Isolation of a murine glucose-dependent insulinotropic polypeptide (GIP) cDNA from a tumor derived cell line (STC₆₋₁₄) and quantification of glucose induced increases in GIP mRNA

This thesis reports the attempts to characterize further the murine neuroendocrine cell line STC₆₋₁₄ with the hope of demonstrating this cell line's potential use for intracellular studies involving GTP. These studies include the identification of a murine GIP cDNA and investigations into the role of glucose in the increase of GIP mRNA within this cell line. Sequence analysis revealed a 537 base pair cDNA clone for murine GIP which was found to encode an open reading frame of432 base pairs. From this sequence a 144 amino acid precursor could be predicted which was shown to code for a 43 amino acid N-terminal extensionwhich includes a 19-21 amino acid signal peptide, a 42 amino acid hormone and a 59 amino acid C-terminal extension. Murine GTP is predicted to differ from the human hormone by three amino acid substitutions: arginine for histidine at position 18, arginine for lysine at position 30 and serine for lysine at position 34. GIP mRNA levels in STC₆₋₁₄ cells incubated in the presence of varying glucose concentrations were investigated using a competitive-PCR method. In the presence of a 5mM glucose stimulus, 1x10⁵ GTP cells were found to contain 3.9 ± 0.59 attomoles (amol) of GIP mRNA while the same number of cells contained 11.6 ± 1.4 amol when subjected to a high (25mM) glucose stimulus (p< 0.0025). Release studies for GIP in the STC₆₋₁₄ cell line were performed in conjunction with the mRNA studies to ascertain the link between GXP mRNA levels within the GTP cell and the amount of mature GIP released into the medium. In response to a low glucose (5 mM) stimulus, 3 x 10⁵ STC₆₋₁₄ cells released 0.84 ± 0.04% of TCC while the same number of cells were found to release 1.11 ± 0.11% of TCC when cultured in a high glucose (25 mM) media. These investigations demonstrated that the STC₆₋₁₄ cell line responds as predicted to a high glucose stimulus with an increase in the level of GIP mRNA but fails to transfer this mRNA increase into a greater GIP peptide release, most likely due to a problem within the posttranslational processes of the GIP cell.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat