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Experimental Cell Research

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Research Article

Vitamin A and insulin are required for the maintenance of hepatic stellate cell
quiescence
Akihiro Yoneda a,n, Kaori Sakai-Sawada a, Yoshiro Niitsu b, Yasuaki Tamura a
a
Department of Molecular Therapeutics, Center for Food & Medical Innovation, Institute for the Promotion of Business-Regional Collaboration, Hokkaido University, West-11, North-
21, Kita-ku, Sapporo 001-0021, Hokkaido, Japan
b
Department of Molecular Target Exploration, School of Medicine, Sapporo Medical University, Japan

ar t ic l e i nf o a b s t r a c t

Article history: Transdifferentiation of vitamin A-storing hepatic stellate cells (HSCs) to vitamin A-depleted myofibro-
Received 4 August 2015 blastic cells leads to liver fibrosis. Vitamin A regulates lipid accumulation and gene transcription, sug-
Received in revised form gesting that vitamin A is involved in the maintenance of HSC quiescence under a physiological condition.
22 January 2016
However, the precise mechanism remains elusive because there is no appropriate in vitro culture system
Accepted 22 January 2016
for quiescent HSCs. Here, we show that treatment of quiescent HSCs with vitamin A partially maintained
the accumulation of lipid droplets and expression of quiescent HSC markers (glial fibrillary acidic protein,
Keywords: peroxisome proliferator-activator receptor-γ and CCAAT/enhancer-binding protein-α) and also the ex-
Hepatic stellate cells pression of myofibroblastic markers (α-smooth muscle actin, heat shock protein 47 and collagen type I).
Vitamin A
On the other hand, combined treatment with vitamin A and insulin sustained the characteristic of HSC
Insulin
quiescence and completely suppressed the expression of myofibroblastic markers through activation of
HSC quiescence
the JAK2/STAT5 signaling pathway and increased expression of sterol regulatory element binding pro-
tein-1. These treated HSCs transdifferentiated to myofibroblastic cells under a culture condition with fetal
bovine serum. The results suggest an important role of vitamin A and insulin in the maintenance of HSC
quiescence under a physiological condition.
& 2016 Elsevier Inc. All rights reserved.

1. Introduction cirrhosis except for liver transplantation, a precise understanding of

Hepatic stellate cells (HSCs) are non-parenchymal cells that are
located perisinusoidally in the space of Disse, in recesses between
hepatocytes (parenchymal cells) [1,2]. HSCs comprise about 5–8% of
total rat liver cells and 1% of the total liver mass. In the liver under a
physiological condition, HSCs are present in a quiescent state and
accumulate 90–95% of total liver vitamin A as retinyl ester in lipid
droplets in their cytoplasm [3,4]. Upon injury or inflammation to
the liver, HSCs undergo a transdifferentiation from quiescent cells to
myofibroblastic cells, termed activation of HSCs. This activation of
HSCs is characterized by the loss of vitamin A in lipid droplets,
progression of cell proliferation and migration, excessive expression
of extracellular matrix proteins including collagen type I, and in-
creased expression of α-smooth muscle actin (α-SMA) [2, 5–8].
Because excessive deposition of extracellular matrix proteins se-
creted by activated HSCs leads to liver fibrosis and cirrhosis, acti-
vated HSCs are thought to be the major contributor to liver fibrosis.
However, since no curative medical treatments are available for
n
Corresponding author.
E-mail address: ayoneda@fmi.hokudai.ac.jp (A. Yoneda).
the characteristics of HSCs is a prerequisite for the development of
specific therapeutic modalities for liver cirrhosis.
Quiescent HSCs are characterized by accumulation of vitamin A
in lipid droplets and expression of glial fibrillary acidic protein
(GFAP) [2]. It has been demonstrated that the activities of adipo-
genic transcription factors such as peroxisome proliferator-acti-
vator receptor-γ (PPAR-γ) and CCAAT/enhancer-binding protein-α
(C/EBP-α) are required for the maintenance of HSC quiescence [9–
11]. Expression levels of PPAR-γ and C/EBP-α proteins are mark-
edly reduced by the activation of HSCs. It has been shown that
treatment with a synthetic ligand of PPAR-γ and overexpression of
PPAR-γ by an adenoviral vector reverse the morphological and
biochemical characteristics of activated HSCs to those of quiescent
HSCs in vivo and in vitro [12,13]. Overexpression of C/EBP-α has
also been reported to inhibit the activation of HSCs, and its in vivo
expression lessens carbon tetrachloride (CCl4)-induced hepatic fi-
brosis in mice [14]. Furthermore, treatment of activated HSCs with
an adipogenic differentiation mixture (isobutylmethylxanthine,
dexamethasone, and insulin) induces up-regulation of PPAR-γ and
C/EBP-α expression and an increase in the accumulation of lipid
droplets [11]. Although these findings suggest that the expression

