Growth hormone (GH) is a poly-peptide hormone produced by the anterior pituitary. Growth hormone not only stimulates body and muscle growth but also inhibits adipose tissue growth. The overall objective of this study was to determine the mechanisms by which GH inhibits adipose tissue growth. Three studies were conducted to achieve this objective. The first study was conducted to determine if GH inhibits fat tissue growth by stimulating lipolysis. In this study, adipose tissue weight and adipocyte size were compared between GH-deficient growth hormone releasing hormone receptor (Ghrhr) homozygous mutant mice (i.e., lit/lit mice), lit/+ mice, and lit/lit mice injected with GH. lit/lit mice had less body weight but more subcutaneous fat and larger adipocytes compared to lit/+ mice at the same ages. GH treatment to lit/lit mice for four weeks partially reversed these differences. These data suggest that GH inhibits adipose tissue growth in mice at least in part by stimulating lipolysis. Additional data from this study suggest that GH indirectly stimulates lipolysis in vivo and this indirect mechanism is independent of " adrenergic receptors in the adipose tissue. The second study was conducted to investigate if GH inhibits fat tissue growth also by inhibiting adipogenesis. In this study, stromal vascular fraction (SVF) cells were isolated from subcutaneous fat of lit/+ and lit/lit mice and were induced to differentiate into adipocytes in vitro. Oil Red O staining and gene expression analysis revealed that the SVF cells from lit/lit mice had greater adipogenic potential than from lit/+ mice. This suggests that GH inhibits adipose tissue growth also through inhibition of adipogenesis. Additional data from this study suggest that GH may inhibit adipogenesis by inhibiting the formation of adipogenic precursor cells in adipose tissue in mice. The third study was conducted to determine the role of the central component of GH receptor signaling, STAT5, in GH inhibition of differentiation of bovine preadipocytes. In this study, preadipocytes were isolated from subcutaneous fat of adult cattle and were induced to differentiate with or without GH. Based on Oil Red O staining, gene expression, glycerol-3-phosphate dehydrogenase (G3PDH) activity and acetate incorporation assays, GH inhibited differentiation of bovine preadipocytes into adipocytes. GH induced phosphorylation of STAT5 in differentiating bovine preadipocytes. Overexpression of constitutively active STAT5 through adenovirus mimicked the effect of GH on differentiation of bovine preadipocytes. These data support a role of STAT5 in mediating the inhibitory effect of GH on differentiation of bovine preadipocytes into adipocytes. Overall, GH inhibits adipose tissue by both stimulating lipolysis and inhibiting adipogenesis; GH stimulates lipolysis through an indirect mechanism that is independent of the " adrenergic receptors; GH inhibits adipogenesis through a direct mechanism that may involve the transcription factor STAT5. / Ph. D.

The highly regulated maturation of the mammary gland is poorly understood. Our studies were designed to further characterize the role of ovarian hormones, growth hormone (GH)/IGF axis proteins and extracellular matrix (ECM) in the growth and development of prepubertal mammary glands. Prepubertal heifers were injected with either exogenous GH or subjected to ovariectomy (OVX). Mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested for DNA, protein, lipid, and western blot analysis. Remaining tissues were preserved for histological staining or snap frozen for quantitative real-time PCR. We examined 13 genes that work in conjunction with the extracellular matrix to regulate mammary proliferation and morphogenesis. Administration of GH, while impacting composition of MFP, had no effect on expression of the selected genes; there was a decrease in expression of fibronectin in PAR. Ovariectomy had no effect on gene expression in MFP but decreased expression of epimorphin, a potent regulator of morphogenesis, in PAR. In both experiments, the presence of a 55 kDa band corresponding to androgen converting enzyme aromatase was detected but its expression was unaffected. In another study, we used in vitro cell culture to evaluate the role of ECM in mammary gland maturation and employed quantitative real-time PCR to evaluate gene expression profiles of select genes involved in proliferation and differentiation. Expression of Rac1 was decreased in response to bovine insulin (BI) but increased on collagen I (Col). Expression of aldehyde dehydrogenase was decreased in BI and serum on plastic and on Col in the presence of BI. Expression of IGF binding proteins (BP) 3, -4, and -6 were decreased in the presence of serum on laminin (LM). Also, IGF-BP2 expression was decreased on Col while IGF-BP6 was increased on LM with BI. Clusterin, a ubiquitous non-adhesive ECM protein was not affected by ECM substrate but did increase over time. In conclusion, we propose that the mammary gland is not able to respond to GH at this age and that while OVX did effect the expression of some genes, the presence of aromatase maintained local estrogen concentrations. Furthermore, ECM alone is insufficient to regulate mammary gland development and growth. / Ph. D.

The present study deals with the comparison and establishment of methods for the detection of antibodies against recombinant human growth hormone (rhGH). Therefore, different methods for the detection of hGH-Abs were evaluated and compared in order to establish a test system that can be used for the detection of neutralizing antibodies against hGH, which could be developed under rhGH treatment. This manuscript describes in detail the validation of a newly developed biological assay, the neutralizing hGH-antibody assay (NAb assay). Therefore, a cell line transfected with the growth hormone receptor, that proliferates in the presence of hGH, was used. This proliferation was quantified by an increase of the optical density (OD/ absorbance) after addition of a colorimetric reagent, whereas the presence of hGH-antibodies leads to an inhibition of cell proliferation. To validate the test system for the detection of hGH-antibodies, we tested serum samples of 4 patients suffering from neurosecretory dysfunction (NSD) and samples taken from 6 patients with growth hormone deficiency (GHD) which were treated with rhGH and were highly suspected for a-hGH antibodies. These samples were tested in two different immunological assays, capable to screen sera for anti-hGH immunreactivity in the case of hGH-insensitivity during GH treatment. Using the NAb assay the neutralizing activity of specific hGH-antibodies was proved in serum samples of NSD and GHD type 1A patients. In case of neutralizing hGH-antibody activity, a clinically based decision can be made whether rhGH therapy should be stopped or the rhGH dosis should be increased. By the use of our test system, we offer the measurement of anti-hGH-antibody activity to other laboratories in cases when secondary hGH-insensitivity is assumed or observed.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21640号 / 医博第4446号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 大森 孝一, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

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published_or_final_version / Zoology / Master / Master of Philosophy