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High glucose inhibits glucose-6-phosphate

dehydrogenase, leading to increased oxidative stress
and ␤-cell apoptosis
Zhaoyun Zhang,*,‡ Chong Wee Liew,* Diane E. Handy,† Yingyi Zhang,†
Jane A. Leopold,† Ji Hu,* Lili Guo,* Rohit N. Kulkarni,* Joseph Loscalzo,†
and Robert C. Stanton*,1
*Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA;
†
Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical
School, Boston, Massachusetts, USA; and ‡Department of Endocrinology and Metabolism, Huashan
Hospital, Shanghai, China

ABSTRACT Patients with type 2 diabetes lose ␤ cells,
but the underlying mechanisms are incompletely under-
stood. Glucose-6-phosphate dehydrogenase (G6PD) is
the principal source of the major intracellular reduc-
tant, NADPH, which is required by many enzymes,
including enzymes of the antioxidant pathway. Previous
work from our laboratory has shown that high glucose
impairs G6PD activity in endothelial and kidney cells,
which leads to decreased cell survival. Pancreatic ␤
cells are highly sensitive to increased ROS. This study
aimed to determine whether G6PD and NADPH play
central roles in ␤-cell survival. Human and mouse islets,
MIN6 cell line, and G6PD deficient mice were studied.
High glucose inhibited G6PD expression and activity.
Inhibition of G6PD with siRNA led to increased ROS
and apoptosis, decreased proliferation, and impaired
insulin secretion. High glucose decreased insulin secre-
tion, which was improved by overexpressing G6PD.
G6PD-deficient mice had smaller islets and impaired
glucose tolerance compared with control mice, which
suggests that G6PD deficiency per se leads to ␤-cell
dysfunction and death. G6PD plays an important role
in ␤-cell function and survival. High-glucose-mediated
decrease in G6PD activity may provide a mechanistic
explanation for the gradual loss of ␤ cells in patients
with diabetes.—Zhang, Z., Liew, C. W., Handy, D. E.,
Zhang, Y., Leopold, J. A., Hu, J., Guo, L., Kulkarni, R. N.,
Loscalzo, J., Stanton, R. C. High glucose inhibits glucose-
6-phosphate dehydrogenase, leading to increased oxida-
tive stress and ␤-cell apoptosis. FASEB J. 24, 1497–1505
(2010). www.fasebj.org
Key Words: diabetes mellitus 䡠 islets 䡠 antioxidants 䡠 pentose
phosphate pathway
Oxidative stress mediates glucose toxicity to cells
and tissues and occurs as a result of an imbalance
between processes that produce reactive oxygen species
(ROS) and processes that reduce ROS (antioxidants).
The major components of the antioxidant system are
catalase, superoxide dismutases (SODs), glutathione
system, and glucose-6-phosphate dehydrogenase (G6PD).
Although any cell is potentially vulnerable to increased
ROS, ␤ cells are particularly vulnerable to ROS cytotox-
icity, which has been attributed to the relatively low
levels of antioxidant enzymes in islets (1–3). For exam-
ple, Tiedge et al. (3) have shown that (cytoplasmic)
Cu/Zn SOD and (mitochondrial) Mn SOD expression
levels in islets were in the range of 30 – 40% of those in
the liver. In other studies, these investigators have
found that glutathione peroxidase-1 (GPx-1) gene
expression was 15% of those in liver and that catalase
gene expression was not detectable in pancreatic
islets (2).
Both type 1 and type 2 diabetes lead to loss of ␤ cells.
In type 1 diabetes, ␤ cells are damaged initially by an
immune-mediated process (4). In type 2 diabetes, ␤-cell
function decreases gradually over years. Moreover, ␤-cell
mass diminishes over time (5). No definitive causes for
loss of ␤ cells have been determined, but it is likely that
chronic exposure to elevated blood glucose contributes
to decreased ␤-cell survival. As ␤ cells are highly sensi-
tive to increased ROS, it is likely that increased ROS
play a role in the loss of ␤ cells. Indeed, many in vivo
and in vitro studies have shown that treatments target-
ing oxidative stress improve both ␤-cell function and
survival (5–7).
Although all components of the antioxidant system
are important for cell survival, G6PD has a unique role,
as it is the principal source of NADPH, which is the
main intracellular reductant that promotes the antiox-
idant action of peroxidases (8 –11). G6PD is the rate-
limiting enzyme in the pentose-phosphate pathway,
which produces ribose-5-phosphate and NADPH. Al-
though other sources for NADPH exist, studies by our
laboratory and others have shown that G6PD is the
1
Correspondence: Renal Division, Joslin Diabetes Center,
One Joslin Pl., Boston, MA 02215, USA. E-mail: robert.
stanton@joslin.harvard.edu
doi: 10.1096/fj.09-136572

