Proceedings of The Physiological Society

University of Manchester (2006) Proc Physiol Soc 2, PC29

Poster Communications

Fatty acid and particle sensing mechanisms in epithelial and endocrine cells

Anthony David Jackson1, Andrew J Higgins1, R Maynard Case1, John T McLaughlin1

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

The murine enteroendocrine cell line STC-1 responds to fatty acids (FA) by secreting biologically active CCK, via an elevation of intracellular calcium, [Ca2+]i. This therefore represents a tractable model of FA sensing. Previous work has suggested that the formation of aggregates by FA in physiological media is an important factor in lipid sensing by STC-1 cells (Kazmi et al. 2003). Synthetic latex microspheres (LM) of similar size to FA aggregates (~90-300nm) also stimulate STC-1 cells causing CCK secretion, raising the possibility that these are sensed by the same cellular mechanism as FA. The cellular mechanism(s) underlying FA sensing remain largely uncharacterized, although one candidate is the newly assigned FA receptor, GPR40, first described in pancreatic beta cells, but which is also expressed by STC-1 cells. We therefore evaluated (1) if LM activate GPR40, and (2) if other cell models sensitive to LM are also FA sensitive. Real-time changes in [Ca2+]i were measured using fura-2-loaded cells challenged with FA (linoleic acid, C18:2) and LM, by measuring a rise in the fura-2 340/380 ratio value which corresponds to a rise in intracellular calcium. Initially, RT-PCR confirmed expression of GPR40 in STC-1 but not in (rat) neuroendocrine PC12 cells. mGPR40 was therefore subcloned from STC-1 cells and stably overexpressed in PC12 cells. PC12 sensitivity to FA was increased 2.5-fold in PC12 mGPR40+ cells versus wild type cells (P=0.005, Mann-Whitney U test); PC12 mGPR40+ cells responded to 500µM C18:2 with a rise in the fura-2 340/380 ratio value of 0.4±0.05 (n=5) compared to 0.16±0.02 (n=5) in PC12 WT cells. As with STC-1 cells, PC12-WT cells also responded to LM with a 0.23±0.06 (n=6) rise in 340/380 ratio value, but LM sensitivity was not significantly altered in mGPR40+ PC12 cells. Furthermore, the [Ca2+]i response to LM was entirely abolished in the absence of extracellular calcium, whilst the FA-induced [Ca2+]i response was preserved. Finally, a [Ca2+]i response was also induced by LM in both MDCK (canine renal tubule epithelial model) and CaCo-2 (human intestinal epithelial model) cell lines, but not induced by FA in either. In conclusion, cell stimulation by LM is not via GPR40, nor is it a property restricted to FA-sensitive epithelial cells. This indicates that LM are not mimicking FA aggregates by activating specific FA-sensing mechanism(s). The mechanisms by which LM gate intracellular calcium entry remain to be elucidated. Nonetheless, LM induce CCK secretion in enteroendocrine cells, so may provide a novel method by which to modify upper gastrointestinal function or satiety, employing a non-nutrient signal that is retained within the lumen of the GI tract.

Where applicable, experiments conform with Society ethical requirements