Biochemical and Biophysical Research Communications 476 (2016) 204e211

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

ROS-mediated glucose metabolic reprogram induces insulin resistance

in type 2 diabetes
Kelei Dong, Hua Ni, Meiling Wu, Ziqing Tang, Michael Halim, Dongyun Shi*
Department of Biochemistry and Molecular Biology, Shanghai Medical College of Fudan University, Free Radical Regulation and Application Research Center
of Fudan University, Shanghai 200032, Peoples Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Oxidative stress is known to contribute to insulin resistance in diabetes, however the mechanism is not
Received 11 May 2016 clear. Here we show that reactive oxygen species (ROS) could reprogram the glucose metabolism through
Accepted 16 May 2016 upregulating the pentose pathway so as to induce insulin resistance in type 2 diabetes (T2DM). By using
Available online 17 May 2016
streptozotocin-high fat diet (STZ-HFD) induced T2DM in rats, we show that diabetic rats exhibited high
level of oxidative stress accompanied with insulin resistance. Hypoxia inducible factor (HIF-1a) protein
Keywords:
expression as well as its downstream target glucokinase (GK), were upregulated; The glycogen synthesis
Reactive oxidative stress
increased accordingly; However the glycolysis was inhibited as indicated by decreased
Insulin resistance
Pentose pathway
phosphofructokinase-1 (PFK-1), pyruvate kinase (PK), phospho-PFK-2/PFK-2 (p-PFK-2/PFK-2) ratio,
Glycolysis lactate dehydrogenase (LDH) and pyruvate dehydrogenase kinase (PDK); Pyruvate dehydrogenase (PDH)
Type 2 diabetes mellitus which promotes pyruvate to generate acetyl-CoA declined as well. While phospho-acetyl-CoA carbox-
ylase/acetyl-CoA carboxylase (p-ACC/ACC) ratio increased, meaning that lipid beta-oxidation increased.
The pentose pathway was activated as indicated by increased G6PD activity and NADPH level. Our results
suggest that diabetic rats countervail ROS stress through increasing pentose pathway, and reprogram the
energy metabolic pathway from glycolysis into lipid oxidation in order to compensate the ATP
requirement of the body, which causes insulin resistance.
2016 Elsevier Inc. All rights reserved.

1. Introduction with vitamin E, vitamin C, or glutathione improves insulin sensi-

In recent years, diabetes has been characterized as a global
epidemic, with numbers expected to reach 300 million by 2025 [1].
T2DM is considered to be attributable to peripheral insulin resis-
tance. Insulin resistance plays a pivotal role in the process of T2DM
and it is the main pathological process of T2DM, however its
mechanisms are still unclear [2].
Oxidative stress is a phenomenon that arises when an imbal-
ance occurs to the production and elimination of ROS and it is
contributed to the occurrence and development of T2DM. Oxidative
stress has been shown to be related to insulin resistance. Some
studies suggest that oxidative stress is responsible for altering
intracellular signaling pathways, which thereby inducing the pos-
sibility of insulin resistance [3]. In vitro studies and in animal
models, antioxidants have been shown to improve insulin sensi-
tivity [4]. Several clinical trials have demonstrated that treatment
* Corresponding author. Tel.: 86 21 54237299; fax: 86 21 54237897.
E-mail address: dyshi@fudan.edu.cn (D. Shi).
tivity in insulin-resistant individuals [4,5]. The recent nding that
insulin resistance is associated in humans with reduced intracel-
lular antioxidant defense also support that oxidative stress is
involved in insulin resistance [6].
It is well known that excessive calorie intake, combined with
little physical activity will lead to a situation of insulin resistance.
When caloric intake exceeds the energy expenditure, the substrate-
induced increase in citric acid cycle activity generates an excess of
mitochondrial NADH (mNADH) [7]. When excessive NADH cannot
be dissipated by oxidative phosphorylation (or other mechanisms),
the mitochondrial proton gradient increases and single electrons
are transferred to oxygen, leading to the formation of free radicals,
particularly superoxide anion [8]. To protect themselves against
ROS damage, cells would reduce ROS generation or increase ROS
removal. By inhibiting the uptake of insulin stimulating nutrient
and preventing high energy compounds (pyruvate, fatty acids)
from entering into the mitochondria, mNADH accumulation is
hindered and this eventually reduced ROS generation. This may be
one of the reasons as to how ROS causes insulin resistance,
nevertheless, the mechanism is still unclear.

