Eucommia ulmoides Cortex, Geniposide and Aucubin Regulate Lipotoxicity through the Inhibition of Lysosomal BAX
Geniposide and Aucubin Regulate Lipotoxicity through the
Inhibition of Lysosomal BAX. PLoS ONE 9(2): e88017. doi:10.1371/journal.pone.0088017
Eucommia ulmoides Cortex, Geniposide and Aucubin Regulate Lipotoxicity through the Inhibition of Lysosomal BAX
Geum-Hwa Lee 0
Mi-Rin Lee 0
Hwa-Young Lee 0
Seung Hyun Kim 0
Hye-Kyung Kim 0
Hyung-Ryong Kim 0
Han-Jung Chae 0
Giovanni Li Volti, University of Catania, Italy
0 1 Department of Pharmacology and Cardiovascular Research Institute, Medical School, Chonbuk National University , Jeonju , Republic of Korea, 2 College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University , Incheon , Korea , 3 Department of Dental Pharmacology and Wonkwang Dental Research Institute, School of Dentistry, Wonkwang University , Iksan , Republic of Korea
In this study we examined the inhibition of hepatic dyslipidemia by Eucommia ulmoides extract (EUE). Using a screening assay for BAX inhibition we determined that EUE regulates BAX-induced cell death. Among various cell death stimuli tested EUE regulated palmitate-induced cell death, which involves lysosomal BAX translocation. EUE rescued palmitate-induced inhibition of lysosomal V-ATPase, a-galactosidase, a-mannosidase, and acid phosphatase, and this effect was reversed by bafilomycin, a lysosomal V-ATPase inhibitor. The active components of EUE, aucubin and geniposide, showed similar inhibition of palmitate-induced cell death to that of EUE through enhancement of lysosome activity. Consistent with these in vitro findings, EUE inhibited the dyslipidemic condition in a high-fat diet animal model by regulating the lysosomal localization of BAX. This study demonstrates that EUE regulates lipotoxicity through a novel mechanism of enhanced lysosomal activity leading to the regulation of lysosomal BAX activation and cell death. Our findings further indicate that geniposide and aucubin, active components of EUE, may be therapeutic candidates for non-alcoholic fatty liver disease.
Funding: This study was supported by the National Research Foundation (2012R1A2A1A03001907 and 2008-0062279). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Non-alcoholic fatty liver disease (NAFLD) is an increasingly
recognized form of chronic liver disease that can progress to
endstage liver disease [1,2]. A net retention of lipids within
hepatocytes, mostly in the form of triglycerides, is a prerequisite
for the development of NAFLD . Proposed mechanisms for
cellular dysfunction include increased production of reactive
oxygen species (ROS), de novo ceramide biosynthesis, nitric oxide
generation, and caspase activation. It has also been reported that
free fatty acids in hepatocytes lead to the translocation of BAX to
lysosomes, lysosomal destabilization, and the resultant expression
of nuclear factor-kappa B-dependent tumor necrosis factor-alpha
. Separately, it was reported that saturated free fatty acids
induce mitochondrial dysfunction and increased ROS production
downstream of lysosomal permeabilization and the resultant
cathepsin B release in both human and murine hepatocytes .
However, the pathogenesis of NAFLD, and in particular the
mechanisms responsible for liver injury and disease progression,
remain poorly understood.
Eucommoia ulmoides is a species of small tree native to China. It
belongs to the genus Eucommia, the only genus of the family
Eucommiaceae. E. ulmoides has been used in traditional oriental
medicine to improve the tone of the liver and kidney, increase
longevity, and reduce blood pressure . More recently, E.
ulmoides cortex extract has been widely used to improve liver
steatosis and has become considered a functional health food [6
8]]. E. ulmoides has been reported to contain polyphenolics,
flavonoids, and triterpines as its chemical constituents .
Flavonol glycosides present in this plant including quercetin and
kaempferol have been reported to possess glycation inhibitory
activity and prevent diabetes . Recently, a controlled pilot study
has also shown efficacy of a herbal mixture containing E. ulmoides
by demonstrating its regulatory effect on alanine aminotransferase
(ALT) in patients with non-alcoholic steatohepatitis (NASH) .
However, the mechanism by which E. ulmoides extract affects liver
physiopathologic status needs to be studied.
To clarify how the Eucommia ulmoides extract (EUE) regulates the
NAFLD condition we examined the effects of EUE on
palmitateinduced cell death through the regulation of BAX and related
cathepsin B-induced cell death in hepatic cells, an in vitro lipotoxicity
model. EUE was also applied to an animal high-fat diet model to
determine its regulatory effects on pathologic phenomena including
lipid accumulation, lipid peroxidation, and tissue damage.
Korea). We followed an ethanol extraction method in the
preparation of E. ulmoides Oliver extracts, as described previously
. Briefly, E. ulmoides Oliver cortex (100 g) was treated with
80% ethanol (1 g in 8 ml) and boiled twice under reflux for 1 h.
After filtration, the supernatant was vacuum dried at 50uC,
redissolved in distilled water, filtered, and vacuum-dried at 50uC.
After freeze-drying, the final E. ulmoides Oliver extracts were stored
For animal experiments, E. ulmoides cortex was purchased from
SAMHONG HANYAKJAE (Seoul, Korea). Voucher specimens
(YP-001) documenting these collections have been deposited in the
College of Pharmacy, Yonsei University. The powdered sample
was weighed and extracted with 25% ethanol and water,
respectively, for 2 hours at 90uC using a reflux. After
freezedrying, the final EUE was stored at 4uC. The pulverized extract
was kept at 4uC.
Phosphate-buffered saline (PBS) was purchased from Invitrogen
and trypan blue was purchased from Sigma (St Louis, MO).