http://dx.doi.org/10.1016/j.yexcr.2016.01.012
0014-4827/& 2016 Elsevier Inc. All rights reserved.

Please cite this article as: A. Yoneda, et al., Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence,
Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.01.012i
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of GFAP, PPAR-γ and C/EBP-α is important for the maintenance of
HSC quiescence, the molecular mechanism by which the expres-
sion of these genes is induced remains unclear.
Vitamin A regulates multiple physiological activities, such as
vision, reproduction, morphogenesis, cell proliferation and cell
differentiation [15–17]. Vitamin A acquired from the diet binds to
retinol-binding protein (RBP) in the bloodstream, and it is taken
up initially by liver cells, transferred to HSCs via the RBP receptor
and stored in cytoplasmic lipid droplets [18–21]. It has been de-
monstrated that vitamin A induced the accumulation of lipid
droplets and the expression of GFAP and PPAR-γ in activated HSCs
and adipocytes [22–24]. Furthermore, treatment with vitamin A
has been reported to ameliorate liver fibrosis in bile duct-ligated
and CCl4-induced model rats [25], suggesting that vitamin A
achieves the phenotypic reversal of activated HSCs to quiescent
cells. Despite extensive studies on the transdifferentiation of
quiescent HSCs and reversal of activated HSCs to quiescent HSCs,
the mechanism by which quiescent HSCs are maintained in the
liver under a physiological condition remains elusive because
there is no appropriate in vitro culture system. In the present
study, we investigated whether vitamin A regulates the expression
of quiescent HSC-related genes in quiescent HSCs under a culture
condition with serum replacement (heat-inactivated bovine serum
albumin, transferrin, and insulin). We also examined the effect of
the adipogenic differentiation factor insulin on the maintenance of
HSC quiescence induced by vitamin A.
2. Materials and methods
2.1. Materials
Vitamin A, serum replacement (SR) and insulin were purchased
from Sigma-Aldrich (St. Louis, MO). An antibody against GFAP was
purchased from DAKO (Glostrup, Denmark), and an antibody
against α-SMA was obtained from Sigma-Aldrich. Antibodies
against heat shock protein 47 (Hsp47), collagen type 1, PPAR-γ, C/
EBP-α, JAK2, phospho-JAK2, STAT5, phospho-STAT5, sterol reg-
ulatory element binding protein-1 (SREBP-1) and GAPDH were
obtained from Abcam (Cambridge, UK). An antibody against bro-
modeoxyuridine (BrdU) was from Medical & Biological Labora-
tories (Nagoya, Japan).
2.2. Animals
Sprague-Dawley male rats (300–400 g in body weight) were
purchased from Charles River Laboratories Japan (Yokohama, Ja-
pan). The feeding, maintenance, and use of the animals were
performed in accordance with the guidelines of the Experimental
Animal Ethics Committee of Hokkaido University.
2.3. Isolation of hepatic stellate cells
HSCs were isolated from the livers of Sprague-Dawley male rats
by the method using enzymatic digestion of liver tissue and were
enriched by density gradient centrifugation as described pre-
viously [26]. HSCs were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, Sigma-Aldrich) containing 10% fetal bovine ser-
um (FBS, Hyclone Laboratories, Ins., South Logan, UT), 100 U/ml
penicillin and 100 μg/ml streptomycin at 37 °C under 5% CO2 in air.
Cells cultured for 24 h were used as quiescent HSCs and cells
cultured for 168 h (7 days) were used as activated HSCs.
Quiescent HSCs were washed three times with serum-free
