The effect of gliadin digest is independent of FFAR1 and MyD88, but is abrogated by diazoxide treatment.

<p>A. Fatty acid receptor FFAR1 downregulation using siRNA, did not affect insulin secretion during 24 hours of gliadin digest stimulation (P = 0.48, n = 4). Following MyD88 downregulation, glucose induced insulin secretion was reduced to 61% (0.6±0.3X, P = 0.02, n = 4). Though not significant, gliadin digest was still able to increase insulin secretion in the cells with downregulated MyD88 (P = 0.06, n = 4). B. No significant cell death was observed after transfections, except for cells treated with FFAR1 siRNA (P = 0.05, n = 3). C. Efficiency of the MyD88 silencing was confirmed using qPCR, which downregulated MyD88 expression as low as 25% of baseline expression levels (0.25±0.38X, P = 0.01, n = 3). D. Efficiency of the FFAR1 silencing was confirmed using qPCR, which downregulated FFAR1 expression as low as 25% of baseline expression levels (0.25±0.28x, P<0.01, n = 3). In low-glucose medium, FFAR1 expression was increased by 63% (1.63±0.17x, P<0.01, n = 4). E. Cells treated with gliadin digest for 24 hours prior to stimulation with palmitate and glucose for 30 min secreted 13% more insulin than palmitate-stimulated control cells (1.13±0.12x, P = 0.04, n = 5). F. Gliadin digest did not increase ATP content in INS-1E cells after 24 hours, as compared to controls (P = 0.34, n = 5). G. No significant increase in intracellular ATP was detected in cells stimulated with glucose and gliadin digest for 30 min compared to controls (P = 0.35, n = 4). H. Insulin secretion was increased by 31%, when cells were stimulated with both forskolin and gliadin digest compared to forskolin alone (1.31±0.26x, P = 0.03, n = 4). I. Addition of 100 µM diazoxide to medium with 11 mM glucose, reduced insulin secretion to 43% of normal secretion levels (0.43±0.13x, P = 0.0008, n = 4), similar to levels secreted in low glucose medium. However, when diazoxide was added to cells stimulated with gliadin digest and glucose, insulin secretion was restored to the levels observed for 11 mM glucose alone (P = 0.87, n = 4).