Author’s Accepted Manuscript

Vitamin C deficiency aggravates tumor necrosis factor

α-induced insulinresistance

Zhou Qing, Wu Xiao-Hui, Wu Xi-Mei, Zou Chao- Chun

www.elsevier.com/locate/ejphar

PII: S0014-2999(18)30210-3
DOI:
https://doi.org/10.1016/j.ejphar.2018.0
3.044 Reference: EJP71748
To appear in: European Journal of Pharmacology
Received date: 15 February 2018
Reviseddate: 26March2018
Accepted date: 29 March 2018
Cite this article as: Zhou Qing, Wu Xiao-Hui, Wu Xi-Mei
and Zou Chao-Chun, Vitamin C deficiency aggravates
tumor necrosis factor α-induced insulin r e s i s t a n c
e , European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.03
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Vitamin C deficiency aggravates tumor necrosis
factor α-induced insulin resistance

Zhou Qinga: PhD, Wu Xiao-Huia: PhD, Wu Xi-Meib,

M.D., PhD, Zou Chao-Chuna, M.D.

aDepartment of Endocrinology of the affiliated

Children’s Hospital, b
Department of
Pharmacology, Zhejiang University School of
Medicine

Running title: Vitamin C in insulin resistance

Corresponding author:

Zou Chao-Chun, M.D., Professor

3333 Binsheng Road, Hangzhou 310051, China

Tel: +86-15067123060

E-mail: zcc14@zju.edu.cn Fax: +86-571-87033296


Abstract
Chronic low-grade inflammation plays a major
role in the development of insulin resistance. The
potential role and underlying mechanism of vitamin
C, an antioxidant and anti-inflammatory agent, was
investigated in tumor necrosis factor-α
(TNF-α)-induced insulin resistance. Gulonolactone
oxidase knockout (Gulo-/-) mice genetically unable
to synthesize vitamin C were used to induce insulin
resistance by continuously pumping small doses of
TNF-α for seven days, and human liver
hepatocellular carcinoma cells (HepG2 cells) were
used to induce insulin resistance by treatment with
TNF-α. Vitamin C deficiency aggravated TNF-α-
induced insulin resistance in Gulo-/- mice,
resulting in worse glucose tolerance test (GTT)
results, higher fasting plasma insulin level, and the
inactivation of the protein kinase B NH2-terminal kinase (JNK), p38 and extracellular
(AKT)/glycogen synthase kinase-3β (GSK3β)
pathway in the liver. Vitamin C deficiency also
worsened liver lipid accumulation and
-/-
inflammation in TNF-α-treated Gulo mice. In
HepG2 cells, vitamin C reversed the TNF-α-
induced reduction of glucose uptake and glycogen
synthesis, which were mediated by increasing
GLUT2 levels and the activation of the insulin
receptor substrate (IRS-1)/AKT/GSK3β pathway.
Furthermore, vitamin C inhibited the TNF-α-
induced activation of not only the mitogen-
activated protein kinase (MAPKs), but also nuclear
factor-kappa B
(NF-κB) signaling. Taken together, vitamin C is
essential for preventing and improving insulin
resistance, and the supplementing with vitamin C
may be an effective therapeutic intervention for
metabolic disorders.
Keywords: vitamin C; inflammatory cytokine;
insulin resistance; hepatocytes
1. Introduction
Insulin resistance is a principal feature of type-
2 diabetes mellitus (Goldstein, 2002), which has
been implicated in the development of
cardiovascular disease,
non-alcoholic fatty liver disease and cognitive
impairment in the metabolic syndrome (Bender et
al., 2013; Kim & Feldman, 2015; Wang et al.,
2017). The disturbance of energy nutrients
metabolism is a major characteristic of insulin
resistance. The liver plays a crucial role in the
maintenance of glucose homeostasis. Under normal
insulin action in the liver, the combination of
insulin to its receptor on hepatocytes recruits and
activates insulin receptor substrate (IRS-1), which
subsequently activates the downstream
phosphatidylinositol 3-kinase (PI3K)/protein
kinase B (AKT) signaling pathway, and regulates
gluconeogenesis and glycogenesis, in order to
maintain glucose homeostasis (Kim et al., 2015).
Chronic inflammation is a major contribution
to the development of insulin resistance
(Nandipati et al., 2017; Xu et al., 2003). The two
main inflammatory pathways, mitogen-activated
protein kinase (MAPKs) and nuclear factor-kappa
B (NF-κB), play crucial roles in the progression of
insulin resistance. MAPKs are a family of three
distinct mammalian protein kinase termed c-Jun
signal-related kinase (ERK). The activation of
MAPKs induces the serine phosphorylation of
IRS-1, which result in the impairment of the
downstream PI3K-AKT pathway, leading to a
decrease in insulin signaling transduction
(Fujishiro et al., 2003). The mediation of pro-
inflammatory cytokines in the activation of the
inhibitor of IκB kinase (IKK)-β has been
considered as a core mechanism that connects
metabolic inflammation and insulin resistance
(Arkan et al., 2005). Studies have indicated that
TNF-α induces the activation of IKK-β, which
directly targets the serine residue of IRS-1 or
rapidly removes tyrosine phosphate groups from
IRS-1 in a NF-κB-dependent manner, finally
leading to insulin resistance (Fernandez-Veledo et
al., 2006; Gao et al., 2002; Zabolotny et al., 2008).
Hence, inhibiting chronic inflammation may
protect against insulin resistance.
Vitamin C is a soluble antioxidant and
enzyme cofactor that participates in various
biological functions in vivo (Lykkesfeldt et al.,
2014). Vitamin C has been recognized as an anti-
inflammatory molecule due to its ability to
downregulate
pro-inflammatory cytokines induced by oxidative
stress (Burek & Rose, 2008; Gren, 2013). Studies
have shown that vitamin C supplementation
reduces the rate of congenital malformations in the
offspring of diabetic rats (Siman & Eriksson,
1997), and the daily intake of vitamin C attenuates
 the exacerbation of cerebral ischemic injury in a
diabetic state (Iwata et al., 2014). Furthermore,
low plasma vitamin C concentration has been
