Eucommia ulmoides Oliver Extract, Aucubin, and Geniposide Enhance Lysosomal Activity to Regulate ER Stress and Hepatic Lipid Accumulation
and Geniposide Enhance Lysosomal Activity to
Regulate ER Stress and Hepatic Lipid Accumulation. PLoS ONE 8(12): e81349. doi:10.1371/journal.pone.0081349
Eucommia ulmoides Oliver Extract, Aucubin, and Geniposide Enhance Lysosomal Activity to Regulate ER Stress and Hepatic Lipid Accumulation
Hwa-Young Lee 0
Geum-Hwa Lee 0
Mi-Rin Lee 0
Hye-Kyung Kim 0
Nan-young Kim 0
Seung-Hyun Kim 0
Yong-Chul Lee 0
Hyung-Ryong Kim 0
Han-Jung Chae 0
Giovanni Li Volti, University of Catania, Italy
0 1 Department of Pharmacology and Institute of Cardiovascular Research, Medical School, Chonbuk National University , Jeonju, Chonbuk , Republic of Korea, 2 College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University , Incheon , Korea , Republic of Korea, 3 Department of Internal Medicine, Chonbuk National University Medical School , Jeonju , Korea , 4 Department of Dental Pharmacology, School of Dentistry, Wonkwang University , Iksan, Chonbuk , Republic of Korea
Eucommia ulmoides Oliver is a natural product widely used as a dietary supplement and medicinal plant. Here, we examined the potential regulatory effects of Eucommia ulmoides Oliver extracts (EUE) on hepatic dyslipidemia and its related mechanisms by in vitro and in vivo studies. EUE and its two active constituents, aucubin and geniposide, inhibited palmitateinduced endoplasmic reticulum (ER) stress, reducing hepatic lipid accumulation through secretion of apolipoprotein B and associated triglycerides and cholesterol in human HepG2 hepatocytes. To determine how EUE diminishes the ER stress response, lysosomal and proteasomal protein degradation activities were analyzed. Although proteasomal activity was not affected, lysosomal enzyme activities including V-ATPase were significantly increased by EUE as well as aucubin and geniposide in HepG2 cells. Treatment with the V-ATPase inhibitor, bafilomycin, reversed the inhibition of ER stress, secretion of apolipoprotein B, and hepatic lipid accumulation induced by EUE or its component, aucubin or geniposide. In addition, EUE was determined to regulate hepatic dyslipidemia by enhancing lysosomal activity and to regulate ER stress in rats fed a high-fat diet. Together, these results suggest that EUE and its active components enhance lysosomal activity, resulting in decreased ER stress and hepatic dyslipidemia.
Funding: This study was supported by the National Research Foundation (2012R1A2A1A3001907 and 2008-0062279) and Korea Health Industry Development
Institute (A121931-1211-0000200). 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.
Nonalcoholic fatty liver disease (NAFLD) is one of the most
common chronic liver disorders . NAFLD is clearly associated
with features of Metabolic Syndrome including obesity, type 2
diabetes, hypertension, and dyslipidemia. Hepatic steatosis is
considered to be the first stage of NAFLD and often leads to more
severe complications including steatohepatitis, cirrhosis, and
hepatocellular carcinoma . Thus, a growing number of
studies looking at the mechanism of hepatic steatosis are focused
on the causative role of ER stress.
When the ER receives extracellular stress signals, the unfolded
protein response (UPR) relieves stress from protein misfolding in
the ER. Specifically, the expression of protein kinase-like ER
kinase (PERK) and the phosphorylation of eukaryotic initiation
factor 2a (p-eIF2a) are increased during chronic ER stress,
attenuating new protein synthesis . The UPR regulates genes
involved in the transport of unfolded proteins out of the ER as well
as in the degradation of these unfolded proteins by ER-associated
degradation (ERAD) . The ERAD I is a proteasome/
ubiquitination pathway, while the ERAD II pathway is a
lysosomal activity pathway . The ERAD mechanism increases
the protein folding capacity by reducing protein folding loads
[7,8], implying that ERAD is a physiological pathway that can
regulate ER stress responses [8,9]. Events that disturb ER protein
folding and induce the UPR include an altered redox state,
calcium equilibrium, and protein degradation. Likewise,
accumulation of fatty acids or triglycerides is related to alteration of
secretory apo-lipoproteins such as ApoB, which can also induce
the UPR and cause hepatic steatosis.
The secretion of ApoB-containing lipoproteins involves co- and
post-translational processes. Unassembled or aberrantly expressed
ApoB retained in the ER is typically degraded, and, under mild
physiological stress, the degradation process is highly activated as
an adaptive response that involves both ER resident molecular
chaperones such as calnexin and calreticulin as well as ER
proteases such as ER 60 [10,11]. However, under pathological ER
stress conditions not regulated by the adaptive response, the
physiological degradation machinery does not function efficiently,
leading to accumulation of unfolded proteins including ApoB .
