Progress 07/01/99 to 06/30/05
Outputs
Despite anabolic responses in mammals, growth hormone (GH) administered to rapidly growing domestic poultry does not improve muscle deposition or improve feed efficiency. Studies in our laboratory for the last five years have begun to elucidate the underlying reasons for this significant discrepancy. By administering GH in a physiologically appropriate pulse pattern and over a comprehensive dose response range to late post-hatch broilers, it was determined that exogenous GH suppresses hepatic Type III iodothyronine deiodinase (DIII) activity and gene expression, which degrades both metabolically active triiodothyronine (T3), and thyroxine (T4). This effect was determined to be a direct effect of the hormone, as verified in cultured LMH hepatoma cells. IGF-1 was also determined to suppress DIII gene expression, but this effect was not additive with that of GH, suggesting a common signaling pathway. Suppression of DIII resulted in a hyper-T3 state, with reduced net
skeletal muscle accretion and impaired hepatic IGF-1 release (necessary for an anabolic response), despite significantly elevated hepatic tissue IGF-1. This effect was determined to be developmentally regulated, as GH administered to older broilers approaching sexual maturity did not result in elevated T3, and did allow for increased circulating IGF-1, which was elevated over fourfold above control levels. Growth hormone administration was also associated with a 19% reduction in voluntary feed intake (FI), and a 50% decrease in hypothalamic neuropeptide Y (NPY) in the infundibular nuclei and midline region. NPY is likely a secondary factor mediating the GH-induced reduction in FI, as hepatic leptin gene expression was significantly increased with GH administration and is known to regulate NPY production in the hypothalamus. These responses in hypothalamic NPY and hepatic leptin are not secondary to reduced FI, as birds pair-fed to the reduced FI level of GH-treated birds did not
exhibit related changes in NPY and leptin. In order to realize any potential anabolic effect of exogenous GH in the broiler, the hyper-T3 state and excessive reduction in voluntary FI, mediated via GH-induced suppression of DIII and induction of leptin in the liver, would both need to be selectively eliminated. This would coordinate FI and metabolic (thyroid) hormone responses to allow for a potentially anabolic framework to be realized, and upon which GH-induced stimulation of circulating IGF-1 would be predicted to elicit positive and significant effects on net skeletal muscle deposition. To do so necessitates an understanding of the pathway of GH signaling in the avian liver, and studies in our laboratory now and for the immediate future are focused on doing so, using the LMH cell line as a model system.
Impacts
Although the anabolic effects of GH are well recognized in mammals, fish and in children suffering from primary pituitary insufficiency, and its clinical use has extended to catabolic states including AIDS and burn victims, the metabolic effects of the hormone are less recognized and in many cases misunderstood. For example, the biomedical literature reflects associated changes in thyroid hormone status in patients treated with GH for non-growth deficient states, but the assumption underlying these changes is an effect of GH on Type I iodothyronine deiodinase (DI), which converts the metabolically inactive T4 to active T3. Our work clearly demonstrates that the mechanism by which GH increases T3 is by suppression of hepatic DIII, with no effect on DI, which is a significant distinction in that it impacts the hypothalamic-pituitary feedback loop of thyroid hormone regulation in an opposite manner to a DI-mediated effect. The recognition that GH dramatically suppresses
voluntary FI via alterations in peripheral leptin and central NPY gene expression may have significant implications for controlling human obesity and its associated health risks (e.g., Type II Diabetes, osteoarthritis and related joint diseases, cardiovascular disease, etc.). In total, the work conducted for this project has provided clear evidence of an important role for GH in metabolic regulation, which has not been previously well recognized in contrast to the growth-promoting effects of the hormone, and may lead to useful strategies for improving food animal productivity and human metabolic disorders.
Publications
- Zhou, Y., Wang, X.-H., Hadley, J., Corey, S. J. and Vasilatos-Younken, R. 2005. Regulation of JAK2 protein expression by chronic, pulsatile GH administration in vivo: A proposed mechanism for ligand enhancement of signal transduction. Gen. Comp. Endocrinol. 144:128-139.