found to be associated with insulin resistance
(Donin et al., 2016). These evidences indicate that
vitamin C has a protective effect on diabetes, and
vitamin C deficiency may be closely correlated to
the progression of insulin resistance. However, the
direct effects of vitamin C on the amelioration of
insulin resistance remain unclear, and the
underlying mechanism by which vitamin C
protects against insulin resistance is far from clear.
In the present study, we attempted to
determine whether vitamin C deficiency
exacerbates TNF-α-induced insulin resistance in
Gulo-/- mice, and identify the underlying
mechanism in human HepG2 cells, Focus was
given on the link between TNF-α-associated
inflammation and hepatic insulin resistance.
2. Materials and Methods
2.1 Animals experiment
Gulonolactone oxidase (Gulo-/-) knockout
mice, which lack terminal enzyme Gulo in the
synthesis pathway of vitamin C, is also absent in
humans, and depends completely on dietary
vitamin C (Maeda et al., 2000), were obtained from
the Mutant Mouse Regional Resource Center
(University of California, Davis, CA). Serum
vitamin C levels were set within the normal or
insufficient range by the supplementation of 3.3
g/L and 0.33 g/L of vitamin C in drinking water,
respectively. Then, 9-week-old male mice, which
were paired for body weight and glucose level,
were randomly divided into four groups: LD group
(0.33 g/L of vitamin C), TNF-α + LD group (TNF-
α + 0.33 g/L of vitamin C), HD group (3.3 g/L of
vitamin C), and TNF-α + HD group (TNF-α + 3.3
g/L of vitamin C); Each group comprised of eight
mice, and all mice were fed with a standard
laboratory diet in a
temperature-controlled (20-24°C) and humidity-
controlled (45-55%) environment. A 12 h/12 h
light/dark cycle was maintained. At nine weeks,
all mice had an ALZET osmotic pump (Durect,
Cupertino, CA, USA), which was implanted into
the interscapular subcutaneous space. Mice in the
TNF-α treatment group were implanted with a
pump filled with 6.44 μg/ml of TNF-α diluted in
the carrier (0.9% NaCl and 0.1% BSA), with a 7-
day pumping capacity and an infusion rate of 1
μl/h. Mice in the control group were implanted
with a pump filled with the carrier only.
All mice were housed at the Animal Care
Facility of Zhejiang University according to the
institutional guidelines for laboratory animals, and
all the protocols were approved by the Zhejiang
University Institutional Animal Care and Use
Committee.
2.2 Tolerance test
At the end of the 7-day TNF-α exposure, an
intraperitoneal glucose tolerance test (GTT) was
performed in mice. Mice were fasted overnight for
14 h and intraperitoneally injected with glucose (1
g/kg of body weight). Blood glucose
concentrations were measured from tail blood
samples at 0, 30, 60 and 120 min after glucose
injection using a glucometer (Onetouch Ultra,
Johnson).
2.3 Blood and tissue sampling
On the next day of the GTT experiment, after
fasting overnight (12 h), blood samples were
collected from the medial canthus of the eye using
a capillary tube, centrifuged at 1,400 g for 10 min,
and stored at -80 ºC. At the same time, liver tissues
were surgically removed, portions of the liver
tissue were immediately frozen in liquid nitrogen
for subsequent biochemical analysis, and portions
of the liver tissue were fixed with 4%
formaldehyde in PBS for histopathological
examination.
2.4 Biochemical analyses and histopathological
examinations
Blood samples were used to carry out the
analysis of insulin using a mouse insulin enzyme-
linked immunosorbent assays (ELISA) kit,
according to manufacturer’s instructions (R&D
Systems, Minneapolis, USA). The homeostasis
model assessment of insulin resistance (HOMA-
IR) index was calculated using the following
formula: HOMA-IR= [FBS (mmol/l) × Fins
(mIU/l)] / 22.5. Hepatic glycogen levels were
measured with a glycogen assay kit (Biovision,
USA), according to manufacturer’s instructions.
Hepatic triglyceride and cholesterol contents were
measured with a commercial assay kit (Nanjing
Jiancheng Bioengineering Institute, China),
according to manufacturer’s instructions. Hepatic
IL-1α and IL-6 levels were determined using
mouse ELISA kits, according to manufacturer’s
instructions (IL-6: eBioscience, CA; IL-1α:
Abcam, USA). The hematoxylin and eosin (H&E)
staining and immunohistochemistry staining of
NF-κBp65 were performed, as previously
described (Ji et al., 2017). The primary antibody
 against NF-κBp65 (1:800, CST, USA) was used in
the experiment. Histological analyses were
performed on five independent mice in a blinded
manner.
2.5 Cell cultures
HepG2 cells were purchased from ATCC
(Manassas, VA) and maintained in Dulbecco's
Modified Eagle Medium supplemented with 10%
fetal bovine serum (Gibco, USA) and 1%
penicillin-streptomycin at 37°C with 5% CO 2. In
order to check insulin signaling molecules, cells
were treated with 100 nM of insulin for 10 min
before the protein was collected, as previously
reported (Lin & Lin, 2008; Nakajima et al., 2000;
Zang et al., 2004). Cellular vitality was measured
by cell counting kit-8 (Beyotime, China),
according to manufacturer’s instructions. Human
umbilical vein endothelial cells (HUVEC) were
primary cultured and preserved in our laboratory,
as described in a previous publication (Yao et al.,
2015). The cells from passages 2 to 5 were used in
the experiment.
2.6 Glycogen measurement, VEGF protein
secretion and glucose uptake
Glycogen levels were measured in HepG2
cells incubated with 100 nM of insulin for 3 h
using a glycogen assay kit (Biovision, USA),
according to manufacturer’s instructions.
In order to evaluate the VEGF protein
secretion, the HUVECs culture supernatant was
collected for determination of VEGF release using