During this type of ER stress, hepatic lipid synthesis and secretion
may also be affected by the alteration of secretory ApoB protein
folding processes . Therefore, it is necessary to study ER
stresses to determine how to control pathological ER stress
phenomena such as hepatic steatosis.
Eucommia cortex obtained from the bark of 1520-year-old E.
ulmoides Oliver trees  is a traditional medicine used in Korea,
Japan, and China. According to ancient records, roasted Eucommia
cortex is recommended for reinforcing muscles and lungs,
lowering blood pressure, preventing miscarriages, improving the
tone of the liver and kidneys, and increasing longevity .
Duzhong (E. ulmoides Oliver) leaves containing many of the same
components as the Eucommiae cortex have recently become a focus
of medical research . Indeed, E. ulmoides Oliver tea, an
aqueous extract of E. ulmoides Oliver leaves, is known as a
functional health food and is commonly used for the treatment of
hypertension . Likewise, extracts of E. ulmoides Oliver leaves
have been suggested to have recuperative effects for
hypercholesterolemia and fatty liver disease . E. ulmoides Oliver contains
many phytochemicals such as polyphenolics, flavonoids, and
triterpenes . Flavonol glycosides from E. ulmoides Oliver have
been reported to inhibit glycation and to prevent diabetes [20,21].
Yen and Hsieh  reported that water extracts of E. ulmoides
Oliver leaves have antioxidant activity toward various lipid
peroxidation models, with a good correlation between the
polyphenol content of water extracts and observed antioxidant
Based upon these observations, we examined the potential
regulatory effects of E. ulmoides Oliver on hepatic dyslipidemia. We
found that E. ulmoides Oliver significantly regulated hepatic lipid
accumulation both in vitro and in vivo. Our study results suggest that
the regulatory mechanism of E. ulmoides Oliver and its active
constituents, aucubin and geniposide [22,23], toward hepatic
dyslipidemia involves regulation of ER stress and associated
Materials and Methods
E. ulmoides Oliver extracts (EUE) were obtained from the Korea
Research Institute of Bioscience & Biotechnology (Daejeon,
Korea). Extracts were prepared by ethanol extraction of E.
ulmoides Oliver extracts (200 g) by sonicating with 100% methanol
(1g: 8 mL) for 3 days. After filtration, the solvent phase of the
filtrate was concentrated by freeze-drying, and the final EUE was
obtained and stored at 24uC. For animal experiments, powdered
EUE was weighed and re-extracted with 25% ethanol for 2 hours
at 90uC using a reflux apparatus. The extract was then filtered,
evaporated under vacuum, pulverized, and stored at 4uC.
Bafilomycin, aucubin, and geniposide were obtained from Sigma
Chemical Company (St. Louis, MO). Antibodies against the 20S
core proteasome subunit and
carbobenzoxy-Leu-Leu-Glu-7-amino-4-methylcoumarin (Z-LLE-AMC) were from Enzo Life
Sciences (Farmingdale, NY). LysoTracker Red DND-26 was from
Molecular Probes (Eugene, OR). Kits for measuring total levels of
cholesterol and triglyceride were from Asan Pharmaceutical
Company (Seoul, Korea). Phosphate-buffered saline (PBS) was
purchased from Invitrogen (Carlsbad, CA). Poly-(I:C) and trypan
blue dye were from Sigma-Aldrich (St. Louis, MO). All other
chemicals were of analytical grade and were purchased from
Human hepatocellular carcinoma cells (HepG2) were cultured
in Dulbeccos modified Eagle Medium (DMEM) (Invitrogen) with
10% fetal bovine serum (FBS) (Biomeda Corp, Foster City, CA)
and penicillin-streptomycin (penicillin: 10,000 U/mL,
streptomycin: 10,000 mg/mL) (Invitrogen). Freshly trypsinized HepG2 cells
were suspended at 56105 cells/mL in standard HepG2 culture
medium and seeded at 106 cells per well in standard six-well tissue
culture plates. Cells were incubated at 37uC in a 90% air/10%
CO2 atmosphere, and 2 mL of medium was exchanged every
other day. HepG2 cells were cultured in standard medium for 2
3 days to 90% confluency before being treated with free fatty acids
and other additives. HepG2 cell viability was determined by
trypan blue dye exclusion using a hemocytometer.
Animal Treatment and Care
Female Sprague-Dawley rats weighing 250270 g were
obtained from Damul Science Co (Daejeon, Korea). Rats were
maintained on a 12:12-hours 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. Rats were
fed an appropriate diet with free access to water and were weighed
weekly. All animal procedures for this study were performed in
accordance with the regulations of the care and use of laboratory
animals guide of Chonbuk National University and were approved
by the Chonbuk National University laboratory animal center of
the Institutional Animal Care and Use Committee (IACUC, CBU
20130015), and all efforts were made to minimize animal
suffering. The control group (n = 10) was fed a standard diet, while
the high-fat diet (HFD) group (n = 12) was fed a calorie-rich diet
composed of 1% cholesterol, 18% lipid (lard), 40% sucrose, 1%
AIN-93G vitamins, and 19% casein. The fiber and mineral
contents were the same in both the control and high-fat diets. Rats
in the EUE-25 group (n = 10) and control group (n = 10) were fed
an HFD with 0.25, 0.5, or 1 g/kg EUE-25. Experiments were
terminated after 10 weeks, and serum samples were collected and
measured for cholesterol, triglyceride, and liver lipid contents. In
addition, tissues were homogenized for Western blot analysis.
