The insulin-like growth factor (IGF) system is composed of IGF, IGF binding proteins (IGFBP-1 to -10) and the acid labile subunit (ALS). IGF exists as two isoforms, IGF-I and IGF-II. IGF-I is the major circulatory form and is primarily secreted by the liver. It functions to regulate proliferation and differentiation in a number of different cell types and elicits an insulin-like metabolic effect. As well as being regulated at levels of transcription and translation, IGF-I activities are also regulated through formation of complexes in circulation. IGF complexes form as binary complexes, such as the IGFBP-1 complex, and ternary complexes containing IGF-I, IGFBP-3 and ALS. Binary and ternary IGF complexes function to maintain stable pools of bioactive IGF-I. They also function to increase IGF half-life and sequester IGF in the bloodstream. <p> ALS and IGFBP-1 are well characterized and exist as 85 kDa and 32 kDa proteins, respectively. They are expressed primarily in liver hepatocytes. Circulating ALS binds the IGF-I-IGFBP-3 complex and increases IGF half-life from 10 min in the IGFBP-3 binary complex to 10-15 hr in the ternary complex. IGFBP-1 binds IGF-I and increases the half-life from 10 min to 30 min. The ternary complex is the predominant IGF-I binding protein complex found in circulation. The IGFBP-1 complex represents only a small fraction of circulating IGF complexes. <p> In this thesis ALS and IGFBP-1 regulation were investigated in terms of expression related to metabolic modulators and streptozotocin (STZ)-induced diabetes. Results from rat studies showed a decreased liver ALS gene expression in STZ-induced diabetic rats. STZ-treatment in rats mimics type-I diabetes with no change in secreted insulin with increase of circulatory glucose. The administration of insulin into the STZ-induced diabetic rats brought ALS levels to that of the untreated controls. ALS expression was positively regulated by insulin in H4IIE hepatoma cells. Growth hormone (GH), glucose, dexamethasone also positively regulated ALS gene expression while cAMP (2-b-cAMP) acted as a negative regulator in H4IIE cells. HepG2 cells expressing constitutively active protein kinase B (PKB) (HepG2-PKB-CA) increased ALS gene expression to levels 20% higher then parental HepG2. Insulin treatment of these cells unexpectedly increased ALS levels in both parental and PKB-CA HepG2. This may have indicated a partial regulatory role of the mitogen activated protein (MAP) kinase pathway as PKB was thought to be over-expressed therefore rendering the insulin signal redundant. Inhibition of the phosphoinositol-3 (PI-3) kinase and MAP kinase pathways through wortmannin and PD98059 incubation, respectively, suggested a possible interplay or crosstalk between the two pathways in insulin signaling. PKB is known to be activated through the PI-3 kinase pathway. Results suggested possibility that PKB may interact through the MAP kinase pathway in regulation of ALS gene expression. The activity of cAMP on ALS gene expression may occur through interaction with the PI-3 kinase pathway as inhibition enhanced the negative effect of cAMP on ALS expression. <p> The secretion of IGFBP-1 was positively regulated by glucose and GH and negatively regulated by insulin in H4IIE cells. HepG2-PKB-CA cells showed significantly lower IGFBP-1 secretion as compared to parental HepG2 cells. The involvement of the PI-3 and MAP kinase pathways in the modulator-mediated effect on IGFBP-1 secretion were. As observed for ALS expression, the effect of insulin on IGFBP-1 secretion may also be affected through interplay or crosstalk between the PI-3 kinase and MAP kinase pathways. Glucose and GH effected IGFBP-1 expression and secretion independent of these pathways although glucose expression may interact in some way through the PI-3 kinase pathway. Our investigation of hepatic regulation of IGFBP-1 secretion and ALS gene expression has shown regulatory roles for the metabolic hormones tested, especially insulin. Mechanisms of cell signaling have also been approached with the use of pathway inhibiters and HepG2-PKB-CA cells. Much work is yet to be done to fully understand the effects of insulin and other hormones on the secretion and expression of IGFBP-1 and ALS.

Thesis (Ph.D.)--University of Adelaide, Department of Animal Science, 1994.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1994.

Tübingen, Univ., Diss., 2003.

Tübingen, Univ., Diss., 2007.

Techn. University, Diss., 2005--München.

Universiẗat, Diss., 2003--München.

München, Techn. Univ., Diss., 2004.

