Figure 1.
Chemical structure of 1,1,1-trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18).
Figure 2.
Comparison of Inhibition of Cytosol-Associated iPLA2β by BEL and FKGK18 in INS-1 OE Cells.
Cytosol was prepared from INS-1 OE cells and the ability of FKGK18 to inhibit cytosol-associated iPLA2β activity was compared with that of S-BEL and R-BEL. The radioactivity enzymatic assay was performed using 30 µg protein aliquots and the data are presented as mean ± SEM of residual activity in the presence of an inhibitor, relative to activity measured in the presence of only the vehicle.
Figure 3.
Cytosol-Associated iPLA2β Activity in Myocardium of WT and iPLA2β-KO Mice.
Cytosol was prepared from hearts isolated from wild type (WT) and iPLA2β-deficient (KO) mice. The iPLA2β enzymatic radioactivity assay was preformed using 30 µg protein aliquots in the absence and presence of ATP (10 mM) and FKGK18 (10−6 M) either alone or in combination as indicated. The data for each group are presented as mean ± SEM of fold-change in activity in the presence of an inhibitor, relative to activity measured in the presence of only vehicle. (*Significantly different from WT Control group, p<0.05 and †significantly different from WT+ATP group, p<0.05).
Figure 4.
Inhibition of Membrane-Associated iPLA2 Activity in Hearts from WT and iPLA2β-KO Mice by R-BEL and FKGK18.
Membrane fractions were prepared from hearts isolated from WT and iPLA2β-deficient (KO) mice and iPLA2β activity was assayed in 30 µg protein aliquots. The data are presented as mean ± SEM of residual activity in the presence of an inhibitor relative to activity measured in the presence of only vehicle. A. WT membrane-associated activity. Residual activity was assayed in the absence and presence of FKGK18, S-BEL, or R-BEL. B. KO membrane-associated activity. Residual activity was assayed in the absence and presence of FKGK18 or R-BEL.
Figure 5.
Inhibition of Myocardial Cytosol- and Membrane-Associated iPLA2 Activity by FKGK18.
A. Organelle localization of iPLA2β and iPLA2γ. Cytosol and membrane fractions were prepared and processed for immunoblotting analyses using primary antibody against iPLA2β (Top panel), iPLA2γ (Middle Panel), and loading control GAPDH (Bottom Panel). B. Summary plots of residual activity in cytosol (iPLA2β) and membrane (iPLA2γ) the presence of FKGK18. Cytosolic and membrane fractions were prepared from WT hearts and iPLA2β activity was assayed in 30 µg protein aliquots. The data are presented as mean ± SEM of residual activity in the presence of the inhibitors expressed, relative to the activity measured in the presence of vehicle alone. The estimated IC50 of each is shown.
Figure 6.
Comparison of S-BEL and FKGK18 Effects on Alpha-Chymotrypsin Activity.
BSA (2 µg) was digested with trypsin (T) + α-chymotrypsin (C) (final concentration of each <100 nM) in the absence and presence of S-BEL and FKGK18 (20 µM) for 15 min at 37°C. The peptide digests were dried down to 20 µl and loaded onto 4–12% Bis-Tris gel and peptide fragments were separated for 35 min at 200V constant and visualized by overnight colloidal blue staining. Duplicate Lanes 1, S-BEL + FKGK18; 2, T + C; 3, T + C + FKGK18; and 4, T + C + S-BEL.
Figure 7.
Temporal Effects of FKGK18 Exposure on iPLA2β Activity in INS-1 OE Cell Cytosol.
INS-1 OE cells were treated with FKGK18 (10−5 M) for 2 to 48 h. Cytosol was then prepared and iPLA2β activity was assayed in 30 µg protein aliquots in the absence and presence of ATP (10 mM). The data are presented as mean ± SEM of activity, relative to that measured in Control groups.
Figure 8.
Effects of FKGK18 on Glucose-Stimulated Insulin Secretion (GSIS) and PGE2 Generation in Human Pancreatic Islets.
Human pancreatic islets (30/condition) were incubated in KRB containing 5 mM glucose (5G) for 1 h at 37°C under 5%CO2/95% air atmosphere. The medium was then replaced with 5G + DMSO or 5G + FKGK18 (10−6 M) and the islets were incubated for 1 h. The islets were then exposed to KRB medium containing 5G+DMSO, 5G+FKGK18, 20G+DMSO or 20G+FKGK18. Medium was collected after 1 h and insulin and PGE2 contents in the medium were measured by ELISA. The islets were washed in PBS (3×) and islet protein concentration was determined. The data were normalized to total protein content. A. GSIS. (*20G group significantly different from other groups, p<0.01.) B. PGE2 generation. (*20G group significantly different from other groups, p = 0.001).
Figure 9.
Effects of FKGK18 on ER Stress-Induced NSMase2 Expression in INS-1 OE Cells.
INS-1 OE cells were treated with vehicle only or with thapsigargin (1 µM) for 8 or 24 h in the presence of FKGK18 (0.10, 1.0, or 10 µM). Total RNA was then prepared and cDNA generated and used to determine NSMase2 mRNA. The data are presented as fold-change in message, relative to vehicle-treated group only. A. 8 h. B. 24 h. (*Significantly different from vehicle group, p<0.001, †significantly different from Thaps group, p<0.003, and #significantly different from Thaps group, p = 0.03).
Figure 10.
Effects of FKGK18 on ER Stress-Induced INS-1 OE Cell Apoptosis.
INS-1 OE cells were treated with vehicle only or with thapsigargin (1 µM) for 24 h in the presence of FKGK18 (10−7–10−5 M). The incidence of apoptosis was then assessed by TUNEL staining using a flow cytometry protocol. The data in each group are plotted as fold-change in apoptosis, relative to vehicle treated cells. (*Significantly different from vehicle group, p<0.01 and †significantly different from Thaps group, p<0.05, n = 6).