UBC Theses and Dissertations
Alternative signaling pathways of the glucose-dependent insulinotropic polypeptide (GIP) receptor Ehses, Jan A.
Glucose-dependent insulinotropic polypeptide (GIP) regulates pancreatic β -cell function by binding to its cognate Family B, G protein-coupled receptor and elevating intracellular cAMP and Ca2 + . The main rationale for the studies described in this Thesis was that multiple interacting intracellular signal transduction pathways were proposed to mediate GIP's pleiotropic actions on the pancreatic β-cell. Moreover, the ability of GIP to potentiate insulin secretion is blunted in some type 2 diabetics, implying potential defects at the receptor signaling level. Therefore, the major aim was to characterize further the intricate network of GIP receptor signaling pathways underlying β-cell processes using β-cell models. Using GIP receptor-transfected CHO-K1 cells and βTC-3 and INS-1 tumour cell lines, it was possible to correlate GIP receptor activation with the regulation of lipid signaling (arachidonic acid release), K + ATP channel-independent events, MAP kinase signaling (Raf->Mekl/2->ERKl/2->p90RSK and p38 MAPK), and CREB signaling. These events were demonstrated to be functionally relevant for insulin secretion, cell growth and survival (MAPK signaling), and insulin gene transcription (CREB signaling) respectively. Through insulin secretion studies and pharmacological approaches, GIP was shown to regulate the secretion of insulin via activation of PLA2 and K+ATP channel-independent mechanisms in βTC-3 cells. In an attempt to elucidate novel signals coupled to the GIP receptor, we examined the expression of 75 protein kinases and 25 protein phosphatases in CHO-K1, βTC-3, and INS-1 cells. This has allowed the partial mapping of intracellular signal transduction pathways for these cell models. From this, GIP receptor coupled signaling events were studied using phospho-specific antibodies, transfection techniques, pharmacological inhibitors, and gene reporter assays. Studies in CHO-K1 cells expressing the GIP receptor and in INS-1 B-cells have implicated cAMP/PKA signaling in the regulation of the mitogenic ERK 1/2 module, Raf->Mekl/2->ERKl/2->p90RSK. Results further suggest that the GIP receptor is coupled to β-cell survival via cAMP mediated inhibition of p38 MAPK and caspase-3 activity. Finally, coupling of the GIP receptor to rat insulin promoter activity was shown to occur via cAMP/PKA and a CREB family transcription factor. These events were found to be independent of phosphoregulation of S133 CREB/S117 CREM/S63 ATF-1, and suggest that the tightly regulated phosphorylation of these transcription factors by GIP may be involved in novel signals regulating β-cell function.
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