Rats were anaesthetized with diethyl ether (Sigma) and
sacrificed by cervical dislocation. Tissues and blood samples were
collected from all sacrificed animals. Whole blood was
immediately placed on ice in a 1.5 mL centrifuge tube for 15 to 30 min
and spun at 8,000 rpm for 10 min. Sera were then transferred to
fresh 1.5 mL centrifuge tubes and stored at 280uC. All harvested
tissues were immediately placed in liquid nitrogen and stored at
Liver samples were fixed in 10% formalin and embedded in
paraffin. Liver sections were incubated for 10 min in 0.5%
thiosemicarbazide, stained with 0.1% Sirius red F3B in saturated
picric acid for 1 hours, and washed with acetic acid (0.5%).
Sections were visualized using a Nikon Eclipse E600 microscope
(Kawasaki, Kanagawa, Japan) at 406 magnification, and relative
Figure 3. Aucubin and geniposide inhibit palmitate-induced ER stress response. Cells were treated with 300 mM palmitate in the absence
or presence of 10 ug/mL aucubin or geniposide for 0, 3, 6, 9, 12, 18, or 24 hours. Immunoblotting was performed using antibodies against GRP78,
PERK, p-PERK, CHOP, IRE1a, p-eIF2a, eIF2a, or b-actin. Quantification of immunoblot data is shown (lower panel). *p,0.05, significantly different from
cells treated with palmitate alone at the corresponding time point. Pal, palmitate; EUE, E. ulmoides Oliver extracts.
areas of fibrosis (% positive areas for Sirius red staining) were
quantified by histomorphometry using a computerized image
analysis system (AnalySIS, Soft Imaging System, Munster,
Germany). Hepatic steatosis was assessed by Oil Red O staining.
Briefly, liver cryosections were fixed for 10 min in 60%
isopropanol followed by staining with 0.3% Oil Red O in 60%
isopropanol for 30 min and were then washed with 60%
isopropanol. Sections were counterstained with Gills hematoxylin,
washed with acetic acid (4%), and mounted with an aqueous
solution. Stained sections were quantified by histomorphometry.
HepG2 cells were washed twice with cold PBS and lysed in
300 mL/well CelLytic M cell lysis buffer (Sigma-Aldrich)
supplemented with protease inhibitor cocktail (Roche Applied Science,
Indianapolis, IN). Cell lysates were clarified by centrifugation at
12,000 rpm for 30 min, and the supernatant was collected. Total
protein was quantified using a BCA assay kit (Pierce Inc.,
Rockford, IL). Lysates (45 mg) were resolved by SDS-PAGE
(Bio-Rad) and then transferred to nitrocellulose membranes.
Membranes were blocked for 1 hours with 5% skim milk in
Tris-buffered saline (0.137 M NaCl, 0.025 M Tris, pH 7.4)
containing 0.1% Tween-20 (T-TBS). Primary antibodies consisted
of mouse anti-amylase, mouse anti-eIF2a, rabbit anti-ATF6a,
mouse anti-GADD153/C/EBP homologous protein (CHOP),
mouse anti-GRP78, b-actin (Santa Cruz Biotechnologies, Inc.,
Santa Cruz, CA), and rabbit anti-phospho-eIF2 and rabbit
antiIRE1a (Cell Signaling. Technologies, Inc., Danvers, MA).
Antibodies were diluted according to the manufacturers
recommended protocols. Protein signals were visualized using enhanced
chemiluminescence (ECL) reagent (SuperDetectTM ECL Western
Blotting Detection Reagent, DaeMyung Science Co., Ltd, Seoul,
Korea). Finally, membranes were exposed to imaging film (Kodak
BioFlexEcono Scientific Supplies, Citrus Heights, CA) and
developed using a Kodak X-OMAT 1000A Processor.
Oil Red O Staining
To measure cellular neutral lipid droplet accumulation, HepG2
cells were stained using Oil Red O. After treatment, cells were
washed three times with ice cold PBS and fixed with 10% formalin
for 60 min. After fixation, cells were washed and stained with Oil
Red O solution (stock solution, 3 mg/mL in isopropanol; working
solution, 60% Oil Red O stock solution and 40% distilled water)
for 60 min at room temperature. After staining, cells were washed
with water to remove unbound dye. To quantitate Oil Red O
content levels, isopropanol was added to each sample, and samples
were shaken at room temperature for 5 min. Oil Red O levels
were determined by spectrophotometry at 510 nm.