Geniposide was from Sigma. Aucubin was isolated from EUE by
serial chromatographic separations using silica gel column
chromatography and semi-preparative HPLC. All other chemicals
were purchased from Sigma. The purity of all reagents was at least
Cell Culture and Viability Analysis
Human hepatocellular carcinoma (HepG2) cells were cultured
in Dulbeccos modified eagle medium (DMEM) (Invitrogen,
Carlsbad, CA) with 10% fetal bovine serum (Invitrogen) and
penicillin-streptomycin (Invitrogen). HepG2 cells were cultured
with or without various agents, including EUE as indicated, and
cell viability was assessed by trypan blue dye exclusion using a
Animal Care and Treatment
Female Sprague-Dawley rats weighing 250270 g were
obtained from Damul Science Co (Daejeon, Korea). Rats were
maintained on a 12 h:12 h light:dark cycle (lights on at 06:00) in
stainless steel wire-bottomed cages and acclimated under
laboratory conditions for at least 1 week before experiments. The control
group (n = 10) was fed a standard diet, whereas the high-fat diet
(HFD) group (n = 12) was fed a calorie-rich diet of 1% cholesterol,
18% lipid (lard), 40% sucrose, 1% AIN-93G vitamins, and 19%
casein, with the same fiber and minerals as the control groups
diet. Rats in the EUE-25 groups (n = 10) were fed 0.25, 0.5, or
1 g/kg EUE-25 with the HFD. Experiments were terminated after
10 weeks. Rats were anaesthetized with diethyl ether (Sigma) and
cervical dislocation was performed. Tissues were immediately
harvested as needed for each experiment. First, whole blood was
obtained by cardiac puncture, immediately placed on ice in a
1.5mL centrifuge tube for 15 to 30 minutes, and then centrifuged at
8,000 rpm for 10 minutes. Serum was transferred to a fresh
1.5mL centrifuge tube and stored at 280uC before measurement of
cholesterol, triglyceride, and liver lipid content. Next, liver tissues
were harvested, immediately placed in liquid nitrogen, and stored
at 280uC. Tissues were homogenized for Western blotting. All
animal procedures for this study were approved by the
Institutional Animal Care and Use Committee of Chonbuk National
University laboratory animal center (IACUC, CBU 2013-0018)
and all efforts were made to minimize animal suffering.
For immunoblotting, cells or tissues were lysed on ice by direct
addition of RIPA lysis buffer [50 mM TrisHCl (pH 7.4),
150 mM NaCl, 0.25% sodium deoxycholate, 1% NP40, 1 mM
ethylenediaminetetraacetic acid (EDTA), 0.1% sodium dodecyl
sulfate (SDS)] plus protease inhibitor cocktail set III and
phosphatase inhibitor cocktail set II (EMD Biosciences, La Jolla,
CA, USA). Lysates were then transferred to microtubes and
incubated for 30 min on ice, followed by centrifugation at
12,000 rpm for 10 min at 4uC, and supernatants were collected
to obtain protein extracts. Protein extracts were added to sample
buffer, boiled in a water bath for 5 min, and stored at 20uC until
use. Ten micrograms of extracted proteins were run on a
polyacrylamide gel and transferred to a nitrocellulose membrane,
which was blocked with Tris-buffered saline (TBS) solution
containing 0.05% Tween-20 for 30 min at room temperature.
Rabbit anti-Bax, rabbit anti-hsp60, rabbit anti-caspase-3, rabbit
anti-caspase-9, mouse anti-Bid, rabbit anti-PDI, and rabbit
anti-atubulin were purchased from Cell Signaling Technologies, Inc.
(Danvers, MA). Mouse anti-Lamp-1, rabbit anti-COX II, rabbit
anti-cathepsin B, and mouse anti-b-actin antibodies were obtained
from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA). The blots
were probed overnight at 4uC with antibodies, washed, and
probed again with species-specific secondary antibodies coupled to
horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA).
Chemiluminescence reagents (GE Healthcare) were used for
Measurement of Caspase-3 Activity
For the detection of caspase-3 activity, PBS-washed cell pellets
were resuspended in extract buffer [25 mM HEPES (pH7.4), 0.1%
Triton X-l00, 10% glycerol, 5 mM DTT, protease inhibitor
cocktail] and extracts were centrifuged at 14,000 rpm at 4uC for
15 min. Soluble cytosolic protein (40 mg) was mixed with 100 mM
of the caspase-3-specific substrate Ac-DEVD-AFC
(Sigma-Aldrich) in a final volume of 100 ml and incubated at 37uC.
Caspase3 activity was measured continuously by monitoring the release of
fluorogenic AFC at 37uC. Subsequently, substrate cleavage was
monitored at 405 nm using a SPECTRAmax 340 microplate
reader and analyzed using SOFTmax PRO software (Molecular
Devices, Sunnyvale, CA, USA).
Oil Red O Staining
To measure fat accumulation in HepG2 cells, cell grown in
culture dishes were washed with cold PBS and fixed in 4%
paraformaldehyde. After two washes with 60% isopropanol,
OilRed-O agent was added with agitation for 10 min, followed by
washing in 50% isopropanol. For each dish, three images were
photographed and a representative image is shown.
For immunostaining of LAMP-1, BAX, or cathepsin B, the cells
were fixed with acetone/methanol (50:50) for 3 min at 220uC.
Fixed cells were blocked with 10% fetal bovine serum in PBS for
1 hour, incubated for 24 h at 4uC with antibody against
LAMP1 or cathepsin B at a 1:100 dilution in PBS containing 3% bovine
serum albumin, washed with PBS and incubated for an additional
hour at room temperature with Cy2-conjugated anti-rabbit IgG at
a dilution of 1:800 in PBS containing 3% bovine serum albumin.
The cells were then viewed under a fluorescence microscope and
images were acquired using an Olympus IX 70 equipped with
NanomoverH and Softworx DeltaVision (Applied Precision,
EUE for 24 hours and cell viability was analyzed (A). Cells were treated with 500 mM palmitate in the presence or absence of 100 mg/mL EUE for 0, 6,
12, 24, or 48 hours. Cell viability (B) or caspase-3 activity (C) was analyzed and immunoblotting with anti-caspase-3, caspase-9, or b-actin antibody was
performed (D). Immunoblotting of lysosome and cytosol fractions was performed with antibody against cathepsin B, LAMP-1, or tubulin (E).
