Insulin secretion ngml

1
Insulin secretion (ng/ml)
Glucose (mM) 3 20 3 3
3 3 H2O2 (mM) - -
1 10 - -HNE (10 mM) -
- - - -
SIN-1 (200 mM) - - - -
-
Fig. S1. Effects of H2O2, HNE and SIN-1 on
insulin secretion in INS-1 (832/13) cells. n
3-10. , p lt 0.05 compared with 3 mM glucose
alone.
2
Insulin secretion (ng/ml)
Glucose (mM) 3 20 3 3 3 3 H2O2
(µM) - - 1 2 4 8
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 Krebs 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.
3
B/B0
Log (ng/ml)
Fig. S3. NAC and DEM do not interfere with the
insulin RIA.
4
A
B
C
of control
HNE (µM)
MGO (µM)
Arsenite (µM)
D
of control
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.
5
A
B
H2O2 levels (fluorescence intensity)
Glucose (mM)
Incubation time (min)
Fig. S5. Glucose autoxidation is a powerful
source of H2O2 generation in vitro. A) Glucose
dose-dependently increases H2O2 production in PBS
or Krebs buffer. Different concentrations of
glucose were incubated in PBS or Krebs buffer at
37C for 30 min. The H2O2 levels were measured
using Amplex Red Hydrogen Peroxide/ Peroxidase
Assay Kit (Molecular Probes) and the fluorescence
intensity (?Ex 545 nm ?Em 590 nm) was
measured by VICTOR 3 1420 Multilabel Counter
(Perkin Elmer, Finland). Krebs, glucose was
incubated in Krebs buffer PBS, glucose was
incubated in PBS KrebsCAT, glucose was
incubated in Krebs buffer containing 20 U/ml of
catalase PBSCAT, glucose was incubated in PBS
containing 20 U/ml of catalase. B) Glucose
time-dependently increases H2O2 production in
PBS. 20 mM glucoseCAT, 20 mM glucose was
incubated in PBS containing 20 U/ml of catalase.
6
A
B
3 mM glucose
3 mM glucose
20 mM glucose
20 mM glucose
Cellular ATP (nmol/mg protein)


H2O2 accumulation (Fluorescence intensity)










Cont Rot Ant Oli
Cont Rot Ant Oli
C
3 mM glucose
20 mM glucose

Insulin secretion (ng/ml)



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 Krebs 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 lt 0.05
compared with 3 mM glucose alone , p lt 0.05
compared with 20 mM glucose alone.
7
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.
8
Table S1. Induction of Nrf2 downstream genes in
INS-1 (832/13) cells
Genes
Treatments
HO-1 GCLC NQO-1 GPx2
SOD2
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 lt 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.