Measurement of Total Lipid, Triglyceride, and Cholesterol
For lipid determination, cell or rat liver homogenates were
extracted according to a modified Bligh and Dyer procedure
. Samples were homogenized with
chloroform-methanolwater (8:4:3), shaken at 37uC for 1 hours, and centrifuged at
1,1006g for 10 min. The bottom layer was collected and
suspended for hepatic lipid analysis. Triacylglycerol, total
Figure 5. E. ulmoides Oliver extract, aucubin, and geniposide enhance lysosomal activity. (A) Cells were treated with 100 mg/mL EUE,
10 mg/mL aucubin, or 10 mg/mL geniposide in the presence or absence of 300 mM palmitate for 12 hours, followed by exposure to 5 mM LysoTracker
and image acquisition and quantification (lower panel). (B) Cells were fixed and immunostained with antibodies for cathepsin B or Lamp-1.
Quantification of fluorescence is shown (lower). (C) The activities of a-mannosidase, b-galactosidase, and b-glucuronidase were analyzed from
lysosomal extracts of cells treated for the indicated time periods. *p,0.05, significantly different from cells treated with palmitate alone (A, B);
*p,0.05, significantly different from cells treated with palmitate alone at each corresponding time point (C). DIC, differential interference contrast
microscopy; Pal, palmitate; EUE, E. ulmoides Oliver extract.
cholesterol, and total lipid contents were measured using kits
from Randox Laboratories (Antrim, UK) in accordance with the
Lysosomal isolation was performed according to a published
protocol . Briefly, cells were rinsed in cold STE buffer (0.25 M
sucrose, 0.01 M Tris-HCl, 1 mM EDTA, and 0.1% ethanol) and
scraped into a dish containing 1 mL STE buffer and protease
inhibitors (Sigma-Aldrich). The cell suspension was disrupted in a
Kontes cell disruption chamber using three 20 min passes at 150
psi. This method consistently disrupted .95% of cells while
leaving the lysosomes intact. The resulting suspension was
centrifuged at 10006g to separate post-nuclear supernatant and
nuclear pellet. Post-nuclear supernatant density was increased to
1.15 g/mL with sucrose and applied to a sucrose density gradient
from 1.28 to 1.00 mg/mL, which was then centrifuged at
64,0006g for 4 hours at 4uC to isolate the lysosomal fraction.
Microscopic Assessment of Lysosome Activity
A time course of lysosomal activity was determined by staining
with LysoTracker Red (577/590 nm). Cells were grown in cell
culture dishes, 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, followed by
washing with PBS. Red fluorescent images were acquired using a
digital CCD color video camera CCS-212 (Samsung, Seoul,
Korea) and transferred to a computer with a WinFast 3D S680
frame grabber (Leadtek, Taipei, Taiwan). Fluorescence values of
100 randomly selected cell images were measured for each
treatment condition. Lysosome fluorescence intensity was
expressed as a ratio of the average fluorescence of 100 treated cells to
the fluorescence of 100 control cells. Localization of cathepsin B
and LAMP-1 was determined by immunofluorescence confocal
microscopy. Cells were cultured and fixed with methanol for
10 min at room temperature. After permeabilization with Triton
X-100 (0.1%), cells were incubated with PBS containing 3% BSA
for 30 min at room temperature. Cells were then double-stained
for 2 hours at room temperature in PBS-3% BSA with primary
antibodies: anti-LAMP-1 rabbit (1:100, Millipore, MA) or
anticathepsin B (15 mg/mL, R&D Systems, MN).
Immunofluorescence staining was performed with secondary antibodies in
PBS0.5% BSA for 60 min at room temperature. Slides were mounted
with Aqua Poly/Mount (Polysciences), and images were acquired
using a Delta Vision Spectris Image Deconvolution System on an
Olympus IX70 microscope with Softworx Explorer software from
Lysosomal Enzyme Activity Assays
Lysosomal enzyme assays were performed at 35uC with
pnitrophenyl-derivatized monosaccharide substrates, as previously
described . Enzymatic reactions were terminated by the
addition of an equal volume of 1 M Na2CO3. Released
pnitrophenol was measured by spectrophotometry at 420 nm, with
activity units defined as nanomoles of p-nitrophenol released per
Results are presented as the mean 6 SEM. MicroCal Origin
software (Northampton, MA) was used for statistical calculations.
Differences were tested for significance using one-way analysis of
variance (ANOVA) with Duncans multiple range test. Statistical
significance was set at p,0.05.
EUE inhibits palmitate-induced ER stress response
E. Oliver extracts (EUE) are known to have anti-hyperlipidemic
properties; however, the mechanisms by which EUE mediates
these effects are unknown . In this study, EUE was applied to a
model of palmitate-induced ER stress and its associated lipid
accumulation. To confirm that non-toxic concentrations and time
periods were chosen for palmitate treatment, HepG2 cells were
exposed to palmitate at 300 or 500 mM for 0, 12, 24, 36, or
48 hours. Treatment with 300 mM palmitate for 12 hours was
selected as the optimal nontoxic condition for studying lipid
metabolism (Figure S1). Among the ER stress responses,
phosphorylation of PERK and eIF2a was markedly increased in cells
treated with 300 mM palmitate. Pretreatment with EUE inhibited
phosphorylation of PERK and expression of its downstream
effectors, eIF2a and CHOP (Figs. 1 A and B). However, other ER
stress signaling proteins, namely ATF6, IRE-1a, and GRP78, were
not affected by treatment with palmitate.
