Protective effects of piceatannol on methylglyoxal-induced cytotoxicity in MC3T3-E1 osteoblastic cells

Article abstract of DOI:10.1080/10715762.2018.1467010

Methylglyoxal (MG) is a reactive α-oxoaldehyde that increases under diabetic conditions and subsequently contributes to the complications associated with this disease. Piceatannol is a naturally occurring analogue of resveratrol that possesses multiple biological functions. The present study investigated the effects of piceatannol on MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells. Piceatannol significantly restored MG-induced reductions in cell viability and reduced lactate dehydrogenase release in MG-treated MC3T3-E1 osteoblastic cells, which suggests that it suppressed MG-induced cytotoxicity. Piceatannol also increased glyoxalase I activity and glutathione levels in MG-treated cells, which indicates that it enhanced the glyoxalase system and thus cellular protection. The present study also showed that piceatannol inhibited the generation of inflammatory cytokines and reactive oxygen species and ameliorated mitochondrial dysfunction induced by MG. Furthermore, piceatannol treatment significantly reduced the levels of endoplasmic reticulum stress and autophagy induced by MG. Therefore, piceatannol could be a potent option for the development of antiglycating agents for the treatment of diabetic osteopathy.

Full text of DOI:10.1080/10715762.2018.1467010

Free Radical Research
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: http://www.tandfonline.com/loi/ifra20
Protective effects of piceatannol on methylglyoxal-
induced cytotoxicity in MC3T3-E1 osteoblastic cells
Kwang Sik Suh, Suk Chon & Eun Mi Choi
To cite this article: Kwang Sik Suh, Suk Chon & Eun Mi Choi (2018) Protective effects of
piceatannol on methylglyoxal-induced cytotoxicity in MC3T3-E1 osteoblastic cells, Free Radical
Research, 52:6, 712-723, DOI: 10.1080/10715762.2018.1467010
To link to this article: https://doi.org/10.1080/10715762.2018.1467010
Published online: 24 May 2018.
Submit your article to this journal
Article views: 4
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ifra20

FREE RADICAL RESEARCH
2018, VOL. 52, NO. 6, 712–723
https://doi.org/10.1080/10715762.2018.1467010
ORIGINAL ARTICLE
Protective effects of piceatannol on methylglyoxal-induced cytotoxicity
in MC3T3-E1 osteoblastic cells
ꢀ
ꢀ
Kwang Sik Suh , Suk Chon and Eun Mi Choi
Department of Endocrinology and Metabolism, School of Medicine, Kyung Hee University, Dongdaemun-gu, Republic of Korea
ABSTRACT
ARTICLE HISTORY
Received 6 February 2018
Revised 16 April 2018
Accepted 17 April 2018
Methylglyoxal (MG) is a reactive a-oxoaldehyde that increases under diabetic conditions and sub-
sequently contributes to the complications associated with this disease. Piceatannol is a naturally
occurring analogue of resveratrol that possesses multiple biological functions. The present study
investigated the effects of piceatannol on MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells.
Piceatannol significantly restored MG-induced reductions in cell viability and reduced lactate
dehydrogenase release in MG-treated MC3T3-E1 osteoblastic cells, which suggests that it sup-
pressed MG-induced cytotoxicity. Piceatannol also increased glyoxalase I activity and glutathione
levels in MG-treated cells, which indicates that it enhanced the glyoxalase system and thus cellu-
lar protection. The present study also showed that piceatannol inhibited the generation of inflam-
matory cytokines and reactive oxygen species and ameliorated mitochondrial dysfunction
induced by MG. Furthermore, piceatannol treatment significantly reduced the levels of endoplas-
mic reticulum stress and autophagy induced by MG. Therefore, piceatannol could be a potent
option for the development of antiglycating agents for the treatment of diabetic osteopathy.
