c mice treated with growth hormone

Comparative Biochemistry and Physiology Part A 132 (2002) 247–256

Review

Body growth and substrate partitioning for fat and protein gain in weaned BALByc mice treated with growth hormone ´ ˜ ´ A. Agis-Torres*, M.E. Lopez-Oliva, M.T. Unzaga, E. Munoz-Martınez ´ Departamental de Fisiologıa, ´ Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain Seccion Received 3 August 2001; received in revised form 4 February 2002; accepted 6 February 2002

Abstract Previously we have found that recombinant human growth hormone (rhGH) (GH; 74 ng g body wt.y1) administration to weaned BALByc male mice (fed 12% or 20% protein diet) induced a growth lag and subsequent repletion similar to the catch-up growth process. We studied the partitioning of feed and protein intakes between adipose and protein body stores through the linear relationships among them. The non-linear relationship of protein intake with body fat gainy protein gain (FGyPG) ratio was especially adequate in determining the partitioning of substrates. rhGH induced an increase in feed and protein intake utilization for body weight gain (50%) and fat gain (75–140%) over saline; macronutrient utilization was the greatest in rhGH-treated mice fed 20% protein. However, growth recovery of rhGH mice was anomalous and protein intake was derived primarily for fat gain. Mice fed 12% protein (treated and control) also derived protein intake in preference to fat stores. Treatment and diet had a cumulative effect with the result that rhGH-treated animals fed 12% protein showed the greatest FGyPG ratio (1.6), and therefore, the lowest efficiency to gain protein. Weaning is a critical stage in mice when treating with rhGH, as this could provoke a growth lag. The study showed that a high protein level is required to surpass the rhGH-induced lag, but it is not enough to obtain an enhanced protein deposition. Feeding a 12% protein diet was even worse as mice did not improve on the growth lag and substrates were directed mainly to body fat. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Growth hormone; Nutrient partitioning; Protein-energy malnutrition; Weaning; Catch-up growth; Mice

1. Introduction Partitioning agents, such as growth hormone (GH) or b-adrenergic agonists change the flow of dietary energy for fat synthesis to protein synthesis (Bergen and Merkel, 1991) resulting in an improved lean to fat ratio. GH administration causes vast anabolic effects on muscle and carcass such as dramatic reduction of fatty acid synthesis in adipose tissue and increased protein deposition (Etherton, 2000; Douglas et al., 1991). GH and *Corresponding author. Tel.: q34-91-394-1838; fax: q3491-394-1838. E-mail address: [email protected] (A. Agis-Torres).

insulin-like growth factor (IGF)-I constitute the somatotropic (or GH-IGF-I) axis (Butler and Le Roith, 2001), whose integrity is necessary for normal growth and which is highly sensitive to changes in nutritional status (Brameld et al., 1999). The interaction between nutritional status and GH plasma concentration has been recognized in many animal species. However, the pattern of GH secretion with a superimposed low nutritional status is different among species (Vance et al., 1992); thus protein deprivation nearly abolishes secretion of the hormone in rats (Harel and Tannenbaum, 1993), whereas malnourished pigs presented an

1095-6433/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 0 8 5 - 5

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elevation of serum concentrations of GH (Kasser et al., 1981). However, hepatic GH receptor and IGF-I gene expression appear to be reduced in pigs (Brameld et al., 1996) and rats (Straus and Takemoto, 1990) suffering from low nutritional status. It has been demonstrated that body composition and protein accretion rate in GH-treated animals depend on intake of both energy and protein (Campbell et al., 1991), since an insufficient supply of nutrient could suppress the capacity ` et al., 1993), of cells to synthesize protein (Seve thereby conditioning the anabolic effect of the hormone. Brameld et al. (1996) attributed to energy, in the form of glucose, the capacity to control expression of GH receptor, interacting with the effects of glucocorticoids and thyroid hormones; and to protein the control of GH-stimulated IGF-I expression. The capacity of GH to redirect nutrients towards specific body stores, as well as the nature of the deposit itself, is highly dependent on the developmental stage of the animal (Turner et al., 1998). Etherton and Bauman (1998) signaled that the increase in growth rate and the effects on protein and lipid deposition in animals treated with GH are significantly greater in the later stage of growth. However, developmental regulation of IGF-I and GH receptor appears to be tissue specific with different patterns of expression, as Shoba et al. (1999) found in rats from 17 days of gestation to 17 months of age. Weaning is a crucial stage with drastic nutritional and hormonal changes. An abrupt reduction in voluntary feed intake and body growth has been found in weaning piglets, which results in a weaning growth lag (LeDividich and ` Seve, 2000). The weaning growth lag seems to be related to the hormonal and nutritive status of animals, as weaned piglets show a well-characterized endocrine profile with high serum GH levels and low serum concentration of IGF-I and IGF-II; this profile has also been observed during undernutrition or feed deprivation (Matteri et al., 2000). Previously we found that exogenous administration of recombinant human growth hormone (rhGH) to weaned BALByc male mice induced a biphasic response on feed intake and body weight, resulting in the delay of the growth process from ´ 25 to 30 days of age (Lopez-Oliva et al., 2000). During the second stage (from 35 to 50 days of age), hyperphagic behavior allowed the mice fed a 20% protein diet, but not the mice fed a 12% protein diet, to recover their body weight to control