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Progress 01/01/04 to 12/31/04
Outputs
GH has profound effects in poultry including regulation of metabolic enzymes (such as hepatic type III iodothyronine deiodinase (5DIII)), and a variety of other proteins (including IGF-I). Using the LMH (Leghorn Male Hepatoma) cell line, we have previously demonstrated that GH affects hepatic 5DIII activity and gene expression in vitro in the same manner as in vivo (addition of GH decreases 5DIII which normally degrades the metabolically active thyroid hormone, T3, resulting in elevated levels of T3). In other experiments conducted previously, we have determined that both GH and IGF-I individually suppress 5DIII gene expression. However, the effects of both GH and IGF-I together are not additive, suggesting a common signaling pathway is being activated. Previous in vivo and in vitro experiments have demonstrated that JAK2 is activated following GH binding to the GH receptor; and that the effect of GH on JAK2 phosphorylation may be influenced by the age of the bird, or
analogously, the growth phase of the cells, suggesting that GH may have differential effects on JAK2 activation (and 5DIII gene expression) at different stages of growth. Recent in vitro experiments with the LMH cell line have shown that JAK2 activation is rapid, and GH dose-dependent. Additional experiments with the LMH cell line have recently been conducted to assess the effects of GH on signaling components that may be part of the cascade that impacts deiodinase activity in the liver. These experiments indicate that GH activates several signaling pathways, including Erk1/2 (p44/42 MAP kinase), and STAT (signal transducers and activators of transcription). For other species/ cell lines it has been determined that GH-activated JAK2 phosphorylates at least four members of the STAT family. In vitro experiments with LMH have shown that STAT5b is activated by GH in a dose-dependent and time-dependent manner, as is p44/42 MAP kinase. Other JAK (e.g., JAK1, Tyk), STATs (e.g., STAT1,3,5a)
and MAP kinases (e.g., p38 MAP kinase) have yet to be examined in LMH. Additional experiments with additional GH dosages and treatment times, as well as those utilizing specific signaling component inhibitors, and those conducted with both GH and IGF-I will be needed to determine which of the activated pathways is crucial for the regulation of 5DIII. These experiments, and experiments with LMH in various phases of growth, are currently being conducted.
Impacts
Work in our laboratory has demonstrated that GH has a significant impact on thyroid hormone activity through regulation of the expression of the key enzyme, 5DIII, that degrades the metabolically active form of thyroid hormone, T(3), as well as its precursor, T(4). Thyroid hormones play a central role in growth, metabolism, and almost every biological function, as evidenced by the number of genes with regulatory sites for thyroid hormone in their sequence. Whereas GH has been recognized to have important applications in enhancing growth and lactation in food animals, as well as therapeutic uses in the biomedical arena, its regulation of T(3) activity suggests even broader biological implications.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Previous in vivo work done in this laboratory has demonstrated that growth hormone (GH) has profound metabolic effects in poultry, including regulation of the metabolically active thyroid hormone 3,3'5-triiodothyronine (T(3)), by decreasing hepatic type III iodothyronine deiodinase (5DIII) (which degrades T(3) and T(4)), resulting in elevated levels of T(3). Through experiments with the Leghorn Male Hepatoma (LMH) cell line previously described, we have demonstrated that GH affects deiodinase activity and gene expression in vitro in the same manner as in vivo. In addition to a direct impact on the thyroid hormone axis, it was determined in vivo that GH increases hepatic IGF-I protein content and mRNA levels, but does NOT increase circulating IGF-I levels, suggesting an impairment of IGF-I release possibly due to the hyper-T(3) state. In order to begin to understand the relationship between GH, IGF-I and the thyroid hormone axis, recent in vitro experiments with LMH
have been conducted to determine if IGF-I alone, or in conjunction with GH, also plays a role in regulating T(3) through 5DIII. These experiments indicate that IGF-I does have an impact on 5DIII activity; but that the relationship with GH on 5DIII is highly complex. Additional experiments conducted with LMH cells (which do not constitutively express IGF-I), and LMH cells that have been retrovirally transfected to express IGF-I, indicate that both GH and IGF-I individually suppress 5DIII gene expression. However, preliminary data indicate that the effects of both GH and IGF-I do not appear to be additive, suggesting they may elicit effects through a common pathway. Current experiments to confirm and further examine these results are underway. Additional experiments are also being conducted to assess the effects of GH on signaling components that are part of a cascade that impacts deiodinase activity in the liver, especially JAK2. The results of preliminary experiments suggest that the
effect of GH on JAK2 phosphorylation may be influenced by the age of the bird, or analogously in vitro, the passage number of the cells. This suggests that GH may have differential effects on JAK2 activation and 5DIII gene expression at different stages of growth.
