Progress 10/01/12 to 03/31/13
Outputs
Target Audience: animal physiologists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During this reporting period, we conducted experiments toidentify the signaling pathways mediating the effects of IGF-I on muscle cell proliferation, protein synthesis, and protein degradation in bovine satellite cells.We isolated muscle satellite cells from adult cattle and expanded them as myoblasts or induced them to form myotubes in culture. We determined the effects of IGF-I on proliferation of myoblasts and protein synthesis and degradation in myotubes in the presence or absence of specific signaling inhibitors. Our data suggest that both the MEK/ERK and PI3K/AKT pathways mediate the stimulatory effect of IGF-I on myoblast proliferation and that the PI3K/AKT pathway mediates this effect through cyclin D2. Our data also suggest that both the MEK/ERK and PI3K/AKT pathways mediate the stimulatory effect of IGF-I on protein synthesis through p70S6K and that the PI3K/AKT pathway mediates the inhibitory effect of IGF-I on protein degradation through FoxO3a.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ge X, Zhang Y, Jiang H. Signaling pathways mediating the effects of insulin-like growth factor-I in bovine muscle satellite cells. Mol Cell Endocrinol. 2013 Jun 15;372(1-2):23-9.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Jia D and Jiang, H. Growth hormone stimulates liver growth by increasing the size of hepatocytes. 2013 ADSA-ASAS Loint Annual Meeting.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Jiang, H and Ge X. Mechanisms of growth hormone and IGF-I stimulation of skeletal muscle growth in cattle. 2013 ADSA-ASAS Joint Annual Meeting.
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Progress 04/01/08 to 03/31/13
Outputs
Target Audience: Animal physiologists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate students: Yu, J., Eleswarapu, S., Zhao, L., Ge, X. (Animal Science) Postdoctoral research associate: Wang, A., Ge, X. (Animal Science). How have the results been disseminated to communities of interest? Through publications in peer-reviewed journals and presentations at professional conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Growth hormone is a major regulator of growth and metabolism in agriculturally important animals such as cattle. The biological effects of growth hormone include stimulation of growth and milk production and inhibition of fat deposition. Recombinant bovine growth hormone or bST has been used to increase milk production in the dairy industry. However, despite this application, the biological mechanisms of growth hormone actions are not fully understood. Through this project, we have gained new understanding of the molecular mechanism by which growth hormone regulates the expression of genes in the liver, how growth hormone stimulates skeletal muscle growth, and how nutritional intake modulates the action of growth hormone in cattle. This new knowledge helps explains why recombinant growth hormone can be used to improve productivity and production efficiency in cattle. We used a microarray analysis to identify genes that are regulated by growth hormone in bovine liver. The analysis revealed 392 and 25 genes that were upregulated and downregulated, respectively, by growth hormone in bovine liver. Whereas 60% of the growth hormone-upregulated genes contained at least one STAT5 binding site in their 2-kb promoters, only 30% of the growth hormone-downregulated genes contained putative STAT5 binding sites in their promoters. This difference suggests that whereas growth hormone upregulation of gene expression in bovine liver is probably mainly mediated by STAT5, growth hormone downregulation of gene expression in bovine liver is perhaps mediated by other transcription factors. Many of the identified growth hormone-regulated genes were previously not known to be regulated by growth hormone. We selected two of such genes, FOXA3, which is also known as hepatocyte nuclear factor 3 gamma, and FGF21, which is a recently discovered metabolic hormone, and studied their regulation by growth hormone in detail. Using real-time RT-PCR, we confirmed their upregulation by growth hormone in bovine liver. Using EMSA and ChIP-PCR, we validated STAT5 binding to the STAT5 binding sites in the promoters of these genes. Using co-transfection and reporter gene assays, we demonstrated the importance of these STAT5 binding sites in mediating growth hormone-induced gene transcription in cultured cells. The effects of growth hormone on growth and milk production in cattle depend on the nutritional status of the animals. Overfed cattle have greater blood IGF-I (another potent growth-stimulating hormone, the production of which is under the control of growth hormone), growth, and milk production responses to growth hormone administration than underfed cattle or those fed ad libtium. Given the potential role of circulating IGF-I in mediating the effects of growth hormone on animal growth and metabolism, we have conducted experiments to understand the mechanisms by which nutrition modulates the action of growth hormone on serum IGF-I in cattle. We have found that growth hormone injection caused greater increases in both serum IGF-I concentration and liver IGF-I mRNA expression in cows fed concentrates than in those fed hay only. In cows fed concentrates, the magnitude of growth hormone-induced increase in serum IGF-I concentration was, however, much greater than that in liver IGF-I mRNA expression. This difference was not because growth hormone-induced IGF-I mRNA was translated more efficiently. Growth hormone injection also induced greater increases in serum concentrations of IGFBP-3 and ALS in cows fed concentrates than hay. The injection, however, did not induce a greater increase in IGFBP-3 or ALS mRNA in cows fed concentrates than fed hay. These data and the fact that the ternary complex of IGF-I/IGFBP-3/ALS stabilizes each of the components in the blood, suggest that in cows with increased nutrition enhances serum IGF-I response to GH not only by increasing IGF-I mRNA response to growth hormone in the liver but also by elevating IGFBP-3 and ALS concentrations in the blood.