values. Nevertheless, rhGH-treated male mice fed 12% or 20% protein increased their body fat gain at the end of the experiment by approximately 40% and 90%, respectively, over control mice, indicating an anomalous nutrient partitioning (unpublished results). The characteristic GHinduced nutrient redistribution in normal animals is expected to enhance both dietary protein and dietary energy efficiencies for lean deposition (Etherton and Bauman, 1998). The purpose of this work was to determine the effects of exogenous rhGH administration to BALByc male mice fed 12 or 20% protein diets on the modification of body substrate distribution during the growth lag and subsequent recovery of body weight described above. This was achieved through the study of the efficiency of utilization of protein and energy intake to body fat and protein gain. In addition, we used the relationship between body fat gainyprotein gain (FGyPG) ratio vs. protein intake to ascertain the priority for directing the substrates towards one or other deposit. 2. Materials and methods 2.1. Experimental design The animal protocol was approved by the Ethical Animal Experimental Committee of the Faculty of Pharmacy, Universidad Complutense de Madrid, Spain. One hundred and twenty weaned (21 days of age) male BALByc mice (Faculty of Pharmacy, Breeding Unit) were caged individually in metabolic cages maintained at a temperature of 23"1 8C with 12-h lightydark cycles. Mice were assigned randomly to four groups of 30 mice each: fed either 12% or 20% protein (casein) isocaloric diets (isocaloricity was achieved by varying the sucrose content of the diets) supplemented with 3 g D,L-methionine per kg diet, and administered with either saline (s; 9 gyl NaCl) or rhGH (GH; 74 ng g body wt.y1, in 20 ml of saline) (donated by Novo Nordisk Pharma S.A., Madrid) every 2 days. Feed and water were available ad libitum. Body weight and feed intake were recorded daily. Diets were formulated according to the American Institute of Nutrition recommendations (Reeves et al., 1993) and analyzed following the methods of the Association of Official Analytical Chemists (AOAC, 1990). Six partial experimental periods were established within the total experimental period of 29 days. The initial day of every partial

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period started at 21 days of age, whereas the final days were at age 25, 30, 35, 40, 45 or 50 days. Thus, the duration of the partial experimental periods was 4, 9, 14, 19, 24 or 29 days. On the final day of every partial experimental period, five mice from each group were killed by decapitation; viscera were removed, soaked in saline and stored in liquid nitrogen. 2.2. Measurements and calculations The eviscerated bodies of mice were frozen in liquid nitrogen and ground to a homogeneous mixture. Aliquots were used to determine dry matter, ether extract, protein and ash (AOAC, 1990). Water content was calculated as the difference between initial and final weight dried of the aliquot to constant weight at 100 8C in a dryingchamber (P-SELECTA, Madrid, Spain). Lipid extraction was performed with an automated Soxhlet system (Soxhtec System 1040, TECATOR, Sweden). The ether extract was assumed to represent the fat content of the aliquot. Carcass nitrogen was determined by a Kjeldahl automated system (Kjeltec 1030, TECATOR), and the protein content was expressed as total nitrogen=6.25. Ash was determined by burning one aliquot in a muffle furnace at 525 8C for 15 h (Select-Horn furnace, P-SELECTA, Madrid). Results from water, fat, protein and ash content of the aliquots were used to calculate absolute and relative content of the eviscerated body weight. All composition data within this study are based on the eviscerated body weight; however, the word ‘eviscerated’ may not always be explicitly stated. Feed intake (FI) (g) was the cumulative value, minus spillage, for the partial experimental period of each mouse. Protein intake (PI) (g) was obtained by multiplying the calculated feed intake by the dietary protein content of each diet (0.2 or 0.12 g per g diet). Body weight gain (BWG) was computed as the difference between the body weight at the beginning and at the end of the partial experimental period of every mouse. Allometric relationships between eviscerated body fat or protein deposits (Y) and body weight (X) were calculated for each mice group (data not shown). The allometric function (Ysa=X b) was fitted by linearizing the function in the form Logn YsLogn aqX=b. Fat and protein gains were calculated as the difference between the initial and final fat or protein content, which were