a human VEGF Quantikine ELISA kit obtained
from R&D Systems (Minneapolis, USA),
according to manufacturer’s instructions.
For the glucose uptake measurement, the
medium of cells was replaced with DMEM (low-
glucose, 1g/L) for 12 h, followed by DMEM (high-
glucose, 4.5g/L) containing 2-[N-(7-nitrobenz-2-
oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose
(2-NBDG, a fluorescent glucose analog, Sigma;
100 μM final concentration) for 30 min. After
washing twice with PBS to remove free 2-NBDG,
cells were collected. Then, 2-NBDG uptake
activity was measured using a FACS flow
cytometer (BD Biosciences, USA) and analyzed
using the CellQuest software.
2.7 Quantitative real-time PCR
Total RNA from HepG2 cells and mice liver
homogenates were extracted using TRIzol reagent
(Takara Biotechnology, China), according to
manufacturer’s instructions. The mRNA levels
were determined by quantitative RT-PCR, as
previously described (Tang et al., 2016). The
relative amounts of mRNA levels of the target gene
were normalized to β-actin (for mice) or GAPDH
(for human), and the differences in mRNA levels
were calculated using the 2-△△Ct method. The
primer sequences for the target genes (IL-1α, IL-6,
ACC1, FAS, PEPCK and G6P) used for real-time
PCR are listed in Table 1 (Supplementary Table 1).
2.8 Western blot
Nuclear fractions from the mice liver
homogenate and cytosolic, and membranal and
nuclear fractions from HepG2 cells were extracted
using a Nuclear, Membrane and Cytoplasmic
Protein Extraction Kit (Beyotime, China),
according to manufacturer's instructions. Next, 80
μg of protein was separated by 8-10%
SDS-PAGE, transferred onto a nitrocellulose
membrane, probed with antibodies at 4 °C
overnight, and incubated in IRDye 680 and 800
second antibodies (Lincoln, Nebraska) for 1 h at
room temperature. The immunoreactive signals
were visualized using the Odyssey Infrared
Imaging System (LI-COR, Lincoln, NE).
Antibodies against P-IKKα/β, IKKβ, P-JNK, JNK,
P-ERK, ERK, P-p38, p38, P-AKT, AKT,
P-GSK-3β, P-NF-κBp65, P-ACC and ACC were
all purchased from Cell Signal Technology
(USA). Antibodies against IκBα, P-IκBα, Sodium
Potassium ATPase (Na+/K+-ATPase), GLUT2,
IRS-1, P-IRS-1, VEGFA, PEPCK, G6P and FAS
were all
obtained from Abcam (USA). Antibodies against
GAPDH and GSK-3β were purchased from Santa
Cruz Biotechnology (USA). Antibodies against α-
tubulin and Histone H3 were purchased from
HuaAn Biotechnology (China). Histone H3,
α-tubulin and Na+/K+-ATPase were used as the
internal standards for the nuclear, cytosolic and
membranal protein, respectively. GAPDH was
used as the internal standard for the total protein.
2.9 Immunofluorescent staining
Cells were cultured in a 12-well plate covered
with coverslips. After treatment, cells were fixed
with 4% paraformaldehyde for 10 min, followed by
permeabilization with 0.1% Triton X-100 for 30
min. After incubation with blocking buffer for
30min, the coverslips were incubated with primary
antibodies overnight at 4 °C. After washing with
TBST, cells were incubated with Alexa Fluor 546-
tagged secondary antibody (Life Technologies).
Then, the nuclei were counterstained with DAPI.
Imaging was performed using a Zeiss fluorescent
microscope equipped with an epifluorescence and
 Axiocam camera system coupled with the
axiovision software (Carl Zeiss, Oberkochen,
Germany).
2.10 Statistical analysis
Numerical data were expressed as mean ±
S.E.M. One-way ANOVA and Dunnett’s multiple
comparison tests were used to determine the
statistical significance. P<0.05 was considered
statistically significant.
3. Results
3.1 Vitamin C deficiency aggravated TNF-α-
induced insulin resistance in Gulo-/-mice
In order to determine the effect of vitamin C
deficiency on glucose metabolism in TNF-α-
treated Gulo-/- mice, the fasting serum insulin level,
hepatic glycogen level and hepatic mRNA
expression of genes involved in gluconeogenesis
(phosphoenolpyruvate carboxykinase [PEPCK],
glucose-6-phosphatase [G6P]) were assessed, and a
GTT was performed. The plasma glucose levels of
Gulo-/- mice were higher in the TNF-α + LD group
(vitamin C deficiency group) than in the TNF-α +
HD group (vitamin C normal group), especially at
30 and 60 min after glucose injection (Fig. 1A).
The difference was indicated by the area under the
glucose curve (AUC) of the GTT (Fig. 1B). The
fasting serum insulin level and HOMA-IR of Gulo-
/-
mice significantly increased in the TNF-α + LD
group than in the TNF-α + HD group (Figs. 1C and
1D). Meanwhile, a decrease in glycogen level and
an increase in PEPCK and G6P mRNA levels were
found in liver tissues of Gulo-/- mice in the
TNF-α + LD group when compared with the TNF-
α + HD group (Figs. 1E and 1F). These in vivo data
validates that vitamin C deficiency exacerbated
insulin resistance in TNF-α-treated Gulo-/- mice.
3.2 Vitamin C deficiency aggravated TNF-α-
triggered hepatic lipid accumulation in Gulo-/-
mice
Hepatic lipid metabolism is closely related
with hepatic insulin resistance. Hence, the hepatic
lipid accumulation in these Gulo-/- mice was
examined. First, the liver histologic analysis from
mice in the TNF-α + LD group indicated
prominent vacuolization, when compared with
mice in the TNF-α + HD group (Fig. 2A).
Consistent with the result from the H-&-E staining,
livers from mice in the TNF-α + LD group
exhibited increased triglyceride and cholesterol
content (Figs. 2B and 2C) and lipogenesis-related
gene mRNA expression (acetyl CoA carboxylase 1
[ACC1] and fatty acid synthase [FAS]), compared
with that in the TNF-α + HD group (Fig.2D).
These data demonstrate that vitamin C deficiency
aggravated TNF-α-triggered lipid accumulation in
the liver of Gulo-/- mice.
3.3 Vitamin C deficiency increased the TNF-α-
induced inactivation of the