Impacts
There is a large volume of accumulated evidence for the role that thyroid hormones play in regulating GH, particularly in poultry. Work in our laboratory has shown that the reverse is also true: GH has a significant impact on thyroid hormone activity through regulation of gene expression of the key enzyme, 5DIII, that degrades the metabolically active form of thyroid hormone, T(3), as well as its precursor, T(4). In addition, preliminary experiments indicate the IGF-I may also contribute to T(3) regulation. Given that thyroid hormones play such a key role in somatic growth and metabolism, as well as commercially relevant functions such as pipping in poultry during the hatching process, an understanding of complexity of thyroid hormone regulation is of considerable importance.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Growth hormone (GH) has profound metabolic effects in poultry, including regulation of thyroid hormones. We have previously demonstrated that, in vivo, GH decreases hepatic type III iodothyronine deiodinase (5DIII) (which normally degrades 3,3'5-triiodothyronine (T3)), resulting in elevated levels of T3. In addition, while GH increases hepatic IGF-I protein content and mRNA levels, circulating IGF-I is not increased, suggesting an impairment of IGF-I release, possibly due to the hyper-T3 state. Recent experiments were conducted in an in vitro system utilizing the Leghorn Male Hepatoma (LMH) cell line to confirm that alterations in hepatic 5DIII gene expression and enzyme activity observed with GH administration in vivo were direct effects of GH. We have recently determined that this cell line retains hepatocyte characteristics, including glycogen production, and 5DIII activity; expresses all of the components necessary for GH signaling, including GH receptors and key
elements in the signaling pathway; and expresses both the type I IGF receptor and a 28 kDa IGF binding protein, but does not express IGF-I itself (so any response to GH is not confounded by a secondary response to IGF-I). We have demonstrated that GH affects deiodinase activity and gene expression in vitro in the same manner as in vivo (increasing GH decreases 5DIII enzyme activity, and down regulates 5DIII gene expression). Current experiments with LMH retrovirally transfected to express IGF-I, non-transfected LMH, and non-transfected LMH to which IGF-I has been added, are being conducted to assess whether IGF-I influences 5DIII activity and gene expression independently or synergistically with GH.
Impacts
The current work in our laboratory is the first to define an in vitro system capable of quantitatively discerning a GH biological response, and to characterize a hepatoma cell line for which the deiodinase pathway has been defined. This will allow for considerable opportunities to further investigate factors controlling the pathway of thyroid hormone metabolism, as well as the specificity of the effect of GH on suppressing 5DIII gene expression. Given the pervasive role of thyroid hormones in overall somatic growth and metabolism, further understanding these is of considerable importance to improving animal growth and nutrient utilization, as well as for providing potential insights into optimal use of GH in non-growth hormone deficient states for which GH is a therapeutic adjunct.
Publications
- Farhat, A., Maddox, C. W., Edwards, M. E., Costell, M. H., Hadley, J. A., and Vasilatos-Younken, R. 2002. Oral lavage with polyethylene glycol reduces microbial colonization in the gastrointestinal tract of broilers. Poultry Sci. 81: 585-589.
- Farhat, A., Edwards, M. E., Costell, M. H., Hadley, J. A., Walker, P. N., and Vasilatos-Younken, R. 2002. A low residue nutritive supplement as an alternative to feed withdrawal in broilers: efficacy for gastrointestinal tract emptying and maintenance of live weight prior to slaughter. Poultry Sci. 81: 1406-1414.
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Progress 01/01/01 to 12/31/01
Outputs
In previous experiments, chronic chicken GH (cGH) administration to late posthatch broilers resulted in dose-dependent suppression of hepatic Type III iodothyronine deiodinase (DIII) activity, which degrades 3,3'5-triiodothyronine (T3) and thyroxine (T4). Consequently, circulating T3 is markedly increased, with profound effects upon body weight gain and skeletal muscle mass, which were reduced 25 and 12%, respectively. In conjunction with elevated T3, release of hepatic IGF-I into the circulation was impaired, despite large-fold increases in liver IGF-I protein and message. Subsequent experiments suggested a diminution in the induction of the hyper-T3 state by cGH as birds approached sexual maturity. We hypothesized that the hyper-T3 state was responsible for impairment of hepatic IGF-I release, and that circulating IGF-I in response to GH would be proportionately greater as birds approach sexual maturity because of attenuation of the T3 response. Recent experiments
evaluated plasma IGF-I concentrations in 9, 12 and 16-wk-old female broilers receiving daily I.M. injections of cGH for 10 days. Hepatic DIII was reduced and plasma T3 increased by cGH versus controls at every age. However, as previously noted, the increase in T3 was proportionately less as birds approached sexual maturity (16 wks). Plasma IGF-I declined to 21% of 9-wk-old levels in 16 wk controls, but was increased four-fold above controls in GH-treated birds at 16 wks, consistent with this hypothesis.
