73, 477-484 (1989)
Triiodothyronine Inhibition of Thyrotropin-Releasing Hormone- and Growth Hormone-Releasing Factor-Induced Growth Hormone Secretion in Anesthetized Chickens COLIN G.%ANE$AND
Department of Animal Sciences, Rutgers-The State University, New Brunswick, New Jersey 08903, and *Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2%’ Accepted September 19, 1988 The ability of triiodothyronine (T3) to reduce basal and secretagogue-induced growth hormone (GH) release was examined in anesthetized young and adult male chickens. Infusion of T, had no effect on basal plasma concentrations of GH in either young or adult chickens. However, GH secretion following challenge with either thyrotropin-releasing hormone(TRH) or growth hormone-releasing hormone (GRF) was reduced, in a dosedependent manner, by the infusion of T,. In vivo sensitivity to T, inhibition was greater with TRH- than GRF-stimulated GH release in either young (ED,, for TRH-induced GH release, 0.34 +g T,/kg/min; ED,, for GRF-induced GH release, 0.49 pg T,/kg/min) or adult chickens (Ed,, for TRH-induced GH release, 0.11 pg T,/kg/min; ED5, for GRF-induced GI-I release 1.89, pg T&g/mm). Moreover, there was an increase in sensitivity of TRH-induced GH release to T, with age. 0 1989Academic PWS, IIIC.
While growth hormone (GH) synthesis in mammals is stimulated by thyroid hormones, in particular triiodothyronine (T3) (Samuels and Shapiro, 1976; Evans et al., 1982), there is strong evidence that thyroid hormones inhibit GH secretion in chickens. Plasma concentrations of GH are elevated in chickens with hypothyroidism due to thyroidectomy (Harvey et al., 1983), to goitrogen administration (Chiasson et al., 1979; Leung et al., 1985), to reduced monodeiodination of thyroxine (T4) in sexlinked dwarf chickens @canes et al., 1983; Stewart et al., 1984; Lilburn et al., 1986), or to autoimmune thyroiditis (Scanes et al., 1986). Moreover, administration of thyroid hormones decreases both basal (Harvey, 1983; Leung et al., 1985), and thyrotropinreleasing hormone (TRH)-stimulated plasma concentrations of GH (Harvey, 1983; Scanes et al., 1986) in conscious chickens. T, is less effective than T3 inhibiting GH release (Harvey, 1983; Leung et ’ To whom correspondence
should be addressed.
al., 1985; Scanes et al., 1986), presumably due to the role of T, as a prohormone, requiring monodeiodation to T, to become active. The T, inhibition of GH secretion may represent a negative feedback as GM has been found to stimulate 5’-monodeiodination in birds (Kuhn et al., 198.5, 1986a, c), mammals (Kuhn et al., 1986b), and fish (deLuze and Leloup, 1984). The present study examines the ability of varying doses of T, (given as an infusion) to affect basal, TRH-stimulated, and, growth hormone-releasing factor (GRF)-stimulated GH release in anesthetized young and aduit chickens. At present, there is little evi-: dence of whether T, can influence GRPinduced GH release or of possible changes in sensitivity to T, inhibition with age. MATERIALS
Male chicks (white Leghorn strain) were obtained at 1 day old from AvianServices (Flemington, NJ). Birds were reared in Pete&me battery cages under a 16L:&D photoperiod. At 8-10 weeks of age, birds were trans-
477 0016~6480/89 $1.50 Copyright 6 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
ferred to floor pens. Feed and water was available ad libitum throughout. Studies employed either young (6 to 7-week-old) or adult (24- to 30-week-old) chickens. One hour prior to experimentation, birds were anesthetized with sodium pentabarbital and pentabarbital anesthesia was maiutained throughout experimentation. The anesthesia decreases plasma concentration of GH and reduces the variability in the GH secretory response to TRH or GRF challenge @canes and Harvey, 1984). In each bird, a brachial vein was cannulated to receive infusions of T, (at doses of 0.03,O. 1, 0.3, 1.0, or 3 pglkglmin) or vehicle (saline, 50 pVkg/min), T, being infused for 60 mitt using a Harvard pump. The contralateral brachial artery was cannulated for serial blood sampling (0.5 ml/sample). Plasma samples were stored at - 20” prior to analysis. After 30 min of T, or vehicle infusion, birds were challenged with bolus intravenous injections of TRH (IO ug/kg) (Sigma, St. Louis, MO) or human GRF (10 &kg) (human GRF 1-44 NH,, kindly donated by Dr. T. Mowles, Hoffman LaRouche, Nutley, NJ). Both batches of hpGRF had similar purity as determined by HPLC, but may have varied in hydration and salt content which had not been determined. The rationale for the doses of TRH and GRF used was to employ approximately the maximally effective dose. The doseresponse relationship for either TRH- or GRFstimulating GH secretion is biphasic with maximal effective doses for TRH being 0.7-10 pg/kg in anesthetized young chicks and l-20 t&kg in anesthetized adult chickens, and for GRF IO pg/kg in anesthetized young chicks and 10-40 &kg in anesthetized adult chickens (Harvey and Scanes, 1984). Plasma concentrations of GH were determined by homologous radioimmunoassay (Harvey and Scanes, 1977). Statistical differences were determined by one-way analysis of variance (ANOVA) (for effect of TX treatment on the increase in plasma concentration of GH, 5 min following secretagogue challenge) or ANOVA for repeated samples (for differences in plasma concentrations of GH with time in a treatment group). Means were separated by LSD as the range test. Analysis of dos+response curves was performed by the method of DeLean and colleagues (1978).