0892-6638/10/0024-1497 © FASEB 1497

major source of NADPH for the antioxidant system and
other critical enzymes (9, 12–18). NADPH is used by
the glutathione and thioredoxin systems to regenerate
reduced forms that will then be used in antioxidant
roles. Catalase, which converts hydrogen peroxide to
water and oxygen, does not use NADPH directly, but an
essential allosteric binding site for NADPH maintains
catalase in its most active tetrameric conformation and
protects it against the toxicity of hydrogen peroxide
(H2O2) (19). The other major component of the
antioxidant system, SOD, which converts superoxide to
hydrogen peroxide, does not use NADPH. However,
the SOD-produced H2O2 is then reduced by either
catalase or GPxs. Hence, SODs become ultimately
dependent on NADPH as lack of it will lead to a
decrease in catalase and the level of reduced glutathi-
one and a resultant increase in hydrogen peroxide
levels. Increased hydrogen peroxide then inhibits SOD
activity by a product inhibition mechanism. Therefore,
decreases in G6PD activity and, as a result, NADPH
level will impair the entire antioxidant system.
Work from our laboratory and others has shown that
high glucose and diabetes decrease G6PD activity in
endothelial cells, kidney, liver, and red blood cells,
which leads to oxidative damage, cellular dysfunction,
and organ damage (20 –22). Previous work has sug-
gested that the inhibition of the pentose phosphate
pathway (G6PD is the rate-limiting enzyme of this
metabolic pathway) leads to ␤-cell dysfunction (23).
Taken together, all of these data led to our hypothesis
that high-glucose-mediated decrease in G6PD would
lead to impaired ␤-cell function and cell death.
MATERIALS AND METHODS
Cell culture and human islet culture
MIN6 ␤ cells were incubated at 37°C and 5% CO2 in DMEM
supplemented with 15% fetal bovine serum, penicillin, and
streptomycin. Human islets were provided by the Islet Cell
Resource Centers Program and cultured in Miami medium
1A containing 5.6 mM glucose (Mediatech Inc., Herndon,
VA,USA).
G6PD activity measurement
G6PD activity of MIN6 cells and islets were measured as
described previously (22).
ROS measurement
ROS production was measured with the dye CM-H2DCFDA
(Invitrogen, Carlsbad, CA, USA). Fluorescence was read with
a microplate fluorometer (Victor2 fluorometer, PerkinElmer,
Wellesley, MA, USA). After the fluorescence reading, cells
were collected to measure protein concentration, which was
used to normalize the readings.
Construction of adenoviral-hG6PD vector
Human G6PD cDNA was excised from pCMV-XL4-G6PD
(OriGene, Rockville, MD, USA) and was confirmed by se-
1498 Vol. 24 May 2010 The FASEB Journal 䡠 www.fasebj.org
quencing. The construction of adenoviral-hG6PD expression
vector was described previously (24). The titer of adenovirus
was determined with a commercial kit (Adeno-XTM Rapid
Titer Kit; Clontech, Palo Alto, CA, USA), following the
manufacturer’s instructions. Empty vector was used for con-
trol experiments. MOI 15 was used for all experiments.
siRNA Transfection
Transfection of siRNA for G6PD was performed according
to the LipofectamineTM RNAiMAX transfection protocol
(Invitrogen), and then the cells were cultured for 48 h. The
sequences designed for inhibiting G6PD gene expression (cat.
no. s66341; Invitrogen) in mouse ␤ cells are AAUCAACUGUC-
GAACCACAtt (sense) and UGUGGUUCGACAGUUGAUUgg
(antisense). A scrambled siRNA (4390846; Invitrogen) without
biological effects was used as control.
Cell-death detection
Histone/DNA complexes released from the nucleus to the
cytosol (DNA fragmentation), a marker for apoptosis, were
measured according to the manufacturer’s instructions (Cell
Death Detection ELISAPlus; Roche, Indianapolis, IN, USA). In
brief, cell lysates were incubated with anti-DNA-peroxidase
and anti-histone-biotin and transferred to streptavidin-coated
96-well plates. Absorbance was measured after addition of the