KEYWORDS
Cytotoxicity; methylglyoxal;
mitochondrial function;
osteoblasts; piceatannol
Introduction
Inflammatory cytokines induce rapid bone loss and
greatly increase the risk of fractures [6]. Of the cyto-
kines, tumour necrosis factor (TNF)-a is an inflammatory
mediator of skeletal damage in cases of arthritis and
postmenopausal osteoporosis [7] in which the balance
between the formation and resorption of the skeleton is
shifted toward resorption and leads to bone loss [8].
TNF-a decreases osteoblastic bone formation through
the suppression of osteoblast proliferation, induction of
osteoblast apoptosis, and inhibition of osteoblast differ-
entiation [9]. Interleukin (IL)-6 is a pleiotropic cytokine
that is commonly produced at local tissue sites and
then released into systemic circulation in almost all sit-
uations involving homeostatic perturbations. IL-6 may
facilitate autoimmune phenomena, amplify acute
inflammation, and promote evolution to a chronic
inflammatory state. Moreover, the generation of ROS
has been implicated in the mechanisms by which TNF-a
increases the apoptosis of osteoblasts [10].
Methylglyoxal (MG) is a major precursor involved in the
formation of advanced glycation end products (AGEs)
that is associated with the pathogenesis of diabetes-
related complications. Excessive glucose levels can
lead to molecular damage via the formation of 1,2-
dicarbonyl compounds, including MG, from the triose
phosphate intermediates of glycolysis [1] or the metab-
olism of acetones generated during ketosis [2].
MG-induced damage to proteins and other aberrant
polypeptides can result in the generation of reactive
oxygen species (ROS), promote mitochondrial dysfunc-
tion, and inhibit proteasomal activity. The important
role of MG in this pathology is further supported by the
existence of the glyoxalase enzyme system [3], which is
a major detoxification system for dicarbonyl com-
pounds in the human body. With reduced levels of
glutathione (GSH) as a cofactor, glyoxalase I can
promptly clear alpha-carbonyl aldehydes, including MG,
and inhibit the formation of AGEs [4]. Deficits in the
glyoxalase degradation pathway render organisms
more susceptible to glycation and subsequent damage,
whereas its overexpression increases lifespan in
Caenorhabditis elegans [5].
Hyperglycemic conditions are associated with mito-
chondrial damage and elevated levels of oxidative
stress that contribute to the pathology of diseases; this
has in part been ascribed to mitochondrial glycation
due to MG [11]. Furthermore, reactive dicarbonyl
CONTACT Eun Mi Choi
cheunmi@khu.ac.kr
Department of Endocrinology and Metabolism, School of Medicine, Kyung Hee University, 1,
Hoegi-Dong, Dongdaemun-gu, Seoul, Republic of Korea
ꢀ
These authors contributed equally to this work.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

FREE RADICAL RESEARCH
713
disrupts mitochondrial function in vitro [12]. Peroxisome
proliferator-activated receptor-a coactivator-1a (PGC-
1a) is the master regulator of mitochondrial biogenesis
and other important molecules that are protective
against ROS generation and damage [13]. PGC-1a
integrates and coordinates the activities of multiple
transcription factors, including nuclear respiratory
factors 1 and 2 (NRF1 and ꢁ2), which control the
transcription of key mitochondrial proteins such as
nuclear encoded respiratory complex proteins and
mitochondrial transcriptional factor [14]. Nitric oxide
(NO) is another important upstream regulator of mito-
chondrial biogenesis that involves both nuclear- and
mitochondrial-encoded genes [15]. Decreases in NO
and increases in ROS lead to loss of mitochondria via
reductions in biogenesis and the removal of dysfunc-
tional mitochondria via mitophagy [16]. Adenosine
monophosphate (AMP)-activated protein kinase (AMPK)
is a multifunctional metabolic and energy sensor that is
activated under conditions of cellular energy depletion
[17]. MG toxicity is associated with energy depletion.