RESULTS The effects of infusion of T, on TRH- and GRF-induced GH release in young and adult chickens are shown in Figs. 1 and 2. During the first 30 min of T, infusion, no changes in basal plasma concentrations of GH were observed with any dose of T, in adult chickens. In young chicks, plasma concentrations of GH declined 28.7% be-
tween 10 min prior to and 29 mm folowing the onset of saline infusion (from 10.0 + (N = 18)SEMl.19 rig/ml to 7.16 + (lQO.92 ng/ ml). In chicks infused with T3 at -3 pg/kg/min, a greater (P < 0.05) decrease (57.4%) in plasma concentrations of GH was observed (decreasing from 11.0 + (18)1.84 rig/ml to 4.7 * (18)0.65 &ml). The decline of the plasma GH concentration in chicks receiving lower doses of T, was intermediate between, and did not significantly differ from, those receiving the high dose of T, or saline. At high doses (0.3, 1.0, and 3.0 &kg/n&), T, reduced the GH secretory response to TRH in young chickens while no effect was observed with lower doses of T3 (0.03 and 0.1 Fg/kg/min) (Figs. I and 3). The magnitude of the inhibitory effect of T, did not differ among 0.3, 1.0, and 3.0 (ug/kg/min) doses. The dose of T, that reduced the GH response to TRH by 50% (ED,,) was estimated at 0.28 p,g/kg/min (Fig. 3). In adults, all doses of T, (0.03,0-l, 0.3, 1, and 3 CLg/kg/min) inhibited but did not abolish TRH-induced GH release (Figs. 1 and 2). The magnitude of the inhibitory effect of T, was dose dependent, since the increase in GH concentration in chickens receiving 1 or 13 pg T,/kg/min was less (P < 0.05) than that induced by the 0..3 t.r.g T,/ kg/min dose which in turn was less (P < 0.05) than that induced by 0.1 p&kg/mm, which was also less (P& 0.05) that of the lowest (0.03) dose. Adult chickens, therefore, appeared to be more sensitive to the inhibitory effect of T, on TRH-induced GH release (EDS,, 0.11 t&kg/mm) than young chicks (ED,, 0.34 &kg/min). Two batches of GRF (bGRF l-44) were employed in studies examining the effect of T, on GRF-induced GH release in chickens. In either young or adults, GRFstimulated GH secretion was observed irrespective of whether saline or T, pretreatment (with the exception of one dose of T, (3.0 pg/kg/min T3) in young chicks and batch 1 of GRF in young chicks where there
T3 INHIBITION I oooc
1. Effect of TT on basal and TRH-induced GH secretion in young (a) and adult chickens (b). Chickens received infusions (solid bars) of vehicle (O), T, at 3 wg/kg/min (V), T, at 1 pg/kg/min (Q), T, at 0.3 pg/kg/min (Cl), or T, 0.1 pg/kg/min (A). The arrows indicate time of TRH challenge (10 p&kg). Vertical bars indicate SEM (N = 6). FIG.