http://dx.doi.org/10.1016/j.bbrc.2016.05.087
0006-291X/ 2016 Elsevier Inc. All rights reserved.
 K. Dong et al. / Biochemical and Biophysical Research Communications 476 (2016) 204e211
It is known that cancer cells exhibit high basal levels of ROS
owning to activation of oncogenes and tumor suppressor genes and
tumor microenvironment deletions. However such substantial
amount of ROS will lead to oxidative stress. Studies have shown
that cancer cells have special ability to counter the oxidative stress
by upregulating the pentose pathway [9]. Our recent study also
found that tumor cells can adaptively generate strong antioxidant
pathways against oxidative stress [10]. Based on the high level of
oxidative stress in diabetes, we speculated that diabetes may also
possess a similar effect, which results in insulin resistance. In this
study, we established STZ-HFD induced type 2 diabetic rat model,
we found that, similar with the cancer cells, diabetic rats exhibited
the upregulated HIF-1a, increased GK as well as enhanced the
pentose pathway. Moreover, the diversion of glucose-6-phophate
(G6P) into the pentose pathway impaired glycolysis, thus repro-
gram the energy metabolic pathway from glycolysis into lipid
oxidation to compensate the ATP requirement of the body, there-
fore resulting insulin resistance. This new point will provide new
ideas for T2DM treatment.
2. Material and methods
2.1. Diabetic animal models
All animal-related procedures were approved by the Fudan
University Institutional Laboratory Animal Ethics Committee. Male
SD rats (150e160 g body weight) were obtained from Fudan Uni-
versity Animal Center (Shanghai, China). Induction of type 2 dia-
betes by STZ (Sigma Aldrich, USA)-high fat diet (STZ-HFD) was
performed as previously described [11,12]. Following a 12 h fasting
period, the rats were randomly divided into ve groups - i. control:
Chow; ii. diabetes: HFD; iii. diabetes: HFD STZ (35 mg/kg); .
diabetes: HFD STZ (50 mg/kg); . diabetes treated with apocynin
(Apo): HFD STZ (50 mg/kg) Apo; (n 8e10). The control group
rats were fed with a standard rodent chow diet whereas the others
received a single intraperitoneal (ip) injection of streptozotocin
(STZ, Sigma) in 0.1 M sodium citrate buffer solution (pH 4.5) and
were fed with HFD diet. Apo was dissolved in the water. For the
HFD STZ (50 mg/kg) Apo groups, rats were pretreated with Apo
for 30 min, and then injected with STZ (50 mg/kg BW). Apo was
administered on a daily basis at 30 mg/kg BW.
Normal chow and high-fat diet (HFD) were purchased from
Shanghai SLRC laboratory animal Company Ltd (Shanghai, China)
and the nutritional composition was shown in Supplementary
Table S1.
After 2 months of treatment, blood samples were drawn for
plasma and serum extraction. Following that, all rats were killed
and their livers were systematically removed and then frozen at
80  C for each index detection and also to perform further assays.
2.2. Oral glucose tolerance test (OGTT), insulin tolerance test (ITT)
and insulin secretion
Measurement of OGTT was performed according to the proce-
dure previously described [13]. Alternatively, after a 12 h fasting
period, rats were administered with glucose of 2.5 g/kg body
weight. ITT was performed according to the procedure previously
described [14]. Accu-Chek Performa (Roche Diagnostics, Germany)
was used to measure blood glucose levels in rats. Insulin secretion
was detected according to the procedure previously described [15].
2.3. Enzyme activities
Liver GK activity was measured as described previously [16].
Glucose-6-phosphate dehydrogenase (G6PD) activity was
205
measured according to the method as described in Ref. [17]. NADPH
oxidase (NOX) activity was measured as previously described [18].
Enzyme activity was standardized using protein quantication.
2.4. GSH/GSSG ratio measurement
Livers were cut into 50 ml 1 M HPO3 for GSH detection. After 2e3
freeze/thaw cycles, the suspension was centrifuged with
12,000 rpm for 10 min at 4  C and then assayed as previously
described [19].
2.5. NADH and NADPH assay
Cells were scraped into 200 ml cold 40 mM NaOH for NADH and
NADPH detection. After 2e3 freeze/thaw cycles, the suspension was
centrifuged with 12,000 rpm for 10 min at 4  C and then assayed as
previously described [20].
2.6. Free fatty acids measurement
Serum and liver free fatty acids (FFA, nonesteried fatty acids)
were measured as previously described in Ref. [21].
2.7. Western blot
Western blot was performed according to standard procedures
and protein extracts from liver was prepared as previously
described [22]. Protein expression was analyzed with antibodies
against GK, PFK-1, PK, GYS-1, HIF-1a, LDH, PDH, PDK and GLUT4
(Proteintech, USA) and p-PFK-2, PFK-2, p-GYS, p-ACC, ACC and b-
actin (Cell Signaling Technology, USA). Protein expression is visu-
alized on Tanon-5200 Chemi-luminescent Imaging System (Tanon
Science Technology).
2.8. MDA measurement
The measurement of plasma malondialdehyde (MDA) experi-
ment was performed according to the manufacturers instructions
of MDA kit (Nanjing Jiancheng Biological Engineering Institute,
Nanjing, China). The measurement principle is the usage of
degradation products of lipid peroxidation MDA with thiobarbituric
acid (TBA) condensation to form a red product which has a
maximum absorption peak at 532 nm, and can be measured by a
colorimetric MDA content.
2.9. TAOC measurement
The measurement of plasma total antioxidant capacity (TAOC)
experiment was performed according to the manufacturers in-
structions of TAOC kit (Nanjing Jiancheng Biological Engineering
Institute, Nanjing, China).
2.10. Statistical analysis
Linear regression statistical analysis was performed using
GraphPad Prism 5.0. Unpaired Student t-tests were used to calcu-
late the signicant difference between two sample groups. For
datasets containing more than two groups that required analysis of
changes overtime, one-way ANOVA and Tukey post hoc test cal-
culations were performed instead. All data are expressed as
mean SD. P values of less than 0.05 were considered to be sta-
tistically signicant between the given data sets.
206 K. Dong et al. / Biochemical and Biophysical Research Communications 476 (2016) 204e211