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estimated using allometric relationships. Linear relationships between body weight gain (BWG), body fat gain (FG) and body protein gain (PG) vs. feed intake (FI) and protein intake (PI) were determined to study the efficiency of FI and PI utilization to body weight gain, fat gain and protein gain. A power relationship between FG:PG ratio and PI (FG:PGsa=PIb ) was fitted by linearizing the function as Logn FG:PGsLogn aqPI=b, in order to determine the partitioning of PI between the gain of fat and protein in each group. 2.3. Statistical methods Statistical analysis and treatment of experimental data were performed with the statistical package SPSS (Version 10.0.6; SPSS Inc. Chicago, Illinois). Coefficients of determination (R 2) and P level were evaluated to judge the fit of the relationships in each group of mice to the experimental data. A given relationship was considered significant if P-0.01. Differences between the slopes of the linear or linearized relationships were analyzed by the dum´ my variables procedure (Lopez-Oliva et al., 1995). Data are expressed as mean"S.E.M. Differences between slopes were considered significant if P0.05. 3. Results The slopes of the linear relationships of body weight gain (BWG), empty body fat (FG) and protein (PG) gains with feed (FI) and protein (PI) intakes are summarized in Table 1. rhGH treatment induced an increase in the feed and protein intakes utilized to gain body fat and body protein; and as a consequence, the utilization of substrates to the increase of body weight was also enhanced (Table 1). Overall, GH-treated mice presented greater feed efficiencies than saline mice within each dietary protein level, as signaled by the slopes of BWG, FG and PG relative to FI and PI, although mice fed 20% protein diet showed greater enhancements. Thus, whereas the slopes of BWG relative to FI and PI were approximately 50% greater in GH-treated than saline mice irrespective of diet, the efficiencies of both feed intakes for FG were different, as 12GH presented approximately a 75%, and 20GH a 140% greater slope than saline mice (Table 1, Figs. 1 and 2). In addition, the slopes of FI and PI relative to PG in

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BWG vs. FI

12s 20s 12GH 20GH

BWG vs. PI

FG vs. FI

FG vs. PI

PG vs. FI

PG vs. PI

Slope

S.E.

R2

Slope

S.E.

R2

Slope

S.E.

R2

Slope

S.E.

R2

Slope

S.E.

R2

Slope

S.E.

R2

0.032 0.063 0.047 0.097

0.006a1 0.007b1 0.005a2 0.007b2

0.501 0.719 0.773 0.884

0.263 0.317 0.390 0.484

0.050a1 0.037a1 0.040a2 0.033b2

0.501 0.719 0.773 0.884

4.52 5.72 7.88 13.71

0.78a1 0.67a1 0.82a2 1.09b2

0.548 0.723 0.769 0.850

0.037 0.028 0.066 0.068

0.006a1 0.003a1 0.007a2 0.005a2

0.548 0.723 0.769 0.850

3.68 7.44 4.68 10.61

0.72a1 0.91b1 0.48a1 0.71b2

0.486 0.704 0.769 0.889

0.031 0.037 0.039 0.053

0.006a1 0.005a1 0.004a1 0.004b2

0.486 0.704 0.769 0.889

Superscripts:

a,b

(diet) and

1,2

(treatment) significantly different, P-0.05 (dummy variables). All linear relationships were significant (P-0.0001).