DMEM and cultured in DMEM supplemented with or without 10%
FBS, SR, 10 μM vitamin A and 50 ng/ml insulin for 7 days at 37 °C
under 5% CO2 in air.
Please cite this article as: A. Yoneda, et al., Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence,
Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.01.012i
2.4. Oil red O staining and immunostaining
To verify the presence of lipid droplets in HSCs, HSCs were
stained with Oil Red O (Sigma-Aldrich). Briefly, HSCs cultured on
glass coverslips were fixed with 4% paraformaldehyde (PFA) for
30 min. The fixed cells were rinsed with 100% propylene glycol. Cells
were stained with 0.7% Oil Red O in propylene glycol for 15 min at
room temperature. After washing with 85% propylene glycol, the
cells were counterstained with hematoxilin. Accumulation of lipid
droplets in HSCs was quantified by BioPix iQ 2.1.8 software.
Expression of GFAP and α-SMA was visualized by using im-
munofluorescent staining. Briefly, cells cultured on glass coverslips
were fixed with 4% PFA for 30 min and permeabilized with 0.5%
Triton X-100 in phosphate buffer saline (PBS) for 3 min at room
temperature. After blocking with 5% goat serum in PBS for 60 min,
antibodies against GFAP and α-SMA in 1% bovine serum albumin
(BSA)-PBS were added and incubated for 60 min. After washing
three times with PBS, the cells were incubated with 1% BSA-PBS
containing Alexa488 or Alexa555-conjugated secondary anti-
bodies for 60 min at room temperature. Prolong Gold Antifade
reagent with DAPI (Invitrogen) was used as a mounting medium.
2.5. Western blot analysis
HSCs were then washed with PBS and were lysed with lysis
buffer. Equal amounts of proteins were separated by SDS-PAGE
and transferred to polyvinylidene difluoride membranes (Milli-
pore, Billerica, MA). The membranes were probed with antibodies
against GFAP, PPAR-γ, C/EBP-α, α-SMA, heat shock protein 47
(Hsp47), collagen type I, and GAPDH followed by incubation with a
horseradish peroxidase-conjugated secondary antibody (Cell Sig-
naling Technology, Danvers, MA). Proteins were detected by a
chemiluminescent method using ECL (GE Healthcare, Waukesha,
WI). Expression levels of Hsp47 and collagen type I proteins were
determined by Image Lab Version 5.2 software (Bio-Rad Labora-
tories, Hercules, CA).
2.6. Cell proliferation assay
HSCs were cultured in a culture medium containing 10 μM
BrdU (Sigma-Aldrich) for 24 h at 37 °C under 5% CO2 in air. The
cells were fixed with 4% PFA for 30 min and permeabilized with
2N HCl in PBS for 10 min at room temperature. After blocking with
5% goat serum in PBS for 60 min, antibodies against BrdU in 1%
BSA-PBS were added and incubated for 60 min. After washing
three times with PBS, the cells were incubated with 1% BSA-PBS
containing secondary antibodies for 60 min at room temperature.
Prolong Gold Antifade reagent with DAPI was used as a mounting
medium.
2.7. Statistical analysis
All data are shown as means 7 SEM. Differences between
groups were tested for statistical significance using Student's t-test
or analysis of variance (ANOVA). Statistical significance was de-
termined at P o0.05.
3. Results
3.1. Effect of vitamin A on accumulation of lipid droplets in rat HSCs
We first examined the effect of vitamin A on accumulation of
lipid droplets in quiescent rat HSCs cultured in DMEM supple-
mented with or without 10% FBS and vitamin A. As shown in
Fig. 1A and B, the morphologic feature of quiescent HSCs was
 A. Yoneda et al. / Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