was no GRF-induced GH release). In young chicks, the infusion of T,, at a dosage of 1 or 3 pg/kg/min, reduced the GH secretory response to GRF (batch 1, P < 0.001; batch 2, P < 0.01). Lower doses were without effect on the response to GRF (Figs. 2 and 4). Similarly, in adult chickens the increment in GH concentration induced by GRF was decreased (P < 0.01) by the infusion of T, at doses of 1 and 3 &kg/min but not by lower doses (Figs. 2 and 4). In young chicks, the second batch of GRF evoked a larger (P < 0.05) increase in plasma concentrations of GH than did the first batch (Fig. 2). A similar trend was observed in adults; This batch difference in activity/potency presumably reflects differences in hydration and/or salt content in the two preparations. Figure 4 compares the
GH responses to GRF in the presence of varying doses of T3 in young and adult chickens. The data from the two batches of GRF are either combined as the absolute increase in plasma concentration of GM (AGHGRF in nglml) or were expressed as a percentage relative to the increase in plasma concentration of GH induced by GRF (in the absence of T,) (% AGHGRF). Infusion of T, (at either 1 or 3 l&kg&&) doses reduced the absolute GH response (AGHGRF in ngCml)to similar but low levels at the two ages (Fig. 4). However, the GH secretory response to GRF in young chicks appears to be more sensitive to Tj inhibition than that in adults (young EI& 0.49 bg T&g/min; adult ED,,, 1.89 ng ,T,J kg/min). The relative GH secretory responses (% AGHGRF) were decreased (P
(b) GRF GRF
Time (minutes) 2. Effect of TS on basal or GRF-induced GH release in young (a, c) or adult chickens (b, d). Chickens received infusions (solid bars) of vehicle (O), T3 at 3 pg/kg!min (V), T, at 1 p.g/kg/min (O), or T3 at 0.3 pg/kg/min (0). The arows indicate time of GRF challenge with lot A (a, b) or lot B (c, d) of GRF (10 pg/kg). Vertical bars indicate SEM (N = 6). FIG.
< 0.05) by the infusion of 0.3, 1, or 3 pg T,/kg/min in either young or adult chickens. The inhibitory effect of 1 or 3 Fg T,/kg/min was greater than the 0.3 pg/kg/min dose in young, but not adult chicks. DISCUSSION
In the present study with anesthetized chickens, T, had little effect on basal concentrations of GH in either young or adult birds. This is in contrast to the situation in
conscious chicks where a bolus injection of T3 reduces plasma concentrations of GH (Harvey, 1983). This may reflect the presumed lack of endogenous TRH and/or GRF in the anesthetized chicken. This is the first report that GRFstimulated GH release ia viva can be inhibited by T,; this acute effect of T, being observed in both young and adult chickens (Figs. 2 and 4). Previous work might have suggested that T, would inhibit GRFstimulated GH release. Chronic hypothy-
T3 @g/kg/mid FIG. 3. Dose-response inhibition of TRH-induced GH release of T, infused into young (x) (0) chickens. The increase in plasma concentrations of TRH-stimulated GH (AGH), 5 min TRH (10 p,g/kg) is the index of GH release. The increase in plasma concentrations of percentage of that in the absence of T, (0 dose) (top), the increase in plasma concentrations rig/ml (bottom). Vertical bars indicate SEM (N = 6).
roidism (in sex-linked dwarf chickens which are T, deficient) has been found to increase the secretory response to GRF (young chicks: Huybrechts et al., 1987; adults: Harvey et al., 1984). However, sexlinked dwarf chickens have other endocrine abnormalities, including reduced plasma concentrations of immunoreactive somatomedin C (Huybrechts et al., 1985) or somatomedin bioactivity (Hoshino et al., 1982) and reduced somatomedin C feedback could account for the increased response to GRF in dwarf chickens. Alternatively, even if a nonspecific strain difference is precluded and hypothyroidism is
and adult following GH as a of GH in
assumed to be the cause of the increased response to GRF in dwarf chicks, this may represent a chronic effect of hypothyroidism. For instance, hypothyroidism may result in somatotroph hypertrophy or hypoplasia. As in previous studies, administration of T, either acutely or chronically decreased the GH secretory response to TRH in young chicks (Harvey, 1983; Leung et a&, 198.5; Scanes et al., 1986). While conscious adult chickens do not increase CH seqetion in response to TRH, the response ‘is restored following anesthesia @canes and Harvey, 1984) and is also observed in (con-
I I T3
( g/kg/mid I-’
FIG. 4. Dose-response inhibition of GRF-induced GH release by T, infusion in young (x) and adult (0) chickens. The index of GRF-induced GH release was the increase ih plasma concentration of GH (AGH), 5 min following GRF injection (10 CLglkg). (a) Increase (AGH) as a percentage of that in the absence of T, (0 dose) and (b) increase (AGH) in rig/ml. Vertical doses indicate SEM (N = 6).