3. Results
3.1. HFD combined with STZ treatment induces insulin resistance in
diabetic rats
In our experiment, we induced type 2 diabetic rats by combining
STZ and HFD treatment. In HFD treated group, fasting and post-
prandial blood glucose did not alter signicantly (Fig. 1A), but in-
sulin levels are signicantly increased (Fig. 1B), hence showing a
marked hyperinsulinemia situation, nonetheless overt diabetes are
not manifested. In HFD-STZ combination treated rats, the glucose
tolerance was impaired in a STZ dose-dependent manner (Fig. 1C).
In the 35 mg/kg STZ group, although fasting blood glucose did not
show signicant increase, postprandial blood glucose increased
signicantly (Fig. 1D). Meanwhile insulin tolerance test also
showed that insulin resistance may occur in diabetic rats (Fig. 1E).
With increasing doses of STZ, blood glucose increased in both fas-
ted and fed condition. 50 mg/kg STZ treated group exhibited overt
diabetes (Fig. 1D).
3.2. Oxidative stress in diabetic rats compensatorily enhanced
pentose pathway
Our results show that MDA level increased and NOX activity
which causes cytosolic ROS, was activated (Fig. 2A-B), while TAOC
level declined in diabetic rats (Fig. 2C), suggesting these diabetic
rats were underwent oxidative stress. Furthermore, NADH content
also increased signicantly in diabetic rats (Fig. 2D), which impli-
cates impaired oxidation phosphorylation and mitochondrial ROS
generation. The expression of glucose transporter GLUT4 decreased
in diabetic rats (Fig. 2E). Interestingly, although diabetic rats
exhibited oxidative stress, GSH/GSSG ratio did not show signicant
change (Fig. 2F). We found that G6PD increased signicantly in
those diabetic rats (Fig. 2G). This suggests that pentose phosphate
pathway was activated in response to oxidative stress. NADPH
increased in parallel (Fig. 2H), presumably to maintain the GSH
levels which can protect against ROS stress. However, the ratio of
NADPH/NADP was declined (Fig. 2H). We supposed that NADPH
was consumed to offer reducing equivalents to GSSG, and NADPH
itself being converted to NADP, thus caused the declined NADPH/
NADP ratio. Our results suggest that enhanced PPP generates
NADPH to countervail oxidative stress in diabetic rats.
3.3. Enhanced G6PD diverts G6P into pentose pathway from
glycolysis
It is known that HIF-1a is an important transcription factor in
regulating glycolysis. Our previous study showed that Hif-1a could
be stabilized by hypoxia or ROS in tumor [23]. We speculated Hif-1a
might also be upregulated in diabetic rats which were underwent