Cathepsin B activity in the medium was measured (F). Cells were treated with 500 mM palmitate in the presence or absence of 100 mg/mL EUE for 24
hours. Hoechst staining (G) and cathepsin B immunostaining (H) were performed, and the diffuse staining pattern was quantitatively analyzed (H;
lower). Co-immunostaining of cathepsin B and LAMP-1 was performed (I), and the overlap of staining was quantified (right). *p,0.05, significantly
different from palmitate-treated condition, Pal.; palmitate, EUE; Eucommia ulmoides Oliver extract.
Figure 3. EUE regulates palmitate-reduced lysosomal activity. Cells were treated with 500 mM palmitate in the presence or absence of
100 mg/mL EUE for 24 hours followed by exposure to 5 mM LysoTracker and image acquisition (A). Lysosomal fluorescence was quantified (A; lower).
Lysosomal V-ATPase activity was measured as described in Materials and Methods (B). Acridine orange solution and valinomycin were added to cell
monolayers and intravesicular H+ uptake was initiated by the addition of Mg-ATP (C); fluorescence was quantified at 24 hours (C; right). Cells were
treated with 500 mM palmitate in the presence or absence of 100 mg/mL EUE for 0, 6, 12, 24, or 48 hours, and levels of a-galactosidase,
amannosidase, and acid phosphatase were measured (D). *p,0.05, significantly different from palmitate-treated condition. DIC; differential
interference contrast microscopy, Pal.; palmitate, EUE; Eucommia ulmoides Oliver extract.
Figure 4. Lysosomal V-ATPase inhibitor bafilomycin blocks the effect of EUE on lysosomal BAX location and cell death. Cells were
treated with 500 mM palmitate in the presence or absence of 100 mg/mL EUE after pretreatment with 1 mM bafilomycin for 24 hours. Lysosomal
VATPase activity was measured (A). Acridine orange solution and valinomycin were added to cell monolayers and intravesicular H+ uptake was initiated
by the addition of Mg-ATP (B); the fluorescence was quantified at 24 hours (B; right). Cell viability assay (C) and caspase-3 activity analysis (D) were
performed. Immunostaining was performed with anti-BAX or LAMP-1 antibody and the co-localized BAX was quantified as the percent of
lysosomaltranslocated BAX (E). Immunoblot analysis of lysosome fractions with antibody against BAX, t-Bid, PDI, COX II, or LAMP-1 (F). *p,0.05, significantly
different from EUE-treated condition in the presence of palmitate. Con; control, Pal.; palmitate, EUE; Eucommia ulmoides Oliver extract, Bafi;
Assessment of Lysosome Activity
LysoTracker probes have high selectivity for acidic organelles
and are effective for the labeling of live cells . HepG2 cells
were grown in a dish, rinsed with PBS, and stained with 100 nm
LysoTracker Red DND-26 (Molecular Probes, Eugene, OR) in
serum-free medium for 30 min at room temperature. Cells were
then washed with PBS. Lysosome size and staining intensity were
analyzed by fluorescence microscopy at 488 nm. The cells were
viewed and analyzed using Softworx DeltaVision software
(Applied Precision, Issaquah, WA, USA).
Isolation of Lysosomes
Lysosomes were isolated from a light mitochondrial-lysosomal
fraction in an OptiPrep density gradient (Lysosome Isolation Kit,
Sigma) according to the manufacturers instructions.
Assessment of Lysosomal V-ATPase Activity in Lysosome
For measurement of lysosomal V-ATPase activity, an acridine
orange uptake assay was performed with lysosome vesicles [12,13].
The activation buffer contained 6 mM acridine orange, 150 mM
KCl, 2 mM MgCl2, and 10 mM Bis-Tris-propane. After a steady
spectrofluorometric baseline was achieved, V-ATPase was
activated by the addition of ATP (1.4 mM final concentration) and
valinomycin (2.5 mM, pH 7.0) to promote the movement of K+
from the inside to the outside of the lysosome to generate a
membrane potential. V-ATPase-driven pumping of hydrogen ions
into lysosomes (acridine orange dye uptake) was measured by
stimulation of intralysosomal fluorescence (excited at 495 nm and
recorded at 530 nm), which was measured using a fluorescence
system (Photon Technology International). Separately, isolated
lysosomes were placed in a cuvette containing the same activation
buffer. Extralysosomal quenching of acridine orange fluorescence
was determined using a fluorescence system (Photon Technology
International). To confirm V-ATPase activity, stimulation of
intralysosomal fluorescence and the quenching of the
extralysosomal fluorescence were measured with and without 1 mM
bafilomycin, a specific inhibitor of the vacuolar type H+-ATPase.
Results are presented as mean 6 SEM from at least three
independent experiments, unless stated otherwise. MicroCal
Origin software (Northampton, MA) was used for statistical
calculations. Differences were tested for significance using
oneway analysis of variance (ANOVA) with Duncans multiple range
tests. Statistical significance was set at p,0.05.
EUE Protects against BAX-induced Cell Death in Yeast
Human BAX is not endogenously expressed in yeast. For
screening genes or materials against cell death stimuli, a human
BAX screening system has been used [14,15]. The system includes
a galactose (Gal) promoter containing the BAX promoter. For the
experiments, yeast containing YEp51-BAX were grown in
glucose-containing medium (SDMM) to repress BAX expression,
and the cells were transferred to the culture medium with galactose
(SGMM) to induce expression of BAX. The expression of BAX
was shown in Supplementary Fig. 1A (left). The functional
relevance of BAX expression was also measured. Yeast cells were
separately spread on solid media containing glucose, called
SDMM, or galactose, called SGMM, and the agar plates were
incubated for three days at 30uC. As shown in Figure S1 (right),
cell death was clearly induced on SGMM plate. Based upon a
screening assay with BAX, yeasts containing YEp51-Bax were
grown in glucose-containing medium to repress BAX expression,
then the yeasts were diluted 10-fold, and 50 mg/mL of EUE was
added to galactose-containing medium and incubated for 16
hours. EUE significantly and reproducibly protected cells against
BAX (Figure S1B). In the solid medium condition, the protective
effect of EUE was also confirmed in a dose-dependent manner
(Figure S1C), indicating EUE regulates BAX-induced cell death at
least in Yeast system.
