ infusion (75-120 min) (6). Therefore, we used ¹³CO₂ in expired breath to estimate whole body CO₂ production rate (see APPENDIX) during metabolic steady state of NaH¹³CO₃ infusion (75-120 min). Isotopic enrichments of ¹³CO₂ in expired breath and [1-¹³C]phenylalanine in arterial blood during the phenylalanine infusion (270-360 min) plus the CO₂ production rate were used to estimate whole body phenylalanine oxidation (see APPENDIX).

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View larger version (28K): [in this window] [in a new window]   Fig. 2.   Whole body phenylalanine turnover in fed somatotropin-treated and control pigs. Values are means ± SD in control (n = 7) and somatotropin-treated (n = 9) pigs. * Different from control, P  0.05.

Phenylalanine kinetics across the HQ. Isotope-labeled tracers (i.e., [²H₅]phenylalanine and NaH¹³CO₃) and caudal aorta blood flow measurements were utilized to estimate phenylalanine kinetics across the HQ during isotopic steady state (Fig. 1). Isotopic steady state was achieved in the artery and vena cava for [²H₅]phenylalanine (Fig. 3). Blood flow to the HQ was significantly (P < 0.01) increased (+63%) with ST treatment compared with controls (Table 2). ST treatment increased (P < 0.03) mass balance in the HQ, and thus the utilization of phenylalanine by the HQ was significantly (P < 0.01) increased (+44%). The extraction of phenylalanine by the HQ was similar between treatment groups (ST, 0.17 ± 0.04 vs. control, 0.23 ± 0.03 µmol · kg¹ · h¹ uptake for µmol · kg¹ · h¹ input). The majority of the phenylalanine extracted by the HQ was retained for protein synthesis and deposition, mediated largely by the significant increase in blood flow, not extraction rate. Although we did not quantify the rate of phenylalanine conversion to tyrosine, the net release of ¹³CO₂ by the HQ was negligible. The rate of utilization of phenylalanine for protein synthesis in the HQ was significantly (P < 0.01) greater (+43%) in the ST-treated pigs than in controls. The calculated rate of proteolysis was not significantly affected by ST treatment in growing pigs. ST treatment increased (+120%; P < 0.03) protein deposition rates compared with controls. There was a positive net protein balance in both the ST and control pigs, as expected in pigs studied in the fed state, owing largely to the significant increase in protein synthesis. Thus 7 days of ST treatment significantly increased the utilization of amino acids by the HQ, resulting in an increase in protein deposition.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Isotopic enrichments (IE) of arterial (A), portal venous (B), and vena cava (C) [1-13C]- and [2H5]phenylalanine (Phe) at steady state during [1-13C]- and [2H5]phenylalanine infusions. Duration of tracer infusion was 120-360 min, with steady-state conditions reached between 270 and 360 min. APE, atom percent excess.

Phenylalanine kinetics across the PDV. Isotope-labeled tracers (i.e., [1-¹³C]- and [²H₅]phenylalanine and NaH¹³CO₃) and portal venous blood flow measurements were utilized to estimate phenylalanine kinetics across the PDV (Fig. 1). Portal and arterial isotopic steady state was achieved for [1-¹³C]- and [²H₅]phenylalanine (Fig. 3). In contrast to the HQ, portal venous blood flow was unaffected by ST treatment in growing pigs (Table 2). Phenylalanine utilization by the PDV was estimated from combined enteral and arterial sources of phenylalanine (Fig. 1). The phenylalanine tracer was infused intraduodenally and had the potential to recycle to the PDV via the systemic circulation. Thus the phenylalanine kinetics across the PDV were corrected for recycling of the [1-¹³C]phenylalanine tracer by accounting for the fractional uptake of the [²H₅]phenylalanine tracer infused systemically, assuming similarity in kinetics between the two different phenylalanine tracers. In both ST-treated and control pigs, 35-45% of the enteral source of phenylalanine (206 µmol · kg¹ · h¹) was utilized by the mucosa. The remaining 55-65% of enteral phenylalanine was absorbed and available for the remaining tissues in the body. Approximately 10% of arterial phenylalanine influx or input was utilized by the PDV. There was no significant effect of ST treatment on PDV mass balance with ST treatment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

The results of this study indicate that 7 days of exogenous ST administration in growing pigs enhanced protein anabolism in the fed state by altering protein turnover in the whole body and amino acid kinetics in the HQ and PDV. ST treatment in growing pigs reduced whole body phenylalanine flux, oxidation, and protein degradation without altering whole body protein synthesis. In the HQ and PDV, ST treatment increased protein deposition by increasing the utilization of phenylalanine for protein synthesis, but it had no effect on protein degradation or oxidation rates. Given the fact that protein synthesis rates exceeded proteolysis rates, a positive net protein balance was attained in both treatment groups at both the whole body and tissue-bed level in young, growing swine in the fed state.