Figure 7. E. ulmoides Oliver extract regulates ER stress and hepatic lipid accumulation and enhances lysosome activity in
high-fatdiet rats. Rats were fed a normal or high-fat diet with 0, 0.25, 0.5, or 1 g/kg EUE for 10 weeks, after which livers were isolated. (A) Immunoblotting
was performed with antibodies against GRP78, PERK, p-PERK, CHOP, IRE1-a, p-eIF2a, eIF2a, or b-actin. (B) Lysosome fractionation was performed
using liver samples, and the activities of a-mannosidase, b-glucuronidase, and b-galactosidase were subsequently determined. (C) Livers were stained
with Oil Red O dye, and images were obtained at 200X magnification to observed hepatic fat accumulation. (D) Triglyceride and cholesterol levels
were measured in both the liver and plasma. (E) Immunoblotting was performed with ApoA1 or ApoB antibodies using liver and plasma samples.
*p,0.05, significantly different from the control group at each corresponding time point. CV, central vein; Con, control; HFD, high-fat-diet; EUE, E.
ulmoides Oliver extract.
EUE regulates lipid metabolism
To investigate the ability of EUE to regulate lipid accumulation,
HepG2 cells were treated with palmitate to induce hepatic lipid
accumulation. Treatment of HepG2 cells with EUE and a
nontoxic concentration of palmitate (300 mM) significantly
inhibited palmitate-induced cellular lipid accumulation (Fig. 2A). To
investigate the mechanism of this inhibition, apolipoprotein
pathways were examined because dysregulation of apolipoprotein
secretion is related to ER stress and hepatic accumulation .
Expression of ApoA1 and ApoB was analyzed after treatment with
100 mg/mL EUE with or without palmitate. Expression of ApoB
but not ApoA1 was increased in cell lysates after palmitate
treatment (Fig. 2B). Likewise, treatment with EUE regulated the
change in ApoB expression. Specifically, the level of secreted
ApoB in the medium was decreased in the presence of palmitate
without EUE but increased in a time-dependent manner after
treatment with EUE. Levels of triglycerides and cholesterol were
measured for cell lysates and media from palmitate-treated cells
with or without EUE treatment. The levels of intracellular
triglyceride and cholesterol were strongly increased in
palmitatetreated cells, whereas this increase was prevented by EUE
(Fig. 2C). In media samples, triglyceride and cholesterol levels
were significantly decreased by treatment with palmitate and,
consistent with our ApoB expression results, were recovered by
treatment with EUE.
Aucubin and geniposide from EUE inhibit
palmitateinduced phosphorylation of PERK and eIF2a
We next examined the effects of aucubin and geniposide, the
major active constituents of EUE, on palmitate-associated lipid
metabolism. First, we co-treated cells with palmitate and either
aucubin or geniposide. Neither aucubin nor geniposide affected
cell viability in the presence of palmitate (Figure S2). We then
exposed cells to palmitate in the presence or absence of 10 mg/mL
of aucubin or geniposide and examined the ER stress response.
Phosphorylation of PERK and eIF2a was markedly increased in
cells treated with 300 mM palmitate, while expression of other ER
stress signaling proteins, namely ATF6, IRE-1a and GRP78, were
not affected. Conversely, co-treatment with aucubin or geniposide
inhibited phosphorylation and downstream signaling of PERK,
eIF2a, and CHOP (Fig. 3).
Aucubin and geniposide regulate lipid metabolism
We examined the effects of aucubin and geniposide on hepatic
lipid accumulation. Treatment with aucubin and geniposide in
the presence of 300 mM palmitate significantly inhibited
palmitate-induced cellular lipid accumulation as determined by Oil
Red O staining (Fig. 4A). Expressions of ApoA1 and ApoB were
also analyzed after treatment with aucubin or geniposide in the
presence or absence of palmitate. Co-treatment of cells with
palmitate and either aucubin or geniposide inhibited
palmitateinduced expression of ApoB (Fig. 4B). In culture media,
expression of ApoB but not ApoA1 was increased in a
timedependent manner by 10 mg/mL aucubin or geniposide under
palmitate-treatment conditions. The levels of triglycerides and
cholesterol are shown in Fig. 4C; these levels were strongly
increased in 300 mM palmitate-treated cells but were ameliorated
by 10 mg/mL aucubin or geniposide. Consistent with the ApoB
expression results, levels of triglycerides and cholesterol in media
were significantly decreased by palmitate, whereas treatment with
aucubin or geniposide prevented this decrease (Fig. 4B).
