0021-972X/91/7206-1302$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 72, No. 6 Printed in U.S.A.
Effects of Growth Hormone on Neonatal Growth in Nursing Rhesus Monkeys* MARK E. WILSON, THOMAS P. GORDON, KATHY CHIKAZAWA, DEBORAH GUST, JAMES M. TANNER, AND CHRISTOPHER G. RUDMAN Yerkes Primate Research Center, Field Station, Emory University (M.E.W., T.P.G., K.C., D.G.), Lawrenceville, Georgia 30243; Genentech, Inc. (C.G.R.), South San Francisco, California 94080; the Institute of Child Health, University of London (J.M.T.), London, England; and School of Public Health, University of Texas (J.M.T.), Houston, Texas 77225
ABSTRACT. The effects of recombinant human GH treatment of either nursing mothers or their infants on neonatal growth in rhesus monkeys was determined. Growth rates of infants treated daily from birth with GH (INFGH; n = 9; 100 fig/ kg, sc) were compared to those of infants given saline (INFc; n = 10), infants whose mothers received saline from the second trimester of pregnancy through 7 weeks postpartum (CON; n = 9), infants of mothers who received GH during pregnancy only from the second trimester to parturition (PRG; n = 8), infants of mothers who received GH during lactation only from parturition through 7 weeks postpartum (LAC; n = 9), and infants of mothers who received GH during the second trimester of pregnancy through 7 weeks postpartum (PRG/LAC; n = 8). Mothers receiving GH were given 250 Mg/kg, sc, Monday, Wednesday, and Friday. Infants were allowed to nurse ad libitum. Although infant birth weights were similar among the six groups, body weights at 7 weeks of age were significantly greater in PRG/ LAC infants (0.77 ± 0.03 kg) compared to those in CON (0.66 ± 0.02 kg), INFc (0.62 ± 0.03 kg), LAC (0.62 ± 0.04 kg), and INFGH infants (0.62 ± 0.01 kg), with infants of PRG mothers
L
OW BIRTH weight and slow growth rates compound the problems of preterm infants. Consequentially, nutritional supplementation has been used to enhance postnatal weight gain (1). Recent data suggest that growth in preterm infants is accelerated when they are fed milk derived from mothers delivering preterm infants (2-5) or milk or formula fortified with protein (4-6). Milk from females delivering preterm infants facilitates growth due to a higher protein content (7, 8). Neonatal growth rates are positively related to protein intake up to a ceiling beyond which no additional growth is obtained (8, 9). Although growth rates are enhanced by increased energy intake (8,9) simple supplementation of milk with fat does not affect growth in babies (6, 8). Received September 10,1990. Address all correspondence and requests for reprints to: Mark E. Wilson, Yerkes Primate Research Center, Field Station, Emory University, 2409 Taylor Road, Lawrenceville, Georgia 30243. * This work was supported by a grant from Genentech, Inc., and in part from NIH Grants HD-16305, HD-18120, and RR-00165.
intermediate (0.71 ± 0.02 kg) between them. By 35 weeks of age, after infants had been weaned by their mothers, body weights were similar among all groups. Serum concentrations of insulin-like growth factor-I (IGF-I) rose significantly in all infants during the study period. Although IGF-I levels did not vary significantly among the treatment groups, average concentrations of IGF-I were significantly related to weight gains. Analyses of milk composition revealed that total protein, lactose, and IGF-I levels were similar among groups, whereas the percentage of fat in the milk was significantly higher in PRG/LAC mothers. Milk protein content was significantly related to weight gain. These data suggest that neonatal body weight gain can be accelerated in nursing infants whose mothers have received GH from at least the second trimester of pregnancy through the lactational interval. Since infants of mothers receiving GH during lactation only were not different from controls, the effect of GH in this treatment paradigm may be mammogenic rather than galactopoietic per se. (J Clin Endocrinol Metab 72: 13021307,1991)
In contrast, neonatal growth of mice pups is positively related to the concentration of fat in milk (10). Improving the quantity and/or the quality of a mother's milk would be beneficial to nursing preterm or low birth weight infants. The galactopoietic properties of GH in ruminants are well established, as treatment of lactating females with GH increases milk yields (11-13). GH may also be lactogenic in primates, as evidenced by an increase in a-lactalbumin production in cultures of primate mammary tissue (14). By increasing the quantity of milk produced, GH administration to ruminants increases the yield of fat, protein, and lactose; nevertheless, the composition of nutrients in the milk is not altered by GH administration (11-13). GH treatment also elevates serum concentrations of insulin-like growth factorI (IGF-I) (11), and infusion of IGF-I into an artery of the mammary gland increases milk yield in goats (15). Since IGF-I is detected in milk (16, 17), stimulates growth in rat pups (18), and is positively associated with
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EFFECTS OF GH ON NEONATAL GROWTH rate of growth in preterm infants (19), it is possible that, in addition to milk nutrients, IGF-I in milk is important for infant growth. Importantly, piglets suckling sows treated with GH gain more weight during the lactational period (13). The present study examined the effects of administration of recombinant human GH to either mothers or nursing infants on neonatal growth rate in rhesus monkeys. The studies were designed to determine whether GH could influence infant growth by treating infants directly with GH or by administering GH to mothers during pregnancy and/or lactation.
Materials and Methods Subjects for these studies were rhesus monkeys (Macaca mulatto) from the breeding colony at the Yerkes Primate Research Center (Lawrenceville, GA). Animals were randomly assigned to a specific treatment group, as described below. All infants remained with their mothers throughout the study period and were thus allowed to nurse ad libitum. Animals were housed in outdoor compounds with attached shelters and were members of social groups containing adult males, other adult females, and juvenile male and female monkeys. All mothers were between 5-14 yr of age and had from 0-10 previous pregnancies. The effect of parity as a function of treatment condition is statistically evaluated in Results. Animal husbandry and handling for the collection of physiological data have been described previously (20). Animals were fed Wayne 25% Primate Diet 8663 (Madison, WI) ad libitum twice daily and received fresh fruit once daily. The primate diet contained more than 25% protein, more than 5% fat, and less than 5% fiber.