peroxidase substrate ABTS (2,2⬘-azino-bis-3-ethylbenzthiazo-
line-6-sufonate).
Measurement of cellular proliferation
MIN6 cells were seeded in a 96-well plate, and siRNA trans-
fection was done the next day. Then cells were incubated for
48 h in DMEM medium with 10% FBS. The cellular prolifer-
ation was measured with cell proliferation ELISA BrdU assay
(Roche Diagnostics) according to the manufacturer’s instruc-
tions.
Ki67 staining
Min6 cells were seeded in coverslips, and the next day siRNA
transfection was done. After 48 h, cells were fixed with
methanol and washed. Cells were blocked with 10% BSA for
20 min. Subsequently, the cells were incubated overnight with
Ki67 antibody (BD Pharmingen, San Jose, CA, USA) in
antibody buffer solution (10% BSA/PBS). Excess primary
antibodies were washed off with 10% BSA/PBS solution. The
cells were then incubated with Alexa-conjugated secondary
antibody (Molecular Probes, Eugene, OR, USA) for 1 h.
Highly cross-adsorbed Alexa594 goat anti-mouse IgG (H⫹L)
(Invitrogen) was used. The coverslips were mounted with
antifade mounting medium. To determine nonspecific bind-
ing, staining with normal IgG was also performed. Slides were
viewed under a fluorescence microscope.
Glucose-stimulated insulin secretion (GSIS) in MIN6 cells
and isolated islets
MIN6 cells were grown in 12-well plates until 80% confluent
and were then starved with DMEM supplemented with 0.1%
bovine serum albumin and 2.8 mM glucose for 14 h. Cells
were washed with PBS and starved again with KRB buffer (125
mM NaCl, 4.74 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 1.2
mM MgSO4, 5 mM NaHCO3, and 25 mM HEPES, pH 7.4)
supplemented with 2.8 mM glucose for 1 h. Medium was then
changed to KRB buffer with either 2.8 mM or 16.7 mM
ZHANG ET AL.
glucose. Medium was collected after 1 h and centrifuged. The
supernatant was used for insulin assay. Cells were lysed to
measure protein concentration. Insulin was measured with an
ELISA kit (Crystal Chem Inc., Downers Grove, IL, USA), and
the results were normalized with protein concentrations.
Islets isolated from mice were cultured in RPMI 1640 medium
containing 10% FBS. The same GSIS procedure was done as
described for the MIN6 cells.
Western blotting
Protein (40 ␮g/ lane) was size-fractionated electrophoreti-
cally using 10% SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membranes blocked with a 5%
nonfat milk solution. The membranes were incubated with
antibodies to G6PD (Bethyl Laboratories, Montgomery, TX,
USA) and actin (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) and visualized with the ECL detection system (Amer-
sham, Piscataway, NJ, USA).
Animal model
All animal experiments were approved by the Institutional
Animal Care and Use Committee at Harvard Medical School
and conducted in accordance with the National Institutes of
Health Guidelines for the Care and Use of Laboratory
Animals. The G6PD-deficient mouse model was recovered in
the offspring of 1-ethyl-nitrosourea-treated male mice on a
C3H murine background by Pretsch et al. (25). Later, Sanders
et al. (26) showed that there is a single-point mutation (A to
T transversion) in the 5⬘ splice site consensus sequence at the
3⬘ end of the exon1. The mice were bred at Harvard Medical
School from frozen embryos obtained from the Medical
Research Council (Harwell, U.K.). G6PD is an X-linked
gene, and thus heterozygotes and homozygotes are female
and hemizygotes are male. Wild-type C3H control mice and
hemizygous G6PD-deficient male mice were studied. Ani-
mals were genotyped and characterized as described pre-
viously (14).
Islet isolation
Pancreatic islets were isolated by the collagenase digestion
method (27). After injection of collagenase (2 mg/ml) into
pancreatic ducts, pancreases were harvested and incubated at
37°C in a water bath for 12 min to digest. Ficoll gradient
solution (Sigma, St. Louis, MO, USA) was used to separate
islets from exocrine tissues. Islets were hand-picked under a