EUE, aucubin, and geniposide enhance lysosomal activity
We were interested in how EUE regulates ER stress (Fig. 1B).
Because enhanced protein degradation has been demonstrated to
relieve ER stress , we investigated proteasomal and lysosomal
degradation. In palmitate-treated cells, proteasomal activity was
not affected by EUE, aucubin, or geniposide (Figure S3). We then
used lysoTracker dye to evaluate lysosomal activity in HepG2
cells, which decreased significantly after treatment with palmitate
(Fig. 5A). Treatment with EUE, aucubin, or geniposide clearly
reversed the decrease in fluorescence in palmitate-treated cells
To evaluate the effect of EUE on lysosomal degradation, we
used the representative lysosomal enzyme Cathepsin B .
Immunostaining analysis with antibodies to cathepsin B and
lysosomal-associated membrane protein 1 (Lamp-1), a lysosomal
marker, revealed decreased fluorescence of cathepsin B in
palmitate-treated cells (Fig. 5B). The decreased fluorescence of
cathepsin B was reversed by co-treatment with EUE and aucubin
or geniposide. Colocalization of cathepsin B with Lamp-1 was
observed in all treatment groups, suggesting that palmitate affected
lysosome function but did not influence lysosomal membrane
permeability. To confirm these results, we evaluated several other
lysosomal enzymes, namely, b-galactosidase, a-mannosidase, and
b-glucuronidase. These enzymes were inhibited by palmitate
treatment in a time-dependent manner (Fig. 5C). However, in cells
exposed to EUE, aucubin, or geniposide in the presence of
palmitate, the activities of a-mannosidase, b-glucuronidase, and
bglucuronidase were relatively stable compared to levels observed
after treatment with palmitate alone.
The lysosome inhibitor bafilomycin reverses the effects
of EUE on the ER stress response and lipid metabolism
V-ATPase maintains the acidic pH of the lysosome and is a
representative lysosomal protein . To understand the role of
lysosomes in ER stress-associated dyslipidemia, we used the
VATPase inhibitor bafilomycin. Specifically, we evaluated the effect
of bafilomycin on the ER stress response in palmitate-exposed
HepG2 cells. Treatment of cells with bafilomycin and EUE
significantly reversed the effect of EUE against the ER stress
response as determined by measuring the expression of p-PERK,
p-eIF-2a, and CHOP (Fig. 6A). Likewise, bafilomycin markedly
reversed EUE-induced cellular lipid accumulation, as shown by
Oil Red O staining (Fig. 6B). Bafilomycin also reversed
EUEinduced intracellular ApoB accumulation (Fig. 6C). Treatment
with 10 nM bafilomycin decreased the levels of secreted ApoB but
not ApoA1 in the media of cells co-treated with EUE and
palmitate (Fig. 6C, lower). We consistently observed increased
accumulation of intracellular triglyceride and cholesterol with
bafilomycin treatment in cells co-treated with EUE and palmitate
compared to cells not treated with bafilomycin (Fig. 6D, left). The
levels of triglycerides and cholesterol secreted into the culture
media decreased significantly after treatment with bafilomycin,
confirming that the lipid secretion pathways were dysregulated by
the lysosomal V-ATPase inhibitor (Fig. 6D, right). The V-ATPase
inhibitor, bafilomycin, similarly reversed the component of EUE,
aucubin or geniposide-induced regulation against lipid
accumulation processes in palmitate-treated cells. Together, these data
suggest that enhanced V-ATPase activity is necessary for EUE to
diminish the ER stress response and associated hepatic lipid
EUE reduces hepatic lipid accumulation and stimulates
lysosome activity in high-fat-diet-fed rats
To examine the physiological relevance of our in vitro
observations, we examined the effect of EUE on hepatic
dyslipidemia in high-fat-diet (HFD)-fed rats. For in vitro
experiments, E. ulmoides cortex was re-extracted with various ethanol/
water mixtures (25, 50, 75, or 100% ethanol v/v) by reflux. The
aucubin and geniposide contents in the extracts were measured by
HPLC to determine the amount of extract to use for animal
experiments. We found no significant difference in the content of
geniposide extracted (Figure S4) according to the amount of
ethanol. Conversely, we measured the highest content of aucubin
in the 25% ethanol extract, suggesting a positive correlation with
triglycerides and total cholesterol secretion activity, especially for
the 25% ethanol extract in palmitate-treated hepatic cells
(Figure S5). Based on these results, the EUE with the highest
abundance of active compound was orally administered (0.25, 0.5
and 1 g/kg/day) to HFD rats. Body weight was reduced in
EUEtreated HFD rats compared to HFD-only controls (Figure S6).
The liver weights exhibited tended to be less in EUE-treated HFD
rats compared to HFD-only controls (Figure S6). We next
examined whether EUE affects the HFD-induced ER stress
response in vivo. The ER stress response was diminished by EUE as
determined by expression of p-PERK, p-eIF2a, and CHOP
(Fig. 7A). Lysosomal activity was also analyzed in vivo to identify a
possible mechanism for regulation of ER stress by EUE.