Fig. 1. HFD combined with STZ treatment induces insulin resistance in diabetic rats. AeB: Wild-type male SD rats were fed with HFD for 2 months and then blood glucose and
plasma insulin level were measured. A: Fasting and postprandial blood glucose levels of Chow and HFD groups. B: Fasting and postprandial plasma insulin levels of Chow and HFD
groups. C-E: Wild-type male SD rats were fed with Chow or HFD STZ (35 and 50 mg/kg, respectively) for 2 months and then killed for further analysis. C: OGTT. D: Fasting and
postprandial blood glucose levels in Chow and HFD STZ groups. E: ITT. n 8e10 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Chow group.
 K. Dong et al. / Biochemical and Biophysical Research Communications 476 (2016) 204e211 207

Fig. 2. Oxidative stress in diabetic rats compensatorily enhanced pentose pathway. A: Measurement of plasma MDA level in rats. B: NOX activity analysis in rat livers. C: Mea-
surement of plasma TAOC level in rats. D: NADH content analysis. E: Protein expression of GLUT4 in rat livers. F: GSH/GSSG ratio analysis in rat livers. G: G6PD activity analysis in rat
livers. H: Measurement of NADPH and NADPH/NADP ratio in rat livers. n 8e10 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Chow group.

oxidative stress. Our results showed that HIF-1a expression was
signicantly increased in diabetic rat livers (Fig. 3A). Its down-
stream target GK (HK), the key enzymes in the glycolytic pathway,
was also upregulated in diabetic rats (Fig. 3A). Increased GK activity
could produce more gluocose-6-phosphate (G6P) and should in-
crease glycolysis. Interestingly, the glycolysis was inhibited. We
found that the expression of PFK-1 PK, LDH and PDK, the enzymes
in the glycolytic pathway, was signicantly lower in diabetic rats
(Fig. 3C). The phospho-PFK-2 is the bifunctional enzyme that syn-
thesizes fructose 2,6-bisphosphate (F2,6BP), the allosteric regulator
of PFK-1. We found that phospho-PFK-2/PFK-2 (p-PFK-2/PFK-2)
ratio which implicates the glycolytic rates signicantly decreased
(Fig. 3C). Moreover, PDH which catalyze pyruvate to form acetyl-
CoA was also inhibited (Fig. 3C). We also analyze the synthesis of
glycogen. As shown in Fig. 3B, the p-GYS-1/GYS-1 decreased which
indicates the increased glycogen synthesis. These results suggest
that the ux of G6P into PPP and glycogen synthesis enhanced,
however, the ux of G6P into the glycolysis was inhibited. Therefore
the energy generation derived from glucose oxidation impaired in
diabetes.
3.4. ROS reprogram energy pathway into lipid oxidation
Since ROS impaired energy generation derived from glucose, we
208 K. Dong et al. / Biochemical and Biophysical Research Communications 476 (2016) 204e211

