Expression of free fatty acid receptor 1 enhances palmitate induced cell death in HEK293 cells

Victoria Stubbs, Nick Holliday. University of Nottingham, Nottingham, United Kingdom.

It is unknown whether the G protein coupled receptor FFAR1 (Itoh et al., 2003) contributes to selective long term actions of free fatty acids (FFAs), such as increased pancreatic β-cell death after palmitate (C16:0) but not oleate (C18:1) treatment (Maedler et al., 2003). Here we compare FFAR1 signalling after palmitate, or oleate, and effects of sustained agonist exposure on HEK293 cell viability. We established an inducible 293TR FFAR1 cell line to allow receptor expression to be initiated by 18 h 1 μg / ml tetracycline (tet) treatment. Ca2+ responses (Flexstation, 1.5μM Fluo-4) and ERK1/2 activation (Alphascreen, Perkin Elmer) were measured in media containing 0.1 % fatty acid free BSA, with pEC50s calculated from pooled data (Prism v5.01). Viability experiments used triplicate treatments with vehicle or FFA for up to 24h, added to previously tet-treated or control cells. After LIVE/DEAD assay (Invitrogen), nuclei (H33342), viable (calcein AM) or dead cell (Ethidium homodimer-1, EthD) images were acquired on an IX Ultra platereader. Scoring classified cells with each staining combination (only % EthD positive (+) shown here) for 4 fields of view /well. In tet induced (tet+) 293TR FFAR1 cells, oleate and palmitate were full agonists when measuring Ca2+ responses (respective pEC50 values 4.97 ± 0.04, n = 12 and 4.61 ± 0.07, n = 5) and both FFAs produced sustained increases in ERK1/2 phosphorylation over 30 min (pEC50 at 5 min of 4.27 ± 0.08 for oleate (n = 4) and 4.41 ± 0.10 for palmitate, n = 5). No agonist responses were observed in control cells without tet induction (tet-). Inducing FFAR1 expression reduced total adherent cell count in the LIVE/DEAD assay (at 24 h, vehicle controls tet+: 357 ± 26 cells / image, 0.2 ± 0.1 % EthD+ compared to tet-: 420 ± 55 cells / image, 0.1 ± 0.1 % EthD+, n = 4). 100 μM oleate treatment did not impair viability in cells with or without FFAR1 induction (24 h: 1.3 ± 0.6 % EthD+ in tet+ cells, compared to 0.1 ± 0.1 % in tet-, n = 4). In contrast 100 μM palmitate increased cell death, but only in cells expressing FFAR1 (15.3 ± 7.3 % EthD+ cf. 0.3 ± 0.1 % in tet- cells; P < 0.05, one way ANOVA / Bonferroni post test). Thus while both FFAs elicit FFAR1 Ca2+ or ERK1/2 responses with similar acute characteristics, expression of FFAR1 selectively enhances cell toxicity of saturated palmitate.

Itoh, Y et al., (2003) Nature 422,173
Maedler, K et al. (2003) Diabetes 52, 726.

Supported by the University of Nottingham.