Whole body protein turnover effects of ST. In the current study, ST treatment for 7 days in growing swine elicited the metabolic responses that are characteristic of ST treatment in domestic animals. ST treatment increased body weight and improved the efficiency (+35%) with which dietary phenylalanine was utilized for whole body protein deposition and growth, as demonstrated in numerous studies (7, 9, 20, 21, 38, 45, 47). ST treatment also produced the characteristic stimulation of the somatotropic axis (4, 9, 19, 45, 47, 49), as indicated by a fivefold elevation in IGF-I concentration, and a diabetogenic response (10, 16, 46, 48), as indicated by the rise in plasma glucose concentration.

Evaluation of the dual stable isotope tracer/mass transorgan balance technique. Several assumptions were made with this study design. First, we assumed that the metabolism throughout the body of the [1-¹³C]- and [²H₅]phenylalanine tracers would not be different simply because of their isotopic label (i.e., no isotope effect). Second, we have assumed that all [1-¹³C]phenylalanine taken up by the tissues (either HQ or PDV) and not oxidized to CO₂ was incorporated into protein. A consequence of this assumption was that we might have underestimated the rate of irreversible loss of phenylalanine, since the rate of conversion to tyrosine was not quantified.

Amino acid kinetics in the HQ. A major objective of this study was to identify the responses of protein synthesis and degradation in the HQ to 7 days of exogenous ST administration. In the process of measuring the phenylalanine kinetics by the HQ, we observed that ST markedly increased blood flow to the HQ by 80%. The substantial increase in blood flow contributed to the increase in the amount of phenylalanine utilized by the HQ in ST-treated pigs. This finding is consistent with previous studies and is believed to be a function of local tissue metabolism (4, 16, 22, 24, 25, 33, 45). The localized increase in blood flow to the HQ may be mediated also by the vasodilator properties of IGF-I (28), which can stimulate the production of endothelial nitric oxide and induce endothelium-dependent vasodilation (3, 23, 28).

Amino acid kinetics across the PDV. A secondary objective of this study was to compare amino acid kinetics in the HQ with those in the PDV. To determine amino acid metabolism in the PDV, we infused two separate isotope tracers of phenylalanine via the intraduodenal ([1-¹³C]phenylalanine) and intravenous ([²H₅]phenylalanine) routes. We did this because the intraduodenally infused [1-¹³C]phenylalanine tracer, once absorbed through the small intestine, is recycled back to the PDV through the systemic circulation.

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Perspectives. Few in vivo studies have been performed to estimate amino acid kinetics in different areas of the body by use of the dual stable isotope tracer/mass transorgan balance technique that was utilized in this current study. The technique allowed us to investigate the tissue-specific metabolism of an individual essential amino acid in the fed state in pigs administered ST for 7 days. On the whole body level, we observed a decrease in total phenylalanine flux, oxidation, and protein degradation with ST treatment that was similar to our previous results with a leucine tracer (45). However, at the individual tissue level, these observations were somewhat different. The results show that ST increased the utilization of phenylalanine for protein synthesis by the HQ and PDV. The increase in amino acid utilization and protein synthesis in the HQ, but not in the PDV, was mediated largely by the regional increase in blood flow. Interestingly, recent studies have shown that adipose tissue protein synthesis is highly responsive to nutrient stimulation (31, 32). Because ST induces a repartitioning of nutrients away from adipose tissue and toward lean tissue deposition (19), we postulate that the lack of ST-induced stimulation of whole body protein synthesis in the fed state may, in part, be due to an ST-induced suppression of the feeding-induced stimulation of protein synthesis in adipose tissue. However, other mechanisms may be involved. We further found that ST treatment had no effect on protein degradation in either the HQ or PDV. Whether the reduction in whole body degradation with ST treatment can be accounted for by a reduction in proteolysis in liver and other visceral tissues remains to be determined. Phenylalanine was not oxidized by the HQ and thus was unaffected by ST treatment, and although a significant portion of phenylalanine utilized by the mucosa was oxidized, this was unaffected by ST treatment. Because ST treatment does not alter urea cycle enzyme activity in the intestine but reduces urea cycle activity in the liver (6), we postulate that the reduction in whole body phenylalanine oxidation is due largely to a reduction in phenylalanine oxidation by the liver.