Fig. 1. Accumulation of lipid droplets in quiescent HSCs treated with vitamin A. (A) Quiescent HSCs (qHSCs) were cultured in DMEM supplemented with 10% FBS and with or
without vitamin A at concentrations of 1, 5, 10 and 50 μM for 168 h. Accumulation of lipid droplets was determined by Oil Red O staining. Representative images are shown.
Scale bars: 100 μM. (B) Quantification of lipid droplets in HSCs treated with vitamin A. Area (μm2) of lipid droplets in HSCs was quantified by BioPix iQ 2.1.8 software. *
Po 0.05. n.s: not significant.

transformed to that of activated HSCs and lipid droplets dis-
appeared with the culture in DMEM containing 10% FBS. On the
other hand, the morphological feature of quiescent HSCs was
converted to that of activated HSCs and some of the lipid droplets
were stored with culture in DMEM supplemented with 10% FBS
and vitamin A at each concentration of 1, 5, 10 or 50 μM. Accu-
mulation of lipid droplets in HSCs treated with 10 μM vitamin A
was not significantly different from that in HSCs treated with
50 μM vitamin A (Fig. 1B), and we therefore used vitamin A at a
concentration of 10 μM for the further studies.

Fig. 2. Accumulation of lipid droplets in quiescent HSCs treated with serum replacement and vitamin A. (A) Quiescent HSCs (qHSCs) were cultured in DMEM supplemented
with or without 10% FBS, serum replacement (SR), and 10 μM vitamin A for 168 h. Accumulation of lipid droplets was determined by Oil Red O staining. Representative
images are shown. Scale bars: 100 μM. (B) Quantification of lipid droplets in HSCs treated with SR and vitamin A. Area (μm2) of lipid droplets in HSCs was quantified by BioPix
iQ 2.1.8 software. * P o0.05. n.s: not significant.

Please cite this article as: A. Yoneda, et al., Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence,
Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.01.012i
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To remove the effects of growth factors and cytokines in FBS on
the morphological change of quiescent HSCs, the cells were cul-
tured in serum-free DMEM with or without SR (heat-inactivated
BSA, transferrin, and insulin) and 10 μM vitamin A. When quies-
cent HSCs were cultured in serum-free DMEM supplemented with
or without vitamin A, lipid droplets were not observed, though the
morphological feature of the cells was similar to that of quiescent
HSCs (Fig. 2A and B). On the other hand, the morphological feature
of quiescent HSCs and the accumulation of lipid droplets were
sustained by treatment with DMEM supplemented with SR and
vitamin A. These results suggested that factors other than vitamin
A are required for the accumulation of lipid droplets in HSCs.
3.2. Expression of quiescent HSC-related genes in rat HSCs treated
with vitamin A
We next examined the effect of vitamin A on expression of
quiescent HSC-related genes (GFAP, PPAR-γ and C/EBP-α) and an ac-
tivated HSC-related gene (α-SMA) in quiescent HSCs. As shown in
Fig. 3A, immunofluorescent staining against GFAP and α-SMA showed
the expression of GFAP in HSCs treated with 10 μM vitamin A re-
gardless of whether FBS and SR were added or not. Expression of α-
SMA was observed in HSCs cultured in DMEM with or without 10%
FBS and vitamin A. On the other hand, the expression of α-SMA was
suppressed in HSCs under the culture condition of DMEM with SR and
vitamin A. Expression patterns of GFAP and α-SMA in HSCs cultured in
DMEM with SR and vitamin A were similar to those in quiescent HSCs.
Furthermore, western blot analysis showed the expression of PPAR-γ
and C/EBP-α in quiescent HSCs treated with SR and vitamin A but not
in activated HSCs and HSCs cultured in DMEM with SR alone (Fig. 3B).
3.3. Expression of heat shock protein 47 and collagen type I in HSCs
treated with vitamin A
Since activated HSCs are well known to express and deposit
collagen type I, expression of the collagen-specific chaperone heat
shock protein 47 (Hsp47) and collagen type I was examined in HSCs
cultured in DMEM with or without 10% FBS, SR and vitamin A. As
shown in Fig. 3C and D, western blot analysis revealed that there
was no significant difference in the expression levels of Hsp47 in
HSCs cultured in DMEM with or without 10% FBS and vitamin A.
The expression level of Hsp47 in HSCs cultured in DMEM supple-
mented with SR was also similar to that in HSCs cultured in DMEM
with 10% FBS. On the other hand, the expression level of Hsp47 in
HSCs cultured in DMEM with SR and vitamin A was significantly
lower than the expression level with 10% FBS and SR, which was
similar to that in quiescent HSCs (Fig. 3C and D).
Expression levels of collagen type I were not significantly dif-
ferent in HSCs cultured in DMEM containing 10% FBS in the pre-
sence and absence of vitamin A (Fig. 3C and E). In HSCs cultured in