With this feeding regimen, all mothers could eat to satiety. To address the effects of recombinant human GH treatment on neonatal growth of infants, animals were randomly assigned to one of six treatment groups: 1) control (CON; n = 9) mothers received physiological saline from the second trimester of pregnancy through 7 weeks postpartum; 2) mothers received GH 3 days/week (Monday, Wednesday, and Friday) during pregnancy only (PRG; n = 8) from the second trimester to parturition; 3) mothers received GH 3 days/week during lactation only (LAC; n = 9) from parturition through 7 weeks postpartum; 4) mothers received GH 3 days/week during pregnancy and lactation (PRG/LAC; n = 8) from the second trimester of pregnancy through 7 weeks postpartum; 5) infants of GH-naive mothers received daily injections of saline (INF C ; n = 10) from birth through 7 weeks postpartum; and 6) infants of GH-naive mothers received daily injections of GH (INF G HI n = 9) from parturition through 7 weeks postpartum. Pregnant and lactating mothers receiving GH three times each week were administered 250 Mg/kg-injection (750 jug/week), sc. Infants receiving daily GH were administered 100 /xg/kg-day (700 jug/week), sc, a treatment regimen approximating that used to treat GHdeficient children (21). The distribution of male and female infants was similar within each treatment group (percentage of males: CON, 56%; PRG, 75%; LAC, 44%; PRG/LAC, 63%; INFc, 50%; and INF G H , 56%). Maternal body weights were obtained weekly from the second
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trimester of pregnancy through 7 weeks postpartum. Infant body weights were obtained at birth and weekly thereafter through 7 weeks of age. Posttreatment weights were obtained on all subjects at 35 weeks of age. Serum samples were obtained from infants every 2 weeks for the determination of IGF-I and insulin. Serum samples were obtained from the mothers 48 h after a GH injection each week for the determination of PRL. Serum biochemistries were determined in infants at 6 weeks of age. Breast milk was manually expressed from a subsample of mothers (CON, LAC, and PRG/LAC) 2 and 5 weeks postpartum, 48 h after the previous GH or saline injection, for the determination of milk fat, total protein, lactose, and IGF-I. Mothers were anesthetized with ketamine hydrochloride (10 mg/kg, im) and given 2.0 IU oxytocin (Peninsula Laboratories, Belmont, CA), iv, to facilitate the collection of milk. Analyses Serum PRL (22) and IGF-I concentrations (23) were determined by previously validated RIA procedures. Rather than using a pool of normal human serum for the IGF-I standard curve (23), recombinant human IGF-I (Amgen Corp., Thousand Oaks, CA) was used as the standard, and all results were expressed as micrograms of IGF-I per L (24). Inter- and intraassay coefficients of variation for both assays were less than 8.0%. Unextracted milk was analyzed using the same IGF-I assay. All milk IGF-I determinations were performed in the same assay. Serial dilutions of unextracted rhesus milk were parallel to the IGF-I standard curve (Fig. 1). Unknown samples were assayed at volumes of 0.5 pL equivalents to 20 /xL. The intraassay coefficient of variation averaged 5.2%. Concentrations of milk lactose were determined using a kit from Boehringer Mannheim (no. 176 303, Indianapolis, IN). Concentrations of total protein in milk were determined with a kit from Sigma Chemical Co. (St. Louis, MO). The amount of fat in the milk was determined by the creamatocrit method (25). Serum biochemistries were determined by Smith-Kline Bioscience Laboratories (Tucker, GA). Data were expressed as the mean ± sem and were analyzed
1
10 100 1000 log pg IGF-1 or uL milk FIG. 1. Displacement curves for purified human IGF-I standard and serial dilutions of rhesus monkey milk samples in the RIA of IGF-I. B/ Bo, Bound to free ratio.
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WILSON ET AL.
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with an analysis of variance model for repeated measures. Specific comparisons among treatment groups used Duncan multiple range tests. Linear relationships among variables were evaluated by linear regression. Statistical tests yielding P < 0.05 were considered significant. Results
As illustrated in Fig. 2, birth weights were indistinguishable among the six treatment groups (F5;46 = 1.55; P > 0.05). In contrast, 7 weeks postpartum the PRG/ LAC infants were significantly heavier than the CON, LAC, INFc, and INFGH infants, with the infants of PRG mothers being intermediate (F5,46 = 4.28; P < 0.05). The CON, LAC, INFc, and INF G H infants were indistinguishable from one another. This overall difference in body weight for the PRG/LAC group was reflected in significantly greater weekly weight gain compared to other groups, with the PRG infants again being intermediate (F5i46 = 2.87; P < 0.05). By 35 weeks of age, these differences in body weight (kilograms) among the infants were abolished (CON, 1.57 ± 0.06; LAC, 1.67 ± 0.09; PRG, 1.54 ± 0.04; PRG/LAC, 1.69 ± 0.07; INFC, 1.61 ± 0.13; INFGH, 1.71 ± 0.17; F5,46 = 1.35; P > 0.05). Despite these differences in postnatal body weight gain, serum concentrations of IGF-I during the treatment period did not vary significantly among the six groups (F5i46 = 1.10; P > 0.05) at any time during week 5 or 7 of treatment. Serum concentrations of IGF-I (micrograms per L) did rise significantly in all infants from 5-7 weeks of age (23.8 ± 2.3 vs. 33.2 ± 3.0; n = 52; F1>46 = 19.57; P < 0.05). Serum concentrations of IGF-I (micrograms per L) at 7 weeks of age averaged 38.5 ± 11.3 for CON, 41.5 ± 10.9 for LAC, 37.5 ± 8.8 for PRG, 35.8 ± 7.9 for PRG/ LAC, 31.0 ± 8.1 for INFC, and 16.1 ± 2.8 for INFGH.