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Table 1 Slope, standard error (S.E.) and R 2 of the linear regression equations of body weight gain (BWG) (g), body fat gain (FG) (mg) and body protein gain (PG) (mg) vs. feed intake (FI) (g) and protein intake (PI) (g) of BALByc mice fed 12% (12) or 20% (20) protein diet and subjected to saline (s) or rhGH treatment (GH) from 21 to 50 days of age

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Fig. 1. Linear regression between body fat gain and feed intake of BALByc mice fed 12% (12) or 20% (20) protein diets and subjected to saline (s) or rhGH treatment (GH) from 21 to 50 days of age.

mice fed 12% protein diet were unaffected by treatment, while an increase of 43% in FI and PI utilization for PG was induced by GH-treatment in 20GH over 20s (Table 1, Figs. 3 and 4). These results indicated the additive effects of a higher dietary protein level and rhGH administration as both factors led to higher fat and protein gains. The 20% dietary protein level improved FI and PI utilization for BWG, FG and PG over 12% protein diet in GH-treated mice (differences between the slopes of both groups ranged from 24 to 106%), except for the PI directed towards FG which did not differ (Table 1). Only the slopes of FI with BWG and FI with PG were significantly greater in 20s over 12s mice (Table 1). Thus, a higher dietary protein level, specially in GHtreated mice, enhanced feed utilization for storage. Table 2 and Fig. 5 show the power relationship of the body fat gainyprotein gain (FGyPG) ratio with PI. Treatment with rhGH induced a clear increase of protein partitioning into body fat gain. As a result, body protein deposit was impaired in spite of body fat. Therefore, the FGyPG ratio in GH-treated mice fed both diets maintained a trend above saline mice for the whole range of PI. Thus, 20GH mice showed an initial ratio (for 2.5 g of PI) of 0.84 and a final ratio (for 22.5 g of PI) of 1.16 whereas 12GH mice had an initial ratio of

1.22 and a final ratio of 1.60, indicating the tendency to gain more fat (Table 2, Fig. 5). Moreover, the diet with the lower protein content also derived protein intake more for FG than for PG in both GH-treated and saline mice, resulting in an additive effect with GH-treatment. The groups fed 20% protein diet showed higher efficacy of protein gain compared with the 12% groups, and this effect was greater in the saline mice. 4. Discussion 4.1. Weaning growth lag induced by GH treatment Between 35 and 50 days of age, BALByc male mice administered with rhGH since weaning showed a growth pattern similar to that found when recovering from a dietary restriction. This growth, known as catch-up growth (Dulloo and Girardier, 1992), results in an anomalous body composition, with a higher tendency to gain body fat than body protein. The depletion-recovery process was due to the initial fall in the feed intake provoked by rhGH administration between 21 and 35 days of age. This led to a protein-energy malnutrition resulting in a ‘weaning growth lag’ (Matteri et al., 2000) followed by the catch-up

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Fig. 2. Linear regression between body fat gain and protein intake of BALByc mice fed 12% (12) or 20% (20) protein diets and subjected to saline (s) or rhGH treatment (GH) from 21 to 50 days of age.

growth. These events were concurrent with the adaptation of mice to a solid diet characteristic of ` the post-weaning period (LeDividich and Seve, 2000).

The self-regulated increase of feed intake that appeared from 35 days of age to the end of the experiment allowed 20GH mice to recover body weight, from the previous lag, to that of 20s (20s:

Fig. 3. Linear regression between body protein gain and feed intake of BALByc mice fed 12% (12) or 20% (20) protein diets and subjected to saline (s) or rhGH treatment (GH) from 21 to 50 days of age.

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20.0 g; 20GH: 20.42 g), through an increased efficiency of feed and protein utilization to body protein gain, demonstrating the GH anabolic effect. A higher anabolic effect of the hormone was also found by Azain et al. (1995a) in mature female rats, without previous growth deficiencies (225 g body weight), subjected to pGH, which attained a five-fold increase of protein accretion. However, rhGH administration to the 12GH mice was ineffective in restoring body weight to control values (12s: 18.6 g; 12GH: 15.6 g) from the previous growth lag, due to the inefficient feed and protein intake utilization to body protein gain. Weaning piglets show a typical endocrine profile with increased serum concentrations of GH and decreased concentrations of IGF-I and IGF-II (Matteri et al., 2000) that is also found during nutritional deprivation (Vance et al., 1992). Decreases in the levels of plasma IGF-I and IGFI mRNA expression can lead to the reduction of growth promotion since the protein anabolic effects of GH are believed to be largely mediated by IGFI (Zhao et al., 1995; Brameld et al., 1999; Gautsch et al., 1999). Specifically, local IGF-I synthesis has been identified as a greater determinant of muscle growth than its circulating concentrations (Matteri et al., 2000). An appropriate dietary protein level is also required for the complete

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Table 2 Power relationship of body fat gainyprotein gain (FGyPG) ratio with protein intake (PI) of BALByc mice fed 12% (12) or 20% (20) protein diets and subjected to saline (s) or rhGH treatment (GH) from 21 to 50 days of age