Fig. 3. Effect of vitamin A on the maintenance of HSC quiescence. (A) Quiescent HSCs (qHSCs) were cultured for 168 h in DMEM supplemented with or without 10% FBS,
serum replacement (SR) and 10 μM vitamin A. Expression of GFAP and α-SMA was examined by immunofluorescent staining. Representative images are shown. Scale bars:
100 μM. (B-E) Expression of PPAR-γ and C/EBP-α proteins was detected by western blotting (B). Expression of GFAP, α-SMA, Hsp47, and collagen type I proteins was
determined by western blotting (C). Expression levels of Hsp47 (D) and collagen type I (E) proteins were quantified. * P o 0.05. n.s: not significant.

Please cite this article as: A. Yoneda, et al., Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence,
Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.01.012i
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Fig. 4. Quiescent HSCs treated with serum replacement and vitamin A are transdifferentiated by treatment with exogenous stimuli. (A) Quiescent HSCs (qHSCs) were
cultured in DMEM supplemented with or without 10% FBS, serum replacement (SR), and 10 μM vitamin A for 168 h (7 days). HSCs cultured in DMEM supplemented with SR
and vitamin A were re-cultured in DMEM containing 10% FBS for 120 h (5 days). Expression of GFAP and α-SMA was evaluated by immunofluorescent staining. Re-
presentative images are shown. Scale bars: 100 μM. (B) Expression of collagen type I was determined by western blotting. (C) Quiescent HSCs were cultured in DMEM
supplemented with or without 10% FBS, SR and 10 μM vitamin A for 7 days. Cell proliferation was examined by the incorporation of BrdU. * Po 0.05. n.s: not significant.

serum-free DMEM supplemented with or without vitamin A and
in DMEM with SR, the expression level of collagen type I was
lower than that in HSCs cultured in DMEM containing 10% FBS.
Furthermore, expression of collagen type I in HSCs cultured in
DMEM with SR and vitamin A was slightly detected, and the ex-
pression level was similar to that in quiescent HSCs.
A was increased by treatment with DMEM containing 10% FBS for
5 days, similar to that of HSCs cultured in DMEM containing 10%
FBS.
3.5. Effects of vitamin A and insulin on accumulation of lipid droplets
in quiescent HSCs

3.4. Transdifferentiation of HSCs treated with serum replacement
and vitamin A
We next investigated whether quiescent HSCs treated with SR
and vitamin A were transdifferentiated to activated HSCs by exo-
genous stimuli. As shown in Fig. 4A, expression of GFAP detected
in HSCs treated with SR and vitamin A disappeared when the cells
were treated with DMEM containing 10% FBS for 5 days, while the
expression of α-SMA was detected. Furthermore, western blot
analysis showed that expression of collagen type I in HSCs treated
with DMEM containing SR and vitamin A was increased by treat-
ment with DMEM containing 10% FBS for 5 days (Fig. 4B).
Proliferation of activated HSCs was previously reported to be
stimulated by signal transduction of integrin-collagen interaction
[26,27]. Proliferation of HSCs cultured in DMEM with SR and vi-
tamin A was examined. As shown in Fig. 4C, the proliferation rate
of HSCs cultured in DMEM supplemented with SR was lower than
that of HSCs cultured in DMEM containing 10% FBS regardless of
the presence or absence of vitamin A. On the other hand, the
proliferation rate of HSCs cultured in DMEM with SR and vitamin
We clarified the effects of vitamin A and the adipogenic dif-
ferentiation factor insulin on the accumulation of lipid droplets in
quiescent HSCs. As shown in Fig. 5A and B, the morphological
feature of HSCs cultured in serum-free DMEM was similar to that
of quiescent HSCs, but accumulation of lipid droplets in HSCs was
not detected, regardless of the culture condition with or without
10 μM vitamin A or 50 ng/ml insulin. On the other hand, combined
treatment of quiescent HSCs with vitamin A and insulin sustained
the morphological feature, and the increase of lipid droplet con-
tent in HSCs was dependent on the concentration of insulin (5, 10
and 50 ng/ml).
3.6. Expression of quiescent HSC-related genes in HSCs treated with
vitamin A and insulin
The expression of quiescent HSC-related genes (GFAP, PPAR-γ
and C/EBP-α) and activated HSC-related genes (α-SMA, Hsp47, and
collagen type I) in quiescent HSCs was determined when the HSCs
were cultured in serum-free DMEM supplemented with or with-
out 10 μM vitamin A and 50 ng/ml insulin. As shown in Fig. 6A,