scious) adult sex-linked dwarf chickens (Harvey et al., 1984). Administration of T, reduced the response to TRH in anesthetized adults (Figs. 1 and 2). Similarly, T3 decreases the response to TRH in sexlinked dwarf chickens @canes, 1987). The mechanism by which T, inhibited both TRH- and GRF-induced GH secretion may involve: (a) down-regulation of both TRH and GRF receptors, (b) uncoupling of the receptors to the intracellular messanger system(s), and (c) direct effects of T, on GH synthesis and/or release. In view of the short time required for the T, effect, being apparent after only 30 min infusion, it may be considered unlikely for T3 to be exerting its effect via either protein synthesis or receptor down-regulation. However, these possibilities cannot be precluded. There appears to be an influence of age on the relative ability of T, to inhibit GRFand TRH-induced GH release. In young chicks, TX exerted a relatively similar inhibitory effect on either TRH- or GRF-induced GH release (EDSo for TRH, 0.20 kg/kg/mm; cf. ED5, for GRF, 0.49 pg/kg/min) with T3
reducing secretagogue-induced GH release at infusion doses of 0.3 or 3 pg/kg/min. In contrast, in the adult TRH-induced GH secretion is more sensitive to T, inhibition (ED,, for TRH, 0.11 pg T,/kg/min; cf. EDSo for GRF, 1.89 pg/kg/min) with TRHinduced GH release being inhibited by the low dose levels of T, (0.03 kg/kg/min), which had no effect on GRF-induced GH secretion. It might be speculated that the lack of a response to TRH in conscious adult chickens (Harvey et al., 1981; Scanes et al., 1981) reflects endogenous T, inhibition of TRH-induced GH release (due to higher sensitivity of the TRH-induced GH release to T3 in the adult). This is supported by previous studies in the conscious hypothyroid sex-link dwarf adult (Harvey et al., 1984; Scanes, 1987). The ability of T, to inhibit secretagogueinduced GH release in chickens is in marked contrast to the situation in mammals (see introduction for references). While T, is required for GH synthesis in mammals (e.g., Evans et al., 1982), it is not clear as to the locus of the inhibitory effects
of T, in the chicken. It is possible that T, affects receptors to secretagogues, the signal tranduction mechanism(s), or GH synthesis. It is unlikely, however, that GH synthesis requires T, in chickens in view of the prolonged elevation of plasma concentrations of GH in hypothyroid birds (see introduction for references). At present, the effects of thyroid hormones on GH release in lower vertebrate species have not been reported. ACKNOWLEDGMENTS This is a paper of the Journal Series, New Jersey State Agricultural Station (Project 18141) supported by State and Hatch Act funds and grants from the National Science Foundation (DCB 86-00447) and NSERC of Canada A3617. The authors are grateful to Mrs. H. Mozolic for her expert assistance and to Hoffman-LaRoche, Inc. for the gift of hpGRF l-44.
REFERENCES Chiisson, R. B., Sharp, P. J., Klandorf, H,, Scanes, C. G., and Harvey, S. (1979). The effect of rapeseed meal and methimazole on levels of plasma hormones in growing broiler cockerels. PO&. Sci. 58, 1575-1583. DeLean A., Munson, P. J., and Rodbard, D. (1978). Simultaneous analysis of families of sigmoidial curves: Application to bioassay, radioligand assay, and physiological dose-response curves. Amer. J. Physiol. 235, E97-E102. deluze, J., and Leloup, J. (1984). Fish growth hormone enhances peripheral conversion of thyroxine to triiodothyronine in the eel (Anguilla anguilla L.). Gen. Camp. Endocrinol. 56, 308-312. Evans, R. M., Birnberg, N. C., and Rosetield, M. G. (1982). Gluoocorticoid and thyroid hormones translationally regulate growth hormone gene expression. Proc. Natl. Acad. Sci. USA 79, 76597663. Harvey, S. (1983). Thyroid hormones inhibit growth hormone secretion in domestic fowl (Gallus domesticus).
Harvey, S., and Scanes, C. G. (1977). Purification and radioimmunoassay of chicken growth hormone. J. Endocrinol. 73, 321-329. Harvey, S., and Scanes C. G. (1984). Comparative stimulation of growth hormone secretion in anesthetized chickens by human pancreatic growth hormone-releasing factor (hpGRF) and thyrotro-
phin-releasing hormone (TRH). Newoendocrinology 39, 314-320. Harvey, S., Scanes, C. G., and Marsh, J. A. (1984). Stimulation of growth hormone secretion in dwarf chickens by thyrotrophin-releasing hormone (TRH) or human pancreatic growth-hormonereleasing factor (hpGRF). Gen. Comp. Endocrinol.
