Growth Hormone Increases Whole-Body Protein Turnover in Growing Pigs' L

F. M. Tomas, R. G. Campbellz, R. H. Kings, R. J. Johnsons, C. S. Chandler, and M. R. Taverners

CSIRO, Division of Human Nutrition, Adelaide, South Australia 5000

ABSTRACT: Ten pigs with a n average initial live weight of 65 kg were used to investigate the effects of daily exogenous porcine pituitary growth hormone (pGH; . 1 mg .kg-l. d-ll for a 13-dperiod on N retention and whole-body protein turnover. Feed intake was restricted to both the control (treated with excipient) and pGH-treated groups to ensure that animals in each group consumed equal amounts. Whole-body protein turnover was estimated from the excretion of 15N in urinary urea and ammonia after a single oral dose of [15Nlglycine.Nitrogen balance and whole-body N

flux were increased by 35 to 40% with pGH treatment (P c .0011. Protein synthesis and breakdown were increased by 56 and 59% (P c . O O l l , respectively, in pGH-treated pigs relative to controls. These higher rates of protein turnover seemed to lower slightly the efficiency of the metabolic process for protein deposition. However, the absolute increment in protein synthesis rate was greater than that for breakdown, leading to the increased net N retention. Thus, pGH treatment improved the utilization of dietary amino acids for protein deposition.

Key Words: Pigs, Somatotropin, Protein Turnover, Nitrogen Balance J. Anim. Sci. 1992. 70:3138-3143

Introduction The metabolic effects of porcine growth hormone (pGHI lead to effective partitioning of nutrients between tissues. Thus, exogenous pGH administration enhances protein accretion and reduces fat deposition (Campbell et al., 1988, 1989; Boyd and Bauman, 1989; Verstegen et al., 19901. The mechanism for the observed effects on lipid deposition have been investigated and characterized as predominately a reduction in lipogenesis (Walton and Etherton, 1986; Walton et al., 1987; Pell et al., 1990; Dunshea et al., 1992). However, little information exists on the mechanisms leading to increased protein deposition. Pell and Bates (1987) administered recombinant growth hormone

'The authors are grateful to Shari Madden and Judy Burgoyne for their skilled laboratory work and to the staff a t the Victorian Dept. of Agric. and Rural Affairs, h i m . Res. Inst., Werribee, for their assistance with the experimental animals. 2Bunge Meat Industries Limited, P. 0. Box 78, Corowa, New South Wales 2646, Australia. 3Animal Research Institute, Werribee, Victoria 3030,Australia. Received November 22, 1991. Accepted May 19, 1992.

to lambs and showed increased protein synthesis rates in muscle. Net deposition of protein was increased because the increase in protein synthesis rates exceeded the concomitant increase in protein breakdown rates. These results were confirmed with acute studies in lambs using ovine growth hormone (Crompton and Lomax, 1989). Thus, the mechanism of the protein anabolic effects of growth hormone in the muscle of lambs seems to be different from that of other anabolic agents such as P-adrenergic agonists and many steroid-based compounds, which act principally via reducing the rates of protein breakdown (Buttery and Dawson, 19881. The anabolic effects of exogenous pGH in pigs are unequivocal, but there is no published information on the changes in protein synthesis and breakdown that lead to this response. This information is important to increase understanding of the action of pGH and for predicting the likely effect of its use in production on the dietary N and amino acid requirements for optimum growth. The objectives of this study were to characterize the effects of pGH on N metabolism and wholebody protein turnover in pigs under the defined conditions of matched feed intakes.