AKT/GSK3β pathway in Gulo-/- mice
In order to further explore the potential targets
of vitamin C on the amelioration of insulin
resistance, the specific protein expression of the
insulin signaling pathway in liver tissues of TNF-α-
treated Gulo-/- mice were analyzed. Figure 3 shows
that the phosphorylation of AKT and GSK3β
decreased in mice in the TNF-α + LD group, but
significantly increased in mice in the TNF-α + HD
group. These data suggest that vitamin C
deficiency aggravated insulin resistance by
increasing the TNF-α-induced inactivation of the
AKT/GSK3β pathway in the liver of Gulo-/- mice.
3.4 Vitamin C deficiency aggravated TNF-α-induced
inflammation in Gulo-/- mice
The production of pro-inflammatory cytokines
in the liver has been implicated in the development
of hepatic insulin resistance. In order to evaluate
the effect of vitamin C deficiency on TNF-α-
triggered inflammation, the mRNA and protein
level of IL-1α and IL-6 in liver tissues of Gulo-/-
mice were measured. The results revealed that the
mRNA and protein levels of IL-1α and IL-6
significantly increased in the TNF-α + LD group
than that in the TNF-α + HD group (Figs. 4A and
4B). Moreover, immunohistochemistry revealed
that NF-κBp65 was widely expressed in the nuclei
of hepatocytes in mice in the TNF-α + LD group,
when compared with the TNF-α + HD group (Fig.
4C), which was further confirmed by western blot
(Fig. 4D). These data indicate that TNF-α
treatment induced a significantly hepatic
inflammation, and vitamin C deficiency increased
the TNF-α-triggered inflammation in liver tissues
of Gulo-/- mice.
3.5 Vitamin C improved TNF-α-induced
glucose and lipid metabolic disorder in HepG2
cells
Since the animal models confirmed that
vitamin C supplementation can improve insulin
resistance in TNF-α-treated Gulo-/- mice, the
investigators subsequently explored the
underlying mechanism by which vitamin C
supplementation improves insulin resistance.
Focus was given on the effects of vitamin C on
glycogen synthesis, glucose uptake, and the
mRNA and protein levels of ACC1, FAS, PEPCK
and G6P. First, the cell viability of HepG2 treated
with different concentrations of vitamin C for 24 or
48 h were quantified by cell counting kit-8 assays
to exclude the cytotoxicity of vitamin C, and result
indicated that no cytotoxicity was observed after
exposure of HepG2 cells to vitamin C (100 μM,
24h; Fig. 5A). Next, glycogen level, 2-NBDG
uptake and mRNA and protein levels of ACC1,
FAS, PEPCK and G6P were tested, since these
indicators are manifestations of insulin resistance.
The results revealed that vitamin C reversed the
TNF-α-induced reduction of glycogen synthesis in
a dose and time-dependent manner (Figs.5B and
5C), and vitamin C also increased
TNF-α-impaired 2-NBDG uptake in HepG2 cells
(Fig. 5D). Moreover, vitamin C inhibited the
mRNA expression of ACC1, FAS, PEPCK and G6P
genes (Figs. 5E and 5F), downregulated the protein
expression of PEPCK, G6P, FAS, and upregulated
the phosphorylation of ACC in HepG2 cells
induced by TNF-α (Fig. 5G), since
phosphorylation at 79 serine residue of ACC
inactivate the enzyme, indicating that vitamin C
inhibited gluconeogenesis and lipogenesis in
insulin resistant HepG2 cells treated by TNF-α.
Taken together, these data demonstrate that
vitamin C improved glucose and lipid metabolic
disorders in TNF-α-induced insulin resistant
HepG2 cells.
3.6 Vitamin C restored TNF-α-negated
GLUT2 levels and the IRS-1/AKT/GSK3β
pathway in HepG2 cells
IRS-1 phosphorylation plays a switch role for
insulin signaling transduction. The serine
phosphorylation of IRS-1 is a negative regulator of
the insulin-PI3K pathway. The serine
phosphorylation of IRS-1 not only reduces
tyrosine phosphorylation, but also accelerates IRS-
1 degradation and the dissociation between insulin
receptor-IRS-1 and IRS-1-PI3K, leading to the
inactivation of the AKT pathway (Moeschel et al.,
2004; Yu et al., 2002). GSK3β phosphorylation in
residue Ser9 plays a positive role in glycogen
synthesis, and GLUT2 is a predominant glucose
transporter in the plasma membrane of
hepatocytes. In order to explore whether vitamin C
reversed the TNF-α-mediated reduction of
glycogen synthesis involved in the activation of
the IRS-1/AKT/GSK3β pathway, and whether
vitamin C improved the glucose uptake involved
in increasing GLUT2 level, the effects of vitamin
C on the TNF-α-triggered inactivation of the IRS-
1/AKT/GSK3β pathway and GLUT2 protein
expression were evaluated. The results revealed
that vitamin C significantly increased AKT
phosphorylation at position Ser473, GSK3β
phosphorylation at position Ser9, and IRS-1
tyrosine phosphorylation at position Tyr632, and
was accompanied by reduced IRS-1 serine
phosphorylation at position Ser312, in
TNF-α-induced insulin resistant HepG2 cells (Fig.
6A). Moreover, vitamin C also reversed the TNF-
α-mediated reduction of GLUT2 protein expression
(Fig. 6B). The above results demonstrate that
vitamin C can attenuate TNF-α-induced insulin
resistance by improving the IRS-1/AKT/GSK3β
pathway and enhancing GLUT2 levels in HepG2
cells.
3.7 Vitamin C upregulates the high glucose-
negated VEGF expression in HUVECs
Diabetes mellitus is accompanied by a series
of diabetic macro-angiopathy and micro-
angiopathy, such as atherosclerosis, retinopathy and
nephropathy. Furthermore, vascular endothelial
growth factor (VEGF) plays an important role in
diabetic vascular complication. Therefore, the
effect of vitamin C on synthesis and the secretion
of VEGF in primary HUVEC incubated with high
glucose for 24 h were explored. The results
revealed that high glucose inhibited VEGF protein
expression and secretion, while vitamin C
upregulated the high glucose-negated VEGF