Fig. 3. Enhanced G6PD diverts G6P into pentose pathway from glycolysis. A: Protein expression of HIF-1a and GK and GK activity analysis in rat livers. B: Protein expression of p-
GYS in rat livers. C: Protein expression of PFK-1, PK, p-PFK-2, LDH, PDH, PDK in rat livers. n 8e10 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Chow group.

speculate the other pathway should be upregulated in order to 4. Discussion

meet the ATP requirement in diabetic rats. We found p-ACC/ACC
ratio increased which means lipid beta-oxidation in diabetic rats
increased (Fig. 4A). The FFA levels were signicantly increased
(Fig. 4B). After inhibition cellular ROS by Apo treatment, FFA levels
in diabetic rats decreased signicantly (Fig. 4C). This suggests ROS
involved in reprograming glycolysis into lipid oxidation. Apo
treatment also improved GLUT4 expression and postprandial blood
glucose (Fig. 4D and E), suggesting that ROS induced insulin resis-
tance could be alleviated by antioxidant intervention.
Insulin resistance is an important decisive factor in T2DM
metabolic abnormalities. Oxidative stress plays an important role in
inducing insulin resistance, but the mechanism behind it remains
unclear.
In our experiment, we established the model of T2DM by HFD-
STZ. Studies shown that prior to insulin resistance, the body may
exhibit at near normal blood glucose level owing to hyper-
insulinemia. Our studies revealed that in HFD group, fasting and
 K. Dong et al. / Biochemical and Biophysical Research Communications 476 (2016) 204e211 209

Fig. 4. ROS reprogram energy pathway into lipid oxidation. A: Protein expression of p-ACC in rat livers. B: Measurement of liver and serum FFA levels in diabetic rats. C: Mea-
surement of liver and serum FFA levels in Apo intervention rats. D: Protein expression of GLUT4 in Apo intervention rat livers. E: Postprandial blood glucose levels in Apo
intervention rats. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Chow group except in (C-E, HFD STZ (50 mg/kg) group).