Consistent with our in vitro data, the activities of the lysosomal
enzymes a-mannosidase, b-glucuronidase, and b-galactosidase
were reduced in HFD rats and were restored by treatment with
EUE (Fig. 7B). Analysis of hepatic lipid accumulation was also
determined. Oil Red O staining indicated that hepatic lipid
accumulation was reduced in EUE-treated HFD rats (Fig. 7C).
Consistent with these results, EUE markedly decreased hepatic
lipid content as determined by hepatic triglyceride and cholesterol
levels (Fig. 7D). Conversely, EUE increased total plasma
triglyceride and cholesterol levels. Expression of hepatic ApoB,
the lipoprotein for LDL and triglycerides, was increased in
HFDfed rats, while levels of ApoA1 were unaffected (Fig. 7E). Further,
in plasma, ApoB expression was decreased in HFD-fed rats, while
the level of ApoA1 was not affected. Lastly, treatment with EUE
reversed the dysregulated secretion of ApoB in both the liver and
In this study, we examined the effects of EUE on hepatic
dyslipidemia both in vitro and in vivo. EUE diminished the
palmitate-induced ER stress response, expression of associated
lipogenic genes, and apolipoprotein secretion through
enhancement of lysosomal V-ATPase activity. Consistent with these
observations, aucubin and geniposide influenced fatty
acidinduced ER stress and hepatic dyslipidemia through a similar
mechanism, namely, lysosome activation and suppression of ER
The expression of p-eIF2a and CHOP increased in cells
exposed to palmitate (Fig. 1B, Fig. 3), suggesting activation of a
PERK-dependent pathway among the ER stress response signal
transductions. Although significant activation of additional ER
stress pathways has been observed in pancreatic beta-cell system
, activation of components of the ER stress pathway by
palmitate has previously been thought to be primarily confined to
a PERK-dependent process . Indeed, the specific
PERKeIF2a-CHOP ER stress arm was stimulated without affecting
expression of GRP78, IRE-1a, or ATF6. More specifically, PERK
siRNA has been shown to abrogate lipid accumulation . The
application of a nontoxic concentration of palmitate in our
experiments (Figure S1) may explain the specific PERK-ER stress
arm activation. The PERK-eIF2a-CHOP axis may be the
primary mechanistic target of EUE against hepatic dyslipidemia.
EUE regulates the ER stress response, alters apolipoprotein
secretion, and influences hepatic lipid accumulation in
palmitatetreated cells (Fig. 2A). ER stress-associated alteration of ApoB
secretion may explain the relationship between ER stress and
hepatic steatosis. In our study, we utilized the specific conditions of
palmitate-associated ER stress altering ApoB secretion, which, in
turn, lead to hepatic lipid accumulation. The alteration of
apolipoprotein secretion and subsequent lipid accumulation were
inhibited by EUE (Figs. 2B, 2C). It has been suggested that ER
stress-associated hepatic lipid accumulation is related to a
reduction of ApoB secretion . Accumulating evidence suggests
an association between ER stress and secretory protein alterations
. Thus, EUE-induced ER stress regulation may also contribute
to reduction of lipid accumulation through alteration of
Our observations of lysosomal activity may explain how EUE
regulates the ER stress response and dysregulation of ApoB.
Lysosomal activity was significantly decreased upon exposure to
palmitate; however, in the presence of EUE, lysosomal activity was
only slightly affected by exposure to free fatty acids (Fig. 5C).
Thus, the role of EUE on reestablishing ER homeostasis by
enhancing lysosomal activity may lead to the amelioration of ER
stress and subsequently alleviate hepatic dysfunction, e.g., lipid
Lysosome-associated protein degradation also functions as a
cytoplasmic quality control mechanism to eliminate protein
aggregates and damaged organelles [28,29]. Although
nonlysosomal functions are required for the degradation of
shortlived proteins in the cytosol as well as for the stress-induced
enhancement of degradation of cellular proteins within lysosomes
, lysosomal functions appear to reflect a reduced ER stress
response under EUE-treated conditions. Throughout this study,
we observed high lysosomal activity that explained how EUE
regulates the ER stress response. Basally increased lysosomal
function is a key phenomenon of the EUE single-treatment
condition (Figs. 5A, B). When exposed to free fatty acids, EUE
may reduce the folding load of altered/damaged proteins through
the highly activated ERAD II pathway, leading to the
amelioration of ER stress and hepatic lipid accumulation. Defects in the
ERAD II system enhance the ER stress response, leading to
dyslipidemia . ER stress itself has been correlated with hepatic
steatosis, but the basic mechanism regarding induced ER stress
response has not been elucidated. Puri and colleagues 
recently examined a potential role of ER stress in human NAFLD
and found that the degree of UPR activation in liver biopsies from
patients with NAFLD is variable compared to those from subjects
with metabolic syndrome or normal liver histology. Further, they
determined that human NASH was specifically associated with the
activation of C-jun N-terminal kinase (JNK), an ER
stressassociated stress protein. However, any induction mechanism or
regulatory pathway of the ER stress in hepatic dyslipidemia has
not been determined. An alteration of ERAD II activity needs to
be considered as a possible mechanism for hepatic ER stress, as
strategies capable of maintaining lysosomal activity appear to be
necessary for successful treatment of hepatic lipid accumulation.
