* * Insulin secretion (ng/ml) Glucose (mM) H 2 O 2 (  M) HNE (10  M) SIN-1 (200  M) Fig. S1. Effects of H 2 O 2, HNE and SIN-1 on insulin secretion in INS-1 (832/13) cells. n = *, p < 0.05 compared with 3 mM glucose alone.

Glucose (mM) H 2 O 2 ( µ M) Insulin secretion (ng/ml) Fig. S2. Ca 2+ -free conditions prevent H 2 O 2 -stimulated insulin secretion and GSIS in INS-1(832/13) cells. Cells were pre-incubated with Ca 2+ -free Kreb’s buffer (140 mM NaCl, 30 mM HEPES, 4.6 mM KCl, 1 mM MgSO 4, 0.15 mM Na 2 HPO 4, 5 mM NaHCO 3, 100 µM EGTA and 0.05% BSA, pH 7.4) containing 3 mM glucose for 30 min. Then the buffers were replaced with Ca 2+ -free Krebs’ buffer containing secretagogues and followed by a 30 min-incubation. n = 4.

% B/B0 Log (ng/ml) Fig. S3. NAC and DEM do not interfere with the insulin RIA.

% of control MGO (µM) HNE (µM) Arsenite (µM) Fig. S4. Cytotoxicity of HNE, MGO and arsenite in INS-1 (832/13) cells and isolated mouse islets. Cell viability was measured by MTT assay. (A-C) INS-1 (832/13) cells were cultured on 96-wells plate overnight and followed by treatment with the agents for 24 hrs; D. Isolated islets (20 islets/well) were incubated with arsenite for 24 hrs and viability was measured. ABC % of control D Arsenite (µM)

H 2 O 2 levels (fluorescence intensity) Glucose (mM) Fig. S5. Glucose autoxidation is a powerful source of H 2 O 2 generation in vitro. A) Glucose dose-dependently increases H 2 O 2 production in PBS or Kreb’s buffer. Different concentrations of glucose were incubated in PBS or Kreb’s buffer at 37˚C for 30 min. The H 2 O 2 levels were measured using Amplex Red Hydrogen Peroxide/ Peroxidase Assay Kit (Molecular Probes) and the fluorescence intensity (λ Ex = 545 nm; λ Em = 590 nm) was measured by VICTOR Multilabel Counter (Perkin Elmer, Finland). Kreb’s, glucose was incubated in Kreb’s buffer; PBS, glucose was incubated in PBS; Kreb’s+CAT, glucose was incubated in Kreb’s buffer containing 20 U/ml of catalase; PBS+CAT, glucose was incubated in PBS containing 20 U/ml of catalase. B) Glucose time-dependently increases H 2 O 2 production in PBS. 20 mM glucose+CAT, 20 mM glucose was incubated in PBS containing 20 U/ml of catalase. Incubation time (min) AB

Cont Rot Ant Oli 3 mM glucose 20 mM glucose Insulin secretion (ng/ml) C Cellular ATP (nmol/mg protein) Cont Rot Ant Oli * # # # * * * * # # # 3 mM glucose 20 mM glucose A H 2 O 2 accumulation (Fluorescence intensity) 3 mM glucose 20 mM glucose Cont Rot Ant Oli * * * # # Fig. S6. Block of mitochondrial electron transport chain results in H 2 O 2 accumulation, decreased ATP generation and impaired GSIS in INS-1 (832/13) cells. Rot, rotenone, a complex I inhibitor; Ant, antimycin A, a complex III inhibitor; Oli, oligomycin, a blocker of mitochondria ATPase and phosphoryl group transfer. The cells were treated with rotenone (1 µM), antimycin A (2 µM), or oligomycin (1 µg/ml) in Kreb’s buffer with 3 mM glucose for 30 min and followed by glucose stimulation (3 mM vs. 20 mM) in the same buffer system for 30 min. *, p < 0.05 compared with 3 mM glucose alone; #, p < 0.05 compared with 20 mM glucose alone. B

Fig. S7. Simulation results showing pre-existing oxidative stress can suppress H 2 O 2 signaling triggered by a subsequent stimulus. The degree of suppression is positively correlated to the stress level.

Table S1. Induction of Nrf2 downstream genes in INS-1 (832/13) cells Genes HO-1 GCLC NQO-1 GPx2 SOD2 Treatments Control 100 ± ± ± ± ± 8.0 HNE 172 ± 15 * 211 ± 4.5 * 181 ± 18 * 131 ± ±12 MGO 179 ± 18 * 303 ± 9.0 * 112 ± ± ± 1.7 Arsenite 276 ± 4.8 * 196 ± 8.1 * 210 ± 9.0 * 187 ± 7.2 * 167 ± 12 * Data shown as mean ± SEM (n = 3). *, p < 0.05 compared with control. Treatments: the cells were exposed to HNE (20 µM), MGO (0.4 mM) and arsenite (5 µM) for 6 hours.