dissection microscope. Exocrine tissue was collected for West-
ern blot experiments.
Relative islet mass calculation and immunostaining of
pancreas
Mice were anesthetized, and the pancreas was immediately
dissected and fixed in Z-fix solution. Three sections (sepa-
rated by 25 ␮m) were obtained from each pancreas. Immu-
noperoxidase staining of sections was done with a guinea pig
polyclonal antibody against mouse insulin (Zymed, Burlin-
game, CA, USA). Relative islet mass was evaluated by point-
counting morphometry. Images were acquired using a BX60
microscope (Olympus, Melville, NY, USA) and were analyzed
with ImageJ software (National Institutes of Health, Bethesda,
MD, USA). Immunofluorescence staining of pancreas sec-
tions of the mice was done with a rabbit polyclonal antibody
against mouse G6PD (Bethyl Laboratories).
HIGH GLUCOSE DECREASES G6PD ACTIVITY IN ␤ CELLS
Glucose tolerance test (GTT)
GTT was performed on animals that had been deprived of
food overnight for 16 h. Glucose levels were measured from
blood collected from the tail immediately before and at 15,
30, 60, and 120 min after i.p. glucose injection (2 g/kg body
weight).
Statistical analysis
Data were expressed as means ⫾ sd. Comparison between
groups was performed by Student’s paired 2-tailed t test.
ANOVA was used for comparisons across groups with post hoc
analysis using the Newman-Keuls test. Values of P ⬍ 0.05 were
considered significant.
RESULTS
High glucose inhibits G6PD activity and protein
expression
To determine whether high glucose affects G6PD ex-
pression and activity in ␤ cells, human islets and mouse
islets were cultured in 5.6 and 25 mM glucose for 72 h.
As compared to the islets in normal glucose, G6PD
protein expression was decreased by high glucose (Fig. 1A)
in both models, and G6PD activity was also reduced
(Fig. 1B). These data show that high glucose leads to a
decrease in G6PD activity and protein level in islets,
which likely contributes to increased ROS generation.
Inhibition of G6PD leads to increased ROS and
apoptosis
To determine further whether decreased G6PD activity
could contribute to increased ROS in ␤ cells, MIN6
cells were transfected with either specific siRNA tar-
geted to G6PD or scrambled siRNA as control (Fig. 2A).
Specific knockdown of G6PD protein in MIN6 cells led
to a significant increase of ROS in MIN6 cells (Fig. 2B),
illustrating that inhibition of G6PD per se can lead to
increased ROS.
As increased ROS may lead to cell death, and since
research from our laboratory has shown an important
role for G6PD and NADPH in preventing ROS-medi-
ated cell death (28), studies on cell survival were also
performed. As a marker for apoptosis, histone/DNA
complexes released from the nucleus to the cytosol
were measured (DNA fragmentation). Cell death was
increased significantly by inhibiting G6PD (Fig. 2C).
Caspase 3 is an important mediator of apoptosis, and
cleaved caspase 3 protein is observed in apoptosis (29).
Inhibition of G6PD increased cleaved-caspase 3 protein
in MIN6 cells, suggesting that inhibition of G6PD
promotes apoptosis of ␤ cells (Fig. 2D).
Inhibition of G6PD decreases cellular proliferation
and insulin secretion in MIN6 cells
In addition to playing an important role in cell death,
previous research from our laboratory has shown that
1499
Figure 1. High glucose decreases G6PD expression and activity in human and mouse islets. Human and mouse islets were
exposed to either 5.6 or 25 mM glucose for 72 h, after which islets were collected for protein extraction. G6PD activity was
measured, and Western blot was performed to detect G6PD protein expression. A) G6PD protein expression was lower in islets
exposed to 25 mM glucose as compared to 5.6 mM glucose. A total of 200 islets was used for each sample; n ⫽ 3. *P ⬍ 0.05 vs.
5.6 mM. B) G6PD activity of both human islets and mouse islets exposed to high glucose was lower than with 5.6 mM glucose.
n ⫽ 4. *P ⬍ 0.05 vs. 5.6 mM.

