Background and aims: Therapies based on the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are widely used in the treatment of diabetes. Clinically used agents are either injectable peptidergic GLP-1 receptor agonists or orally available dipeptidylpeptidase-4 inhibitors, which prolong the half-life of the endogenous hormones. Enhancing endogenous hormone secretion has not yet been exploited, but appears promising, as enhanced postprandial GLP-1 secretion after Roux-en-Y gastric bypass surgery correlates with substantially improved glycaemia. Two potential pharmaceutical targets to increase GLP-1 secretion are the free fatty acid (FFA) receptors 1 and 4, present in both GLP-1 secreting L-, and GIP secreting K-cells. In this study we investigated the role of FFA1 and 4 in lipid stimulated GIP and GLP-1 secretion in mice.Methods: Secretion, expressed as % of total hormone content, was assessed in 2 h incubations of murine duodenal and colonic cell cultures treated with post prandial micelles (PPM) (containing (mM): oleic acid (0.2), 2-monooleoyl-glycerol (0.07), L-α-lysophosphatidylcholine (0.07), cholesterol (0.017), taurocholate (0.7)) or with taurocholate (2 mM) +/- oleic acid (0.4 mM). Responses were compared between wild-type and FFA1 or FFA4 knock-out (-/-) animals. In FFA4-/- and +/+ mice plasma hormone was assessed after an intragastric lipid gavage of 1:1 corn and olive oils. All animal experiments were carried out with ethical approval and in accordance with UK legislation. Results: In duodenal cultures PPM increased GIP secretion from 1.6±0.2% to 13.4±1.0% (n=32), and GLP-1 secretion from 5.3±0.4% to 47.9±1.8% (n=24). PPM stimulated secretion was reduced in both FFA1-/- (GIP: 1.1±0.1% to 7.5±0.7% (n=11); GLP-1: 3.9±0.3% to 20.5±0.8% (n=11)) and FFA4-/- cultures (GIP: 1.5±0.3% to 7.4±0.9% (n=24) and GLP-1: 2.9±0.4% to 31.3±2.3%; (n=13) respectively). Taurocholate increased GIP and GLP-1 secretion similarly from wild type and knockout cultures, but the additional presence of oleic acid, which increased GIP and GLP-1 secretion a further 1.7 and 2.2-fold from wild type cultures, was ineffective in FFA1-/- and FFA4-/- cultures. Somewhat surprisingly, PPM stimulated GLP-1 secretion similarly from colonic cultures established from wild-type or FFA1-/- or FFA4-/- mice, perhaps reflecting the stimulatory actions of other PPM components. A significant reduction in plasma GIP elevation was observed in FFA4-/- animals after lipid gavage (n=21), whilst plasma GLP-1 elevations tended to be lower, but this failed to reach significance.Conclusions: Both FFA1 and FFA4 underlie lipid stimulated incretin secretion and knock-out of either receptor impaired both GLP-1 and GIP secretion with limited receptor redundancy.