JCE & M • 1991 Vol 72 • No 6
Although groups were not differentiated on the basis of circulating IGF-I concentrations, average serum IGF-I values during the 7 weeks postpartum predicted overall body weight gain (F M 6 = 19.95; P < 0.05; r2 = 0.28). Serum biochemistries obtained at 6-7 weeks of age were similar among the infants of the six treatment groups (Table 1; F5,46 < 2.15; P > 0.05). Maternal body weights (kilograms) were not affected by treatments, either at parturition (CON, 8.14 ± 0.33; LAC, 8.26 ± 0.57; PRG, 7.95 ± 0.33; PRG/LAC, 8.36 ± 0.33; INFc, 7.53 ± 0.29; INFGH, 7.61 ± 0.49; F5,46 < 1.00; P > 0.05) or at the completion of the treatment 7 weeks postpartum (CON, 7.62 ± 0.25; LAC, 7.88 ± 0.57; PRG, 7.73 ± 0.32; PRG/LAC, 8.01 ± 0.28; INFC, 7.92 ± 0.38; INFGH, 8.05 ± 0.39; F5,46 < 1.00; P > 0.05). Maternal parity also had no effect on infant growth under the
present treatment conditions (covariance analysis of variance; parity by infant weight gain F li50 = 1.24; P > 0.05; r = 0.03; n = 52). Maternal serum PRL concentrations for the CON, PRG, LAC, and PRG/LAC groups rose significantly in all groups in the week before parturition and remained elevated throughout the 7-week study period (Fig. 3; F10>290 = 15.39; P < 0.05). PRL concentrations did not vary among these four groups of lactating females (F3,29 = 1.64; P > 0.05) during the study period (F30>290 < 1.00; P > 0.05). Assessment of milk samples revealed no differences in any parameter between 2 and 7 weeks of lactation, so the two time points were averaged for analysis (Fig. 4). The percentage of fat in the milk was significantly greater in PRG/LAC compared to either CON or LAC mothers (F2>23 = 4.57; P < 0.05). In contrast, concentrations of total protein (F2,23 = 1.59; P > 0.05), lactose (F2 23 < 1.00; P > 0.05), and IGF-I (F2)23 = 1.69; P > 0.05) did
1.00 D CON ffl LAC
FIG. 2. Mean ± SEM body weights (kilograms) of infants in each treatment group at birth and at the completion of treatment at 7 weeks of age. Bars with different letters indicated groups are significantly different from one another using the Duncan multiple range test (P < 0.05).
0.75-
•
PRG
•
PRG/LAC
QH INF
0 a
o 0.50
a
0.25
Birth
Age (weeks)
Seven
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EFFECTS OF GH ON NEONATAL GROWTH TABLE 1. Mean (±SEM) values of auxological measures for each group in Exp 1 at 12 weeks of age Auxological measure Rx group Crown-rump Tibia (cm) (cm)
Tail (cm)
8.6 (0.1) 8.7 (0.1) 9.0 (0.1)
14.3 (0.4) 14.3 (0.3) 15.5 (0.8)
CON GHi GHm
24.0 (0.4) 24.6 (0.3) 25.6 (0.2)
Skin fold Bone age (% of adult) (mm) 39.2 (1.8) 39.9 (0.7) 39.5 (1.3)
1.5 (0.1) 1.3 (0.1) 1.8 (0.2)
not differ significantly in the milk of the three treatment groups. Furthermore, the concentration of total protein in the milk was significantly associated with the overall weight gain of the infants, regardless of treatment group (Fli24 = 11.89; P < 0.05; r2 = 0.30), whereas neither lactose, fat, nor IGF-I content in the milk was related to individual infant body weight gain. In addition, total protein concentrations in milk were significantly associated with infant serum IGF-I concentrations (Fi>24 = 6.15; P < 0.05; r2 = 0.0.20).
Discussion These results indicate that neonatal increases in body weight were significantly advanced in nursing infants whose mothers received GH treatment during both pregnancy and lactation. Birth weights were similar among all subjects, suggesting that GH treatment of mothers during pregnancy does not affect fetal growth. Infants
themselves treated with GH did not grow faster than untreated control subjects. Similarly, treatment of mothers with GH only during lactation had no effect on
FIG. 3. Mean ± SEM serum PRL concentrations in mothers from 4 weeks before parturition through 7 weeks postpartum. Data for INFC and INF G H mothers are not shown.
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neonatal growth rates. In contrast, mothers treated with GH during the last two trimesters of pregnancy had infants whose growth rates were statistically intermediate between those of PRG/LAC and the remaining groups, suggesting that GH treatment during pregnancy alone can augment postpartum weight gains in infants that subsequently nurse. Although the present study was designed only to determine whether GH administration to infants or mothers would augment neonatal growth, the results do address the question of whether the effect of GH was mammogenic or galactopoietic. Mothers treated with GH during pregnancy and lactation had significantly higher amounts of fat in milk than did either control mothers or mothers treated only during lactation. Although group differences in total protein in milk were not evident, the amount of milk protein was significantly related to the amount of weight gained. These data are consistent with reports in humans that milk protein is essential for enhanced weight gains in neonatal preterm infants (2-6, 8, 9). Although fat content in milk may not be related to weight gain in human infants (6, 8), growth rates in mice are predicted from milk fat concentration (10). The observation that milk fat content was increased in PRG/ LAC females contrasts with data from dairy cows (12) and sows (13) which indicate that daily administration of GH does not affect the composition of milk (12). Although serum IGF-I concentrations in infants were not different as a function of treatment condition, average IGF-I concentrations were significantly associated with the weight gain of individual infants. These data support the observation that IGF-I secretion is important for maintaining normal neonatal growth in rats (18).