postprandial blood glucose did not change signicantly, however,
insulin levels became signicantly higher, herein showing a
marked hyperinsulinemia situation, but overt diabetes did not
appear. We know that STZ is widely used to induce experimental
diabetes in animals. It was found to generate reactive oxygen spe-
cies and reactive nitrogen. STZ enters the beta cell via a glucose
transporter (GLUT2) and causes the formation of superoxide radi-
cals. Consequently, hydrogen peroxide and hydroxyl radicals are
also generated [24]. Essentially, STZ-induced oxidative stress occurs
in the incidence of T2DM. In HFD-STZ group we show that as STZ
dose increases, glucose tolerance decreases in diabetic rats. In the
35 mg/kg STZ group, although there is no signicant increase in
fasting blood glucose, postprandial blood glucose increased
signicantly. Meanwhile insulin tolerance test also showed that
insulin resistance may occur in diabetic rats. When islet b cells are
no longer able to through increasing insulin compensate for insulin
resistance, impaired glucose tolerance appeared. With increasing
doses of STZ, 50 mg/kg STZ group showed overt diabetes, where
hyperglycemia is apparent. Our experiments show that in diabetic
rats, the expression of GLUT4 is signicantly decreased, which
further proves that insulin resistance occurs in diabetic rats. Insulin
resistance leads to a reduction in the amount of glucose that is
transported into the muscle cells, and lipolysis increased, which
explains why the FFA levels are signicantly increased.
In the HFD model, owning to high fat diet intake, energy surplus
occurs, this causes a lot of substrate to enter the citric acid cycle,
which in turn leads to an excessive mitochondrial acetyl-CoA and
NADH production [7]. Acetyl-CoA, derived either from glucose
through pyruvate or from beta-oxidation of FFA, combines with
oxaloacetate to form citrate, which enters the citric acid cycle and is
converted to isocitrate. NAD-dependent isocitrate dehydrogenase
generates NADH. The accumulation NADH could cause ROS gener-
ation [7]. The persistence of such an imbalance between caloric
intake and expenditure can lead to accumulation of ROS, which can
consequently lead to overt diabetes. Therefore diabetes caused by
obesity generally required a long period of time. In our model, by
increasing the oxidative stress, it can promote the accumulation of
ROS to a certain threshold, which can in turn promote overt dia-
betes occurring ahead of time.
According to our study, in addition to the signicant increase in
oxidative stress in diabetic rats, NADH content also increased
signicantly, especially in the 50 mg/kg STZ group. Mitochondrial
ROS could activate PKC and NF-kB and favor the overexpression of
NOX, which could further generate a great amount of superoxide.
Our results show that NOX activity increased, which resulted in a
vicious cycle of ROS in vivo, thus leading to rising levels of MDA and
declining levels of TAOC. ROS can damage DNA. DNA damage is an
obligatory stimulus for the activation of the nuclear enzyme poly
(ADP-ribose) polymerase, resulting in reduced GLUT4 expression
and the subsequent insulin resistance. In our experiments, GLUT4
210 K. Dong et al. / Biochemical and Biophysical Research Communications 476 (2016) 204e211
increased after being subjected to Apo intervention accompanied
by improvement in postprandial blood glucose, these results sug-
gest that oxidative stress could through impairing GLUT4 lead to
insulin resistance.
HIF-1a is a hypoxia-inducible factor, rst discovered in
mammalian cells, where oxygen concentration is low. Recent
studies reveal that HIF-1a expression does not only occur in the
absence of oxygen, oxidative stress can also increase the expression
of HIF-1a [23]. Owning to the fact that T2DM exhibits high levels of
oxidative stress, we speculate that there is a high expression of HIF-
1a. Our results show that diabetic rats had oxidative stress micro-
environment and increased HIF-1a level. The expression of HIF-1a
enhanced with increasing concentration of STZ. We then analyzed
the downstream targets of HIF-1a. We found that GK which cata-
lyzes glucose into G6P was signicantly activated. G6P can ow into