For example, Gugliucci [18] proposed that MG causes a
modification of AMPK that attenuates the activation of
this critical fuel sensor, which could produce metabolic
dysregulation. Furthermore, Jiang et al. [19] reported
that MG-induced AMPK inactivation is associated with
the induction of apoptotic cell death in PC12 cells.
[23]. The autophagic pathway is also involved in mito-
chondrial dysfunction, in which it serves as a crucial
pathogenic mechanism [24].
Piceatannol (trans-3,4,30,50-tetrahydroxystilbene) is a
hydroxylated analogue of resveratrol that is found in
various types of plants, including sugar cane, berries,
passion fruit seeds, peanuts, grapes, wine, and white
tea [25]. Based on its structural similarity to resveratrol
and its formation through the in vivo metabolism of
resveratrol [26], it has been hypothesised that piceatan-
nol may have biological activities similar to those of
resveratrol. Piceatannol is thought to exert stronger
antioxidant activities than resveratrol due to its extra
hydroxyl (OH) group [27]. Recently, piceatannol has
been receiving more attention regarding age-related
diseases due to its anticarcinogenic, antiviral, antioxida-
tive, neuroprotective, and estrogenic effects [28].
Additionally, piceatannol displays a variety of properties
including the ability to induce sirtuin activity in human
monocytes [29], enhance vasorelaxant effects in endo-
thelium-intact aortas [30], aid in the upregulation of
endothelial NO synthase (NOS) expression in endothe-
lial cells [31], promote collagen synthesis in dermal
fibroblast cells, inhibit melanogenesis in melanoma cells
[32], and protect the skin from ultraviolet B (UVB) irradi-
ation [33].
Son et al. [34] reported that piceatannol is capable of
protecting neuronal cells against glutamate-induced
cell death by enhancing Nrf2-dependent heme oxygen-
ase-1 expression. Piceatannol also possesses anti-inflam-
matory properties that suppress the activation of
nuclear transcription factor nuclear factor kappa B (NF-
jB) by inhibiting the phosphorylation of IjBa kinase
and p65 [35]. Chang et al. [36] found that piceatannol
increases bone morphogenetic protein-2 synthesis by
inducing osteoblast maturation and differentiation,
which is followed by increases in bone mass. Thus, the
present study investigated the protective effects of
piceatannol against glycative stress in osteoblastic
MC3T3-E1 cells with a focus on MG-induced alterations
in inflammatory cytokines, oxidative stress, mitochon-
drial function, the MG detoxifying system, and ER
stress/autophagy.
The endoplasmic reticulum (ER) is a central organelle
engaged in lipid synthesis as well as protein folding
and maturation. A variety of toxic insults can disturb ER
function and result in ER stress [20]. Regardless of
the stimulus, elevated ER stress is characterised by
increased concentrations of unfolded or misfolded pro-
teins within the ER, which triggers the unfolded protein
response (UPR) in an attempt to restore protein-folding
homeostasis. The UPR evolved to counter the stresses
that occur in the ER due to misfolded proteins. This
sophisticated quality control system attempts to restore
homeostasis via the actions of a number of different
pathways that are coordinated in the first instance by
ER stress-sensor proteins, such as inositol-requiring pro-
tein 1a (IRE1), activating transcription factor 6 (ATF6),
and protein kinase RNA-like endoplasmic reticulum kin-
ase (PERK) [21]. Connective tissue cells such as chondro-
cytes, osteoblasts, and fibroblasts are also included in
this category of strongly secretory cells and may be par-
ticularly sensitive to the induction of ER stress. ER stress
is a potent inducer of autophagy, which may facilitate
the removal of unfolded or misfolded proteins [22].
Autophagy widely occurs in eukaryotic cells and is an
evolutionarily conserved catabolic process involved in
the elimination and recycling of nonessential or abnor-
mal organelles and long-lived proteins by lysosomes
Materials and methods
Materials
Piceatannol was purchased from ChromaDex Inc.