J3 PL,
-
2
0
2
4
Weeks from Parturition
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JCE & M • 1991 Vol 72 • No 6
FIG. 4. Mean ± SEM values of percent fat, total protein, lactose, and IGF-I in milk from CON, LAC, and PRG/LAC mothers. Values represent the means from samples collected at weeks 2 and 7 postpartum. Bars with different letters indicate groups that are significantly different from one another (P < 0.05).
CON
LAC Treatment Group
Preterm, low birth weight infants have lower circulating concentrations of IGF-I (19), but it is not known how milk supplementation which stimulates accelerated growth in these infants affects circulating levels of IGFI. Nutrient intake, in particular protein content, regulates IGF-I secretion in a variety of species (26-28) and is primarily important for regulating IGF-I concentrations during the neonatal period (29). The significant relationship observed between milk protein and serum IGF-I concentrations in nursing infants suggests that milk protein may have influenced neonatal growth by regulating IGF-I production in neonates. Although differences in milk quality may account for the differences in body weight gain, it is possible that the accelerated weight gain of PRG/LAC infants was due in part to increased maternal milk yields at each nursing bout. A consistent effect of GH administration to lactating nonprimate animals is a significant increase in milk yields and, thus, an increased yield of milk nutrients (11, 12). This galactopoietic effect of GH may be mediated through IGF-I, as infusion of IGF-I directly into a mammary artery (15), but not into the peripheral circulation (11), increases milk yield in goats. Furthermore, IGF-I does enhance PRL-induced increases in casein production in rabbit mammary cultures (30). We were not able to quantify milk yields in the present experiment given the difficulty in standardizing the collection procedure and, thus, were not able to directly determine the galactopoietic properties of GH. Nevertheless, the effect of GH on neonatal growth was not simply due to the galactopoietic properties of GH, as infants of mothers treated with GH during lactation only (LAC) had growth rates similar to those of control infants. A daily regimen of GH treatment during lactation (11-13), rather than 3 days/week, might have produced a different effect.
PRG/LAC
CON
LAC Treatment Group
PRG/LAC
A more likely hypothesis for the present finding is that mammogenesis was affected by GH treatment during pregnancy and that this, in turn, enhanced milk yields, particularly with continued GH administration through lactation. Milk yield is influenced by the number of mammary gland cells as well as the secretory capacity of the cells (31). In this regard, mammary gland development is enhanced by placental lactogens (30), which bind to GH receptors on mammary tissue (32). In mice, hypophysectomy during pregnancy does not affect mammary gland development due to the presence of high concentrations of circulating PRL-like placental lactogens, but does reduce protein synthesis and, probably, the secretory capacity of the mammary gland (33). Treatment of hypophysectomized nonpregnant monkeys with GH does not induce mammary growth (34). On the other hand, mammary gland cell proliferation in most species takes place during pregnancy, with some increase in cell number occurring during lactation (31, 35). These data suggest that GH treatment during pregnancy amplifies mammary gland growth and the consequent increase in its secretory capacity. The fact that the infants of mothers that received GH during pregnancy exhibited weight gains during the neonatal period that were intermediate between those of the PRG/LAC and CON groups suggests that this treatment regimen may have affected mammary growth and its secretory capacity. It is important to note that the differences in body weight among the various treatment groups were no longer evident at 35 weeks of age, 4-5 months after the termination of treatment and at a time when infant rhesus monkeys are typically weaned from their mothers and rely almost exclusively on solid food (36). Thus, the GH treatment simply accelerated weight gains and did not produce bigger infants.
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EFFECTS OF GH ON NEONATAL GROWTH
Acknowledgments 17.
The technical assistance of Erika Seres and Beth Wellington is appreciated. Reagents for the PRL assay and the IGF-I antibody were kindly provided by the National Hormone and Pituitary Program. All assays were performed in the Yerkes RIA Laboratory. We wish to thank the Department of Veterinary Medicine at the Primate Center for assisting in the collection of blood samples from the infants. These projects were approved by the Institutional Animal Care and Use Committee of Emory University. The Yerkes Primate Research Center is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.
20.