EUE Inhibits Palmitate-induced Cell Death
Based on the results from a yeast-BAX study with EUE
(Supplementary Fig. 1), we examined the protective effects of EUE
on HepG2 cell viability after treatment with various cell death
stimuli such as etoposide, staurosporine, or palmitate. Treatment
of HepG2 cells with these agents significantly reduced cell viability
(Fig. 1A). EUE showed a significant protective effect only against
cell death induced by palmitate, which involves a lysosomal cell
death pathway . Consistent with this result, among the three
stimuli lysosomal BAX localization was observed only in the
palmitate-treated cells, and the palmitate-induced lysosomal BAX
localization was specifically inhibited by EUE (Fig. 1B). The purity
of the lysosomal fraction was confirmed by lack of expression of
the mitochondrial protein HSP60. Although EUE did not affect
the expression of BAX, it regulated the lysosomal localization of
BAX at 24 and 48 hours after treatment (Fig. 1C). The expression
of truncated BID (t-BID), a downstream molecule in the signaling
pathway of lysosomal BAX localization , in the lysosomal
fraction was also delayed by treatment with EUE. The purity of
lysosomal protein was confirmed by positive expression of the
lysosomal marker LAMP-1 and failure to detect PDI, an ER
protein, or COXII, a mitochondrial protein, on the same blots.
The regulation of lysosomal BAX was confirmed by
immunostaining with antibody to endogenous BAX. The co-localization of
BAX and LAMP-1 was clearly inhibited in the presence of EUE
(Fig. 1D). Supplementary Results.
EUE Protects against Palmitate-induced Hepatic Cell
Death through Regulation of Lysosomal Cathepsin B
Next, we analyzed the characteristics of palmitate-induced cell
death. First, we confirmed hepatic lipid accumulation in
palmitatetreated cells (Fig. 2A, left). In the presence of a high concentration
of palmitate (500 mM) cell viability was approximately 6065% at
analyzed. Immunoblotting was performed with antibody against active caspase-3, caspase-9, or b-actin (C). Cells were incubated with 500 mM
palmitate in the presence or absence of 10 mg/mL aucubin or geniposide for 0, 6, 12, 24, or 48 hours. Cell viability (D) and caspase-3 (E) activity were
analyzed. Cells were incubated with 500 mM palmitate in the presence or absence of 10 mg/mL aucubin or geniposide for 24 or 48 hours.
Immunoblotting was performed with antibody against active caspase-3, caspase-9, or b-actin (F). Cells were incubated with 500 mM palmitate in the
presence or absence of 10 mg/mL aucubin or geniposide for 24 hours and stained with Hoechst (G). Cells were incubated with 500 mM palmitate in
the presence or absence of 10 mg/mL aucubin or geniposide for 24 or 48 hours. Immunoblotting was carried out with antibody against BAX,
cathepsin B, LAMP-1, or tubulin (H). Cathepsin B activity in the medium was measured (I). Cells were incubated with 500 mM palmitate in the presence
or absence of 10 mg/mL aucubin or geniposide for 24 hours. Immunostaining was performed with anti-LAMP-1 antibody and subsequently with
anticathepsin B antibody. The degree of overlap in staining was quantified (J). *p,0.05, significantly different from palmitate-treated condition Pal.;
24 hours. EUE showed a dose-dependent (Fig. 2A, right) and
timedependent (Fig. 2B) protective effect against palmitate-induced cell
death. Consistent with these findings, caspase-3 activity was
regulated by EUE (Fig. 2C). The active forms of caspase-3 and
9 were expressed at relatively low levels in the presence of EUE,
compared with treatment with palmitate alone (Fig. 2D). Because
the localization of BAX in lysosomes induces lysosome
permeabilization and subsequent leakage of the acidic protease cathepsin,
which leads to cell death [17,18], we investigated the expression of
cathepsin B. In lysosome fractions, the expression of cathepsin B
was decreased by treatment with palmitate, but the decrease was
delayed by co-treatment with EUE (Fig. 2E). The expression of
cathepsin B in cytosol fractions was similarly regulated by EUE.
LAMP-1 and tubulin were used as lysosome and cytosol markers,
respectively . The activity of cathepsin B in the medium was
also regulated by treatment with EUE (Fig. 2F). In microscopy
studies, bright Hoechst dye staining of cells, which indicates cell
death, was also decreased by EUE treatment (Fig. 2G). A diffuse
cellular expression pattern of cathepsin B was confirmed in the
presence of palmitate, and was also regulated by EUE (Fig. 2H),
indicating regulation of lysosomal BAX translocation and the
resultant cathepsin B leakage from lysosomes. Co-localization of
cathepsin B and LAMP-1 was clearly observed in cells that were
co-treated with EUE and palmitate (Fig. 2I), demonstrating that
the integrity of lysosomes was maintained in the presence of EUE.
