GLP-1 effects on pancreatic b-cell lines
The aims of this thesis were to establish a suitable model system for the study of glucose and nutrient regulation of insulin secretion and biosynthesis. This would serve as a basis for investigating the GLP-1 effects on pancreatic b-cell biology. This thesis shows that two b-cell model systems, namely MIN6 and INS-1 cells, respond to increasing concentrations of glucose, by increasing insulin secretion and cell proliferation in a physiological manner. The MIN6 cell line also responds to other cellular nutrients, L-arginine and L-leucine in a manner similar to primary islet cells. The MIN6 cells however, fail to consistently increase insulin secretion in response to GLP-1, a known potentiator of insulin secretion in b-cells, despite the presence of the GLP-1 receptor. Incubation of GLP-1 with INS-1 cells, increases insulin secretion in a glucose-dependent manner, and causes a small increase in cell proliferation. GLP-1 is also known to increase cAMP levels within the cell and interact with cAMP response elements (CRE) via activation protein kinase A (PKA). Using a luciferase reporter gene construct containing 4 copies of the CRE, glucose, GLP-1 and forskolin failed to increase luciferase activity in MIN6 cells, suggesting that a defect in cAMP signalling may explain the inconsistent effect of GLP-1 in MIN6 cells. A stimulatory effect of GLP-1 and forskolin was observed in the INS-1 cells. Using both a rat insulin I and human insulin gene promoter construct, a stimulatory effect of GLP-1 on insulin gene transcription was observed in INS-1 cells. An insight into the signalling pathways involved in GLP-1 stimulation of the rat insulin I gene was gained through the use of protein kinase inhibitors, which inhibit signalling of known signal transduction cascades. It was found that an inhibitor of protein kinase A (H-89) was effective in blocking the increase in insulin promoter activity induced by GLP-1 using the rat insulin I promoter construct. Interestingly, the p38/SAPK2 inhibitor, SB203580, further increased the GLP-1 stimulation of rat insulin I promoter activity, indicating that this pathway usually invokes an inhibitory effect on insulin promoter activity.