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molecular weight binding protein in human milk. Eur J Obstet Gynecol. 1989;30:19-25. Baxter RC, Zaltsman Z, Turtle JR. Immunoreactive somatomedinC/insulin-like growth factor-1 and its binding protein in human milk. J Clin Endocrinol Metab. 1984;58:955-9. Hizuka N, Takano K, Shizume K, Miyakawa M, Tanaka I, Horikawa R. Insulin-like growth factor-I stimulates growth in normal growing rats. Eur J Pharmacol. 1986;125:143-6. Samaan NA, Schultz PN, Johnston DA, Creasy RW, Gonik B. Growth hormone, somatomedin C, and nonsuppressible insulinlike activity levels compared in premature, small, average birth weight, and large infants. Am J Obstet Gynecol.. 1987;157:1524-8. Walker ML, Gordon TP, Wilson ME. Reproductive performance in capture-acclimated female rhesus monkeys {Macaco mulatto). J Med Primatol. 1982;ll:291-302. Frasier SD, Lippe BM. Clinical review 11: the rational use of growth hormone during childhood. J Clin Endocrinol Metab. 1990;71:269-73. Wilson ME, Walker ML, Schwartz SM, Gordon TP. Gonadal status influences developmental patterns of serum prolactin in female rhesus monkeys housed outdoors. Endocrinology. 1985;116:640-5. Osterud EL, Lackey S, Wilson ME. Estradiol increases somatomedin-C concentrations in adolescent rhesus monkeys (Macaca mulatto). Am J Primatol. 1986;ll:53-62. Wilson ME. Gonadal steroids influence somatomedin-C concentrations in prepubertal female rhesus monkeys. Endocrinology. 1986;119:666-71. Lucas A, Gibbs JAH, Luster RLJ, Baum JD. Creamatocrit: simple clinical technique for estimating fat concentration and energy value of human milk. Br Med J. 1978;l:1018-20. Ronge H, Blum J. Insulin-like growth factor 1 responses to recombinant bovine growth hormone during feed restriction in heifers. Acta Endocrinol (Copenh). 1989;120:735-44. Donahue SP, Phillips LS. Response of IGF-1 to nutritional support in malnourished hospital patients: a possible indicator of shortterm changes in nutritional status. Am J Clin Nutr. 1989;50:9629. Fliesen T, Maiter D, Gerard G, Underwood LE, Maes M, Ketelslegers JM. Reduction of serum insulin-like growth factor-1 by dietary protein restriction is age dependent. Pediatr Res. 1989;26:415-19. Phillips A, Drakenberg K, Persson B, et al. The effects of altered nutritional status upon insulin-like growth factors and their binding proteins in neonatal rats. Pediatr Res. 1989;26:128-34. Duclos M, Houdebine LM, Djiane J. Comparison of insulin-like growth factor 1 and insulin effects on prolactin-induced lactogenesis in the rabbit mammary gland in vitro. Mol Cell Endocrinol. 1989;65:129-34. Knight CH, Wilde CJ. Mammary growth during lactation: implications for increasing milk yield. J Dairy Sci. 1987;70:1991-2000. Thordarson G, Talamantes F. Role of the placenta in mammary gland development and function. In: Neville MC, Daniel C, eds. The mammary gland: development, regulation, and function. New York: Plenum Press; 1987;4590-8. Thordarson G, Ogren L, Day JR, Bowens K, Fielder P, Talamantes F. Mammary gland development and a-lactalbumin production in hypophysectomized, pregnant mice. Biol Reprod. 1989;40:517-24. Kleinberg DL, Niemann W, Flamm E, Cooper P, Babitsky G, Valensi Q. Primate mammary development: effects of hypophysectomy, prolactin inhibition, and growth hormone administration. J Clin Invest. 1985;75:1943-50. Forsyth IA. Variation in the endocrine control of mammary growth and function: the roles of prolactin, growth hormone, and placental lactogen. J Dairy Sci. 1986;69:886-903. Wilson ME, Walker ML, Pope NS, Gordon TP. Prolonged lactational infertility in adolescent rhesus monkeys. Biol Reprod. 1988;38:163-74.
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Vol. 72, No. 6 Printed in U.S.A.
Effects of Growth Hormone on Neonatal Growth in Nursing Rhesus Monkeys* MARK E. WILSON, THOMAS P. GORDON, KATHY CHIKAZAWA, DEBORAH GUST, JAMES M. TANNER, AND CHRISTOPHER G. RUDMAN Yerkes Primate Research Center, Field Station, Emory University (M.E.W., T.P.G., K.C., D.G.), Lawrenceville, Georgia 30243; Genentech, Inc. (C.G.R.), South San Francisco, California 94080; the Institute of Child Health, University of London (J.M.T.), London, England; and School of Public Health, University of Texas (J.M.T.), Houston, Texas 77225
ABSTRACT. The effects of recombinant human GH treatment of either nursing mothers or their infants on neonatal growth in rhesus monkeys was determined. Growth rates of infants treated daily from birth with GH (INFGH; n = 9; 100 fig/ kg, sc) were compared to those of infants given saline (INFc; n = 10), infants whose mothers received saline from the second trimester of pregnancy through 7 weeks postpartum (CON; n = 9), infants of mothers who received GH during pregnancy only from the second trimester to parturition (PRG; n = 8), infants of mothers who received GH during lactation only from parturition through 7 weeks postpartum (LAC; n = 9), and infants of mothers who received GH during the second trimester of pregnancy through 7 weeks postpartum (PRG/LAC; n = 8). Mothers receiving GH were given 250 Mg/kg, sc, Monday, Wednesday, and Friday. Infants were allowed to nurse ad libitum. Although infant birth weights were similar among the six groups, body weights at 7 weeks of age were significantly greater in PRG/ LAC infants (0.77 ± 0.03 kg) compared to those in CON (0.66 ± 0.02 kg), INFc (0.62 ± 0.03 kg), LAC (0.62 ± 0.04 kg), and INFGH infants (0.62 ± 0.01 kg), with infants of PRG mothers
L
OW BIRTH weight and slow growth rates compound the problems of preterm infants. Consequentially, nutritional supplementation has been used to enhance postnatal weight gain (1). Recent data suggest that growth in preterm infants is accelerated when they are fed milk derived from mothers delivering preterm infants (2-5) or milk or formula fortified with protein (4-6). Milk from females delivering preterm infants facilitates growth due to a higher protein content (7, 8). Neonatal growth rates are positively related to protein intake up to a ceiling beyond which no additional growth is obtained (8, 9). Although growth rates are enhanced by increased energy intake (8,9) simple supplementation of milk with fat does not affect growth in babies (6, 8). Received September 10,1990. Address all correspondence and requests for reprints to: Mark E. Wilson, Yerkes Primate Research Center, Field Station, Emory University, 2409 Taylor Road, Lawrenceville, Georgia 30243. * This work was supported by a grant from Genentech, Inc., and in part from NIH Grants HD-16305, HD-18120, and RR-00165.