G6PD plays a central role in cell growth (17). To
determine whether inhibition of G6PD would affect
cellular proliferation, two markers of cell proliferation
were studied: BrDu incorporation assay and immuno-
staining of Ki67. Inhibition of G6PD reduced BrDu
incorporation (Fig. 3A), which suggests decreased pro-
liferation. In addition, Ki67, a cell cycle marker of
proliferation (30), was detected using immunofluores-
cence staining. In cells transfected with scrambled
siRNA, a pattern of nuclear localization suggests prolif-
eration (Fig. 3B). However, on knockdown of G6PD,
the pattern changed in the cells to a diffuse staining,
which could indicate no nuclear localization and re-
duced proliferation (Fig. 3B).
Inhibition of G6PD decreases GSIS
To investigate the effects of inhibiting G6PD on pan-
creatic function, GSIS was measured. Specific inhibi-
tion of G6PD using the siRNA construct led to a
significant decrease in insulin secretion (Fig. 3C),
which suggests that G6PD activity plays a role in insulin
secretion.
Overexpression of G6PD lowers ROS level in ␤ cells
High glucose has been shown to cause ROS accumu-
lation in ␤ cells, leading to cell death and dysfunc-

Figure 2. Inhibition of G6PD causes

increased ROS generation and in-
creased apoptosis in MIN6 cells.
A) MIN6 cells were seeded in 6-well
plates, and the next day siRNA
transfection was done. After 48 h,
cells were harvested for protein ex-
traction, after which Western blot
was performed to detect G6PD and
actin. G6PD protein expression de-
creased significantly by specific
siRNA knockdown. n ⫽ 6. *P ⬍
0.01. B) MIN6 cells were prepared
as in A, except that cells were seeded in 24-well plates. ROS accumulation was measured with CM-H2DCFDA. Inhibition of
G6PD led to significant increases in ROS; n ⫽ 12. *P ⬍ 0.05. C) MIN6 cells were prepared as in A. Apoptosis was measured
as described in Materials and Methods. Inhibition of G6PD led to increased DNA fragmentation, a marker of apoptosis; n ⫽
6. *P ⬍ 0.001. D) Cells were prepared as in A. Western blot was performed to detect cleaved caspase-3, total caspase 3, and
actin. Inhibition of G6PD led to increased cleaved caspase-3, a marker of apoptosis. n ⫽ 4. *P ⬍ 0.01.

1500 Vol. 24 May 2010 The FASEB Journal 䡠 www.fasebj.org ZHANG ET AL.
 Figure 3. Inhibition of G6PD causes decreased cellular proliferation and
impaired insulin secretion in MIN6 cells. A) Cells were prepared as in
Fig. 1A. BrDu incorporation was used to measure cellular proliferation.
Inhibition of G6PD significantly decreased cell proliferation; n ⫽ 24.
*P ⬍ 0.001. B) Ki67 staining was performed as another marker of
cellular proliferation. In control cells, Ki67 protein is clearly present in
the nucleus, consistent with proliferating cells. Inhibition of G6PD led to
a diffuse pattern of staining with no clear nuclear localization, consistent
with impaired proliferation. C) GSIS assay was performed in MIN6 cells
as described in Materials and Methods. Inhibition of G6PD led to a
significant impairment in glucose-stimulated insulin release; n ⫽ 8. #P ⬍ 0.05 vs. 2.8 mM. *P ⬍ 0.05 vs. 16.7 mM and
scrambled siRNA.

tion (1–3). As our previous studies (Fig. 2B) deter-
mined that inhibition of G6PD leads to increased
ROS level, we tested whether overexpression of G6PD
would decrease ROS accumulation in ␤ cells exposed
to high glucose. Adenoviral-human G6PD expression
vector was constructed to overexpress G6PD in MIN6
cells and islets. As shown, infection of Ad-G6PD
increased both G6PD protein expression (Fig. 4A, C)
and activity (Fig. 4B, D). In both islets (Fig. 4E) and
MIN6 cells (Fig. 4F), 25 mM glucose significantly
increased ROS level by 20%, compared to 5.6 mM
glucose. Overexpression of G6PD in both models
completely prevented the high-glucose-induced in-
crease in ROS level, which suggested that G6PD is

Figure 4. Overexpressing G6PD decreases ROS levels in Islets and MIN6 cells. Adenoviral-G6PD vector (Ad-G6PD) was
constructed to overexpress human G6PD (see Materials and Methods). Empty adenoviral vector (Ad-C) was used as control.
A–D) Adenoviral overexpression led to increased G6PD protein expression and activity. A, B) Islets were infected with Ad-G6PD
vector or Ad-C for 48 h. G6PD protein expression and activity was measured; n ⫽ 6. *P ⬍ 0.05 vs. Ad-C and no virus controls.
C, D) MIN6 cells were infected with Ad-G6PD vector or Ad-C for 48 h. G6PD protein expression and activity was measured; n ⫽
8. *P ⬍ 0.05 vs. Ad-C and no virus controls. E, F) Islets and MIN6 cells were exposed to either 5.6 or 25 mM glucose for 72 h,
and ROS generation was measured. Data show that 25 mM glucose increased ROS level in both islets (E) and MIN6 cell (F) by
20%, compared with 5.6 mM glucose. In both models, ROS level was reduced by overexpression of G6PD. #P ⬍ 0.05 vs. 5.6 mM.
*P ⬍ 0.05 vs. 25 mM and 25 mM with Ad-C infection.