Harvey, S., Sterling, R. J., and Klandorf, H. (1983). Concentrations of triiodotbyronine, growth hormone, and luteinizing hormone in the plasma of thyroidectomized fowl (Gallus domesticus). Gen. Camp. Endocrinol. 50, 257-281. Harvey, S., Sterling, R. J., and Phillips, J. G. (1981). Diminution of thyrotrophin releasing hormoneinduced growth hormone secretion in adult domestic fowl (Gallus domesticus). J. Endocrinol. 89,405-410. Hoshino, S., Wakita, M., Suzuki, M,, and Yamamoto, K. (1982). Changes in a somatomedin-like factor and immunoassayable growth hormone during growth of normal and dwarf pullets and cockerels. Pouk Sci. 61, 777-784. Huybrechts, L. M., King, D. B., Lauterio, T. J., Marsh, J., and Scanes, C. G. (1985). Plasma concentrations of somatomedin C in hypophysecfomized, dwarf and intact growing domestic fowl as determined by heterologous radioimmunoassay. J. Endocrinol. 104, 233-239. Huybrechts, L. M., Kuhn, E. R., Decuypere, E., Merat, P., and Scanes, C. G. (1987). Plasma concentrations of growth hormone and somatomedin C in dwarf and normal chickens. Reprod. Nutr. Dev.
Kuhn, E. R., Huybrechts, L. M., Decuypere, E., and Merat, P. (1986a). Endocrinological effects dthe sex-linked dwarf gene: III Prolactin and growth hormone fail to increase the liver T,- S-monodeiodinase activity in the sex-linked dwarf chick embryo. Proc. 7th Eur. Poult. Con.. 965-969. Kuhn, E. R., Van Osselaer, P., Siau, O., Decuypere, E., and Moreels, A. (1986b). Thyroid diction in newborn Iambs: influence of prolactin and growth hormone. J. Endocrinol. 109, 215-219. Kuhn, E. R., Verheyen, G., Chiasson, R. B., Huts, C., and Decuypere, E. (1985). Ovine growth hurmone reverses the fasting-induced decrease in plasma T, in adult chickens. IRCS Med. Sci. 13, 451452. Kuhn, E. R., Verheyen, G., Decuypere, E., Huybrechts, L. M., and Igbal, A. (1986~). Growth hormone and thyrotropin releasing hornmne stimulate peripheral conversion of thyroxine into tiiodothyronine and the liver S’monodeiodinase activity in the adult chicken. 1RCS Med. Sci. 14, 479.
Leung, F. (1985). growth, normal
C., Taylor, J. E., and VanIderstine, A. Effects of dietary thyroid hormones on plasma Ts and T4 and growth hormone in and hypothyroid chickens. Gen. Comp. Endocrinol. 59, 91-99. Lilbum, M. S., Leung, F. C., Ngiam-Rilling, K., and Smith, J. H. (1986). The relationship between age and genotype and circulating concentrations of triiodothyronine (Ta), thyroxine (T,) and growth hormone in commercial meat strain chickens. Proc.
Samuels, H. H., and Shapiro, L. E. (1976). Thyroid hormone stimulates de nova growth hormone synthesis in cultured GH, cells: Evidence for the accumulation of a rate limiting RNA species in the induction process. Proc. Natl. Acad. Sci. USA 73, 3369-3373.
Scanes, C. G. (1987). The physiology of growth, growth hormone, and other growth factors on poultry. CRC Crit. Rev. Poult. Biol. 1, 51-105. Scanes, C. G., and Harvey, S. (1984). Stimulation of growth hormone secretion by human pancreatic growth hormone - releasing factor and thyrotro-
hormone in anesthetized chickens. 56, 198-203. Scanes, C. G., Denver, R. J., and Bowen, S. J. (1986). Effect of thyroid hormones on growth hormone secretion in broiler chickens. Poult. Sci. 65, Gen.
Scanes, C. G., Harvey, S., Morgan, B. A., and Hayes, M. (1981). Effect of synthetic thyrotrophin releasing hormone and its analogues on growth hormone secretion in the domestic fowl (Gallus 453.
Scanes, C. G., Marsh, J. A., Decuypere, E., and Rudas, P. (1983). Abnormalities in the plasma concentrations of thyroxine, triiodothyronine and growth hormone in sex-linked dwarf and autosoma1 dwarf white Leghorn domestic fowl (Gallus domesticus). J. Endocrinol. 97, 127-135. Stewart, P. A., Washburn, K. W., and Marks, H. L. (1984). Effect of the dw gene on growth, plasma hormone concentrations and hepatic enzyme activity in a randombred population of chickens. Growth