glycolysis, PPP pathway and glycogen synthesis, respectively.
Interestingly, other enzymes in the glycolytic pathway such as PFK-
1, PK, p-PFK-2/PFK-2, PDK and LDH were all inhibited. PDH which
catalyzes pyruvate to form acetyl-CoA decreased, this indicates that
oxidation phosphorylation derived from glucose was inhibited. Our
results suggest that both of aerobic and anaerobic respirations
mediated by glycolysis are impaired in diabetic rats.
It is known that the upregulated PPP pathway could through
increasing of NADPH enhance cellular oxidative buffering capacity
in response oxidative stress in tumor, i.e., a phenomenon existed in
the tumor Warburg effect [25]. Here we found, in diabetic rats, liver
cells also use this mechanism to reduce oxidative stress, PPP
pathway obviously enhanced. However, unlike the Warburg effect
which has enhanced glycolysis, in diabetic rats, the upregulated PPP
pathway caused glycolytic substrates to be steered into the pentose
pathway, thereby leading to the declined glycolysis. Our results
show that glycogen synthesis also enhanced in the diabetic rats.
These results suggest that majority G6P ows into glycogen syn-
thesis and PPP pathway rather than glycolysis. We propose ROS
induced upregulation of G6PD diverts G6P into pentose pathway
from glycolysis, causing the declined glycolysis and the emergence
of insulin resistance.
Glycolysis is the foundation of both aerobic and anaerobic
respiration. The impaired glycolysis will lead to energy shortage.
Here we show that lipid oxidation was highly activated to
compensate to energy requirement of the body. Accordingly, the
FFA level signicantly increased in liver cells and serum of diabetic
rats. Inhibition of ROS by Apo interference obviously decreased FFA
level. It suggests that ROS is involved in reprograming energy
pathway into lipid oxidation, which may contribute to the insulin
resistance.
Our results suggest that intracellular redox systems and cellular
glucose metabolism are likely to inuence each other as a balanced
network. Upregulated PPP pathway is the compensatory effect of
the glucoses metabolism towards oxidative stress, and it may be
closely related to many chronic diseases. Mechanism of insulin
resistance is currently not very clear, this study discloses an
important mechanism for insulin resistance: ROS, through
enhancing PPP pathway, reprograms the energy metabolic pathway
from glycolysis into lipid oxidation to produce large amount of FFA,
causing NADH accumulation and ROS generation, which lead to
GLUT4 downregulation. All factors such as impaired glycolysis,
decreased GLUT4 and increased FFA contribute to insulin
resistance.
Our results disclose that diabetic rats countervail ROS stress, and
reprogram the energy metabolic pathway from glycolysis into lipid
oxidation to compensate the ATP requirement of the body, which
causes insulin resistance. This may provide new ideas and ap-
proaches to T2DM treatment: relieving oxidative stress can alle-
viate insulin resistance, but inhibiting insulin resistance before
relieving oxidative stress may not be a wise decision because it may
increase the damage attributable to reactive oxygen species.
Author contributions
K.D. carried out the major experiments; H.N. assisted with the
establishment of diabetic animal models; M.W. carried out the
HPLC measurement; Z.T carried out the GSH/GSSG ratio measure-
ment; M.H. assisted with the western blot and proof reading; D.S.
designed and supervised experiments and wrote the manuscript.
Funding
This work was supported by National Natural Science Founda-
tion of China (31270901, 30970684).
Author disclosure statement
No competing nancial interests exist for any of the authors.
Acknowledgements
The authors are sincerely grateful to Dr. Xiaodong Zhang from
Vero Biotech for his proof reading.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2016.05.087.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.bbrc.2016.05.087.
References
[1] M. Prentki, C.J. Nolan, Islet beta cell failure in type 2 diabetes, J. Clin. Invest.
116 (2006) 1802e1812.
[2] C.R. Kahn, Insulin resistance, insulin insensitivity, and insulin unresponsive-
ness: a necessary distinction, Metabolism 27 (1978) 1893e1902.