EUE Reverses Palmitate-inhibited Lysosomal Activation
LysoTracker staining analysis showed that EUE rescued the
lysosomal dye fluorescence that was decreased by palmitate
(Fig. 3A). The lysosomal fluorescence intensity was also quantified
(Fig. 3A, bottom). Acridine orange has been used to study the
acidification of cytoplasmic vesicles [20,21]. Acridine orange was
loaded into isolated lysosome membranes from cells that were
exposed to palmitate with or without EUE. Extra-lysosomal
fluorescence quenching was measured in the presence of ATP. In
the presence of EUE and palmitate the acridine orange
fluorescence was more significantly quenched than with single
treatment with palmitate (Fig. 3B). Intravesicular H+ uptake in
acridine orange dye-loaded cells was also analyzed by fluorescence
microscopy. H+ uptake was decreased by palmitate, and this
inhibition was rescued by EUE (Fig. 3C). This finding is consistent
with the extralysosomal quenching of acridine orange fluorescence
in the presence of EUE (Fig. 3B). The activities of the lysosomal
enzymes a-galactosidase, a-mannosidase, and acid phosphatase
were also measured. Activity of these enzymes gradually decreased
after treatment with palmitate, but the decrease was mitigated by
EUE (Fig. 3D). To clarify the role of lysosome activity in the
protective effect of EUE, we used the V-ATPase inhibitor
bafilomycin. First, V-ATPase activity was analyzed with and
without bafilomycin. Bafilomycin was used at the relatively low
concentration of 10 nM, the approximate IC50, throughout this
study . Bafilomycin significantly inhibited the rescue of
VATPase activity by EUE (Fig. 4A). In the intravesicular H+ assay,
the EUE-induced recovery of intravesicular H+ uptake was
similarly inhibited by bafilomycin (Fig. 4B). Quantification analysis
is shown in Figure 4B (right). EUE-induced protection against cell
death and reduction of caspase-3 activity were also reversed by
bafilomycin (Figs. 4C, D), indicating a major involvement of
lysosomal activity in EUE-induced protection. The regulation of
lysosomal BAX localization with or without bafilomycin was
examined by confocal analysis (Fig. 4E) and cellular fraction assay
(Fig. 4F). As expected, the regulatory effect of EUE on BAX
localization was suppressed by the V-ATPase inhibitor
Aucubin and Geniposide, Active Constituents of EUE,
Regulate Palmitate-induced Cathepsin B-associated Cell Death through Lysosomal Activation
We next tested the effects of aucubin and geniposide, the main
active components of Eucommia ulmoides cortex , on
palmitateinduced cell death. Cell viability, caspase-3 activity, and the
expression of active caspase-3 and -9 were analyzed in HepG2
cells. Treatment with aucubin or geniposide regulated viability of
palmitate-treated cells in a dose-dependent manner (Fig. 5A). The
activity of caspase-3 (Fig. 5B) and the expression of active
caspase3 and -9 (Fig. 5C) in palmitate-treated cells were also regulated by
treatment with aucubin or geniposide. These results showed
dosedependent protective effects of both agents against palmitate.
Treatment with a specific concentration of aucubin or geniposide,
10 mg/mL, regulated palmitate-induced cell death and caspase-3
activation in a time-dependent manner (Figs. 5D, E). Each
component also suppressed palmitate-induced active caspase-3
and -9 expression at 24 and 48 hours after treatment (Fig. 5F). The
induction of apoptotic cell morphology by palmitate was also
inhibited by co-treatment with aucubin or geniposide (Fig. 5G). To
confirm the regulation of lysosomal localization of BAX and the
subsequent release of cathepsin B in aucubin- or
geniposidetreated cells, lysosome and cytosol fractionation analysis was
performed. Translocation of BAX to the lysosome and leakage of
cathepsin B from the lysosome were clearly reversed by treatment
with aucubin or geniposide (Fig. 5H). Cathepsin B activity in the
medium was inhibited by treatment with each component (Fig. 5I).
Confocal analysis confirmed co-localization of cathepsin B with
LAMP-1. In the presence of palmitate cathepsin B was diffusely
expressed, whereas with addition of aucubin or geniposide the
acidic protease was significantly localized in the lysosome (Figure
S2). Quantification of the percentage of overlap between cathepsin
B and LAMP-1 staining is shown in Figure 5J. Since it has been
reported that lysosomal translocation of BAX and related
cathepsin B release are also related to intracellular ROS ,
intracellular ROS were measured by dihydrodichlorofluorescein
diacetate (DCFDA) analysis. The palmitate-induced ROS
accumulation was diminished by EUE or its active components,
aucubin and geniposide (Figure S3). Because the
palmitateinduced cell death mechanism includes the lysosomal pathway
and EUE regulates cell death through lysosomal activation ,
were added to cell monolayers and intravesicular H+ uptake was initiated by the addition of Mg-ATP (C); the fluorescence was quantified at 24 hours
(C; right). Cells were treated with 500 mM palmitate in the presence or absence of 10 mg/mL aucubin or geniposide for 0, 12, 24, or 48 hours and the
level of a-galactosidase, a-mannosidase, or acid phosphatase was measured (D). *p,0.05, significantly different from palmitate-treated condition.
Con; control, Pal.; palmitate.
we examined the effect of the active components, aucubin and
geniposide, on lysosomal activation. First, lysosomal V-ATPase
activity was analyzed through the acridine quenching method.
Although V-ATPase activity was inhibited by palmitate, either
aucubin or geniposide could reverse that effect (Fig. 6A).
LysoTracker dye analysis revealed that aucubin and geniposide
significantly rescued the reduction in lysosomal integrity that was
induced by palmitate (Fig. 6B). Treatment with aucubin or
geniposide also recovered the palmitate-induced decrease in
intravesicular H+ uptake as measured with the acridine orange
dye loading assay (Fig. 6C). Finally, the activities of the lysosomal
enzymes a-galactosidase, a-mannosidase, and acid phosphatase in
palmitate-treated cells were rescued by co-treatment with aucubin
or geniposide (Fig. 6D).
EUE Reduces Hepatic Lipotoxicity in Rats Fed a High-fat
To examine the physiological relevance of these in vitro
observations, we examined the effect of EUE on hepatic
lipotoxicity in rats fed a high-fat diet. In this study, we selected a
25% ethanol reflux method for EUE, which provides the highest
content of the active constituent aucubin (data not shown). EUE
was orally administered to rats fed a high-fat diet (0.25, 0.5, and 1
EUE/kg/day). Dihydroethidium (DHE) analysis (Fig. 7A) showed
that EUE inhibited the high-fat diet-induced ROS accumulation
in a dose-dependent manner. Similarly, EUE decreased hepatic
lipid peroxidation (Fig. 7B) and inhibited the high-fat diet-induced
increase in caspase-3 activation and active caspase-3 and -9
expression (Figs. 7C, D). Expression of the hepatic toxicity markers
AST and ALT was also down-regulated by the extract (Fig. 7E).