Impacts
Although circumstantial, these data are consistent with a greater relative response to GH with respect to increasing circulating IGF-I concentrations, if the GH-induced hyper-T3 state is attenuated. This suggests the possibility of realizing an IGF-I-mediated effect of GH on increasing muscle deposition if the effect of GH on hepatic DIII gene suppression can be blocked, and also provides new insights into the possible regulation of hepatic IGF-I release.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Growth hormone (GH) has profound metabolic effects in poultry, including regulation of thyroid hormone levels. Growth hormone increases the metabolically active thyroid hormone, 3,3'5-triiodothyronine (T(3)), by decreasing hepatic Type III iodothyronine deiodinase activity, which normally degrades both T(3) and thyroxine (T(4)). We have previously demonstrated that the magnitude of the increase in T3 by GH can be several-fold above normal, and that release of hepatic IGF-1 into the circulation is impaired, despite increases in hepatic IGF-1 protein and mRNA, possible due to this hyper-T(3) state. Recent experiments were conducted to determine if the ability of GH to elevate circulating T(3) concentrations is age-dependent, and could be attenuated (or possibly abolished) in older birds, and if so, whether an increase in circulating IGF-1 would then be observed. Female broilers received daily intramuscular injections of cGH for one week, beginning at 9, 12, and 16 weeks
of age, with the latter group approaching sexual maturity at the time of GH administration. Plasma T(3) was increased versus controls at the end of treatment with cGH for one week in 9-week-old birds, but not in 12 and 16-week-old birds, verifying diminution of the induction of a hyper-T(3) state by GH as birds approach sexual maturity. Plasma IGF-1 is currently being evaluated to determine if avoidance of the hyper-T(3) state at the older ages will allow for a positive response in circulating IGF-1 with cGH.
Impacts
Growth hormone has a variety of clinical applications in non-growth hormone deficient states (eg., Turner's syndrome, burn victims, etc.), but the magnitude of any beneficial effect is often limited for undefined reasons, and subsequent GH withdrawal leads to observed thyroid hormone imbalances of unknown etiology. The work conducted in our laboratory would suggest that the impact of GH in suppressing Type III deiodinase activity, resulting in alterations in thyroid hormone metabolism, and the possibility that a hyper-T(3) state may impair hepatic release of IGF-1, might underlie these observations. Further investigations to determine the mechanism by which hepatic IGF-1 release is inhibited in the presence of elevated T(3) concentrations will be important for developing strategies to control this, and thereby optimize the potential anabolic effects of GH.
Publications
- Vasilatos-Younken, R., Zhou, Y., Wang, X. H., McMurtry, J. P., Decuypere, E., Buys, N., Darras, V. and Tomas, F. 2000. Altered chicken thyroid hormone metabolism with chronic growth hormone (GH) enhancement in vivo: Consequences for skeletal muscle growth. J. Endocrinol. 166:609-620.
- Wang, X. H., Day, J., Zhou, Y., Beard, J. and Vasilatos-Younken, R. 2000. Evidence of a role for neuropeptide Y and monoamines in mediating the appetite suppressive effect of growth hormone. J. Endocrinol. 166:621-630.
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Progress 07/01/99 to 12/31/99
Outputs
This project was only recently initiated, and is an extension of our previous Project# 3409. We have continued analysis of the metabolic responses to GH in the broiler chicken, and confirmed that GH results in increased circulating triiodothyronine (T3) concentrations, directly related to a decrease in hepatic type III deiodinase activity. This appears to be at the pre-translational level, as confirmed by collaborative work with V. Darras and E. Decuypere, Leuven, Belgium. We have also confirmed that GH reduces hypothalamic levels of neuropeptide Y at high physiological dosages which, through our collaborative work with J.P. McMurtry at the USDA Growth Biology Lab in Beltsville, is found to involve alterations in hepatic leptin expression, which may mediate this effect.
Impacts
Chicken is the primary muscle food consumed in the U.S., with over 8 billion broilers raised annually. This research will further our understanding of the role of GH in regulation of metabolism and voluntary feed intake in the broiler, which are major determinants of growth. By understanding how GH brings about biological effects, strategies may be developed to selectively regulate them.
Publications
- Ashwell, C. M., McMurtry, J. P., Wang, X.-H., Zhou, Y. and Vasilatos-Younken, R. 1999. Effects of growth hormone and pair-feeding on leptin mRNA expression in liver and adipose tissue. Dom. Anim. Endocrinol. 17:77-84.
- Vasilatos-Younken, R., Wang, X.-H., Zhou, Y., Day, J. R., McMurtry, J. P., Rosenbrough, R. W., Decuypere, E., Buys, N., Darras, V., Beard, J. L. and Tomas, F. 1999. New insights into the mechanism and actions of growth hormone (GH) in poultry. Dom. Anim. Endocrinol. 17:181-190.
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