intermediate (0.71 ± 0.02 kg) between them. By 35 weeks of age, after infants had been weaned by their mothers, body weights were similar among all groups. Serum concentrations of insulin-like growth factor-I (IGF-I) rose significantly in all infants during the study period. Although IGF-I levels did not vary significantly among the treatment groups, average concentrations of IGF-I were significantly related to weight gains. Analyses of milk composition revealed that total protein, lactose, and IGF-I levels were similar among groups, whereas the percentage of fat in the milk was significantly higher in PRG/LAC mothers. Milk protein content was significantly related to weight gain. These data suggest that neonatal body weight gain can be accelerated in nursing infants whose mothers have received GH from at least the second trimester of pregnancy through the lactational interval. Since infants of mothers receiving GH during lactation only were not different from controls, the effect of GH in this treatment paradigm may be mammogenic rather than galactopoietic per se. (J Clin Endocrinol Metab 72: 13021307,1991)
In contrast, neonatal growth of mice pups is positively related to the concentration of fat in milk (10). Improving the quantity and/or the quality of a mother's milk would be beneficial to nursing preterm or low birth weight infants. The galactopoietic properties of GH in ruminants are well established, as treatment of lactating females with GH increases milk yields (11-13). GH may also be lactogenic in primates, as evidenced by an increase in a-lactalbumin production in cultures of primate mammary tissue (14). By increasing the quantity of milk produced, GH administration to ruminants increases the yield of fat, protein, and lactose; nevertheless, the composition of nutrients in the milk is not altered by GH administration (11-13). GH treatment also elevates serum concentrations of insulin-like growth factorI (IGF-I) (11), and infusion of IGF-I into an artery of the mammary gland increases milk yield in goats (15). Since IGF-I is detected in milk (16, 17), stimulates growth in rat pups (18), and is positively associated with
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EFFECTS OF GH ON NEONATAL GROWTH rate of growth in preterm infants (19), it is possible that, in addition to milk nutrients, IGF-I in milk is important for infant growth. Importantly, piglets suckling sows treated with GH gain more weight during the lactational period (13). The present study examined the effects of administration of recombinant human GH to either mothers or nursing infants on neonatal growth rate in rhesus monkeys. The studies were designed to determine whether GH could influence infant growth by treating infants directly with GH or by administering GH to mothers during pregnancy and/or lactation.
Materials and Methods Subjects for these studies were rhesus monkeys (Macaca mulatto) from the breeding colony at the Yerkes Primate Research Center (Lawrenceville, GA). Animals were randomly assigned to a specific treatment group, as described below. All infants remained with their mothers throughout the study period and were thus allowed to nurse ad libitum. Animals were housed in outdoor compounds with attached shelters and were members of social groups containing adult males, other adult females, and juvenile male and female monkeys. All mothers were between 5-14 yr of age and had from 0-10 previous pregnancies. The effect of parity as a function of treatment condition is statistically evaluated in Results. Animal husbandry and handling for the collection of physiological data have been described previously (20). Animals were fed Wayne 25% Primate Diet 8663 (Madison, WI) ad libitum twice daily and received fresh fruit once daily. The primate diet contained more than 25% protein, more than 5% fat, and less than 5% fiber.
With this feeding regimen, all mothers could eat to satiety. To address the effects of recombinant human GH treatment on neonatal growth of infants, animals were randomly assigned to one of six treatment groups: 1) control (CON; n = 9) mothers received physiological saline from the second trimester of pregnancy through 7 weeks postpartum; 2) mothers received GH 3 days/week (Monday, Wednesday, and Friday) during pregnancy only (PRG; n = 8) from the second trimester to parturition; 3) mothers received GH 3 days/week during lactation only (LAC; n = 9) from parturition through 7 weeks postpartum; 4) mothers received GH 3 days/week during pregnancy and lactation (PRG/LAC; n = 8) from the second trimester of pregnancy through 7 weeks postpartum; 5) infants of GH-naive mothers received daily injections of saline (INF C ; n = 10) from birth through 7 weeks postpartum; and 6) infants of GH-naive mothers received daily injections of GH (INF G HI n = 9) from parturition through 7 weeks postpartum. Pregnant and lactating mothers receiving GH three times each week were administered 250 Mg/kg-injection (750 jug/week), sc. Infants receiving daily GH were administered 100 /xg/kg-day (700 jug/week), sc, a treatment regimen approximating that used to treat GHdeficient children (21). The distribution of male and female infants was similar within each treatment group (percentage of males: CON, 56%; PRG, 75%; LAC, 44%; PRG/LAC, 63%; INFc, 50%; and INF G H , 56%). Maternal body weights were obtained weekly from the second
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trimester of pregnancy through 7 weeks postpartum. Infant body weights were obtained at birth and weekly thereafter through 7 weeks of age. Posttreatment weights were obtained on all subjects at 35 weeks of age. Serum samples were obtained from infants every 2 weeks for the determination of IGF-I and insulin. Serum samples were obtained from the mothers 48 h after a GH injection each week for the determination of PRL. Serum biochemistries were determined in infants at 6 weeks of age. Breast milk was manually expressed from a subsample of mothers (CON, LAC, and PRG/LAC) 2 and 5 weeks postpartum, 48 h after the previous GH or saline injection, for the determination of milk fat, total protein, lactose, and IGF-I. Mothers were anesthetized with ketamine hydrochloride (10 mg/kg, im) and given 2.0 IU oxytocin (Peninsula Laboratories, Belmont, CA), iv, to facilitate the collection of milk. Analyses Serum PRL (22) and IGF-I concentrations (23) were determined by previously validated RIA procedures. Rather than using a pool of normal human serum for the IGF-I standard curve (23), recombinant human IGF-I (Amgen Corp., Thousand Oaks, CA) was used as the standard, and all results were expressed as micrograms of IGF-I per L (24). Inter- and intraassay coefficients of variation for both assays were less than 8.0%. Unextracted milk was analyzed using the same IGF-I assay. All milk IGF-I determinations were performed in the same assay. Serial dilutions of unextracted rhesus milk were parallel to the IGF-I standard curve (Fig. 1). Unknown samples were assayed at volumes of 0.5 pL equivalents to 20 /xL. The intraassay coefficient of variation averaged 5.2%. Concentrations of milk lactose were determined using a kit from Boehringer Mannheim (no. 176 303, Indianapolis, IN). Concentrations of total protein in milk were determined with a kit from Sigma Chemical Co. (St. Louis, MO). The amount of fat in the milk was determined by the creamatocrit method (25). Serum biochemistries were determined by Smith-Kline Bioscience Laboratories (Tucker, GA). Data were expressed as the mean ± sem and were analyzed
1
10 100 1000 log pg IGF-1 or uL milk FIG. 1. Displacement curves for purified human IGF-I standard and serial dilutions of rhesus monkey milk samples in the RIA of IGF-I. B/ Bo, Bound to free ratio.