[3] J.L. Evans, I.D. Goldne, B.A. Maddux, et al., Are oxidative stress-activated
signaling pathways mediators of insulin resistance and beta-cell dysfunc-
tion? Diabetes 52 (2003) 1e8.
[4] G. Paolisso, D. Giugliano, Oxidative stress and insulin action: is there a rela-
tionship? Diabetologia 39 (1996) 357e363.
[5] A. Ceriello, Oxidative stress and glycemic regulation, Metabolism 49 (2000)
27e29.
[6] C.R. Bruce, A.L. Carey, J.A. Hawley, et al., Intramuscular heat shock protein 72
and heme oxygenase-1 mRNA are reduced in patients with type 2 diabetes:
evidence that insulin resistance is associated with a disturbed antioxidant
defense mechanism, Diabetes 52 (2003) 2338e2345.
[7] B.A. Maddux, W. See, J.J. Lawrence, et al., Protection against oxidative stress-
induced insulin resistance in rat L6 muscle cells by micromolar concentra-
tions of alpha-lipoic acid, Diabetes 50 (2001) 404e410.
[8] P. Maechler, L. Jornot, C.B. Wollheim, Hydrogen peroxide alters mitochondrial
activation and insulin secretion in pancreatic beta cells, J. Biol. Chem. 274
(1999) 27905e27913.
[9] R.B. Hamanaka, N.S. Chandel, Cell biology. Warburg effect and redox balance,
Science 334 (2011) 1219e1220.
[10] P. Li, D. Zhang, L. Shen, et al., Redox homeostasis protects mitochondria
through accelerating ROS conversion to enhance hypoxia resistance in cancer
cells, Sci. Rep. 6 (2016) 22831.
[11] M.J. Reed, K. Meszaros, L.J. Entes, et al., A new rat model of type 2 diabetes: the
fat-fed, streptozotocin-treated rat, Metabolism 49 (2000) 1390e1394.
[12] K. Srinivasan, B. Viswanad, L. Asrat, et al., Combination of high-fat diet-fed and
low-dose streptozotocin-treated rat: a model for type 2 diabetes and phar-
macological screening, Pharmacol. Res. 52 (2005) 313e320.
[13] X. Wang, C. Gu, W. He, et al., Glucose oxidase induces insulin resistance via
inuencing multiple targets in vitro and in vivo: the central role of oxidative
stress, Biochimie 94 (2012) 1705e1717.
[14] S. Chen, W. Zhang, C. Tang, et al., Vanin-1 is a key activator for hepatic
gluconeogenesis, Diabetes 63 (2014) 2073e2085.
 K. Dong et al. / Biochemical and Biophysical Research Communications 476 (2016) 204e211
[15] C. Wang, Y. Chi, J. Li, et al., FAM3A activates PI3K p110alpha/Akt signaling to
ameliorate hepatic gluconeogenesis and lipogenesis, Hepatology 59 (2014)
1779e1790.
[16] S.D. Da, P. Ausina, E.M. Alencar, et al., Metformin reverses hexokinase and
phosphofructokinase downregulation and intracellular distribution in the
heart of diabetic mice, IUBMB Life 64 (2012) 766e774.
[17] M. Lamas-Maceiras, E. Rodriguez-Belmonte, M. Becerra, et al., KlGcr1 controls
glucose-6-phosphate dehydrogenase activity and responses to HO, cadmium
and arsenate in Kluyveromyces lactis, Fungal Genet. Biol. 82 (2015) 95e103.
[18] M.C. Castro, F. Francini, G. Schinella, et al., Apocynin administration prevents
the changes induced by a fructose-rich diet on rat liver metabolism and the
antioxidant system, Clin. Sci. (Lond) 123 (2012) 681e692.
[19] P.J. Hissin, R. Hilf, A uorometric method for determination of oxidized and
reduced glutathione in tissues, Anal. Biochem. 74 (1976) 214e226.
[20] C. Bernofsky, M. Swan, An improved cycling assay for nicotinamide adenine
211
dinucleotide, Anal. Biochem. 53 (1973) 452e458.
[21] J.T. Rodgers, P. Puigserver, Fasting-dependent glucose and lipid metabolic
response through hepatic sirtuin 1, Proc. Natl. Acad. Sci. U. S. A. 104 (2007)
12861e12866.
[22] D.M. Erion, S. Yonemitsu, Y. Nie, et al., SirT1 knockdown in liver decreases
basal hepatic glucose production and increases hepatic insulin responsiveness
in diabetic rats, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 11288e11293.
[23] D.Y. Shi, F.Z. Xie, C. Zhai, et al., The role of cellular oxidative stress in regu-
lating glycolysis energy metabolism in hepatoma cells, Mol. Cancer 8 (2009)
32.
[24] T. Szkudelski, The mechanism of alloxan and streptozotocin action in B cells of
the rat pancreas, Physiol. Res. Acad. Sci. Bohemoslov. 50 (2001) 537e546.
[25] D. Anastasiou, G. Poulogiannis, J.M. Asara, et al., Inhibition of Pyruvate Kinase
M2 by reactive oxygen species contributes to cellular antioxidant responses,
Science 334 (2011) 1278e1283.