Abstract
The lysophosphatidylinositol (LPI) has crucial roles in multiple physiological processes, including insulin exocytosis from pancreatic islets. However, the role of LPI in secretion of glucagon-like peptide-1 (GLP-1), a hormone that enhances glucose-induced insulin secretion, is unclear. Here, we used the murine enteroendocrine L cell line GLUTag and primary murine small intestinal cells to elucidate the mechanism of LPI-induced GLP-1 secretion. Exogenous LPI addition increased intracellular Ca2+ concentrations ([Ca2+] i ) in GLUTag cells and induced GLP-1 secretion from both GLUTag and acutely prepared primary intestinal cells. The [Ca2+] i increase was suppressed by an antagonist for G protein-coupled receptor 55 (GPR55) and by silencing of GPR55 expression, indicating involvement of Gq and G12/13 signaling pathways in the LPI-induced increased [Ca2+] i levels and GLP-1 secretion. However, GPR55 agonists did not mimic many of the effects of LPI. We also found that phospholipase C inhibitor and Rho-associated kinase inhibitor suppressed the [Ca2+] i increase and that LPI increased the number of focal adhesions, indicating actin reorganization. Of note, blockage or silencing of transient receptor potential cation channel subfamily V member 2 (TRPV2) channels suppressed both the LPI-induced [Ca2+] i increase and GLP-1 secretion. Furthermore, LPI accelerated TRPV2 translocation to the plasma membrane, which was significantly suppressed by a GPR55 antagonist. These findings suggest that TRPV2 activation via actin reorganization induced by Gq and G12/13 signaling is involved in LPI-stimulated GLP-1 secretion in enteroendocrine L cells. Because GPR55 agonists largely failed to mimic the effects of LPI, its actions on L cells are at least partially independent of GPR55 activation.
Highlights
The lysophosphatidylinositol (LPI) has crucial roles in multiple physiological processes, including insulin exocytosis from pancreatic islets
These findings suggest that TRPV2 activation via actin reorganization induced by Gq and G12/13 signaling is involved in LPI-stimulated glucagon-like peptide-1 (GLP-1) secretion in enteroendocrine L cells
Application of LPI to the cells induced the translocation of TRPV2 to the plasma membrane revealed by total internal reflection fluorescence microscopy. These results suggest that LPI induces GLP-1 secretion from enteroendocrine L cells through the activation of the G protein-coupled receptor 55 (GPR55), Rho-associated kinase (ROCK), and TRPV2 pathways
Summary
To examine the effect of LPI on GLP-1 secretion from enteroendocrine L cells, we first monitored intracellular Ca2ϩ dynamics using the Ca2ϩ-sensing dye Fluo-4 AM in GLUTag cells. Silencing of endogenous GPR55 expression using a specific siRNA significantly suppressed the LPI-induced [Ca2ϩ]i increase (Fig. 2, D and E, and supplemental Fig. S1B) These results suggest that GPR55 functions as a receptor through which LPI induces [Ca2ϩ]i increase in enteroendocrine L cells. This treatment significantly suppressed LPI-induced [Ca2ϩ]i increase and GLP-1 secretion (Fig. 4, E–G, and supplemental Fig. S1D). The increase in the fluorescence intensity of TRPV2-EGFP was significantly suppressed by co-application of O-1918 with LPI (Fig. 5C) These results suggest that LPI-induced TRPV2 translocation to the plasma membrane is involved in the [Ca2ϩ]i increase and GLP-1 secretion. Application of GPR55 agonist, O-1602, had little effect on [Ca2ϩ]i, fluorescence intensity of TRPV2-EGFP, or GLP-1 secretion (Fig. 6). We confirmed expression of TRPV2 mRNA in GLUTag cells by RT-PCR
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