(Irvine, CA, USA), a-modified minimal essential medium
(a-MEM) and fetal bovine serum (FBS) were purchased
from Gibco BRL (Grand Island, NY, USA), and all other

714
K. S. SUH ET AL.
reagents were of the highest commercial grade avail-
able and purchased from Sigma (St. Louis, MO, USA).
Measurement of mitochondrial superoxide levels
Mitochondrial superoxide levels were detected using a
MitoSOXTM Red mitochondrial superoxide indicator
(Invitrogen Molecular Probes, Carlsbad, CA). MitoSOXTM
Red (Ex/Em ¼ 510 nm/580 nm) is a fluorogenic dye used
for the highly selective detection of superoxides in the
mitochondria of cells. The cells were incubated with
2 mM MitoSOXTM Red at 37 ꢂC for 20 min, in accordance
with themanufacturer’s instructions. After washing
the cells, the levels of MitoSOXTM Red fluorescence
were measured.
Cell cultures
The osteoblastic MC3T3-E1 subclone 4 line was
obtained from ATCC (Manassas, VA) and the MC3T3-E1
cells were cultured at 37 ꢂC in a-MEM (Gibco) in an
atmosphere of 5% CO2. Unless otherwise specified, the
medium contained 10% heat-inactivated FBS, penicillin
(100 U/ml), and streptomycin (100 mg/ml). The cells were
treated at confluence with culture medium containing
5 mM b-glycerophosphate and ascorbic acid (50 mg/ml)
to initiate differentiation. After 2 days, the cells were
incubated with piceatannol for 48 h.
Measurement of glyoxalase I activity and
GSH levels
The cells were lysed and homogenised in PBS and then
the homogenates were centrifuged at 13,000 ꢃ g for
15 min at 4 ꢂC. The supernatants were collected and
assessed for glyoxalase I activity and protein content.
Glyoxalase I activity was measured using a modified
version of a previously published method [37]. Briefly,
50 ml of sample and 200 ml of reaction mix [60 mm
sodium phosphate buffer (pH 6.6) containing 4 mm GSH
and 4 mm MG] were loaded onto a UV microplate and
preincubated for 10 min at 37 ꢂC. S-lactoylglutathione
synthesis was assessed by measuring absorbance at
240 nm for 5 min at 25 ꢂC. GSH was measured using a
Glutathione Assay Kit (BioAssay Systems, Hayward, CA,
USA), in accordance with the manufacturer’s instruc-
tions. The determination of GSH was based on the reac-
tion of 50,5-dithiobis-2-nitrobenzoic acid (DTNB) with
GSH, which yields 5-thionitrobenzoic acid (TNB), a
yellow chromophore with a maximum absorbance at
412 nm. The concentrations of GSH were determined
from a freshly prepared standard curve for GSH.
Cell viability
Surviving cells were counted using an MTT assay.
Briefly, 20 ml of MTT in phosphate-buffered saline (PBS;
pH 7.4; 5 mg/ml) was added to each well and the plates
were then incubated for 2 h. After the removal of the
solution from the wells, dimethyl sulfoxide (DMSO) was
added to dissolve formazan products and the plates
were shaken for 5 min. The absorbance of each well
was recorded on a microplate spectrophotometer
at 570 nm.
Lactate dehydrogenase cytotoxicity assay
Cytotoxicity was evaluated by quantifying the degree of
damage to the plasma membrane. Lactate dehydrogen-
ase (LDH) is a stable enzyme present in all cell types
that is rapidly released into the culture medium when
the plasma membrane is damaged. Cell membrane
integrity was evaluated by measuring LDH leakage from
the cells with an LDH Cytotoxicity Assay Kit (BioVision,
Milpitas, CA, USA), in accordance with the man-
ufacturer’s instructions.
Measurements of TNF-a and IL-6
TNF-a and IL-6 contents in the medium were measured
with an enzyme immunoassay system (R&D System Inc,
Minneapolis, MN, USA), in accordance with the man-
ufacturer’s instructions.