Please cite this article as: A. Yoneda, et al., Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence,
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Fig. 5. Accumulation of lipid droplets in quiescent HSCs treated with both vitamin A and insulin. (A) Quiescent HSCs (qHSCs) were cultured for 168 h in serum-free DMEM
supplemented with or without vitamin A (10 μM) and insulin (5, 10 and 50 ng/ml). Accumulation of lipid droplets was determined by Oil Red O staining. Representative
images are shown. Scale bars: 100 μM. (B) Quantification of lipid droplets in HSCs treated with vitamin A and insulin. Area (μm2) of lipid droplets in HSCs was quantified by
BioPix iQ 2.1.8 software. * P o 0.05. n.s: not significant.

immunofluorescent staining against GFAP and α-SMA showed the
expression of GFAP in HSCs cultured in serum-free DMEM sup-
plemented with vitamin A or insulin, but expression of α-SMA was
also observed. Expression of GFAP, but not that of α-SMA, was also
observed in HSCs cultured in serum-free DMEM supplemented
with both vitamin A and insulin, being similar to their expression
pattern in quiescent HSCs. Western blot analysis also revealed the
expression of PPAR-γ and C/EBP-α in HSCs cultured under a con-
dition with both vitamin A and insulin (Fig. 6B). Furthermore, as
shown in Fig. 6C, expression levels of Hsp47 and collagen type I
were not significantly different in HSCs cultured in serum-free
DMEM with and without vitamin A and insulin treatment.
3.7. Transdifferentiation of HSCs treated with vitamin A and insulin
We next examined whether quiescent HSCs treated with both
vitamin A and insulin were transdifferentiated to activated HSCs
by exogenous stimuli. As shown in Fig. 7A, immunofluorescent
staining revealed that expression of GFAP in HSCs cultured in
DMEM with both vitamin A and insulin disappeared when the
cells were treated with DMEM containing 10% FBS for 5 days,
while expression of α-SMA was observed. Furthermore, western
blotting revealed that the expression of collagen type I in HSCs
treated with both vitamin A and insulin was induced by treatment
with DMEM containing 10% FBS for 5 days (Fig. 7B).
Proliferation of HSCs cultured in DMEM with vitamin A and
insulin was examined. As shown in Fig. 7C, the proliferation rate of
HSCs cultured in serum-free DMEM was lower than that of HSCs
cultured in DMEM containing 10% FBS regardless of whether both
vitamin A and insulin were added or not. On the other hand, the
proliferation rate of HSCs treated with both vitamin A and insulin
was increased by treatment with DMEM containing 10% FBS for
5 days, being similar to that of activated HSCs.
3.8. Activation of JAK2/STAT5 and expression of SREBP-1 in HSCs
treated with vitamin A and insulin
We investigated phosphorylation of Janus kinase 2 (JAK2) and
signal transducer and activator of transcription 5 (STAT5) in HSCs
cultured in DMEM supplemented with 10 μM vitamin A and
50 ng/ml insulin. As shown in Fig. 8A, phosphorylation of JAK2 and
STAT5 was not detected in HSCs cultured in DMEM with 10% FBS
(activated HSCs). On the other hand, phosphorylation of JAK2 and
STAT5 in HSCs treated with both vitamin A and insulin was
maintained similar to that in quiescent HSCs. Furthermore, we
examined the expression of sterol regulatory element binding
protein-1 (SREBP-1) in HSCs cultured in DMEM supplemented
with 10 μM vitamin A and 50 ng/ml insulin. Western blotting
showed that the expression level of SREBP-1 in HSCs cultured in
DMEM supplemented with vitamin A and insulin was similar to
that in quiescent HSCs (Fig. 8B).
4. Discussion
Quiescent HSCs are generally characterized by accumulation of
lipid droplets and expression of GFAP and adipogenic transcription
factors PPAR-γ and C/EBP-α. Upon injury or inflammation to the li-
ver, transdifferentiation of HSCs from the quiescent phenotype to
activated phenotype is coincident with the disappearance of lipid