HIGH GLUCOSE DECREASES G6PD ACTIVITY IN ␤ CELLS 1501


Figure 5. Overexpression of G6PD ameliorates the high-
glucose-mediated impairment of insulin secretion in MIN6
cells. MIN6 cells were infected with either Ad-C or Ad-G6PD
and cultured in 5.6 or 25 mM glucose for 48 h. Cells were
then starved, and GSIS was ascertained, as described in
Materials and Methods. High glucose decreased both basal
and stimulated insulin release. Overexpression of G6PD led
to a restoration of GSIS in cells cultured in 25 mM glucose.
n ⫽ 6. *P ⬍ 0.05 vs. 2.8 mM. #P ⬍ 0.05 vs. 16.7 mM and 25
mM with Ad-C.
critical for the regulation of redox homeostasis in ␤
cells.
Overexpression of G6PD enhances insulin secretion
in MIN6 cells
Figure 3C showed that inhibition of G6PD impaired
GSIS. Previous studies have shown a role for the
pentose phosphate pathway (although G6PD was not
specifically studied) in insulin secretion (23, 31, 32);
thus, cells overexpressing G6PD were used to deter-
mine whether overexpression of G6PD would improve
insulin secretion. In MIN6 cells, high glucose decreased
both basal and stimulated insulin release (Fig. 5). Cells
were infected with either empty vector (Ad-C) as a
control or an adenovirus containing G6PD (Ad-G6PD).
Cells were then incubated with either 5.6 or 25 mM
glucose for 48 h. After the 48 h incubation period, GSIS
measurement was done. Cells incubated in 5.6 mM
glucose showed similar GSIS response in Ad-C- or
Ad-G6PD-infected cells. Cells incubated in 25 mM
glucose had decreased insulin release as compared to
cells incubated in 5.6 mM glucose. Cells incubated in
25 mM glucose had a blunted GSIS that was rescued by
overexpression of G6PD, suggesting that the high-
glucose-mediated decrease in GSIS is due, at least in
part, to increased oxidative stress, which can be ame-
liorated by increasing G6PD activity.
Islets express low levels of G6PD as compared to the
exocrine pancreas, and G6PD-deficient mice have
decreased islet mass and impaired glucose tolerance
as compared to mice with wild-type G6PD activity
Lastly, studies were performed in mice (wild-type and
G6PD deficient) to determine whether G6PD defi-
ciency per se leads to changes in pancreatic islets. First,
1502 Vol. 24 May 2010 The FASEB Journal 䡠 www.fasebj.org
the protein expression level of G6PD in ␤ cells was
determined in wild-type mice. Coimmunostaining for
G6PD (red) and insulin (green) showed that islet cells
have very low expression of G6PD, as compared to
exocrine pancreatic cells (Fig. 6A). To further confirm
this, G6PD protein expression was detected by Western
blotting in both isolated islets and exocrine pancreas (Fig.
6B). G6PD protein in islets is significantly lower than in
exocrine pancreas. Compared to wild-type mice, hemizy-
gous G6PD-deficient mice have 15% G6PD activity (14).
The relative islet mass of G6PD-deficient mice was signif-
icantly smaller than that of wild-type mice (Fig. 7A, B).
Furthermore, a GTT showed that the glucose levels in
G6PD-deficient mice were significantly higher than in
wild-type mice (Fig. 7C). Finally, GSIS was assayed in islets
isolated from both wild-type and hemizygous mice. Insu-
lin secretion of islets from hemizygous mice was reduced
compared with wild-type mice (Fig. 7D) These data from
G6PD-deficient mice suggest that G6PD activity plays a
role in ␤-cell survival and function in vivo.
DISCUSSION
The loss of pancreatic ␤-cell function is a central
feature of both type 1 and type 2 diabetes. In type 1
diabetes, an autoimmune process leads to insulitis and
loss of ␤ cells. In type 2 diabetes, loss of ␤ cells occurs
over a period of years due to a process that has not been
elucidated fully. Glucotoxicity and lipotoxicity are
thought to play a major role, at least in significant part
by increasing oxidative stress, leading to apoptosis of ␤
cells (33, 34). High glucose has been shown to increase
Figure 6. Islets have very low levels of G6PD protein, as
determined by coimmunostaining of G6PD (red) and insulin
(green) in pancreas of C3H mice. A) Left panel: there is little
expression of G6PD in the islet, whereas nonislet cells have
much higher levels of G6PD protein. Right panel: insulin
staining localizes islet tissue. B) Western blot shows that G6PD
protein expression in isolated islets (I) is significantly lower
than in exocrine pancreas (Exo); n ⫽ 3.
ZHANG ET AL.
 Figure 7. G6PD deficiency is associated directly with decreased islet mass and
impaired glucose tolerance. Islet mass is decreased significantly in mice express-
ing lower levels of G6PD activity. A) Immunostaining of paraffin-embedded
pancreatic sections (n⫽3⫻3 slides) with antibody against mouse insulin was
performed on pancreas from wild-type control mice and G6PD-deficient mice.
Hemizygous mice have much smaller islets as compared to wild-type mice.
Original view ⫻20. B) Quantitation of relative islet mass. Islet mass was measured
as described in Materials and Methods. Hemizygous mice have significantly
smaller islets as compared to wild-type mice. *P ⬍ 0.05 vs. WT. C) Mice
expressing lower levels of G6PD activity have impaired glucose tolerance.
Intraperitoneal GTTs show that G6PD-deficient mice had impaired glucose tolerance as compared to wild-type control
mice. *P ⬍ 0.01 vs. WT. D) GSIS was measured with islets isolated from wild-type and G6PD-deficient mice. G6PD-deficient
mice had significantly impaired GSIS as compared to wild-type mice. Mice were studied at age of 6 mo. #P ⬍ 0.05 vs. 2.8
mM. *P ⬍ 0.05 vs. 16.7mM WT. WT, wild type; Hemi, hemizygous (15% of WT G6PD activity).