The effects of EUE were also investigated in an HFD-induced
ER stress and liver steatosis model in vivo. Our in vitro results clearly
showed that the geniposide and aucubin components of EUE
contributed significantly to amelioration of intracellular lipid
accumulation, and thus an ethanol extract of EUE with the highest
content of active the compound aucubin was selected for animal
experiments. An HFD can be used to experimentally induce liver
steatosis in an acute setting [36,37]. Lysosome activity was
transiently decreased after administration of an HFD (Fig. 7B).
However, EUE dose-dependently enhanced the activity of
lysosomal enzymes, including a-mannosidase, b-glucuronidase,
and b-galactosidase, in HFD rats, linking it to suppression of ER
stress, induction of associated lipogenic genes, alteration of protein
secretory pathways, and hepatic lipidemia.
In conclusion, the results of our study show that EUE inhibits
liver steatosis. Further, our results suggest that EUE and its active
components, aucubin and geniposide, are ER stress suppressors.
The mechanism of ER stress suppression by EUE and its active
compounds was associated with enhancement of lysosomal
activity, including that of V-ATPase. Through more efficient
protein degradation resulting from treatment with EUE, aucubin,
or geniposide, demands on ER protein folding might be lessened,
leading to a relieved ER stress response. Our findings provide
molecular evidence for the use of EUE as a possible therapy for the
management of hepatic dyslipidemia.
Figure S1 E. ulmoides Oliver extract protects cells
against palmitate. (A) HepG2 cells were exposed to the
indicated concentrations of EUE and cell viability was determined.
(B) Cells were exposed to 300 mM or 500 mM palmitate for 0, 12,
24, 36 or 48 hrs and cell viability was determined by trypan blue
dye exclusion staining. (C) Cells were exposed to the indicated
concentrations of EUE in the presence or absence of 300 mM
palmitate. For the control group, cells were treated with vehicle.
After 12 h, cell viability was determined by trypan blue dye
exclusion staining. *p,0.05, significantly different from
nontreated control cells. Pal, palmitate; EUE, E. ulmoides Oliver
Figure S2 Aucubin and geniposide protects cells against
palmitate. Cells were exposed to 300 mM palmitate and 10 ug/
mL of either aucubin or geniposide. Cell viability was determined
by trypan blue dye exclusion staining analysis. Pal, palmitate.
Figure S3 E. ulmoides Oliver extract, aucubin, and
geniposide do not affect proteasome expression or
activity. (A) Proteasomal protein expression was measured by
immunoblotting using an antibody against the 20 S core
proteasome. (B) Proteasomal activity was determined by
fluorescence. Values are the mean 6 SE of three independent
experiments. Con, control; Pal, palmitate; EUE, E. ulmoides Oliver
Figure S4 A 25% ethanol extract of E. ulmoides Oliver
contains a high amount of Aucubin. (A) LC spectrum of an
aucubin standard. (B) LC spectrum of EUE showing the presence
of aucubin. (C) Concentration of aucubin in EUE as a function of
the concentration of ethanol used for extraction.
Figure S5 A 25% ethanol extract of E. ulmoides Oliver
significantly regulates hepatic cellular lipid
accumulation and contains the highest concentration of aucubin.
Cells were treated with 300 mM palmitate for 12 hrs in the
presence or absence of 100 mg/mL ethanol-extracted E. ulmoides
Oliver in serial dilutions. Triglyceride (A) and total cholesterol (B)
were measured in lysates and media. *p,0.05, significantly
different from cells treated with palmitate alone. Con, control;
Pal, palmitate; EUE, E. ulmoides Oliver extracts; Geni, geniposide.
Figure S6 A 25% ethanol extract of E. ulmoides Oliver
regulates body and liver weight in high-fat-diet-treated
rats. Rats in the EUE group (n = 10) were provided a HFD with
0.25, 0.5, or 1 g/kg EUE for 10 weeks. Body weight was measured
each week over the 10 week period (A). Liver weight was
measured at the end of the 10 week period (B). *p,0.05,
significantly different from the control group at each time point
(A). *p,0.05, significantly different from control rats (B). Con,
control; HFD, high-fat-diet; EUE, E. ulmoides Oliver extract.
Quantitation of aucubin using HPLC-DAD.
Conceived and designed the experiments: HYL GHL. Performed the
experiments: HYL NYK. Analyzed the data: HYL GHL. Contributed
reagents/materials/analysis tools: MRL HKK SHK HRK YCL. Wrote
the paper: HYL HJC.
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