protein synthesis and secretion in HUVECs
(Supplementary Figure 1).
3.8 Vitamin C inhibited the TNF-α-induced
activation of the IKKβ/IκBα/NF-κB pathway in
HepG2 cells
Insulin resistance has been considered as a
chronic low-grade inflammatory response, and the
activation of the IKKβ/NF-κB pathway is involved
in
TNF-α-induced insulin resistance. Hence, it was
further investigated whether vitamin C has an
inhibitory effect on the activation of the IKKβ/NF-
κB signaling pathway.
First, the time for the activation of the IKKβ/IκB-
α/NF-κB pathway induced by TNF-α was
determined. The results revealed that TNF-α
induced IKKβ, IκB-α and NF-κB phosphorylation
maximally by 15 min, and declined by 60 min
(Fig.7A).
Furthermore, western blot analysis revealed that
vitamin C blocked TNF-α-induced NF-κB
activation by inhibiting the p65 translocation from
cytoplasm to nuclei (Fig. 7B), and this inhibitory
effect of vitamin C on NF-κB translocation was
also confirmed by immunofluorescence staining
 (Fig.7C). Since vitamin C has an inhibitory effect
on NF-κB translocation, the effect of vitamin C on
IKKβ and IκBα phosphorylation in the presence of
TNF-α was subsequently tested, which are at the
upstream of NF-κB. As shown in Figure 7D,
vitamin C partially inhibited the
TNF-α-induced activation of IKKβ and IκBα.
Taken together, these results clearly suggest that
vitamin C can inhibit the IKKβ/IκBα/NF-κB
signaling pathway activated by TNF-α in HepG2
cells.
3.9 Vitamin C inhibited the TNF-α-induced
activation of JNK and p38 rather than ERK in
HepG2 cells
Accumulating evidences have shown that the
MAPK pathway is required for the TNF-α-induced
serine phosphorylation of IRS-1. Hence, the impact
of vitamin C on MAPK activation was investigated.
First, the time for the phosphorylation of MAPKs
activated by TNF-α was determined. The results
revealed that TNF-α increased JNK and p38
phosphorylation maximally by 30 min, and
declined by 60 min (Fig.8A).
Furthermore, vitamin C significantly blocked the
TNF-α-triggered phosphorylation of JNK and p38,
but without inducing a significant inhibitory effect
on ERK activation (Fig.8B). These results indicate
that vitamin C can inhibit the TNF-α-induced
activation of JNK and p38 in HepG2 cells.
4. Discussion
Obesity and diabetes mellitus have become a
major public problem worldwide, and both have
been considered as a chronic low-grade
inflammatory state. This
low-grade inflammation is characterized by higher
levels of circulating cytokines and the activation of
pro-inflammatory signaling pathways, which play
a central role in the pathogenesis of insulin
resistance (Khodabandehloo et al., 2016).
Inflammatory cytokines, such as TNF-α, IL-1 and
IL-6, disrupt insulin signaling at the insulin
receptor and IRS levels through multiple signaling
pathways, which finally contribute to the
development of insulin resistance (Asrih &
Jornayvaz, 2013; Dali-Youcef et al., 2013).
Cytokines can activate each other through the
inflammatory pathway, and TNF-α has been
identified as one of the first links between low-
grade inflammation and insulin resistance. Thus,
in the present study, human HepG2 hepatocytes
were treated with 10 ng/ml of TNF-α for 24 h to
induce insulin resistance, and the mouse model
with insulin resistance was established by
continuously pumping small doses
of TNF-α for seven days, as previously described
(Chu et al., 2013; Zhao et al., 2015).
The IKKβ/NF-κB pathway and MAP kinases
JNK, p38 and ERK have been considered as the
two major transcription factors in response to
inflammatory cytokine stimuli. These kinases,
including IKKβ, JNK, p38 and ERK, have been
activated by inflammatory cytokines, which
subsequently induce uncontrolled IRS serine
phosphorylation, finally leading to insulin
resistance (Tanti & Jager, 2009). Multiple studies
have shown that JNK is a crucial mediator of
insulin resistance: mice with JNK-deficient
prevented insulin resistance induced by high fat
diet (Han et al., 2013; Sabio et al., 2008); JNK
activity was significantly increased in liver,
muscle and adipose tissues in both dietary and
genetic (ob/ob) models of obesity; the absence of
JNK1 remarkably improved insulin sensitivity and
enhanced insulin receptor signaling capacity
(Hirosumi et al., 2002). In addition, the IKKβ/NF-
κB pathway also play a major role in connecting
metabolic inflammation and insulin resistance: the
deficiency of IKKβ in adipocytes markedly
alleviated free fatty acid-induced inflammation
and insulin resistance, while IKKβ overexpression
caused inflammation and insulin resistance (Jiao et
al., 2011), and the overexpression of IκBα in the
liver or neurons of mice, which inhibited NF-κB
activation, attenuated high fat diet-induced body
weight gain and insulin resistance (Benzler et al.,
2015; Wang et al., 2014). All these researches
demonstrate that JNK and the IKKβ/NF-κB
pathway are potential targets for the treatment of
insulin resistance.
Vitamin C is an antioxidant with potent
inhibitory action on multiple inflammatory
signaling pathways. For example, Kyaw et al.
(Kyaw et al., 2001) found that vitamin C inhibited
JNK and p38 activation in rat aortic smooth
muscle cells through its anti-oxidative property.
Several studies have also shown that vitamin C
inhibited the activation of the IκBα/NF-κB pathway
in primary HUVECs, protecting against
immunological hepatic injury in mice (Bowie &
O'Neill, 2000; Carcamo et al., 2002; Liang et al.,
2014). Similarly, the present data revealed that
vitamin C inhibited the TNF-α-triggered activation
of IKKβ, IκBα, NF-κB, JNK and p38 (although
without a significant inhibitory effect on ERK
activation) in HepG2 cells, and inhibited the NF-
κB activation and the expression of IL-6 and IL-1α
in liver tissues of