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Jiang, H. and Ge, X. 2014. MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM--Mechanism of growth hormone stimulation of skeletal muscle growth in cattle. J Anim Sci. 92:21-9.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Growth hormone (GH) and insulin-like growth factor I (IGF-I) are two major stimulators of skeletal muscle growth. Since GH can increase the production of both endocrine and paracrine IGF-I, there has been a lot of controversy about the relative contribution of circulating IGF-I, locally produced IGF-I, and IGF-I-independent factors to the effect of GH on muscle growth. Muscle growth in animals can result from an increase in the number of nuclei in the multinucleated myofiber, an increase in the cytoplasmic volume of the myofiber, or both. In postnatal animals, new nuclei of myofibers are provided by satellite cells, the muscle stem cells. The cytoplasmic volume of a myofiber is determined by the balance between protein synthesis and protein degradation. During this reporting period, we have conducted experiments to determine the effects of GH on IGF-I mRNA expression in bovine myoblasts and myotubes, and to compare the effects of GH and IGF-I on proliferation of bovine myoblasts, differentiation of bovine myoblasts into myotubes, and protein synthesis and protein degradation in bovine myotubes. PARTICIPANTS: Honglin Jiang, principal investigator Xiaomei Ge, graduate student Jie Yu, graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We isolated satellite cells from adult cattle skeletal muscle and allowed them to proliferate as myoblasts or induced them to differentiate and fuse into myotubes in culture. Recombinant bovine GH at 10 and 100 ng/ml increased protein synthesis in myotubes, but had no effect on protein degradation in myotubes or on proliferation of myoblasts. Insulin growth factor I at 50 and 500 ng/ml stimulated protein synthesis in bovine myotubes, and this effect was much greater than that of GH at 10 or 100 ng/ml. Besides stimulating protein synthesis, IGF-I at 50 and 500 ng/ml also inhibited protein degradation in bovine myotubes, and IGF-I at 500 ng/ml stimulated myoblast proliferation. Neither GH nor IGF-I affected fusion of bovine myoblasts into myotubes. These data indicate that GH and IGF-I have largely different direct effects on bovine muscle cells. We also found that GH at 10 and 100 ng/ml had no effect on IGF-I mRNA expression in either myoblasts or myotubes. This lack of effect was not because the cultured myoblasts or myotubes were not responsive to GH, as GH receptor mRNA was detectable in them and as the expression of the cytokine-inducible SH2-containing protein (CISH) gene, a well-established GH target gene, was increased by GH in bovine myoblasts. Overall, the data suggest that GH may stimulate skeletal muscle growth in cattle in part through a direct stimulation of protein synthesis in the muscle and that this direct stimulation is not mediated through increased IGF-I mRNA expression in the muscle.
Publications
- Ge X, Yu J, Jiang H. 2012. Growth hormone stimulates protein synthesis in bovine skeletal muscle cells without altering insulin-like growth factor-I mRNA expression. J Anim Sci. 2012 Apr;90(4):1126-33.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Fibroblast growth factor 21 (FGF21) is a recently discovered regulator of glucose homeostasis, lipid metabolism, and insulin sensitivity. Interestingly, FGF21 is also known to inhibit Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling from the growth hormone (GH) receptor in the liver, where FGF21 mRNA is predominantly expressed. During this reporting period, we conducted experiments to test the hypothesis that GH might regulate the expression of FGF21 in the liver. PARTICIPANTS: Honglin Jiang, principal investigator Satyanaryana Eleswarapu, graduate student Jie Yu, graduate student and visiting scholar Xiaomei Ge, graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We found that GH injection to cattle increased FGF21 mRNA expression in the liver. Mapped by a 5'-rapid amplification of cDNA ends assay, transcription of the FGF21 gene in the bovine liver was mainly initiated from a nucleotide 24 bp downstream of a TATA box. The bovine FGF21 promoter contains three putative STAT5 binding sites. Electrophoretic mobility shift assays confirmed the ability of them to bind to liver STAT5 protein from GH-injected cattle. Chromatin immunoprecipitation assays demonstrated that GH administration increased the binding of STAT5 to the FGF21 promoter in the liver. Co-transfection analyses showed that GH induced reporter gene expression from the FGF21 promoter in a STAT5-dependent manner. GH also stimulated FGF21 mRNA expression in cultured mouse hepatocytes. These data together indicate that GH directly stimulates FGF21 gene transcription in the liver at least in part through STAT5. This finding, together with the fact that FGF21 inhibits GH-induced JAK2-STAT5 signaling in the liver, suggest a novel negative feedback loop that prevents excessive JAK2-STAT5 signaling from the GH receptor in the liver.
