Deoxycholic acid (DCA) is a secondary bile acid postulated to be involved in the etiology and the progression of colorectal cancer, but its specific mechanisms are not fully understood. DCA has been shown to induce apoptosis allowing selection for apoptosis-resistant cells, which highlights the importance of understanding the mechanisms of action of DCA. Previously, it has been demonstrated that DCA perturbs the plasma membrane, leading to the activation of receptor tyrosine kinases. Because the insulin-like growth factor-1 receptor (IGF-IR), a receptor tyrosine kinase, is demonstrated to play a significant role in protecting colorectal cancer cells from apoptosis, we hypothesized that DCA modulates IGF-IR functions in colorectal cancer cells. We demonstrated that DCA induced the dynamin-dependent endocytosis of IGF-IR through both clathrin-mediated and caveolin-1-dependent mechanisms. Endocytosis of IGF-IR sensitized cells to DCA-induced apoptosis, which demonstrated that IGF-IR played a role in protecting cells against DCA-induced apoptosis. Since DCA-induced endocytosis of IGF-IR was determined to be a caveolin-1 dependent process, caveolin-1 knockdown in HCT116 (HCT116-Cav1-AS) prevented the DCA-mediated endocytosis of IGF-IR. However, we observed an increased sensitivity of DCA-induced apoptosis in the Cav1-AS cells. This suggested that caveolin-1 knockdown altered the plasma membrane dynamics such that although IGF-IR was maintained at the plasma membrane, it facilitated a pro-apoptotic signal. We demonstrated that DCA induced the activation of the pro-apoptotic p38 signaling pathway in HCT116-Cav1-AS, but not in HCT116-Mock, via IGF-IR. Inhibition of both the IGF-IR and p38 independently in HCT116-Cav1-AS significantly decreased their sensitivity to DCA-induced apoptosis. These observations demonstrated that, in a caveolin-1 dependent manner, IGF-IR played a dynamic role in the DCA-mediated apoptosis. Finally, we provided preliminary evidence demonstrating that autophagy played a central role in protecting DCA-resistant cells from DCA-induced apoptosis.Since resistance to DCA also confers apoptosis-resistance, understanding the mechanisms that lead to or prevent DCA-induced cell death is significant, since they can lead to the development of novel therapeutic strategies to sensitize apoptosis-resistant colorectal cancer cells to undergo cell death.

The effects of insulin on milk production were tested employing two different approaches. Firstly, 12 Holstein cows were used to determine the effects of feeding calcium propionate (Ca prop) on dry matter intake (DMI) and production traits. The experimental design was a switchback with 2 treatments (Ca prop at 0 or 300 g/d). The DMI was lower when animals received Ca prop. Ca prop did not affect the yield of milk and its components; however, Ca prop increased protein content. The (acetate+butyrate)/propionate ratio in rumen fluid 2 h after feeding was lower when cows received Ca prop. Plasma insulin concentration was not different between treatments and the putative effect of propionate as an insulin secretagogue was probably related to the maintenance of insulin levels when DMI was lower. In conclusion, Ca prop is a potential feed ingredient to increase protein content in milk. The second approach consisted of intravenous infusion of insulin. A trial was designed to test the effects of insulin, recombinant bovine somatotropin (rbST) and their interaction in lactating dairy cows. Eight Holstein cows were used in a Latin Square design with 4 treatments: (1) intravenous infusion of saline, (2) infusion of saline and administration of 40 mg of rbST per day, (3) intravenous infusion of 12 mg of insulin per day coupled with glucose infusion and (4) rbST administration combined with insulin and glucose infusion. The theory that rbST causes a peripheral resistance to insulin was confirmed. Insulin infusion increased percent protein, percent casein and decreased milk urea content regardless of rbST administration. For milk yield, protein yield, casein yield, lactose percent and lactose yield, there was an interaction between insulin and rbST administration. Similarly, there was an interaction between insulin and rbST on plasma IGF-I levels. Fat yield was higher, with a higher content of long chain fatty acids, during rbST administration, regardless of insulin infusion. I

Insulin-like growth factor-I (IGF-I) and its principle binding protein (IGFBP-3) are believed to play a role in mediating the anabolic effects of exercise. The purpose of this study was to assess the effect of 4 months of training on IGF-I and IGFBP-3, and to determine if changes in IGF-I or IGFBP-3 were related to changes in training status. Twelve varsity swimmers (5 males, 7 females) were tested pre-season, and again after 8 and 16 weeks of training. Measures included: VO2 max, nutritional status, athletic performance, subjective symptoms of overtraining, and serum levels of IGF-I and IGFBP-3. There was no significant change across time in VO2 max, athletic performance, IGF-I or IGFBP-3. Resting IGFBP-3 was positively correlated to symptoms of overtraining at week 0 (p=0.017), however, this relationship did not persist at week 8 or 16. These findings can not confirm that resting levels of IGF-I and IGFBP-3 are sensitive markers of training status. / ix, 105 leaves : ill. ; 29 cm.