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GROWTH HORMONE AND PROTEIN TURNOVER IN PIGS

Materials and Methods Ten Large White gilts were randomly allocated at 65 kg Orange 62 to 67 kg) between two pGH treatment regimens, 0 and .1 mg of pituitary derived pGH.kgl.d-l. The pigs were kept in metabolism cages and the pGH (USDA-pGH-B14) was administered daily for 13 d a t 0730 by intramuscular injection as described by Campbell et al. (1989). The control group received a n injection of the excipient only (NaC1, 154 mM; Na&03, 25 mM; NaHC03, 25 mM; pH 9.4). All the pigs were given 1.6 kg/d of a single diet in two equal feeds for the first 8 d of the trial and in eight equal feeds a t 0800, 1100, 1400, 1700, 2000, 2300, 0200, and 0500 for the remainder of the trial. The composition of the diet is given in Table 1. The level of intake was chosen to ensure complete consumption of the offered feed by all pigs and took into account the anticipated reduction in feed intake with pGH treatment (Campbell et al., 1989). Water was freely available. After 10 d of treatment each pig was weighed, and a two-way Foley Teflon-coated catheter (Bardco, 5.3 mm 0.d. with 30 mL balloon) was inserted into the bladder under general anesthesia (Halothane). Upon recovery, the pigs were returned to the metabolism cages. The following morning a gelatin capsule containing a n accurately weighed quantity between 300 and 320 mg) of [‘5Nlglycine (MSD Isotopes, 99 atom YO 15N) was placed in the 0800 feed of each pig and was quickly and completely consumed by the pig. Urine and feces were then collected each 12 h for 24 h and then at 24-h intervals from 24 to 72 h after the 15N dosing. Urine was collected into 20 to 40 mL of 6 N HC1 and this was adjusted as necessary on the basis of urine output to ensure a pH value of < 4. A 10% aliquot of each urine collection was taken and stored at -2OOC for further analysis. Fecal and dietary N were measured by the Kjeldahl method. Urinary total N was assayed by the Dumas technique using a Nitrogen Analyser “1500, Carlo-Erba Instruments, Milan, Italy). Urinary urea N and NH3 N were determined using a continuous flow analyzer (Skalar Analytical, Breda, The Netherlands). Urea was reacted with diacetylmonoxime in the presence of thiosemicarbizide under acid conditions essentially as described by Marsh et al. (1965) and the colored product of the reaction was measured a t 520 nm in a flow cuvette. The NH3 analysis was via a modified Berthelot’s reaction (Krom, 19801, and the

4Appreciation is extended to Douglas J. Bolt, Reprod. Lab., h i m . Sci. Inst., Beltsville, MD for his assistance in obtaining pituitaryderived pGH. Downloaded from https://academic.oup.com/jas/article-abstract/70/10/3138/4705815 by University of California School of Law (Boalt Hall) user on 28 July 2018

Table 1. Composition of experimental diet Ingredient Wheat Soybean meal Blood meal Dicalcium phosphate Fat blend L-lysine.HCI DL-methionine Salt (NaCll Mineral/vitamin premixa Nutrient compositionb Digestible energy, MJ/kg Crude protein Lysine Methionine and cystine Threonine Tryptophan Isoleucine Leucine Methionine

YO of Total 73 20 3 1.2 2.0 .2 .1 .2 .3 14.4 183 12.1 6.2 7.2

2.2 7.3 15.2 3.8

&Providedthe following nutrients (milligram/kilogram of airdry diet): retinol, 6.4; calciferol, 8.3; D-tocopherol,22; menadione, 600; riboflavin, 3.3; nicotinic acid, 18.5; pantothenic acid, 5.5; pyridoxine, 1.1; biotin, 56; choline, 110; cyanocobalamin, ,017; Fe, 88; Zn, 55; Mn, 22; Cu,6.6; I, .22; Se, .l. bGrams/kilogram except where indicated.

green-colored indophenolblue complex was measured at 660 nm. The 15N enrichments of urea N and NH3 N were measured with a n isotope ratioing mass spectrometer (VG Micromass 602El after preparation of the samples and adsorption of NH3 onto resin according to the methods described by Read et al. (1982). Briefly, free ammonium N in neutralized urine was adsorbed onto Dowex AG50W-X8 resin (NaK form). The supernatant was removed and treated with urease to convert urea to ammonium, which was adsorbed to another batch of resin. A flask containing the washed resin was attached to the mass spectrometer and nitrogen evolved for analysis from the resin complex with 40% sodium hypobromite. Calculations of N flux (or turnover), protein synthesis, and protein breakdown for the whole body were based on the single-dose 15N end product method and the procedures followed were essentially as described by Fern et al. (19811,which have also since been applied to pigs by Salter et al. (1990). Cumulative excretion of 15N in each of the end products of ammonia and urea was measured in urine collected over the first 12 and 48 h, respectively, after dosing with I15Nlglycine. The rate of nitrogen flux, Q (grams/l2 hl, was calculated from the equation Q = E,.d/e,, where Ex is the rate of excretion of ammonia or urea N Igrams/l2 h),d is the dose of 15N (grams), and e, is the amount of isotope excreted in the urine as urea or ammonia (grams). Rates of whole-body protein (N x 6.25) breakdown and synthesis were

TOMAS ET AL.

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Table 2. Effects of exogenous porcine growth hormone (pGH; .1 mg-kg-lsd-') on average [* SEM) nitrogen excretion and balance (g of N/d) during days 11, 12, and 13 of treatment

Variable Intake* Feces Urine Urea Balance Urea N/urine Nb

48.24 4.54 22.88 20.99 18.84 .92

.42 .83 .58 f 1.0 f .02 f f f

46.24 3.53 17.71 14.52 25.00 .82

f f f f f

Probability of no difference

PGH

Control

PGH

Control

-

1.00 .78 .77 .69 1.33 .E9

.70 .63 .56

.82 .01

NSC < .001 .05. Downloaded from https://academic.oup.com/jas/article-abstract/70/10/3138/4705815 by University of California School of Law (Boalt Hall) user on 28 July 2018

ACCRETION

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TOMAS ET AL.