Consistent with results for BAX localization in vitro, EUE regulated
the lysosomal translocation of BAX in the high-fat diet-treated
condition (Fig. 7F). Hematoxylin and eosin (H&E) staining
revealed structural changes in rats fed a high-fat diet, which were
reduced by co-feeding with EUE (Supplementary Fig. 4). EUE
also markedly decreased the hepatic lipid content, as determined
by hepatic triglyceride and cholesterol levels (Figure S5). These
data demonstrate the function of EUE in a well-developed hepatic
In this study, the protective role of EUE against hepatic
lipotoxicity was examined in hepatic cell and an animal model
system. Based on a BAX-screening assay in yeast cells, EUE was
selected as a regulator of BAX-induced cell death. In the human
hepatic cell line, HepG2, EUE regulated palmitate-induced
lysosomal BAX localization and the cathepsin B-associated cell
death pathway. The active components of EUE, aucubin and
geniposide, showed similar effects to those of EUE. EUE also
regulated high-fat diet-induced hepatic toxicity in rats, in which
the lysosomal location of BAX was inhibited by EUE. Lysosomal
activity was highly enhanced in the presence of EUE. These data
suggest the regulatory mechanism by which EUE protects against
hepatic lipotoxicity in both in vivo and in vitro studies.
Previous studies demonstrated that free fatty acids result in
lysosomal membrane permeabilization and the release of
cathepsin B into the cytosol due to lysosomal translocation of BAX
[4,17]. Our studies showed that lysosomal localization of BAX
induced by palmitate exposure led to lysosomal membrane
permeabilization and a rapid increase in the abundance of cleaved
Bid and stimulation of caspase-3 and -9 activity. These findings are
all in accordance with previously published evidence that
lysosomal BAX is associated with mitochondria-dependent cell
death pathways through the mitochondrial localization of BAX,
Bid cleavage, and associated caspase activation and cell death
[25,26]. Lysosomes contain a large number of hydrolases in an
acidic pH environment and their role in mitochondria-dependent
cell death pathways is well recognized . Similarly, functional
disorders of various organelles other than the mitochondria,
including the ER, Golgi apparatus, and lysosomes, can also trigger
mitochondria-dependent caspase activation and consequent
apoptotic cell death.
Data from a yeast model showed that EUE regulated human
BAX-induced cell death (Figure S1A, B, C). Consistent with these
data, EUE regulated subcellular translocation of BAX in a
GFPBAX-overexpressing cell system, in particular lysosomal BAX
localization induced by free fatty acids (data not shown, Figs. 1B,
C, D), a main finding of this study. We show that EUE regulates
the sequential events in lysosomal cell death signal transduction in
palmitate-treated cultured hepatic cells (Figs. 2E, H, I). Consistent
with previous studies, our results indicate that lysosomal BAX
localization contributes to loss of lysosomal integrity and cathepsin
B release, and show that EUE regulates these events through the
inhibition of lysosomal BAX localization. We further show that the
active components of EUE, aucubin and geniposide, have similar
effects on the regulation of lysosomal BAX-associated cell death to
those of EUE (Figs. 5 and 6).
In this study, we aimed to reveal the importance of lysosomal
activity in controlling lysosomal BAX activation and associated cell
death. Treatment with the V-ATPase inhibitor, bafilomycin,
blocked the EUE-induced protective effect and regulation of
lysosomal BAX and t-Bid locations under conditions of free fatty
acids (Figs. 4C, E, F), indicating that EUE-induced activation of
V-ATPase contributes to the regulation of lysosomal BAX
translocation. These data suggest that modification of lysosomal
activity, such as V-ATPase activity, can affect the localization of
BAX and the related lysosome permeability and plays an
important role in the protective function of EUE against free
fatty acid-induced cell death. The vacuolar H+-ATPase establishes
pH gradients along secretory and degradation pathways.
Considering that progressive acidification is essential for proteolytic
processing in lysosomes , V-ATPase could be a major
contributor to lysosomal function. However, the mechanism by
which EUE induces increases in the activities of lysosomal
VATPase and other lysosomal functions remains unknown.
To investigate whether these in vitro effects of EUE correlate
with its activity in vivo, we applied EUE to a high-fat diet-induced
NAFLD model in rats. Hepatic ROS accumulation, lipid
peroxidation, and their associated cell death phenotypes, including
caspase 3 and 9 activation, were significantly inhibited by EUE
(Figs. 7A, B, C, D). EUE also regulated lysosomal BAX
translocation in the NAFLD rat model (Fig. 7F). Similarly, it has
been reported that EUE decreases serum GOT, GPT, LDH, and
ALP levels in a liver damage model . In previous studies using
a high-fat diet model, hepatic fatty acid synthase and HMG-CoA
Figure 7. EUE reduces hepatic lipotoxicity in rats fed a high-fat diet. Rats were given a normal diet or a high-fat diet with or without 0.25,
0.5, or 1 g/kg EUE for 10 weeks, and serum and livers were harvested. Liver tissues were loaded with 5 mM dihydroethidium and fluorescence image
acquisition was performed (A). Liver tissue was subjected to lipid peroxidation assay (B), caspase-3 activity assay (C), and immunoblotting with
antibody against caspase-3, -9, or b-actin (D). Serum levels of AST and ALT were measured (E). Following subcellular fractionation, immunoblotting
with antibody against BAX, t-Bid, PDI, COX II, or LAMP-1 was performed (F). *p,0.05, significantly different from high-fat diet. HFD; high fat diet, EUE;
Eucommia ulmoides Oliver extract.
reductase activities were significantly decreased by EUE,
demonstrating that the extract exhibits antihyperlipidemic properties,
whereas high levels of erythrocyte superoxide dismutase (SOD),
catalase (CAT), and glutathione peroxidase (GSH-Px) activities
were observed [30,31]. In addition, another EUE extract was
reported to have protective effects against a hepatic injury model
. In a study of patients with NASH and persistently elevated
transaminases, treatment with EUE produced a statistically
significant reduction in ALT values compared with the placebo
group . Although there have been a number of studies on EUE,
its function in terms of lipotoxicity has not been clarified. Based on
the results of this study demonstrating the mechanism of EUE in a
lipotoxicity model, EUE can be suggested as a potential candidate
for the treatment of lipid accumulation-associated toxicity.