No description available.

The alteration of insulin-like growth factor I (IGF-I) and its binding proteins (IGFBP) and their effects on proliferation and differentiation of murine bone marrow-derived macrophage (BMM) precursors were investigated. Bone marrow cells exposed to 20% L929-fibroblast conditioned medium (LCM) were cultured in serum-free medium for 24 h. Western ligand blotting (WLB) analysis detected four bands in all samples. 41-kDa and 30-kDa bands were detected after 12 h and remained constant during BMM differentiation. The 28-kDa and 25-kDa proteins were almost undetectable until day 2, but accumulated significantly from day 3 to day 7. Immunoblotting analysis verified these two bands as IGFBP-4. Northern blotting analyses detected both IGFBP-4 and IGFBP-3 mRNA in the cells. The 41-kDa protein was postulated to be IGFBP-3 in a glycosylated form. The identity of the 30-kDa band is not known. Northern blotting analysis showed that IGF-I mRNA level increased in a time-dependent manner until day 3, and decreased thereafter during BMM differentiation. The effect of IGF-I and its analogs on cell proliferation was studied by ($ sp3$H) thymidine incorporation. IGF-I and its analogs enhanced cell proliferation of freshly isolated bone marrow cells. Both IGF-I and long R$ sp3$ IGF-I, but not des(1-3)IGF-I, continued to exert a stimulating effect on day 1, although to a lesser extent. The effect of IGF-I and its analogs on BMM differentiation was studied by checking morphology, non-specific esterase-1 (NSE-1) activity, and mannose receptor expression. No significant differences in morphology and NSE-1 activity were observed among the treatment groups. There was no difference of mannose receptor expression on day 4 between the IGF-I group and the control cells, whereas long R${ sp3}$ and des(1-3)IGF-I increased the receptor number by 260% and 228% respectively, with less increased K$ rm sb{d}$ values. (Abstract shortened by UMI.)

In the present study, two insulin-like growth factor-binding protein 7 (IGFBP7) C-terminal-peptides were expressed as fusion proteins to bacterial verotoxin pentamerization domain as shown by Western blotting, ELISA and mass spectroscopy. Both in vivo-biotinylated recombinant products were purified from bacterial lysates by IMAC and used directly for panning along with the recombinant IGFBP7 protein using the LAC-M Camelidae naïve single domain antibody (sdAb) library. Target-specific sdAbs to both parental protein and peptide fusions were identified by phage ELISA. Twelve different clones were isolated by phage-ELISA screening and their sdAb genes were sequenced. Soluble sdAbs and their pentameric formats were expressed in TG1 E. coli, purified by IMAC and characterized by ELISA and SPR. Several sdAbs are currently under study, however anti-IGFBP7 (P12/M12) was extensively characterized and exhibited promising anti-tumorigenic effect on PANC-1 cell lines by blocking IGFBP7 promoting activity. This study provides the basis for developing a novel imaging/therapeutic reagent for targeting and treating brain tumor angiogenesis in early stages of tumorogenesis and can also be used as a molecular tool to monitor the degree of angiogenesis in gliomas which may help to improve the clinical management of brain tumors.

Insulin-like growth factor-1 (IGF-1) is essential for oligodendrocyte (OL) development, promoting their survival, proliferation and differentiation. Furthermore, IGF-1 null mutant mice have a decrease in CNS myelination and in the number of OL progenitors (OLPs). IGF-1 interacts with the Type I IGF receptor to activate two main downstream signalling pathways, the PI3K/Akt and the Ras-Raf-MEK/ERK cascades, which mediate survival or proliferation of OLPs. The objective of this study is to elucidate the transduction pathways involved in IGF-I-stimulated protein synthesis, important for growth and differentiation of OLs. In other cellular systems, the PI3K/Akt pathway is involved in protein translation. mTOR and the p70 S6 kinase are downstream effectors that phosphorylate translation initiation factors (e.g. eIF-4E) and their regulators (e.g. 4E-BP1). OLPs were obtained from primary cultures and were treated with IGF-1 with or without inhibitors LY294002 or wortmannin (PI3K), rapamycin (mTOR), Akt III or IV, an adenovirus with a dominant negative form of Akt or PD98059 (ERK). Protein synthesis was assessed by metabolic labeling with [35S]-methionine, and protein phosphorylation by Western blotting. Results from the former showed that IGF-1 stimulates protein synthesis in a dose-dependent manner. Moreover, IGF-1 increases protein synthesis in OLPs through PI3K, mTOR, Akt and ERK activation. Concordantly, Western blot analysis reveals that IGF-1 stimulates phosphorylation of Akt, mTOR, ERK, S6 and 4E-BP 1. Activation of S6 and inactivation of 4E-BP1 occur through phosphorylation and are required for protein synthesis to take place. These events are dependent on the upstream activation of PI3K, Akt and mTOR.