Please cite this article as: A. Yoneda, et al., Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence,
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Fig. 6. Effects of vitamin A and insulin on the maintenance of HSC quiescence. (A) Quiescent HSCs (qHSCs) were cultured for 168 h in DMEM supplemented with or without
10% FBS, 10 μM vitamin A and 50 ng/ml insulin. Expression of GFAP and α-SMA was examined by immunofluorescent staining. Representative images are shown. Scale bars:
100 μM. (B) Expression of GFAP, PPAR-γ and C/EBP-α proteins was detected by western blotting. (C) Expression of Hsp47 and collagen type I proteins was determined by
western blotting.

droplets and reduction of GFAP, PPAR-γ and C/EBP-α expression and
with induction of the expression of α-SMA and collagen type I.
Forced expression of PPAR-γ and C/EBP-α in activated HSCs has been
demonstrated to result in the reappearance of morphologic features
of quiescent HSCs and inhibition of functional parameters for HSC
activation such as increased DNA synthesis and expression of α-
SMA, collagen type I and TGF-β [9–11]. Despite these findings sug-
gesting that the accumulation of lipid droplets and the expression of
GFAP, PPAR-γ and C/EBP-α are required for maintenance of the
quiescent phenotype of HSCs, factors that induce these events have
not been determined. In the present study, accumulation of lipid
droplets and expression of GFAP, PPAR-γ and C/EBP-α proteins in
quiescent HSCs were maintained by in vitro culture under the
condition of the culture medium containing serum replacement and
vitamin A. Combined treatment with vitamin A and insulin also
sustained accumulation of lipid droplets and expression of GFAP,
PPAR-γ and C/EBP-α proteins and suppressed the expression of α-
SMA and collagen type I and cell proliferation in quiescent HSCs.
Furthermore, quiescent HSCs cultured under the condition with both
vitamin A and insulin underwent transdifferentiation to the acti-
vated phenotype (expression of α-SMA and collagen type I and in-
creased cell proliferation) by culture with a standard medium
(DMEM containing 10% FBS). These results suggest that vitamin A
and insulin are required for the maintenance of HSC quiescence
under a physiological condition.
Insulin has been reported to be a pro-fibrotic growth factor and
to trigger activation of HSCs [28,29,30]. Upon injury or in-
flammation to the liver, the concentration of insulin is increased

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Fig. 7. Quiescent HSCs treated with vitamin A and insulin are transdifferentiated by treatment with exogenous stimuli. (A) Quiescent HSCs (qHSCs) were cultured in DMEM
supplemented with or without 10% FBS, 10 μM vitamin A and 50 ng/ml insulin for 168 h (7 days). HSCs treated with vitamin A and insulin were further cultured in DMEM
containing 10% FBS for 120 h (5 days). Expression of GFAP and α-SMA was evaluated by immunofluorescent staining. Representative images are shown. Scale bars: 100 μM.
(B) Expression of collagen type I was determined by western blotting. (C) Quiescent HSCs were cultured in DMEM supplemented with or without 10% FBS, 10 μM vitamin A
and 50 ng/ml insulin for 7 days. Cell proliferation was examined by the incorporation of BrdU. * Po 0.05. n.s: not significant.