In conclusion, this study supports a basic mechanism of fatty
acid-associated lysosomal BAX localization and resultant
cathepsin B leakage and cell death in a NAFLD model. We propose that
EUE and its active constituents, aucubin and geniposide, enhance
lysosomal activity and regulate lysosomal BAX translocation,
leading to resistance against hepatic lipotoxicity. EUE appears to
be a viable treatment strategy to prevent or treat NAFLD and its
associated toxic conditions.
Figure S1 EUE protects against BAX-induced cell death
in both yeast and human cells. Human BAX was
transformed into yeast cells. Immunoblotting was performed with
anti-BAX antibody (A, left). Yeast cells containing YEp51-Bax and
p426-GPD plasmids were grown overnight in SC-U-L/glucose.
Cultures were spread onto SDMM or SGMM plates (A, right).
Yeast cells expressing YEp51-Bax were cultured with 50 mg/mL
EUE for 16 hours, and cell viability assay was performed as
described in Materials and Methods. EUE was serially diluted to
50, 5, 0.5 or 0.05 mg/mL, and 4 mL was dropped onto SDMM or
SGMM plates. After drying, BAX-containing yeast cells were
dropped onto the SDMM or SGMM plates (C). *p,0.05,
significantly different from BAX-expressed condition; CBB,
Coomassie Brilliant Blue; SDMM; SC-U-L/glucose medium,
SGMM; SC-U-L/galactose medium, Pal.; palmitate, EUE;
Eucommia ulmoides Oliver extract, Arrow; indicating
BAXexpressed dead cells (no spreading pattern).
Figure S2 EUE active components geniposide and
aucubin regulate palmitate-induced lysosomal
permeability. Cells were incubated with 500 mM palmitate in the
presence or absence of 10 mg/mL aucubin or geniposide for 24
hours. Immunostaining was performed with anti-LAMP-1
antibody and subsequently with anti-cathepsin B antibody.
Subsequently, secondary FITC or TRITIC antibody was used. The
fluorescence of images was captured by microscope at 200 X
original magnification. Con; control, Pal.; Palmitate.
Figure S3 EUE and the active components, geniposide
and aucubin, regulate palmitate-induced mitochondrial
ROS accumulation. Cells were incubated with 500 mM
palmitate in the presence or absence of 100 mg/mL EUE,
10 mg/mL aucubin or geniposide for 24 or 48 hours. DCF-DA,
100 mg/mL, was loaded into the cells, and after 20 minutes the
fluorescence was measured as described in Materials and Methods.
Con; control, Pal.; Palmitate, EUE; Eucommia ulmoides Oliver
Figure S4 EUE does not induce morphological changes
in rats on a high-fat diet. Rats were administrated a normal
diet or high-fat diet with or without 0.25, 0.5 or 1 g/kg EUE for
ten weeks, and livers were then isolated. Hematoxylin and eosin
staining was performed. Con; control, EUE; Eucommia ulmoides
Oliver extract, HFD; high fat diet.
Figure S5 EUE regulates hepatic lipid accumulation.
Rats were administrated a normal diet or high-fat diet with or
without 0.25, 0.5 or 1 g/kg EUE for ten weeks, and livers were
then isolated. Triglyceride and cholesterol levels were measured in
the liver as described in the Supplementary Materials and
Methods. EUE; Eucommia ulmoides Oliver extract, HFD; high fat
Supplementary Materials and Method.
Conceived and designed the experiments: GHL MRL. Performed the
experiments: GHL MRL HYL. Analyzed the data: GHL MRL.
Contributed reagents/materials/analysis tools: SHK HKK HRK. Wrote
the paper: HJC.
1. Angulo P , Lindor KD ( 2002 ) Non-alcoholic fatty liver disease . J Gastroenterol Hepatol 17 Suppl: S186 - 190 .
2. Vuppalanchi R , Chalasani N ( 2009 ) Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: Selected practical issues in their evaluation and management . Hepatology 49 : 306 - 317 .
3. Li ZZ , Berk M , McIntyre TM , Feldstein AE ( 2009 ) Hepatic lipid partitioning and liver damage in nonalcoholic fatty liver disease: role of stearoyl-CoA desaturase . J Biol Chem 284 : 5637 - 5644 .
4. Feldstein AE , Werneburg NW , Canbay A , Guicciardi ME , Bronk SF , et al. ( 2004 ) Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway . Hepatology 40 : 185 - 194 .
5. Li Z , Berk M , McIntyre TM , Gores GJ , Feldstein AE ( 2008 ) The lysosomalmitochondrial axis in free fatty acid-induced hepatic lipotoxicity . Hepatology 47 : 1495 - 1503 .
6. Peng M , Zhang Y , Shi S , Peng S ( 2013 ) Simultaneous ligand fishing and identification of human serum albumin binders from Eucommia ulmoides bark using surface plasmon resonance-high performance liquid chromatographytandem mass spectrometry . J Chromatogr B Analyt Technol Biomed Life Sci 940 : 86 - 93 .
7. Li H , Chen B , Nie L , Yao S ( 2004 ) Solvent effects on focused microwave assisted extraction of polyphenolic acids from Eucommia ulmodies . Phytochem Anal 15 : 306 - 312 .
8. Kim HY , Moon BH , Lee HJ , Choi DH ( 2004 ) Flavonol glycosides from the leaves of Eucommia ulmoides O. with glycation inhibitory activity . J Ethnopharmacol 93 : 227 - 230 .
9. Hung MY , Fu TY , Shih PH , Lee CP , Yen GC ( 2006 ) Du-Zhong (Eucommia ulmoides Oliv.) leaves inhibits CCl4-induced hepatic damage in rats . Food Chem Toxicol 44 : 1424 - 1431 .