Publications
- Yu, J., Zhao, L., Wang, A., Eleswarapu, S., Ge, X., Chen, D., and Jiang, H. Growth hormone stimulates transcription of the fibroblast growth factor 21 gene in the liver through the signal transducer and activator of transcription 5. Endocrinology. 2011-1591; doi:10.1210/en.2011-1591.
- Ge, X., Yu, J., and Jiang, H. Growth hormone stimulates protein synthesis in bovine skeletal muscle cells without altering IGF-I mRNA expression. J Anim Sci. 2011 Nov 18. [Epub ahead of print].
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Besides stimulating growth and milk production, growth hormone (GH) is also known to increase serum concentrations of insulin-like growth factor I (IGF-I) in cattle. These effects of GH all depend on the nutritional status of cattle. Overfed cattle have greater blood IGF-I, growth, and milk production responses to GH administration than underfed cattle or those fed ad libtium. Given the potential role of circulating IGF-I in animal growth and metabolism, the nutrition-dependent IGF-I response to GH may mediate the nutrition-dependent growth or milk production response to GH in cattle. During this reporting period, we conducted experiments to understand the mechanisms by which nutrition modulates the action of GH on serum IGF-I in cattle. Specifically, we wanted to know whether nutrition-enhanced serum IGF-I response to GH in cattle is due to increased IGF-I gene expression or increased IGF-I mRNA translation in the liver, or increased retention of IGF-I in the circulation as a result of increased insulin-like growth factor binding protein 3 (IGFBP-3) and acid labile subunit (ALS) concentrations in the blood. PARTICIPANTS: Honglin Jiang, principal investigator Satyanaryana Eleswarapu, graduate student Miaozong Wu, graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Cows were fed hay ad libitum or corn-based concentrates in addition to free access to hay for 8 weeks. At week 8, serum concentrations of IGF-I, IGF binding protein 3 (IGFBP-3), and acid labile subunit (ALS) as well as their mRNA levels in liver were determined immediately before and 7 days after an injection of GH formulated for sustained release. Growth hormone injection caused greater increases in both serum IGF-I concentration and liver IGF-I mRNA expression in the cows fed concentrates than in those fed hay. In the cows fed concentrates, the magnitude of the GH-induced increase in serum IGF-I concentration was, however, much greater than that in liver IGF-I mRNA expression. This difference was not because GH-induced IGF-I mRNA was translated more efficiently. Growth hormone injection also induced greater increases in serum IGFBP-3 and ALS concentrations in the cows fed concentrates than fed hay. The injection, however, did not induce greater an increase in IGFBP-3 or ALS mRNA in the cows fed concentrates than fed hay. These data and the fact that the ternary complex of IGF-I/IGFBP-3/ALS stabilizes each of the components in the blood suggest that in cows increased nutrition enhances serum IGF-I response to GH not only by increasing IGF-I mRNA response to GH in the liver but also by elevating IGFBP-3 and ALS concentrations in the blood.
Publications
- Wu, M., Hall, J., Akers, R. M., and Jiang, H., 2010. Effect of feeding level on serum insulin-like growth factor I (IGF-I) response to growth hormone injection. Journal of Endocrinology 206:37-45.
- Jiang, H., Eleswarapu, S., and Wang, Y., 2010. Mechanism of Growth Hormone Regulation of Insulin‐like Growth Factor (IGF)‐I Gene Expression. The Asia-Pacific Journal of Endocrinology. (online).
