Figure 1.
Gliadin digest increases weight in NOD mice.
NOD mice were injected with gliadin digest (blue: 4.5 µg, red: 450 µg, green: controls, displayed as mean±SEM) six times over two weeks. Blood glucose level and weight were measured twice a week. A. Comparison of blood glucose levels. No significant differences were observed between the groups (P = 0.84, n = 15). B. Diabetes incidence. No differences were observed between groups (P = 0.53, n = 15, Mantel-Cox). C. Weight. There was a significant weight increase in mice injected with the 450 µg gliadin digest doses (average 24.9±0.9 g at day 100) compared to controls (average 23.6±0.7 g at day 100) (P<0.0001, n = 15).
Figure 2.
Gliadin digest and a gliadin 33-mer increases insulin secretion in INS-1E cells and rat islets during 24 hours of stimulation.
A. Gliadin digest significantly increased insulin secretion 1.8±0.3fold (P<0.001, n = 6). No effect was observed from heat-inactivated digestion enzymes (P = 0.27, n = 5) or lipopolysaccharide (P = 0.46, n = 4). B. In INS-1E cells, insulin release increased dose-dependently during stimulation for 24 hours with increasing concentrations of gliadin-digest, as compared to glucose stimulation alone (1.5±0.29 fold increase for 30 µg/ml to 2.5±0.4 fold for 600 µg/ml, P<0.0001, n = 6). C. Addition of gliadin digest to cells in low glucose increased insulin secretion by 50% (1.5±0.4X, P = 0.01, n = 4). D. Quantification of live cells with resazurin. Data were normalized relative to the number of cells in 3 mM glucose after 24 hours. No difference was observed between the mass of cells stimulated in 11 mM glucose and that of cells treated with 11 mM glucose and gliadin digest (P = 0.626, n = 4). E. Incubation with 11 mM glucose and enzymatically digested ovalbumin for 24 hours did not increase the insulin secretion significantly (P = 0.22, n = 4). Gliadin digest significantly increased insulin secretion compared to ovalbumin (1.57±0.46x, P = 0.034, n = 3). F. Gliadin digest-stimulated insulin secretion in rat islets of Langerhans was significantly increased up to 1.55±0.35 times compared to controls (P = 0.015, n = 4). G. Stimulation of INS-1E cells with increasing amounts of 33-mer in 11 mM glucose, resulted in up to 1.7 fold dose-dependent increase in insulin secretion compared to the control group (P = 0.03, n = 4). The 19-mer had no effect on insulin secretion at any concentration assayed (P = 0.98, n = 4). H. During 30 min stimulation, INS-1E cells stimulated with 3 mM glucose secreted significantly less insulin than cells stimulated with 11 mM glucose (0.47±0.09, P<0.001, n = 4). Co-incubation with gliadin digest did not increase insulin secretion (P = 0.35, n = 4). Incubation with gliadin digest for 24 hours prior to glucose stimulation did not affect insulin secretion (P = 0.62, n = 4). I. Arginine (1 mM) had no effect on the insulin secretion in INS-1E cells after 24 hours stimulation (P = 0.84, n = 5). The removal of peptides <100–500 Da (by dialysis) and 3000 Da (centrifugal filtration) did not reduce the ability of gliadin digest to stimulate insulin secretion in INS-1E cells (dialysis: P = 0.026, filtration: P = 0.003, n = 3).
Figure 3.
The effect of gliadin digest is independent of FFAR1 and MyD88, but is abrogated by diazoxide treatment.
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).
Figure 4.
Gliadin digest incubation inhibits current through KATP.
Kir6.2 and SUR1 were expressed in HEK-293 cells. The cells were incubated overnight in either 300 µg/ml gliadin digest or a corresponding volume of enzyme mixture. Currents were activated by a ramp protocol every 5 s. A. Representative KATP currents in a cell incubated with the enzyme mixture overnight (1) prior to ATP washout, (2) after ATP washout in the presence of enzyme mix and (3) after 2.5 minutes exposure to gliadin digest. B. Representative currents (traces overlaid in plot) from a cell incubated with gliadin digest overnight, (1) prior to ATP washout, (2) after ATP washout in the presence of the enzyme mixture and (3) after application of the enzyme mixture for 2.5 minutes. C. The time-dependence of the effect of gliadin digest on KATP currents. Representative data from a cell incubated in enzyme mix overnight. The data points represent maximal inward current during washout of endogenous ATP and after application of gliadin digest. D. Summarized current densities in : +ATP, average current density prior to ATP washout in cells incubated in enzyme mix overnight (n = 7), −ATP, average current density after washout (n = 9). Gliadin digest: average current density after 2.5 min exposure to 300 µg/ml gliadin digest (n = 9). The effect of gliadin digest was not significant. In 10/12 cells incubated in gliadin digest overnight there was no expressed KATP current. In the first bar graph all cells are included; in the second bar graph the 2 cells with current are excluded. For cells incubated in the enzyme mixture, 3/12 cells had no current and were excluded from the analysis.
Figure 5.
The 33-mer gliadin fragment inhibits KATP currents.
Kir6.2 and SUR1 were transiently expressed in HEK-293 cells. The cells were incubated with either the 19-mer or 33-mer gliadin fragment in the medium overnight. Currents were activated by a ramp protocol every 5 s as described in Fig. 4. A. Representative KATP currents in a cell incubated with the 19-mer. Trace (1): prior to washout of endogenous ATP, trace (2): after washout of ATP. B. Representative currents from a cell incubated with the 33-mer before and after washout of ATP. C: The time-dependence of the effect of the 19-mer (black circles) or the 33-mer (grey circles) on maximum inward current. The data points represent maximum inward current at the start of the ramp protocol during washout of endogenous ATP. D. A summary of current densities. Non-transfected cells (NT, n = 3), transfected cells incubated in control medium (KATP, n = 7), transfected cells incubated in the 19-mer (KATP, 19, n = 9) or transfected cells incubated in the 33-mer (KATP, 33, n = 9). Average current density before (white) and after (grey) ATP washout is shown.