Because our study was intended to examine specifically the effects of pGH administration per se on protein turnover, the pigs were given a restricted ration, which was designed to prevent indirect perturbations of protein metabolism arising from the pGH-induced reduction in feed intake. The pigs received only approximately 82% of ad libitum intake, and this would have been a major factor leading to the relatively smaller response in N retention compared with earlier studies (Campbell et al., 1989). However, the dietary protein level was relatively high and unlikely to be limiting the growth response (Campbell et al., 1990, 1991). Although it seems likely that the response in protein turnover indicates changes that would occur under conditions of ad libitum intake, further investigation is required to define these relationships under conditions in which energy intake is not limiting. These data show a large difference in the N flux determined from the two excretory end products of I5N, ammonia and urea, This may have arisen as a consequence of a specific compartmentation of glycine metabolism in these rapidly growing pigs, which could result in differential labeling of the urea and ammonia (Fern et al., 1981; Jackson et al., 1981). Another possibility related to these potential metabolic differences is that the growing pig, unlike the adult human, may not excrete the majority of 115NINH3within the first 12 h after the dose of [15Nlglycine (Waterlow et al., 1978). Evidence for this was recently reported by Salter et al. (19901, who found that the plateau for urinary [15NINH3 cumulative excretion did not occur until 1 24 h after 115Nlglycinedosing. Thus, it is possible that our 12-h sample contained a relatively low enrichment of 15N, which would lead to a n overestimate of the calculated flux rate. We have not been able to clarify this further in this experiment because of lack of samples. However, although the flux rates determined from ammonia excretion were higher than those obtained from urea, the same relative response to pGH treatment was obtained from each end product. We chose to include the flux rates based on [15NINH3 in our calculations of protein turnover (Table 3) because we had no direct evidence for their exclusion and also because it made no difference to the interpretation of the results in terms of the effects of pGH on protein turnover. It should be noted, however, that our data obtained from [15Nlurea excretion in the control pigs agree closely with those of Salter et al. (19901 obtained from pigs given adequate nutrition both in terms of cumulative excretion of the label (Figure 1) and in flux rates (1.40 vs 1.42 g N . kg of BW-l. d-l, respectively). For this reason we have also shown the protein turnover rates calculated using only the flux rates derived from 115Nlurea excretion (Figure 2). Downloaded from https://academic.oup.com/jas/article-abstract/70/10/3138/4705815 by University of California School of Law (Boalt Hall) user on 28 July 2018

An increase in the rate of protein deposition occurs if the gap between protein synthesis and degradation rates increases positively. Achieving this positive change through a n increase in both metabolic processes necessarily incurs a cost in terms of energy and of amino acid oxidation and modification. This does not necessarily mean that the added costs are disproportionate to the increase in protein accretion rate, a n aspect that has been discussed by Verstegen et al. (1990) and Reeds and Mersmann (1991). However, our data show that the increment in N retention that accompanies the increments in synthesis and degradation during pGH treatment is roughly onehalf of that predicted from the ratio of these processes observed in the control pigs. Thus, the marginal increase in N retention accruing from pGH treatment is achieved at a relatively lower energetic efficiency than that of the baseline level of protein metabolism measured in the untreated control pigs. This is consistent with the energy metabolism data reported by Verstegen et al. (19901, which indicate that the partial efficiency of energy use for protein deposition is reduced by growth hormone treatment. Of course, the overall conversion of feed N to N gain is increased by pGH treatment in line with improved feed/gain performance (e.g., Campbell et al., 1989). Administration of growth hormone to pigs has been shown to increase circulating insulin-like growth factor I (IGF-I) levels (e.g., Etherton et al., 1987; Owens et al., 1990). Although it is generally accepted that IGF-I is a major mediator of the somatic response to growth hormone, the increased degradation rates in response to growth hormone treatment seen here and in the muscle of sheep (Pel1 and Bates, 1987; Crompton and Lomax, 19891 are not consistent with the reduction in degradation rates observed after the administration of exogenous IGF-I to rats (Jacob et al., 1989; Tomas et al., 1990, 1991a,b) or to cells in vitro (Ballard et al., 1980; Francis et al., 1988). Perhaps the more generalized actions of growth hormone cause metabolic adjustments that predominate over more organ-specific effects of IGF-I (Skottner et al., 19871.

Implications Our experiment shows that the marked increase in nitrogen retention in pigs after porcine growth hormone treatment results from an even greater relative increase in whole-body protein turnover. Knowledge of the nature of this response and its effect on amino acid requirements should allow better formulation of nutritional support for growth hormone-stimulated growth.

GROWTH HORMONE AND PROTEIN TURNOVER IN PIGS

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