Gulo-/- mice. Therefore, vitamin C can be a good
strategy to target chronic inflammation linked to
insulin resistance, and might play a protective role
in the development of insulin resistance. A double-
blind, randomized and placebo-controlled
crossover trial revealed that vitamin C
supplementation decreased the risk for insulin
resistance under an environment of high exposure
to perfluorinated compounds,
which were found to be positively associated with
insulin resistance (Kim et al., 2016). As a result,
the present study investigated whether vitamin C
deficiency exacerbate TNF-α-induced insulin
resistance in Gulo-/- mice. It was found that mice in
the
TNF-α-treated vitamin C deficient group had higher
plasma glucose, increased fasting plasma insulin
levels, decreased hepatic glycogen content, and
higher liver triglyceride and cholesterol levels,
while vitamin C supplementation significantly
improved insulin sensitivity, abnormal glucose and
lipid metabolism in
TNF-α-induced insulin-resistant Gulo-/- mice.
These results are in accordance with a previous
study conducted by Aluwong, in which it was
reported that vitamin C had a positive effect on
diabetes by ameliorating hyperglycemia, oxidative
stress and dyslipidemia in alloxan-induced
diabetic rats (Aluwong et al., 2016). Therefore, it
was concluded that vitamin C deficiency
exacerbates insulin resistance in
TNF-α-treated Gulo-/- mice, which might be
partially the result of losing the anti-inflammation
property.
At the cellular level, hepatic insulin resistance
is characterized by the impaired activation of the
IRS-1/PI3K/AKT signaling pathway. The animal
experiments have demonstrated that vitamin C
deficiency exacerbated TNF-α-induced hepatic
insulin resistance and inflammation. This
prompted the investigators to determine whether
vitamin C could inhibit inflammation-stimulated
IRS-1 serine phosphorylation and restore the
impaired insulin signal transduction pathway. It
was found that vitamin C enhanced the activation
of the IRS-1/AKT/GSK3β pathway, and
downregulated the expression of ACC1, FAS,
PEPCK and G6P in TNF-α-induced insulin
resistant HepG2 cells. This was because PEPCK
and G6P control the rate-limiting steps of liver
gluconeogenesis, while ACC1 and FAS are
involved in the de novo lipogenesis. Decreased
PEPCK, G6P, ACC1 and FAS gene expression
were also observed in liver tissues obtained from
TNF-α-treated vitamin C normal Gulo-/- mice,
further supporting the improvement effect of
vitamin C on insulin resistance. Since GSK3β is an
important factor in the regulation of hepatic
glycogenesis, the upregulation of the AKT/GSK3β
pathway in vitro and in vivo by vitamin C might
be correlated with its positive effect on glycogen
synthesis impaired by TNF-α.
GLUT2 is the major glucose transporter of
hepatocytes, which is responsible for glucose
uptake during the postprandial phase and glucose
release during the fasting phase (Thorens, 2015).
Multiple anti-diabetic drugs have been reported to
recover the diminished level of hepatic GLUT2 in
mice fed on high fat diet (Souza-Mello et al.,
2010). Furthermore, a study revealed that the
reduced GLUT2 level and glucose uptake in
insulin-resistant hepatocytes were recovered when
cells were co-treated with wax apple fruit extract
(Shen et al., 2012). In agreement with the above
studies, GLUT2 levels and glucose uptake
decreased in insulin-resistant HepG2 cells triggered
by TNF-α, but this reductions have been reverted
when cells were co-treated with vitamin C. These
results might partially explain the improvement
effect of vitamin C on glucose tolerance in TNF-α-
induced insulin-resistant Gulo-/- mice.
VEGF is an angiogenic factor for endothelial
cells, which is strongly associated with the
development of diabetic vascular complications.
VEGF level in response to high glucose stimuli is
differentially regulated in different types of cells or
tissues.
The upregulation effect of high glucose stimulation
on VEGF expression is observed in mesangial cells
and retinal pigment epithelial cells (Cai et al.,
2014; Kim et al., 2000), while this effect is the
opposite observed in HUVECs (Yang et al., 2008),
indicating the differential role of VEGF in diabetes-
associated vascular complications. Doronzo et al.
reported that high glucose enhanced VEGF
synthesis and secretion in primary vascular
smooth muscle cells (VSMC) from lean insulin-
sensitive rats, but this effect was attenuated in
VSMC from obese insulin-resistant rats (Doronzo
et al., 2012), indicating that VEGF expression has
a certain correlation with insulin resistance. In
order to explore the effect of vitamin C on VEGF
expression, primary HUVECs treated with high
glucose were cultured, and the results revealed
that vitamin C upregulated the high glucose-
inhibited VEGF protein synthesis and secretion in
 HUVECs. This might be due to the anti-oxidative
stress effect of vitamin C, since high glucose
induces the generation of oxidative stress and
excessive oxidative stress could interfere with
VEGF signaling (Yang et al., 2008). In vivo
studies have shown that vitamin C, through its
antioxidant effect, evidently increased capillary
density in ischemic gastrocnemius tissues of
diabetic rats, accompanied by the upregulation of
VEGF (El-Azab et al., 2012). These findings may
provide a certain basis for vitamin C in the
treatment of diabetic vascular complication.
The limitation of the present study was that
the ameliorative effect of vitamin C on
inflammation-associated insulin resistance in other
insulin responsive tissues such as adipose tissues
and muscle was not examined. The effect of