ROS in many cell types in patients with diabetes due to
a combination of increased production of ROS along
with decreased antioxidant function (1, 21, 33–37).
Many laboratories have shown that pancreatic ␤ cells
are very sensitive to oxidant damage, which has been
attributed to the low expression levels of antioxidant
enzymes (1–3). Thus, ␤ cells are likely at higher risk of
oxidant-mediated cellular damage and death as com-
pared to other mammalian cell types.
The evidence for accumulation of ROS leading to
␤-cell dysfunction and apoptosis has been reviewed
recently (38, 39). Increased ROS occurs due to an
imbalance of processes that produce ROS and pro-
cesses that reduce ROS (antioxidant systems). Possible
sources of increased ROS production in diabetes in-
clude glyceraldehyde autooxidation, increased aldose
reductase reactivity and resultant decrease in NADPH,
increased production of superoxide from mitochon-
dria, and others (1, 33, 34, 38). Examples of decreased
antioxidant function are as follows. Pancreatic ␤ cells
have low levels of GPx, catalase, and SOD activities
under normal glucose conditions as compared to other
cell types. Lenzen et al. (2) have reported levels of
30 – 40% of liver enzyme levels for SOD, 15% of liver
levels for GPx, and nondetectable catalase activity.
Robertson and colleagues (40) have shown that ␤ cells
express very low levels of GPx. Decreased levels of
antioxidant enzymes have been reported in diabetes as
well. In the pancreas of type 2 diabetic patients, SOD
expression was decreased (41). In the pancreas of
diet-induced diabetic mice, GPx was down-regulated
(42). Thus, the combination of high-glucose-mediated
increase in ROS and low levels of antioxidants renders
the ␤ cell to be unusually sensitive to the deleterious
effects of increased ROS as compared to other cell
types. Further support of increased ROS playing an
important role in ␤-cell dysfunction, and cell death has
been demonstrated by studies involving overexpression
of antioxidants. For example, adenoviral-mediated GPx
overexpression protects ␤ cells against oxidative stress
(43). Catalase overexpression protects islets against
oxidative stress (44). Overexpression of GPx provides
protection against the deleterious effects of hypergly-
cemia (45).
Two major roles for G6PD in ␤ cells have been
suggested in the present study: maintenance of ␤-cell
function and preservation of ␤ cells. Figures 3 and 5
show that G6PD activity and ROS likely play impor-
tant roles in GSIS. A role for G6PD and NADPH in
regulating insulin secretion has been suggested pre-
viously through indirect studies. Ammon, Steinke,
and Patel (23, 31, 32) reported that 6-aminonicotin-
amide, a general inhibitor of the pentose phosphate
pathway, decreased insulin release in isolated rat
islets. More recently, a role for ROS in regulating
␤-cell function has been shown by Robertson and
colleagues (46) in HIT-T15 cells cultured in in-
creased glucose concentrations. These studies deter-
mined that the activities of two important regulators
of the insulin promoter, PDX-1 and MafA, were
undetectable (46) and that antioxidant treatment
improved this defect. Recently, a role for NADPH has
also been shown in insulin secretion and another
NADPH producing enzyme, mitochondrial associat-
ed-isocitrate dehydrogenase (ICDH). This form of
ICDH is primarily associated with mitochondria and
is not a source of NADPH for cytoplasmic enzymes.
In this study, mitochondrial-based shuttling mecha-
nisms (ICDH production of NADPH) were deter-
mined to regulate insulin secretion (47). Taken
together, these results suggest that NADPH is impor-
tant for proper ␤-cell function by maintaining proper
ROS levels in the cytoplasm and by providing reduc-
ing power to mitochondrial shuttle reactions.
To treat and prevent diabetes, it is essential to
preserve ␤ cells. The results presented in the present

