Fig. 4.

FASIS in INS-1E cells A) Superoxide (O₂•^–) release into the mitochondrial matrix, monitored under the confocal microscope in INS-1E cells with MitoSOX Red at standard 15 μM palmitic acid (1.3 nM free PA with 15 μM BSA). Relative rates J_m with agents were normalized to standard ones (”% FASIS”; N = 8, n = 12–15). When indicated (n = 3–8), 1 nM, 10 nM, and 100 nM SkQ1 blocked 23 % ± 7 % (N = 8), 28 % ± 8 % (N = 8), and 41 % ± 5 % (N = 3) of the average J_m rates of O₂^•– release. Compare to the negligible effects of Decyl-TPP (“dTPP”; 1 and 10 nM) and the maximum effect of 20 μM rotenone (“Rot”). B) ATP levels established upon FASIS or GSIS after 60 min (conditions of panel A; N = 3; n = 8–20). C) K_ATP-channel closing status in ensemble, taken as glibenclamide-sensitive rate of Tl ⁺ influx after 6th min of cell preincubations (N = 7; n = 3–7; glibenclamide was 100 μΜ) in KRH induced with 15 μM PA (15 μM BSA; 1.3 nM free PA) with no other agents (“PA”) or together with 10 nM SkQ1 (“PA SkQ”), 50 μM cromakalim (“Croma”), and 2.5 μM etomoxir. The effect of SkQ1 without PA was also tested (“only SkQ”). However, upon GSIS with SkQ1, K_ATP remained to be closed (panel C, right). D–F) Insulin secreted and accumulated over the indicated time in representative experiments (N = 3), but with only 1.5 μM BSA and 7.5 μM PA (21 nM free). This is compared to no addition (“No. add.“, i.e. 3 mM glucose; gray triangles in D,F). Etomoxir (red squares) was 2.5 μM; SkQ1 (cyan) was 10 nM. E) GSIS at 25 mM glucose is compared to simultaneous FASIS and GSIS (dark blue). F,G) Cytosolic catalase overexpression – Insulin (F; conditions see panel D) and K_ATP (G, 15 μM BSA; 15 μM PA, i.e. 1.4 nM free) were tested for INS-1E-cells transfected with a control empty-vector (black) vs. catalase vector (brown), the latter blocking FASIS (F) and preventing K_ATP closure of transfected cells (a thallium assay, G) (N = 3; n = 3–7). SkQ1 was 10 nM (cyan/brown semifilled squares); or no PA was present (cyan or brown in G). Tl ⁺ flux suppression upon GSIS is also indicated in panel G, plus the prevention of such suppression in catalase-expressing cells. H,I,K) Carnitine palmitoyltransferase 1 silencing – Insulin (H) and Amplex UltraRed monitoring of H₂O₂release to the INS-1E cell exterior (I,K) for cells transfected with siRNA bearing scrambled sequence (“Scrl”, black) and siRNA CPT1 (red). Insulin FASIS assay was conducted with 75 μM PA in 30 μM BSA (4.55 nM free PA). J,K) H₂O₂release to the INS-1E cell exterior for other FAs at 30 μM BSA: J) exemplar records for 50 μM PA, ωBrPA, and linoleic; 70 μM oleic; 90 μM decanoic, and lauric, plus 180 μM hexanoic acids (for free FA see Table S2). K) Comparison of net H₂O₂ release initial rates with subtracted rates of no FA addition.