vitamin C on the vessel condition in insulin
resistant Gulo-/- mice and the positive effect of
vitamin C on other pro-inflammatory cytokine-
induced insulin resistance need to be clarified
through further studies.
5. Conclusions
In summary, the present study demonstrates that
vitamin C inhibits
TNF-α-associated inflammation by targeting
MAPKs and NF-κB signaling. Vitamin C
supplementation prevents hepatic insulin resistance
caused by TNF-α. The effect of vitamin C in
alleviating hepatic insulin resistance occurs in
association with the prevention of TNF-α-induced
inflammation and corresponding impairment of
hepatic insulin signaling. Therefore, vitamin C
might have potential for use in the treatment of
metabolic disorders.
Acknowledgments
This study was supported by the National
Natural Science Foundation of China (Grant
numbers 81170787 and 81670786), and the
Zhejiang Provincial Program for the Cultivation of
High-Level Innovative Health Talents (2015).
Conflict of interest
The authors declare no conflict of interest with
this work.
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Figure legends:
Figure 1 Vitamin C deficiency aggravated TNF-
α-induced insulin resistance in Gulo-/- mice. (A)
Mice from each group were starved for 14 h,
subsequently intraperitoneally injected with 1 g/kg
of glucose, and blood glucose concentrations were
measured at the indicated time points. (B) Blood
glucose values for the area under the curve (AUC)
were calculated during GTT. (C) Fasting serum
insulin level.
(D) The HOMA-IR index was calculated
according to the formula [FBG (mmol/l) × Fins
(mIU/l)] / 22.5. (E) Hepatic glycogen level. (F)
Relative mRNA levels of PEPCK and G6P in liver
tissues. Data were expressed as mean ± S.E.M
(n=8). *P <0.05 vs. the LD group, #P<0.05 vs. the
TNF-α + LD group. LD: 0.33 g/L of vitamin C;
HD:
3.3 g/L of vitamin C.
Figure 2 Vitamin C deficiency aggravated TNF-
α-triggered hepatic lipid accumulation in Gulo-/-
mice
(A) Hematoxylin and eosin (H&E) staining of
liver tissues (scale bar: 50 µm, magnification:
400×). (B) Liver triglyceride content. (C) Liver
cholesterol content. (D) Relative hepatic mRNA
levels of ACC1 and FAS in liver tissues. Data were
expressed as mean ± S.E.M (n=8). *P <0.05 vs.
the LD group, #P<0.05 vs. the TNF-α + LD group.
Figure 3 Vitamin C deficiency increased the
TNF-α-induced inactivation of the AKT/GSK3β
pathway in Gulo-/- mice
Liver tissue lysates from each group were
prepared and analyzed by western blot.
Representative western blot analysis (left) and
quantification (right) of the expression and
phosphorylation levels of AKT and GSK3β in the
liver homogenate of mice in each group (n=8). *P
<0.05 vs. the LD group, #P<0.05 vs. the TNF-α +
LD group.
Figure 4 Vitamin C deficiency aggravated
TNF-α-induced inflammation in Gulo-/- mice
(A) Relative mRNA levels of IL-1α and IL-6 in liver
tissues of Gulo-/- mice. (B) IL-1α and IL-6 levels
determined by ELISA in liver tissues of Gulo-/- mice.
(C).
NF-κBp65-positive expression (brown) in liver
tissues, as determined by immunohistochemistry
(scale bar: 50 µm, magnification: 200×).
(D).Western blot analysis (left) and quantification
(right) of NF-κBp65 expression in nuclei of the
liver homogenate of mice. Data were expressed as
mean ± S.E.M (n=8). *P <0.05 vs. the LD group,
#P<0.05 vs. the TNF-α + LD group.
Figure 5 Vitamin C improved the TNF-α-
induced glucose and lipid metabolic disorder in
HepG2 cells
(A) Cell viability determined by cell counting kit-8
in HepG2 cells treated with vitamin C (0-200 μM)
for the indicated time (n=8). (B and C) Glycogen
levels determined by the glycogen assay kit in
HepG2 cells treated with vitamin C for the
indicated time and dose, and or TNF-α (10 ng/ml,
24 h; n=5). (D-G) HepG2 cells were cultured with
vitamin C (100 μM) and or TNF-α (10 ng/ml) for
24 h, and
2-NBD glucose uptake were measured using a flow
cytometer (n=5) (D). The relative mRNA levels of
genes were measured by qRT-PCR (n=5, E and F).
The protein expression of PEPCK, G6P, FAS, P-
ACC and ACC were determined by western blot
(n=3, G). The representative western blot (left) and
quantification (right) are shown. Data were
expressed as mean ± S.E.M. *P <0.05 vs. the
Control. #P<0.05 vs. the TNF-α group. VC:
vitamin C.
Figure.6 Vitamin C restored TNF-α-negated
GLUT2 levels and the IRS-1/AKT/GSK3β
pathway in HepG2 cells
HepG2 cells were treated with vitamin C (100 μM)
and or TNF-α (10 ng/ml) for 24 h, and stimulated
with insulin (100 nM) for 10 min. The expression
and phosphorylation of IRS-1, AKT, GSK3β and
GLUT2 were determined by western blot (A and
B). The representative western blot (left) and
quantification (right) are shown. Data were
expressed as mean ± S.E.M (n=3). *P <0.05 vs.
the Control. #P<0.05 vs. the TNF-α group.
Figure.7 Vitamin C inhibited the TNF-α-
induced activation of the
IKKβ/IκBα/NF-κB pathway in HepG2 cells
HepG2 cells were treated with TNF-α (10 ng/ml)
for the indicated time, or treated with vitamin C
(100 μM) for 8 h, before cells were treated with
TNF-α (10 ng/ml) for 15 min. The time for the
activation of the IKKβ/IκB-α/NF-κB signaling
pathway induced by TNF-α were determined by
western blot (A). The expression of
P-NF-κBp65 in the cytoplasm and nuclei were
determined by western blot (B) and
immunofluorescence staining (C). The expression
and phosphorylation of IKKβ and IκBα were
determined by western blot (D). The representative
western blot (left) and quantification (right) are
shown. Data were expressed as means ± S.E.M
(n=3). *P <0.05 vs. controls or 0 min. #P<0.05 vs.
the TNF-α or 30 min group.