HIGH GLUCOSE DECREASES G6PD ACTIVITY IN ␤ CELLS 1503

work show that G6PD is important for ␤-cell prolifera-
tion and prevention of ␤-cell death. Past research has
shown that hyperglycemia stimulates apoptosis in islets
along with expression of proapoptotic genes (38).
Pancreatic ␤ cells from humans with type 2 diabetes
have been shown to have increased expression of the
apoptotic mediators, caspase 3 and caspase 6 (48).
Butler et al. (49) determined that pancreatic tissue from
autopsy specimens from people with type 2 diabetes
showed a 3–10 fold increase in ␤-cell apoptosis as
compared to people without diabetes. The data re-
ported here show that inhibition of G6PD increases the
expression of cleaved caspase 3 and increased histone/
DNA complex release from the nucleus, which are
markers of apoptotic cell death. Our laboratory has
previously shown that G6PD is of central importance in
cell survival in that G6PD is critical for cell growth (17)
and essential for protecting against various forms of cell
death (28). The results in Fig. 3 demonstrated that
decreased G6PD impaired BrdU incorporation and
altered Ki67 staining pattern, both of which are mark-
ers of decreased cell proliferation (50). Furthermore,
the data reported from the G6PD-deficient animals are
particularly interesting, in that islet mass was directly
proportional to G6PD activity. These results suggest
that G6PD activity per se can control islet cell mass.
Thus, considering the central role that G6PD plays in
maintaining the entire antioxidant system by providing
NADPH along with the low level of G6PD expression, it
is intriguing to speculate that enhancing G6PD would
have a highly beneficial, protective effect by enhancing
catalase, glutathione reductase, and SOD, as the han-
dling of hydrogen peroxide by either catalase or the
glutathione system will enhance the flux of partially
reduced forms of oxygen through to water and lipid
alcohols.
G6PD is the main source of NADPH used by the
cellular antioxidant systems, as discussed in the in-
troduction. Information reported in the present
study illustrate, we believe for the first time, that
G6PD protein expression is very low in ␤ cells (Fig.
6). Considering that the entire antioxidant system is
dependent on NADPH (as discussed in the introduc-
tion), even modest decreases in G6PD activity could
significantly increase the ROS level and lead to cell
damage and death. In addition, the data reported
here support the following conclusions. 1) High
glucose impairs G6PD activity and increases ROS in ␤
cells (Fig. 1). 2) Specific inhibition of G6PD alone
leads to decreased ␤-cell survival and decreased ␤-cell
proliferation (Figs. 2 and 3). 3) Overexpression of
G6PD prevents the high-glucose-mediated increase
in ROS (Fig. 4), and overexpression of G6PD restores
GSIS (Fig. 5). 4) The islet mass is directly associated
with G6PD activity in G6PD-deficient mice. Taken
together, these data support a significant role for
decreased G6PD playing a central role in the high-
glucose-mediated increase in ROS, which leads to
␤-cell dysfunction and death. Increasing G6PD activ-
1504 Vol. 24 May 2010 The FASEB Journal 䡠 www.fasebj.org
ity would likely offer a new approach for protecting
pancreatic ␤ cells from dysfunction and death.
The authors thank Dan Kawamori, Xiaodan Wang, Wan-
Chun Li, Jiang Hu, and Hui Li for technical assistance. This
work was supported by U.S. National Institutes of Health
(NIH)/National Institute of Diabetes and Digestive and Kid-
ney Diseases (NIDDK) grant R01-DK54380 to R.C.S.; NIH/
National Heart, Lung, and Blood Institute (NHLBI) grants
HL 61795, N01 HV 28178, P01 HL 81857, and U54 HL
070819 to J.A.L.; and NIH/NHLBI grant HL 089734 to
D.E.H. with Jacob Joseph (Brigham and Women’s Hospital,
Boston, MA, USA). R.N.K. was supported by NIH grants RO1
DK 68721 and RO1 DK 67536; and C.L.W. is supported by
NIH training grant T32DK007260. The Joslin Core Facilities
were used for islet isolation and other measurements, which
are funded by Diabetes Endocrinology Research Center
(DERC) grant P30DK036836. The authors declare no con-
flicts of interests.
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Received for publication June 9, 2009.
Accepted for publication December 3, 2009.
1505