Development of the mammary gland is likely mediated by locally produced growth factors acting in concert with circulating mitogens. To investigate the importance of mammary synthesis of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBP), the initial objective was to evaluate the physiological effects of recombinant IGF-I synthesis in the mouse mammary gland. Expression of recombinant IGF-I was targeted by the mouse mammary tumor virus - long terminal repeat (MMTV-LTR) to the mammary glands of two lines (15 and 29) of transgenic mice. Mammary synthesis of recombinant IGF-I increased the frequency of appearance of mammary alveolar buds (71% vs. 21%) in transgenic compared with non-transgenic CD-1 mice. During lactation, mammary synthesis of recombinant IGF-I reduced the amount of endogenous native IGF-I secreted into milk of transgenic mice. Regardless of transgenesis, a shift in the milk IGFBP profile from predominantly IGFBP-3 to a lower molecular weight IGFBP occurred between d 8 and d 12 of lactation. The altered composition of milk from transgenic line 29 dams reduced by 27% the average daily gain of suckling litters, compared with CD-1 dams. Moreover, mammary glands of transgenic mice were less regressed after weaning than controls and were characterized by the presence of more organized secretory lobules. The second overall objective was to evaluate the regulation and physiological effects of mammary IGF-I and IGFBP synthesis in prepubertal heifers. Serum and extracts of mammary tissue at 5% concentration in media stimulated DNA synthesis 545% and 28%, respectively, in primary mammary epithelial organoids in collagen gel culture. Addition of IGFBP-3 strongly inhibited this growth response. High feeding level tended to increase IGFBP-3 levels in mammary tissue and reduced by 30% the growth response to mammary tissue extracts. Somatotropin increased the mitogenic response to mammary extracts at high feeding level and increased the tissue content of IGF-I by 46%. In summary, local synthesis of IGF-I and IGFBP is influenced by feeding level and exogenous somatotropin and contributes substantially to effects on mammary cell proliferation. Interactions of locally produced IGFBP-3 with IGF-I and other growth factors appear to be especially important when mammary growth is modulated by feeding level. / Ph. D.

The primary aim of this text is to gain insight on how cellular activation by a insulin-like growth factor (IGF-I), in the presence of insulin-like growth factor binding protein-3 (IGFBP-3), is influenced by heparan sulfate proteoglycans (HSPG). Initial research will be presented, assumptions and hypotheses that were included in the development of mathematical models will be discussed, and the future enhancements of the models will be explored. There are many potential scenarios for how each component might influence the others. Mathematical modeling techniques will highlight the contributions made by numerous extracellular parameters on IGF-I cell surface binding. Tentative assumptions can be applied to modeling techniques and predictions may aid in the direction of future experiments. Experimentally, it was found that IGFBP-3 inhibited IGF-I Bovine Aortic Endothelial (BAE) cell surface binding while p9 HS slightly increased IGF-I BAE cell surface binding. IGFBP-3 has a higher binding affinity for IGF-I (3 x 10-9 M) than p9 HS has for IGF-I (1.5 x 10-8 M) as determined with cell-free binding assays. The presence of p9 HS countered the inhibiting effect of IGFBP-3 on IGF-I BAE cell surface binding. Although preliminary experiments with labeled p9 HS and IGFBP-3 indicated little to no cell surface binding, later experiments indicated that both IGFBP-3 and p9 HS do bind to the BAE cell surface. Pre-incubation of BAE cells with either IGFBP-3 or p9 HS resulted in an increase of IGF-I BAE cell surface binding . There was a more substantial increase of IGF-I surface binding when cells were pre-incubated with IGFBP- 3 than p9 HS. There was a larger increase of IGF-I BAE cell surface binding when cells were pre-incubated with p9 HS than when p9 HS and IGF-I were added simultaneously. This suggests that IGFBP-3 and p9 HS surface binding plays key role in IGF-I surface binding, however, p9 HS surface binding does not alter IGF-I surface binding as much as IGFBP-3 surface binding seems to. Experimental work helps further the understanding of IGF-I cellular activation as regulated by IGFBP-3 and p9 HS. Developing mathematical models allows the researcher to focus on individual elements in a complex systems and gain insight on how the real system will respond to individual changes. Discrepancies between the model results and the experimental data presented indicate that soluble receptor inhibition is not sufficient to account for experimental results. The alliance of engineering analysis and molecular biology helps to clarify significant principles relevant to the conveyance of growth factors into tissue. Awareness of the effects of individual parameters in the delivery system, made possible with mathematical models, will provide guidance and save time in the design of future therapeutics involving growth factors. / Master of Science