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ACTIVATION OF THE EXTRACELLULAR CALCIUM-SENSING RECEPTOR (CaSR) STIMULATES PROGLUCAGON TRANSCRIPTION AND GLP-1 SECRETION FROM MURINE ENTEROENDOCRINE CELLS BUT INHIBITS GLP-1 SECRETION FROM HYPOTHALAMIC NEURONS
R Vanner, I Pacheco, R J MacLeod,
GIDRU/Department of Physiology, Queen’s University, Kingston, Ontario
AIM: To assess a) if murine enteroendocrine and hypothalmic cell lines expressed the CaSR and b) determine if CaSR activation influenced GLP-1 homeostasis.
Material and METHODS: Murine enteroendocrine cell lines STC-1, GLUTag and hypothalamic neuron cell line N20/2 were used as models. Expression of CaSR and proglucagon were determined by RT-PCR and Western blotting. A 2.5 kb proglucagon promoter reporter was measured by dual luciferase activity. Activation of MEK1&2 was measured by Western blot and GLP-1 secretion was measured by ELISA.
RESULTS: STC-1, GLUTag cells and N20/2 neurons expressed CaSR as determined by RT-PCR. CaSR Western blot under reducing conditions showed multimeric CaSR protein. In STC-1 cells, 3 mM Ca2+ for 3 or 6h stimulated proglucagon transcription and proglucagon promoter activity was increased 245±32% over basal levels (0.5 mM Ca2+). STC-1 cells treated with forskolin (10uM) in low Ca2+ had 1628±89% activity which addition of 3 mM Ca2+ increased to 2424±107% of basal activity. These increases were reduced by pan-PKC or CAMK11 inhibitors. Activation of the CaSR in N20/2 neurons decreased proglucagon transcription and extinguished GLP-1 secretion within 3h. These effects were blocked by MEK1&2 inhibition. Also, MEK1&2 inhibition stimulated activity under basal conditions and after CaSR activation (3 mM, 173±12% over basal activity). Ca2+ challenge (3 mM) stimulated pERK1&2 production which siRNA duplex against the CaSR reduced.
CONCLUSION: We conclude that CaSR activation of GLP-1 synthesis and secretion in murine enteroendocrine cells occurs via a mechanism requiring CAMK11 which is independent of cAMP. In contrast, activating the CaSR on a hypothalamic neuronal cell line inhibits GLP-1 secretion which requires activation of pERK1&2. Together these are new mechanisms of GLP-1 regulation.