Figure 8 Vitamin C inhibited the TNF-α-

mediated activation of JNK and p38 rather than
ERK in HepG2 cells
HepG2 cells were treated with TNF-α (10 ng/ml)
for the indicated time or treated with vitamin C
(100 μM) for 8 h before cells were treated with
TNF-α (10 ng/ml) for 30 min. The time for the
activation of MAPKs induced by TNF-α were
determined by western blot (A). The expression
and phosphorylation of JNK, p38 and ERK were
determined by western blot (B). The
representative Western blot (left) and
quantification (right) are shown. Data are expressed
as mean ± S.E.M (n=3). *P <0.05 vs. Controls or 0
min. #P<0.05 vs. the TNF-α or 60 min group.

Figure.1 Vitamin C up-regulated high glucose-

negated VEGF expression in HUVECs
(A) HUVECs were treated with vitamin C (100
μM) for 8 hours before the cells were exposed to
25 mM glucose, expression of VEGF was
determined by Western blot (n=3). Representative
Western blot (left) and quantification (right). (B)
VEGF level in culture supernatant was measured
by ELISA (n=5). Data are expressed as means ±
SEM. *P <0.05 versus Control. #P<0.05 versus
HG. Glucose concentration (5.5 mM) as a control
group, HG: 25 mM glucose.
Figure 1A
Figure 1B
Figure 1C
Figure 1D
Figure 1E
Figure 1F
Figure 2A
Figure 2B
Figure 2C
Figure 2D
Figure 3
statistics for Figure 3
Figure 4A
Figure 4B
Figure 4C
Figure 4D
statistics for Figure 4D
Figure 5A
Figure 5B
Figure 5C
Figure 5D
Figure 5E
Figure 5F
Figure 5G
statistics for Figure 5G
Figure 6A
Figure 6B
statistics for Figure 6B
Figure 7A
statistics for Figure 7A
Figure 7B
Figure 7C
Figure 7D
statistics for Figure 7B and 7D
Figure 8A
statistics for Figure 8A
Figure 8B
statistics for Figure 8B
Graphical Abstract