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The overall goal of this project is to understand the molecular and cellular mechanisms of actions of growth hormone (GH) in cattle. During this reporting period, we have conducted experiments to study the mechanisms by which GH stimulates gene expression of insulin-like growth factor I (IGF-I) in bovine liver and by which GH stimulates growth in bovine skeletal muscle. PARTICIPANTS: Honglin Jiang, principal investigator Satyanaryana Eleswarapu, graduate student Xiaomei Ge, graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In studying how GH stimulates the transcription of the IGF-I gene in bovine liver, we focused on the role of hepatocyte nuclear factor (HNF)-3γ, a liver-enriched transcription factor because in a previous study we found that the expression of this gene in bovine liver was increased by GH injection in cattle. A sequence analysis of the bovine IGF-I promoter revealed three putative HNF-3 binding sites, which all appear to be conserved in mammals. Chromatin immunoprecipitation assays showed that GH injection increased binding of HNF-3γ to the IGF-I promoter in bovine liver. Gel-shift assays indicated that one of the three putative HNF-3 binding sites, HNF-3 binding site 1, bound to the HNF-3γ protein from bovine liver with high affinity. Co-transfection analyses demonstrated that this HNF-3 binding site was essential for the transcriptional response of the IGF-I promoter to HNF-3γ in CHO cells and to GH in primary mouse hepatocytes. Using similar approaches, we found that GH increased binding of the signal transducer and activator of transcription 5 (STAT5) to the HNF-3γ promoter in bovine liver, that this binding occurred at a conserved STAT5 binding site, and that this STAT5 binding site was necessary for the HNF-3γ promoter to respond to GH. Taken together, these results suggest that in addition to direct action, GH-activated STAT5 may also indirectly stimulate IGF-I gene transcription in the liver by directly enhancing the expression of the HNF-3γ gene. In studying how GH stimulates skeletal muscle growth in cattle, we focused on the role of locally produced IGF-I. We found that GH injection to cattle increased serum IGF-I concentrations and liver IGF-I mRNA but had no effect on IGF-I mRNA abundance in the longissimus muscle. Growth hormone administration also increased the levels of phosphorylated JAK2 and STAT5, which are known protein components of GH signaling, in the skeletal muscle. Growth hormone at the concentrations of 10 ng/ml and 100 ng/ml did not affect IGF-I mRNA expression in cultures of myoblasts or myotubes derived from bovine skeletal muscle. Taken together, these in vivo and in vitro data do not support the notion that suggest GH stimulates skeletal muscle growth in cattle through the locally produced IGF-I.
Publications
- Eleswarapu S, Ge X, Wang Y, Yu J, Jiang H. 2009. Growth Hormone-Activated STAT5 May Indirectly Stimulate IGF-I Gene Transcription through HNF-3γ. Mol. Endocrinol. 23:2026-2037.
- Ge X and Jiang H. 2009. Growth hormone does not stimulate IGF-I mRNA expression in bovine skeletal muscle, myoblasts, or myotubes. J. Anim. Sci. Vol. 87, E-Suppl. 2 / J. Dairy Sci. Vol. 92, E-Suppl. 1. P242.
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Progress 04/01/08 to 11/30/08
Outputs
OUTPUTS: The overall goal of this project is to understand the molecular and cellular mechanisms of actions of growth hormone (GH) in cattle. During this reporting period, we have conducted studies to determine the mechanism by which growth hormone stimulates the expression of the liver-enriched transcription factor HNF-3g and the role of HNF-3g in growth hormone-stimulated expression of IGF-I in the bovine liver. The results of these studies were reported at two scientific conferences. PARTICIPANTS: Honglin Jiang, principal investigator; Satyanaryana Eleswarapu, graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our studies showed that administration of growth hormone formulated for slow release to cattle caused sustained increases in the expression of IGF-I and HNF-3g mRNAs in the liver. The bovine HNF-3g gene promoter contains two putative binding sites for the transcription factor STAT5, which has been known to be essential for growth hormone regulation of IGF-I gene expression in the liver. The putative proximal STAT5 binding site in the bovine HNF-3g promoter is conserved in other mammals. Chromatin immunoprecipitation assays demonstrated that growth hormone administration increased STAT5 binding to the HNF-3g promoter region containing the two putative STAT5 binding sites in the liver. Electrophoretic mobility shift assays showed that the proximal STAT5 binding site was able to bind specifically to growth hormone-activated STAT5 protein from the bovine liver. Cotransfection analyses indicated that reporter gene activity expressed from the bovine HNF-3g promoter was inducible by growth hormone, and that this response to growth hormone was completely blocked when the proximal STAT5 binding site was mutated. The promoter of the bovine IGF-I gene contains three putative HNF-3 binding sites. In cotransfection analyses, overexpression of HNF-3g markedly enhanced reporter gene expression from the IGF-I promoter. These results together suggest that in addition to the well-established mechanism of direct action, growth hormone-activated STAT5 may also indirectly stimulate IGF-I gene transcription through enhancing HNF-3g gene expression in the bovine liver.
Publications
- Eleswarapu, S., and Jiang, H. 2008 Hepatocyte nuclear factor-3g may mediated growth hormone stimulation of IGF-I gene expression in the liver. The Endocrine Society's 90th Annual Meeting Programs & Abstracts Book, p630.
- Jiang, H., and Eleswarapu, S. 2008 GH and IGF interactions in cattle: Mechanism of GH regulation of IGF-I gene expression. 6th International Congress on Farm Animal Endocrinology, p50.
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