Background and objectives Evidence suggests that circulating IGF-I levels increase mammographic density (a breast cancer risk factor) and breast cancer risk in premenopausal women. The objective of this thesis was to examine the association of genetic variation at the IGF1 gene with IGF-I concentration, mammographic density, breast cancer risk, and related anthropometric measures in premenopausal women. Methods Three IGF1 CA repeat polymorphisms (at the 5′ and 3′ ends, and in intron 2) were genotyped. A cross-sectional design was used to investigate their associations with IGF-I levels, mammographic density, BMI, weight, and height. Families from registries in Ontario and Australia were used to investigate associations with breast cancer risk and also BMI, weight and height. Results In the cross-sectional study, greater number of copies of the 5′ 19 allele were associated with lower circulating IGF-I levels. Greater number of 3′ 185 alleles were associated with greater percentage breast density, smaller amount of non-dense tissue, and lower BMI. Including BMI in regression models removed the association of the 3′ 185 allele with percentage breast density. In the family based study, nominally significant associations (5′ 21 allele, intron 2 212 allele, intron 2 216 allele) with breast cancer risk were observed, but significance was lost after multiple comparison adjustment. There was a stronger association between the intron 2 216 allele and risk under a recessive model, and 5′ allele groupings of length 18 to 20 and 20 or more repeats produced significant positive and negative associations respectively. These associations were not strongly supported in analyses stratified by registry. Results from the family based study did not support an association between genetic variation at IGF1 with BMI, weight or height. Conclusions No specific IGF1 variant influenced each of circulating IGF-I levels, mammographic density, and breast cancer risk. The failure to replicate the association of the 3′ 185 allele with BMI in the family based study suggests that the association of the 3′ 185 allele with percentage breast density is spurious, since this association was mediated through the relationship with BMI (suggesting IGF-I action on body fat). Evidence for an association between IGF1 and breast cancer risk was limited.

The study of diabetes mellitus is vital in this day and age because its incidence is increasing at an alarming rate. Diabetes results in the loss of function of beta-cells within the pancreas. Insulin resistance contributes to diabetes but the human body can compensate in various ways such as increasing the islet cell mass, glucose disposal and insulin secretion, in order to prevent the onset of diabetes. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are two integral hormones important in both glucose homeostasis and islet cell growth. Early studies using cultured islet cells have demonstrated positive regulation of beta-cell growth by both GH and IGF-I. To evaluate their relevance on normal beta-cell growth, compensatory growth, as well as in insulin responsiveness, we have used two mouse models that represent opposite manipulations of the GH/IGF-I axis. Specifically, the growth hormone receptor gene deficient (GHR-/-) and the IGF-I overexpression (MT-IGF) mice, to help understand the role of glucose homeostasis and islet cell growth in the GH/IGF-I axis. GH is essential for somatic growth and development as well as maintaining metabolic homeostasis. It is known that GH stimulates normal islet cell growth. Moreover, GH may also participate in islet cell overgrowth and compensate for insulin resistance induced by obesity. To determine whether the islet cell overgrowth is dependent on GH signaling, we studied the response of GHR-/- mice to high-fat diet (HFD)-induced obesity. We also studied the insulin responsiveness in GHR-/- mice. On the other hand, IGF-I promotes embryonic development, postnatal growth and the maturation of various organ systems. The notion that IGF-I stimulates islet cell growth has been challenged in recent years by results from IGF-I and receptor gene targeted models. We have characterized MT-IGF mice which overexpress the IGF-I gene. / The results of our studies indicate that (1) GH is essential for normal islet cell growth, but not required for compensatory overgrowth of the islets in response to obesity, (2) GHR gene deficiency caused delayed insulin responsiveness in skeletal muscle; in contrast to elevated insulin sensitivity in the liver; (3) although overexpression does not stimulate islet cell growth, a chronic IGF-I elevation caused significant hypoglycemia, hypoinsulinemia, and improved glucose tolerance, (4) finally IGF-I overexpression mice are resistant to experimental diabetes.

The developing chick retina is an extrapituitary site of growth hormone (GH) synthesis and action. GH, GH receptor (GHR) and their mRNAs are present in the neural retina when the neural cells are undergoing proliferation and differentiation during early embryogenesis. It is thus likely that GH acts as an autocrine or paracrine in this location. The present study shows that intra-vitreal injection of a chick GH (cGH) small interfering RNA (siRNA) into the eyes of early embryos [embryonic day (ED) 4] suppresses GH expression in the neural retina and increases the incidence of spontaneous retinal cell death. Our current work also demonstrates a reduction of local IGF-1 expression after retinal GH gene knockdown, suggesting that GH action in retinal cells is regulated through IGF-1 signalling. These results demonstrate that retinal GH is an autocrine/paracrine hormone that acts as a neuroprotective factor in the retina of chick embryos.