Fig. 5.

Extracellular monitoring of redox signaling upon FASIS vs. GSIS and H₂O₂ “rescue” of insulin release without FAs A–M) Extracellular H₂O₂ quantification with Amplex UltraRed and HRP in PIs (A–K) and INS-1E cells (L,M). Assays were calibrated with 2 μl aliquots of 10 μM H₂O₂. A–E) 10 μM Amplex UltraRed plus 5U/ml HRP were added to perifusate samples after their collection from the column containing 200–280 islets; representative data (otherwise N = 7) indicate rates of duplicates at each time point upon FASIS with 0.2 % (30 μM) BSA and with 50 μM PA (A,B), 65 μM stearic, 180 μM hexanoic, and 50 μM ω-bromopalmitic acid (D,E); 70 μM oleic, 90 μM lauric and 50 μM linoleic acid (E); or GSIS with 25 mM glucose (C) for wt or iPLA₂γKO-PIs (green); or with 10 nM SkQ1 (cyan/dark cyan). For free FA calculations, see Table S2. F–K) „Static” incubations of >100 islets in a cuvette with 10 μM Amplex UltraRed plus 5 U/ml HRP. Representative data (F–J; summary for various FA doses see Fig. S6) indicate the accumulated H₂O₂ amount upon FASIS or GSIS (25 mM glucose) for wt vs. iPLA₂γKO-PIs, and upon GSIS for NOX4KO-PIs [36] (panel J, orange) vs. their own backcrossed controls [36] (“wt”). Effects of selected agents are compared in panel K (N = 3–5). When indicated, 10 nM SkQ1 (cyan/dark cyan), 5 μM etomoxir (red/dark red) or 100 μM Trolox (violet) were present (higher concentrations see panel K, where inhibition of net initial rates was calculated with subtracted rates of no addition). L,M) INS-1E cells: representative traces (L) and inhibitory effects after 1-hr pre-incubation (M; 2.5 μM etomoxir; N = 5; n = 5–10) for FASIS with 15 μM PA (1.4 nM free) in 0.1 % (15.5 μM) BSA. For overexpression of the cytosolic catalase (brown), rates with subtracted no addition rates were normalized to cells transfected with an empty vector (M). N–P) Rescue of insulin secretion at low 5.5 mM glucose without FA by 100 μM H₂O₂, i.e. representative perifusions (otherwise N = 3–6), mimicking insulin secretion by the external H₂O₂ present during perifusion of wt (black) and iPLA₂γKO-PIs (green) in the absence and presence of 2.5 μM etomoxir in wt (red) (O) and iPLA₂γKO-PIs (dark red) (P). For a summary of secreted insulin amounts (AUCs), see Fig. S7H.