10. Luo J , Tian C , Xu J , Sun Y ( 2009 ) Studies on the antioxidant activity and phenolic compounds of enzyme-assisted water extracts from Du-zhong (Eucommia ulmoides Oliv.) leaves . J Enzyme Inhib Med Chem 24 : 1280 - 1287 .
11. Trivedi NS , Wang HW , Nieminen AL , Oleinick NL , Izatt JA ( 2000 ) Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy . Photochem Photobiol 71 : 634 - 639 .
12. Cox BE , Griffin EE , Ullery JC , Jerome WG ( 2007 ) Effects of cellular cholesterol loading on macrophage foam cell lysosome acidification . J Lipid Res 48 : 1012 - 1021 .
13. Crider BP , Xie XS ( 2003 ) Characterization of the functional coupling of bovine brain vacuolar-type H(+)-translocating ATPase . Effect of divalent cations, phospholipids, and subunit H (SFD). J Biol Chem 278 : 44281 - 44288 .
14. Chae HJ , Ke N , Kim HR , Chen S , Godzik A , et al. ( 2003 ) Evolutionarily conserved cytoprotection provided by Bax Inhibitor-1 homologs from animals , plants, and yeast. Gene 323 : 101 - 113 .
15. Li A , Harris DA ( 2005 ) Mammalian prion protein suppresses Bax-induced cell death in yeast . J Biol Chem 280 : 17430 - 17434 .
16. Yamasaki M , Miyamoto Y , Chujo H , Nishiyama K , Tachibana H , et al. ( 2005 ) Trans10, cis12-conjugated linoleic acid induces mitochondria-related apoptosis and lysosomal destabilization in rat hepatoma cells . Biochim Biophys Acta 1735 : 176 - 184 .
17. Feldstein AE , Werneburg NW , Li Z , Bronk SF , Gores GJ ( 2006 ) Bax inhibition protects against free fatty acid-induced lysosomal permeabilization . Am J Physiol Gastrointest Liver Physiol 290 : G1339 - 1346 .
18. Oberle C , Huai J , Reinheckel T , Tacke M , Rassner M , et al. ( 2010 ) Lysosomal membrane permeabilization and cathepsin release is a Bax/Bak-dependent, amplifying event of apoptosis in fibroblasts and monocytes . Cell Death Differ 17 : 1167 - 1178 .
19. Zhou J , Tan SH , Nicolas V , Bauvy C , Yang ND , et al. ( 2013 ) Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion . Cell Res 23 : 508 - 523 .
20. Lee GH , Kim HR , Chae HJ ( 2012 ) Bax inhibitor-1 regulates the expression of P450 2E1 through enhanced lysosome activity . Int J Biochem Cell Biol 44 : 600 - 611 .
21. Lee GH , Kim DS , Kim HT , Lee JW , Chung CH , et al. ( 2011 ) Enhanced lysosomal activity is involved in Bax inhibitor-1-induced regulation of the endoplasmic reticulum (ER) stress response and cell death against ER stress: involvement of vacuolar H+-ATPase (V-ATPase) . J Biol Chem 286 : 24743 - 24753 .
22. Kim BH , Park KS , Chang IM ( 2009 ) Elucidation of anti-inflammatory potencies of Eucommia ulmoides bark and Plantago asiatica seeds . J Med Food 12 : 764 - 769 .
23. Werneburg NW , Guicciardi ME , Bronk SF , Kaufmann SH , Gores GJ ( 2007 ) Tumor necrosis factor-related apoptosis-inducing ligand activates a lysosomal pathway of apoptosis that is regulated by Bcl-2 proteins . J Biol Chem 282 : 28960 - 28970 .
24. Li H , Hu J , Ouyang H , Li Y , Shi H , et al. ( 2009 ) Extraction of aucubin from seeds of Eucommia ulmoides Oliv. using supercritical carbon dioxide . J AOAC Int 92 : 103 - 110 .
25. Sobhan PK , Seervi M , Deb L , Varghese S , Soman A , et al. ( 2013 ) Calpain and reactive oxygen species targets bax for mitochondrial permeabilisation and caspase activation in zerumbone induced apoptosis . PLoS One 8 : e59350 .
26. Kagedal K , Johansson AC , Johansson U , Heimlich G , Roberg K , et al. ( 2005 ) Lysosomal membrane permeabilization during apoptosis-involvement of Bax? Int J Exp Pathol 86 : 309 - 321 .
27. Boya P , Gonzalez-Polo RA , Poncet D , Andreau K , Vieira HL , et al. ( 2003 ) Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine . Oncogene 22 : 3927 - 3936 .
28. Sobota JA , Back N , Eipper BA , Mains RE ( 2009 ) Inhibitors of the V0 subunit of the vacuolar H+-ATPase prevent segregation of lysosomal- and secretorypathway proteins . J Cell Sci 122 : 3542 - 3553 .
29. Harish R , Shivanandappa T ( 2010 ) Hepatoprotective potential of Decalepis hamiltonii (Wight and Arn) against carbon tetrachloride-induced hepatic damage in rats . J Pharm Bioallied Sci 2 : 341 - 345 .
30. Park SA , Choi MS , Jung UJ , Kim MJ , Kim DJ , et al. ( 2006 ) Eucommia ulmoides Oliver leaf extract increases endogenous antioxidant activity in type 2 diabetic mice . J Med Food 9 : 474 - 479 .
31. Choi MS , Jung UJ , Kim HJ , Do GM , Jeon SM , et al. ( 2008 ) Du-zhong (Eucommia ulmoides Oliver) leaf extract mediates hypolipidemic action in hamsters fed a high-fat diet . Am J Chin Med 36 : 81 - 93 .
32. Park SA , Choi MS , Kim MJ , Jung UJ , Kim HJ , et al. ( 2006 ) Hypoglycemic and hypolipidemic action of Du-zhong (Eucommia ulmoides Oliver) leaves water extract in C57BL/KsJ-db/db mice . J Ethnopharmacol 107 : 412 - 417 .