compared to that in the liver under a physiological condition,
while the concentration of vitamin A is markedly decreased
compared to that under a physiological condition [31,32,33,34]. It
has been reported that HSCs express patatin-like phospholipase
domain-containing protein 3 in response to insulin, leading to
extracellular release of vitamin A [35,36]. In the present study,
expression of quiescent HSC markers (PPAR-γ and C/EBP-α) was
not sustained and expression of activated HSC markers (α-SMA
and Hsp47) was not suppressed by treatment with vitamin A alone
or with insulin alone. On the other hand, combined treatment of
vitamin A with insulin maintained HSC quiescence and inhibited
the transdifferentiation to activated HSCs, suggesting that the
presence of both vitamin A and insulin is important for the
maintenance of HSC quiescence.
Treatment with vitamin A has been demonstrated to induce the
expression of PPAR-γ through the JAK2/STAT5 signaling pathway,
leading to accumulation of lipid droplets [20,24]. All-trans-retinoic
acid, which is synthesized by the oxidation of vitamin A with re-
tinol dehydrogenases and retinaldehyde dehydrogenases, is gen-
erally considered to be an inducer of GFAP expression, which has
been reported to be mediated by activation of the retinoic acid
receptor (RAR)α and RARγ [37,38]. All-trans-retinoic acid inhibits
the expression of procollagen I, III and IV, fibronectin, laminin and
α-SMA in activated HSCs [39,40]. Treatment with insulin has been
reported to regulate the expression of GFAP in astroglial cells [41].
Furthermore, insulin stimulation of preadipocytes increases the
expression of SREBP-1, leading to expression of PPAR-γ and in-
hibition of α-SMA and collagen type 1 expression [42,43,44]. The
present study demonstrated that combined treatment with vita-
min A and insulin maintained phosphorylation of JAK2/STAT5 and
expression of SREBP-1 in HSCs. Furthermore, the accumulation of
lipid droplets and expression of GFAP, PPAR-γ and C/EBP-α and
inhibition of the transdifferentiation to activated HSCs were sus-
tained by combined treatment of vitamin A with insulin, sug-
gesting that the quiescence of HSCs is regulated by both the vi-
tamin A/JAK2/STAT5 signaling pathway and insulin/SREBP-1 sig-
naling pathway.
Numerous studies have been carried out to elucidate the mo-
lecular mechanism by which HSCs undergo transdifferentiation
from the quiescent phenotype to activated phenotype [2]. The
reversal of activated HSCs to quiescent HSCs has also been in-
vestigated since activated HSCs are thought to be a major con-
tributor to liver fibrosis and cirrhosis [10,22,45,46]. A culture
condition supplemented with FBS has been mainly used for the in

Please cite this article as: A. Yoneda, et al., Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence,
Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.01.012i
 A. Yoneda et al. / Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 8. Phosphorylation of JAK2/STAT5 and expression of SREBP1 in HSCs treated with vitamin A and insulin. (A) Phosphorylation of JAK2 and STAT5 in HSCs cultured in
DMEM supplemented with 10 μM vitamin A and 50 ng/ml insulin for 168 h was examined by western blotting. (B) Expression of SREBP1 in HSCs cultured with 10 μM vitamin
A and 50 ng/ml insulin was determined by western blotting.
vitro culture system of HSCs since there has been no appropriate
in vitro culture system for quiescent HSCs. However, these culture
conditions spontaneously trigger transdifferentiation of quiescent
HSCs to activated HSCs because inducers and stimulators such as
TGF-β for the activation of HSCs are contained in FBS. In the pre-
sent study, combined treatment with vitamin A and insulin
maintained HSC quiescence during the indicated in vitro culture
period, and this treatment may be a powerful tool for analysis of
the molecular mechanism of HSC quiescence.
In conclusion, the present study demonstrated that treatment
of quiescent HSCs with vitamin A maintained the quiescence of
HSCs in combination with insulin and that HSCs treated with both
vitamin A and insulin underwent transdifferentiation to myofi-
bloblastic cells under a culture condition with FBS.
Conflict of interest
The authors have no conflict of interest.
Acknowledgment
Studies described in this manuscript were supported by Grants
from NITTO DENKO Corporation.
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