Calcium mobilisation and CCK secretion induced by modified fatty acids and latex microspheres reveal dual receptor mechanisms for lipid stimulation of STC-1 cells.

How fatty acids stimulate enteroendocrine cells to release cholecystokinin (CCK) is largely unknown. Recently, we proposed that the murine enteroendocrine cell line, STC-1, responds to insoluble fatty acid aggregates rather than fatty acid monomers in solution. This hypothesis led to two testable predictions. First, other insoluble particles of similar size but unrelated to fatty acid may be able to stimulate STC-1 cells in a similar fashion to dodecanoic acid and second, fatty acid sensing in STC-1 cells should be fairly insensitive to chemical modifications of the fatty acid as long as these modifications do not greatly alter the ability of the molecule to form insoluble aggregates. We used several analogues of dodecanoic acid and several varieties of latex microsphere (varying in size and surface charge) to see whether the predictions of our model hold. We found that while there was at least one latex microsphere that could induce CCK secretion and calcium mobilisation in STC-1 cells, there was a very poor correlation between the presence of insoluble aggregates and a cellular response. Instead the most important property, determining the potency of fatty acid analogues as stimulants of CCK secretion, was their amphipathicity. Removal of either the polar head or lipophilic tail completely abolished the ability of a given fatty acid analogue to stimulate STC-1 cells. These data suggested that while fatty acids can stimulate cells as aggregates, they may also be acting in monomeric form with the oil:water partitioning coefficient playing a crucial role. We finally resolved this issue with the observation that the sulfate ion greatly altered the response of STC-1 cells to monomeric dodecanoic acid. In the presence of sulfate, STC-1 cells will only respond to dodecanoic acid aggregates whereas when sulfate is replaced with chloride the cells clearly respond to dodecanoic acid monomers which are completely in solution. In summary, we propose that dodecanoic acid can stimulate STC-1 cells via two separate pathways one involving fatty acid monomers in solution and one involving fatty acid aggregates. Which pathway dominates depends on the presence of sulfate in the extracellular medium.

Fatty acid signalling in a mouse enteroendocrine cell line involves fatty acid aggregates rather than free fatty acids.

Fatty acids induce cholecystokinin (CCK) secretion both in humans and from murine enteroendocrine cell lines. In both cases, only fatty acids above a critical acyl chain length (C(10)) are capable of inducing a response. Using the enteroendocrine cell line STC-1, the aim of this study was to determine whether this acyl chain length dependency is related to the fact that longer chain fatty acids are relatively insoluble in aqueous solutions and, if so, whether it is insoluble aggregates of fatty acids rather than free fatty acids which evoke CCK secretion. Solutions of fatty acids (chain length C(8)-C(14)), which were judged by filtration and Zeta sizer measurement to contain no fatty acid aggregates, never evoked CCK secretion from STC-1 cells. Filtering fatty acid solutions (of chain length C(10), C(12) and C(14)) through polytetrafluoroethylene (PTFE) filters (0.45 microm pore size) revealed a narrow concentration range for each acid over which the amount of fatty acid removed from the solution increased sharply due to the formation of fatty acid aggregates. Filtration experiments, in which suspensions of C(10), C(12) and C(14) fatty acids were passed through pore sizes of 0.2, 0.45 or 1.2 microm, suggested that STC-1 cells did not respond to fatty acid aggregates of greater than 1.2 microm, while at least 50 % of the CCK response was mediated by aggregates which were smaller than 0.45 microm. Fatty acids induce CCK secretion from STC-1 cells by elevating intracellular Ca(2+) concentration ([Ca(2+)](i)). We therefore measured the effects on [Ca(2+)](i) of filtered C(10), C(12) and C(14) fatty acids. In all cases, [Ca(2+)](i) responses were closely correlated with CCK secretion. Interestingly, while filtrates of fatty acid solutions evoked CCK secretion and elevated [Ca(2+)](i), freshly prepared solutions of fatty acids at the same concentration as the filtrates did not. This suggested that fatty acid aggregates were not in equilibrium with the solvent after filtration. The observation that the ability of C(10), C(12) and C(14) filtrates to elevate [Ca(2+)](i) decayed with time was consistent with this hypothesis. Furthermore, sonication of the filtrates abolished their ability to elevate [Ca(2+)](i). These data further suggest that it is a physical property of the fatty acid solution (the presence of insoluble fatty aggregates) which is responsible for the observed cellular responses. We conclude that Ca(2+) mobilisation and CCK secretion in STC-1 cells is driven by a signal transduction mechanism that senses insoluble fatty acid aggregates, rather than free fatty acids in solution.