Regulation of hepatic ALS and IGFBP-1 expression
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. 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. 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. 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.
Acid labile subunit, Insulin-like growth factor binding protein-1, Insulin-like growth factor
Master of Science (M.Sc.)