12s 20s 12GH 20GH

FG:PG (gyg) vs. PI (g)

R2

0.886ØPI0.071"0.012 0.661ØPI0.031"0.008 1.093ØPI0.124"0.018 0.730ØPI0.149"0.018

0.752 0.602 0.801 0.849

a1 b1 a2 a2

Superscripts: a,b(diet) and 1,2(treatment) significantly different, P-0.05 (dummy variables). All relationships were significant (P-0.0001).

expression of the stimulatory effect of pGH on ` et al., 1993). growth (Seve 4.2. Effects of GH treatment and dietary protein on body fat and protein gain Increased fat gain (FG) was observed in rhGHtreated mice on 12 or 20% protein diets, as 20GH and 12GH had 2.4 times and 1.7 times, respectively, higher FG than saline mice per gram of FI and PI (Table 1). Thus treatment with rhGH doubled the efficacy of both feed intakes in depositing fat, suggesting that the effect of the reduction of body fat mediated by GH (Solomon et al., 1994) was

Fig. 4. Linear regression between body protein gain and protein intake of BALByc mice fed 12% (12) or 20% (20) protein diets and subjected to saline (s) or rhGH treatment (GH) from 21 to 50 days of age.

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Fig. 5. Power relationship between body fat gainyprotein gain (FGyPG) ratio and protein intake of BALByc mice fed 12% (12) or 20% (20) protein diets and subjected to saline (s) or rhGH treatment (GH) from 21 to 50 days of age.

abolished irrespective of diet. This led GH-treated mice to an altered efficiency of energy utilization and energy partitioning during body weight recovery following the initial restricted feed intake; redirecting the substrates, at least partially, to fat gain, in spite of protein deposition. A decreased body protein concentration was also found by Zhao et al. (1995) in rhGH treated rat pups refed after feed restriction. The tendency to deposit more fat than protein was revealed by the behavior of the FGyPG ratio, as this ratio increased with the protein intake in mice fed both diets. GH-treatment thus led to a shift of the fitted power function towards higher FG. In addition, a low-protein diet also provoked the shift of the FGyPG ratio towards greater FG in saline and GH-treated mice. Therefore the effects of the low protein concentration in the diet and rhGH administration were added, resulting in a greater inefficiency in the promotion of body protein gain. In contrast, Azain et al. (1995b) indicated that GH administration to lean and fat Zucker rats previously feed-restricted at five weeks (35 days) of age led to a normalization of body composition and growth rate, maintaining body weight, protein accretion, and diminishing lipid accretion. Nevertheless, the age of the beginning of treatment was

a crucial determinant in the growth performance of rhGH-treated animals, as BALByc male mice administered with rhGH from 35 days of age did not show the initial growth lag and subsequent catch-up growth (unpublished results). Gautsch et al. (1999) also found in male rats suffering feed restriction from weaning to 120 days of age, that rhGH treatment promoted whole body growth early in the recovery process (136 days). However, during late growth recovery (167 days), rhGH treatment in combination with a diet enriched in protein and energy resulted in animals with the greatest gains in liver and fat pad. 4.3. Possible mechanisms involved in the anomalous response to GH treatment The expected effect of GH is the improved protein deposition and decreased or non-existent adipose tissue growth (Etherton, 2000). In contrast, we observed in rhGH treated mice, which had previously suffered a GH induced growth lag, an enhanced trend to deposit fat, which revealed an abnormal mechanism of the hormone in the experimental context of this work. Modifications in the gain of body components may be related to changes in the endocrine status and, namely, to elements constituting the somato-