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with an analysis of variance model for repeated measures. Specific comparisons among treatment groups used Duncan multiple range tests. Linear relationships among variables were evaluated by linear regression. Statistical tests yielding P < 0.05 were considered significant. Results
As illustrated in Fig. 2, birth weights were indistinguishable among the six treatment groups (F5;46 = 1.55; P > 0.05). In contrast, 7 weeks postpartum the PRG/ LAC infants were significantly heavier than the CON, LAC, INFc, and INFGH infants, with the infants of PRG mothers being intermediate (F5,46 = 4.28; P < 0.05). The CON, LAC, INFc, and INF G H infants were indistinguishable from one another. This overall difference in body weight for the PRG/LAC group was reflected in significantly greater weekly weight gain compared to other groups, with the PRG infants again being intermediate (F5i46 = 2.87; P < 0.05). By 35 weeks of age, these differences in body weight (kilograms) among the infants were abolished (CON, 1.57 ± 0.06; LAC, 1.67 ± 0.09; PRG, 1.54 ± 0.04; PRG/LAC, 1.69 ± 0.07; INFC, 1.61 ± 0.13; INFGH, 1.71 ± 0.17; F5,46 = 1.35; P > 0.05). Despite these differences in postnatal body weight gain, serum concentrations of IGF-I during the treatment period did not vary significantly among the six groups (F5i46 = 1.10; P > 0.05) at any time during week 5 or 7 of treatment. Serum concentrations of IGF-I (micrograms per L) did rise significantly in all infants from 5-7 weeks of age (23.8 ± 2.3 vs. 33.2 ± 3.0; n = 52; F1>46 = 19.57; P < 0.05). Serum concentrations of IGF-I (micrograms per L) at 7 weeks of age averaged 38.5 ± 11.3 for CON, 41.5 ± 10.9 for LAC, 37.5 ± 8.8 for PRG, 35.8 ± 7.9 for PRG/ LAC, 31.0 ± 8.1 for INFC, and 16.1 ± 2.8 for INFGH.
JCE & M • 1991 Vol 72 • No 6
Although groups were not differentiated on the basis of circulating IGF-I concentrations, average serum IGF-I values during the 7 weeks postpartum predicted overall body weight gain (F M 6 = 19.95; P < 0.05; r2 = 0.28). Serum biochemistries obtained at 6-7 weeks of age were similar among the infants of the six treatment groups (Table 1; F5,46 < 2.15; P > 0.05). Maternal body weights (kilograms) were not affected by treatments, either at parturition (CON, 8.14 ± 0.33; LAC, 8.26 ± 0.57; PRG, 7.95 ± 0.33; PRG/LAC, 8.36 ± 0.33; INFc, 7.53 ± 0.29; INFGH, 7.61 ± 0.49; F5,46 < 1.00; P > 0.05) or at the completion of the treatment 7 weeks postpartum (CON, 7.62 ± 0.25; LAC, 7.88 ± 0.57; PRG, 7.73 ± 0.32; PRG/LAC, 8.01 ± 0.28; INFC, 7.92 ± 0.38; INFGH, 8.05 ± 0.39; F5,46 < 1.00; P > 0.05). Maternal parity also had no effect on infant growth under the
present treatment conditions (covariance analysis of variance; parity by infant weight gain F li50 = 1.24; P > 0.05; r = 0.03; n = 52). Maternal serum PRL concentrations for the CON, PRG, LAC, and PRG/LAC groups rose significantly in all groups in the week before parturition and remained elevated throughout the 7-week study period (Fig. 3; F10>290 = 15.39; P < 0.05). PRL concentrations did not vary among these four groups of lactating females (F3,29 = 1.64; P > 0.05) during the study period (F30>290 < 1.00; P > 0.05). Assessment of milk samples revealed no differences in any parameter between 2 and 7 weeks of lactation, so the two time points were averaged for analysis (Fig. 4). The percentage of fat in the milk was significantly greater in PRG/LAC compared to either CON or LAC mothers (F2>23 = 4.57; P < 0.05). In contrast, concentrations of total protein (F2,23 = 1.59; P > 0.05), lactose (F2 23 < 1.00; P > 0.05), and IGF-I (F2)23 = 1.69; P > 0.05) did
1.00 D CON ffl LAC
FIG. 2. Mean ± SEM body weights (kilograms) of infants in each treatment group at birth and at the completion of treatment at 7 weeks of age. Bars with different letters indicated groups are significantly different from one another using the Duncan multiple range test (P < 0.05).
0.75-
•
PRG
•
PRG/LAC
QH INF
0 a
o 0.50
a
0.25
Birth
Age (weeks)
Seven
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EFFECTS OF GH ON NEONATAL GROWTH TABLE 1. Mean (±SEM) values of auxological measures for each group in Exp 1 at 12 weeks of age Auxological measure Rx group Crown-rump Tibia (cm) (cm)
Tail (cm)
8.6 (0.1) 8.7 (0.1) 9.0 (0.1)
14.3 (0.4) 14.3 (0.3) 15.5 (0.8)
CON GHi GHm
24.0 (0.4) 24.6 (0.3) 25.6 (0.2)
Skin fold Bone age (% of adult) (mm) 39.2 (1.8) 39.9 (0.7) 39.5 (1.3)
1.5 (0.1) 1.3 (0.1) 1.8 (0.2)
not differ significantly in the milk of the three treatment groups. Furthermore, the concentration of total protein in the milk was significantly associated with the overall weight gain of the infants, regardless of treatment group (Fli24 = 11.89; P < 0.05; r2 = 0.30), whereas neither lactose, fat, nor IGF-I content in the milk was related to individual infant body weight gain. In addition, total protein concentrations in milk were significantly associated with infant serum IGF-I concentrations (Fi>24 = 6.15; P < 0.05; r2 = 0.0.20).
