0

30

60

90

120

**

Insu

lin s

ecr

etio

n

(ng/

ml)

Glucose (mM) 3 20 3 3 3 3 H2O2 (M) - - 1 10 - -HNE (10 M) - - - - - + SIN-1 (200 M) - - - - - +

Fig. S1. Effects of H2O2, HNE and SIN-1 on insulin secretion in INS-1

(832/13) cells. n = 3-10. *, p < 0.05 compared with 3 mM glucose alone.

0

20

40

60

80

Glucose (mM) 3 20 3 3 3 3 H2O2 (µM) - - 1 2 4 8

Insu

lin s

ecr

etio

n

(ng/

ml)

Fig. S2. Ca2+-free conditions prevent H2O2-stimulated insulin secretion and GSIS in INS-1(832/13) cells. Cells were pre-incubated with Ca2+-free Kreb’s buffer (140 mM NaCl, 30 mM HEPES, 4.6 mM KCl, 1 mM MgSO4, 0.15 mM Na2HPO4, 5 mM NaHCO3, 100 µM EGTA and 0.05% BSA, pH 7.4) containing 3 mM glucose for 30 min. Then the buffers were replaced with Ca2+-free Krebs’ buffer containing secretagogues and followed by a 30 min-incubation. n = 4.

0

20

40

60

80

100

120

-1.5 -1 -0.5 0 0.5 1 1.5

Normal standard curve

With NAC (0.4 mM)

With DEM (5mM)

% B

/B0

Log (ng/ml)

Fig. S3. NAC and DEM do not interfere with the insulin RIA.

0 1002003004005000

40

80

120

160

0 20 40 60 80 1000

40

80

120

160%

of

cont

rol

MGO (µM)0 10 20 30 40 50

0

40

80

120

160

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.

A

0.0 2.5 5.0 7.5 10.00

50

100

150

B C%

of

cont

rol D

Arsenite (µM)

0 10 200

1000

2000

Kreb'sPBSKreb's+CATPBS+CAT

H2O

2 le

vels

(f

luor

esce

nce

inte

nsity

)

Glucose (mM)

Fig. S5. Glucose autoxidation is a powerful source of H2O2 generation in vitro. A) Glucose dose-dependently increases H2O2 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 H2O2 levels were measured using Amplex Red Hydrogen Peroxide/ Peroxidase Assay Kit (Molecular Probes) and the fluorescence intensity (λEx = 545 nm; λEm = 590 nm) was measured by VICTOR 3 1420 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 H2O2 production in PBS. 20 mM glucose+CAT, 20 mM glucose was incubated in PBS containing 20 U/ml of catalase.

0 10 20 300

1000

2000

10 mM glucose20 mM glucose20 mM glucose+CAT

Incubation time (min)

A B

0

25

50

75

100

Cont Rot Ant Oli

3 mM glucose20 mM glucose

Insu

lin s

ecr

etio

n

(ng/

ml)

0

1

2

3

C

Ce

llula

r A

TP

(n

mo

l/mg

pro

tein

)

Cont Rot Ant Oli

*

# # #

*

* * * ###

3 mM glucose20 mM glucose

0

50

100

150

200A

H2O

2 a

ccu

mu

latio

n

(Flu

ore

sce

nce

in

tens

ity)

3 mM glucose20 mM glucose

Cont Rot Ant Oli

** *

#

#

Fig. S6. Block of mitochondrial electron transport chain results in H2O2 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

0 50 100 150 200100

200

300

400

500

600

700

Time (h)

Intr

ace

llula

r H

2O

2 (

nM

)

Oxidative stress

Stimulus

Oxidative stress

Stimulus

013

013

Stress level

Fig. S7. Simulation results showing pre-existing oxidative stress can suppress H2O2 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 SOD2Treatments

Control 100 ± 10 100 ± 4.5 100 ± 15 100 ± 4.5 100 ± 8.0 HNE 172 ± 15* 211 ± 4.5* 181 ± 18* 131 ± 14 114 ±12 MGO 179 ± 18* 303 ± 9.0* 112 ± 16 116 ± 21 126 ± 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.