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tropic axis. The protein content of the diet can exert strong influences on gene expression for the synthesis of GH and IGF-I and their receptors, but with notable differences among tissues (Brameld et al., 1999). Protein malnutrition in rats has been also found to be linked to shared GH and IGF-I resistance (Jain et al., 1998), suggesting that the high amount of fat accumulated in the mice fed 12% protein could be related to GH resistance, as these animals redirected the highest proportion of their protein and energy intake to body fat gain. Moreover, rhGH treated mice fed the low protein diet did show an even greater failure to gain body protein than the control saline mice fed 12% protein diet. However, rhGH-treated mice fed 20% protein utilized protein and feed intake more efficiently than their control to increase body protein stores but, surprisingly, they also derived many resources to body fat so that the FGyPG ratio remained higher than saline. A reduced number of GH receptors in adipose tissue could lead to diminished lipolysis. Brameld et al. (1999) found reduced GH receptor expression in adipose tissue of GH-treated pigs receiving a high dietary protein intake, whereas expression of GH receptor mRNA was increased with GH treatment in liver and skeletal muscle. Gender is another factor that should be taken into account when evaluating the effects of GH treatment on growth performance. In a previous work, we found that rhGH-treated female mice fed a 20% protein diet showed 34% more protein gain than the corresponding males which, in turn, increased fat gain 91% over females. This dramatically changed the growth pattern of rhGH-treated mice from that of the control mice (Agis-Torres et al., 1996). Although formation of anti-rhGH antibodies may also explain the low anabolic effect of rhGH treatment during the repletion stage (Gause et al., 1983; Gautsch et al., 1999), the differential response obtained in treated male and female mice has to be elucidated. Additional studies of the processes involved are therefore necessary. In conclusion, the present study provides unique data that emphasize the importance of understanding the effect of nutritional and hormonal status at different ages on the ontogeny of the endocrine control of growth. Post-weaning is confirmed as a crucial phase of growth that is highly affected by changes in diet and hormonal status. Thus, rhGH administration to BALByc male mice induced an inversion in the metabolic response to the hor-

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mone, that was found specifically at this stage of growth. The inverted response led to growth similar to the catch-up growth found in rats with restricted feed intake followed by refeeding. Moreover, rhGH treatment and a low protein diet resulted in factors that were, in conjunction or separately, inefficient for the recovery of body protein gain. References ´ ˜ Agis-Torres, A., Lopez-Oliva, M.E., Unzaga, M.T., Munoz´ Martınez, E., 1996. Recombinant human growth hormone modifies the inherent partition of nutrients in growing female and male BALByc mice. Comp. Biochem. Physiol A 115, 317–322. Association of Official Analytical Chemists, 1990. Official Methods of Analysis of the Association of Official Analytical Chemists. fifteenth ed. AOAC, Washington. Azain, M.J., Roberts, T.J., Martin, R.J., Kasser, T.R., 1995. Comparison of daily versus continuous administration of somatotropin on growth rate, feed intake and body composition in intact female rats. J. Anim. Sci. 73, 1019–1029. Azain, M.J., Hausman, D.B., Kasser, T.R., Martin, R.J., 1995. Effects of somatotropin and restriction on body composition and adipose metabolism in obese Zucker rats. Am. J. Physiol. 269, E137–E144. Bergen, W.G., Merkel, R.A., 1991. Body composition of animals treated with partitioning agents: implications for human health. Fed. Am. Soc. Exp. Biol. J. 5, 2951–2957. Brameld, J.M., Atkinson, J.L., Saunders, J.C., Pell, J.M., Buttery, P.J., Gilmour, R.S., 1996. Effects of growth hormone administration and dietary protein intake on insulinlike growth factor-I (IGF-I) and growth hormone receptor (GHR) mRNA expression in porcine liver, skeletal muscle, and adipose tissue. J. Anim. Sci. 74, 1832–1841. Brameld, J.M., Gilmour, R.S., Buttery, P.J., 1999. Glucose and amino acids interact with hormones to control expression of insulin-like growth factor-I and growth hormone receptor mRNA in cultured pig hepatocytes. J. Nutr. 129, 1298–1306. Butler, A.A., Le Roith, D., 2001. Control of growth by the somatotropic axis: growth hormone and the insulin-like growth factors have related and independent roles. Annu. Rev. Physiol. 63, 141–164. Campbell, R.G., Johnson, R.J., Taverner, M.R., King, R.H., 1991. Interrelationships between exogenous porcine somatotropin (PST) administration and dietary protein and energy intake on protein deposition capacity and energy metabolism of pigs. J. Anim. Sci. 69, 1522–1531. Douglas, R.G., Gluckman, P.D., Ball, K., Breier, B.H., Shaw, J.H.F., 1991. The effects of infusion of insulin-like growth factor-1 (IGF-I), IGF-II and insulin on glucose and protein metabolism in fasted lambs. J. Clin. Invest. 88, 614–622. Dulloo, A.G., Girardier, L., 1992. Influence of dietary composition on energy expenditure during recovery of body weight in the rat: implications for catch-up growth and obesity relapse. Metabolism 41, 1336–1342. Etherton, T.D., 2000. The biology of somatotropin in adipose tissue growth and nutrient partitioning. J. Nutr. 130, 2623–2625.

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