Proceedings of The Physiological Society

University of Manchester (2006) Proc Physiol Soc 2, PC30

Poster Communications

Fatty acid sensing by GPR40 heterologously expressed in PC12 cells

Andrew James Higgins1, Anthony D Jackson1, John T McLaughlin1, Craig P Smith1

1. Tissues to Organisms, University of Manchester, Manchester, United Kingdom.

Fatty acid (FA) sensing mechanisms in gut epithelia remain undefined, but enteroendocrine cells (EEC) play a pivotal role. One potential candidate sensor is the recently deorphanised receptor GPR40, originally identified in pancreatic β cells (Itoh et al. 2003). The murine enteroendocrine cell line, STC-1, is known to express GPR40 and is responsive to FA in a chain length-dependent manner. This requires a minimum acyl chain length of 12 carbons, as is observed in vivo in humans. The aim of this study was therefore to express mouse GPR40 in a heterologous system, and study whether the chain length specificity of transferred fatty acid responsiveness is preserved. Initially, mGPR40, obtained by RT-PCR from the STC-1 cell line, was subcloned into the mammalian expression vector pEYFP-N1 in C-terminal fusion with the fluorescent protein EYFP, then stably expressed in the neuroendocrine cell line, PC12. Both wild type (WT) and mGPR40EYFP-expressing PC12 cells were loaded with Fura-2 to monitor [Ca2+]i by fluorescence microscopy. As observed in humans and STC-1 cells, FA of acyl chain length C4-C10 did not increase [Ca2+]i in either PC12-WT or mGPR40EYFP+ cells. However, mGPR40EYFP+ PC12 cells displayed significantly increased [Ca2+]i responsiveness to long chain FA compared to PC12-WT cells. Dodecanoic acid (C12:0) produced no increase in [Ca2+]i in PC12-WT, but a large response was elicited in mGPR40EYFP+ PC12 cells, with an average ratio increase of 0.5±0.08 ratio units (n=5). Linoleic acid (C18:2) induced a small rise in [Ca2+]i in PC12-WT (0.1±0.05 ratio units, n=3) which was amplified in mGPR40EYFP+ PC12 cells (0.4±0.05 ratio units, n=4; P<0.05, Mann-Whitney U test). For comparison, the maximal [Ca2+]i response induced by 70mM KCl was 0.7±0.1 ratio units in PC12-WT and 0.6±0.1 ratio units in mGPR40EYFP+ PC12 cells. Responses were both reversible and reproducible. Data values are mean±SEM. In conclusion, mGPR40 heterologously expressed in the PC12 neuroendocrine cell line retains chain length-dependent FA responses entirely concordant with previous studies in both the STC-1 cell line and in humans, further supporting a functional role for GPR40 in gut FA-sensing by EEC. These data justify further detailed study into the signalling pathways and detailed molecular physiology of the GPR40 receptor in relation to EEC.

Where applicable, experiments conform with Society ethical requirements