Discussion These results indicate that neonatal increases in body weight were significantly advanced in nursing infants whose mothers received GH treatment during both pregnancy and lactation. Birth weights were similar among all subjects, suggesting that GH treatment of mothers during pregnancy does not affect fetal growth. Infants
themselves treated with GH did not grow faster than untreated control subjects. Similarly, treatment of mothers with GH only during lactation had no effect on
FIG. 3. Mean ± SEM serum PRL concentrations in mothers from 4 weeks before parturition through 7 weeks postpartum. Data for INFC and INF G H mothers are not shown.
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neonatal growth rates. In contrast, mothers treated with GH during the last two trimesters of pregnancy had infants whose growth rates were statistically intermediate between those of PRG/LAC and the remaining groups, suggesting that GH treatment during pregnancy alone can augment postpartum weight gains in infants that subsequently nurse. Although the present study was designed only to determine whether GH administration to infants or mothers would augment neonatal growth, the results do address the question of whether the effect of GH was mammogenic or galactopoietic. Mothers treated with GH during pregnancy and lactation had significantly higher amounts of fat in milk than did either control mothers or mothers treated only during lactation. Although group differences in total protein in milk were not evident, the amount of milk protein was significantly related to the amount of weight gained. These data are consistent with reports in humans that milk protein is essential for enhanced weight gains in neonatal preterm infants (2-6, 8, 9). Although fat content in milk may not be related to weight gain in human infants (6, 8), growth rates in mice are predicted from milk fat concentration (10). The observation that milk fat content was increased in PRG/ LAC females contrasts with data from dairy cows (12) and sows (13) which indicate that daily administration of GH does not affect the composition of milk (12). Although serum IGF-I concentrations in infants were not different as a function of treatment condition, average IGF-I concentrations were significantly associated with the weight gain of individual infants. These data support the observation that IGF-I secretion is important for maintaining normal neonatal growth in rats (18).
J3 PL,
-
2
0
2
4
Weeks from Parturition
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JCE & M • 1991 Vol 72 • No 6
FIG. 4. Mean ± SEM values of percent fat, total protein, lactose, and IGF-I in milk from CON, LAC, and PRG/LAC mothers. Values represent the means from samples collected at weeks 2 and 7 postpartum. Bars with different letters indicate groups that are significantly different from one another (P < 0.05).
CON
LAC Treatment Group
Preterm, low birth weight infants have lower circulating concentrations of IGF-I (19), but it is not known how milk supplementation which stimulates accelerated growth in these infants affects circulating levels of IGFI. Nutrient intake, in particular protein content, regulates IGF-I secretion in a variety of species (26-28) and is primarily important for regulating IGF-I concentrations during the neonatal period (29). The significant relationship observed between milk protein and serum IGF-I concentrations in nursing infants suggests that milk protein may have influenced neonatal growth by regulating IGF-I production in neonates. Although differences in milk quality may account for the differences in body weight gain, it is possible that the accelerated weight gain of PRG/LAC infants was due in part to increased maternal milk yields at each nursing bout. A consistent effect of GH administration to lactating nonprimate animals is a significant increase in milk yields and, thus, an increased yield of milk nutrients (11, 12). This galactopoietic effect of GH may be mediated through IGF-I, as infusion of IGF-I directly into a mammary artery (15), but not into the peripheral circulation (11), increases milk yield in goats. Furthermore, IGF-I does enhance PRL-induced increases in casein production in rabbit mammary cultures (30). We were not able to quantify milk yields in the present experiment given the difficulty in standardizing the collection procedure and, thus, were not able to directly determine the galactopoietic properties of GH. Nevertheless, the effect of GH on neonatal growth was not simply due to the galactopoietic properties of GH, as infants of mothers treated with GH during lactation only (LAC) had growth rates similar to those of control infants. A daily regimen of GH treatment during lactation (11-13), rather than 3 days/week, might have produced a different effect.
PRG/LAC
CON
LAC Treatment Group
PRG/LAC
A more likely hypothesis for the present finding is that mammogenesis was affected by GH treatment during pregnancy and that this, in turn, enhanced milk yields, particularly with continued GH administration through lactation. Milk yield is influenced by the number of mammary gland cells as well as the secretory capacity of the cells (31). In this regard, mammary gland development is enhanced by placental lactogens (30), which bind to GH receptors on mammary tissue (32). In mice, hypophysectomy during pregnancy does not affect mammary gland development due to the presence of high concentrations of circulating PRL-like placental lactogens, but does reduce protein synthesis and, probably, the secretory capacity of the mammary gland (33). Treatment of hypophysectomized nonpregnant monkeys with GH does not induce mammary growth (34). On the other hand, mammary gland cell proliferation in most species takes place during pregnancy, with some increase in cell number occurring during lactation (31, 35). These data suggest that GH treatment during pregnancy amplifies mammary gland growth and the consequent increase in its secretory capacity. The fact that the infants of mothers that received GH during pregnancy exhibited weight gains during the neonatal period that were intermediate between those of the PRG/LAC and CON groups suggests that this treatment regimen may have affected mammary growth and its secretory capacity. It is important to note that the differences in body weight among the various treatment groups were no longer evident at 35 weeks of age, 4-5 months after the termination of treatment and at a time when infant rhesus monkeys are typically weaned from their mothers and rely almost exclusively on solid food (36). Thus, the GH treatment simply accelerated weight gains and did not produce bigger infants.
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EFFECTS OF GH ON NEONATAL GROWTH
Acknowledgments 17.
The technical assistance of Erika Seres and Beth Wellington is appreciated. Reagents for the PRL assay and the IGF-I antibody were kindly provided by the National Hormone and Pituitary Program. All assays were performed in the Yerkes RIA Laboratory. We wish to thank the Department of Veterinary Medicine at the Primate Center for assisting in the collection of blood samples from the infants. These projects were approved by the Institutional Animal Care and Use Committee of Emory University. The Yerkes Primate Research Center is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.
20.
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