Measurement of intracellular ROS levels
Intracellular ROS formation was measured using 20,70-
dichlorodihydrofluorescein diacetate (H2DCFDA). To
load the cells with the fluorescent dye, the cells were
incubated with H2DCFDA in Hank’s solution at a final
concentration of 10 mM for 45 min at 37 ꢂC in the dark.
Following a wash with Dulbecco’s phosphate-buffered
saline (DPBS), ROS levels were determined by measur-
ing fluorescence intensity at an excitation wavelength
of 485 nm and an emission wavelength of 530 nm.
Determination of the mitochondrial
membrane potential
A JC-1 Mitochondrial Membrane Potential Assay Kit
(Cayman Chemical Co, Ann Arbor, MI, USA) was used to
measure changes in the mitochondrial membrane
potential (MMP) in cells. JC-1 is a lipophilic and cationic
dye that permeates plasma and mitochondrial mem-
branes. The dye fluoresces red when it aggregates in

FREE RADICAL RESEARCH
715
healthy mitochondria with a high membrane potential,
whereas it appears in a monomeric form and fluoresces
green in mitochondria with a lowered membrane
potential. The cells were incubated with the MMP-
sensitive fluorescent dye for 20 min at 37 ꢂC and then
washed twice in PBS. Thereafter, red (excitation:
550 nm; emission: 600 nm) and green (excitation:
485 nm; emission: 535 nm) fluorescence levels were
diacetate is transformed into a less cell-permeable form
of DAF-FM by cellular esterases, which prevents signal
loss due to the diffusion of the molecule from the cell.
In the presence of oxygen, DAF-FM reacts with NO to
yield the highly fluorescent product triazolofluorescein.
For NO detection [38], the cells were loaded with 5 mM
DAF-FM diacetate (Invitrogen Corporation, Burlington,
ON, Canada) and incubated for 2 h at 37 ꢂC. After
removal of the excess probe, DAF-fluorescence inten-
sity, which reflects intracellular NO levels, was measured
with excitation at 495 nm and emission at 515 nm.
measured using
a fluorescence microplate reader
(Molecular Devices, Sunnyvale, CA, USA). Mitochondrial
depolarisation (i.e. loss of MMP) manifests as a decrease
in the red/green fluorescence ratio.
Measurements of ATF-6 and IRE1
Adenosine triphosphate measurement
The levels of ATF-6 and IRE1 in the cytosol were deter-
mined using an ELISA kit (MyBioSource), in accordance
with the manufacturer’s instructions.
The cells were lysed and homogenised in PBS and then
the homogenates were centrifuged at 13,000 ꢃ g for
15 min at 4 ꢂC. The supernatants were collected and
used for the adenosine triphosphate (ATP) assays and
protein content measurement. ATP concentrations were
Autophagy detection assay
The Autophagy Detection Kit (Abcam, Cambridge, MA,
USA) was used in accordance with the manufacturer’s
protocol, in conjunction with a fluorescence microplate
reader. The 488-nm-excitable green fluorescent detec-
tion reagent supplied in the Autophagy Detection Kit
becomes brightly fluorescent in vesicles produced dur-
ing autophagy and has been validated under a wide
range of conditions known to modulate autoph-
agy pathways.
determined with
a luciferase reaction using an
EnzyLightTM ATP Assay Kit and protein concentrations
were determined using a Bio-Rad protein assay reagent
(BioAssay Systems, Hayward, CA, USA).
Measurements of PGC-1a and NRF-1 levels
The cells were lysed and homogenised in PBS and then
the homogenates were centrifuged at 13,000 ꢃ g for
15 min at 4 ꢂC. The supernatants were collected and
used for enzyme-linked immunosorbent assays (ELISA)
and protein content measurements. PGC-1a was meas-
ured with a Mouse PGC-1a ELISA kit (MyBioSource, San
Diego, CA, USA), NRF1 was measured with an NRF1
ELISA kit (Cusabio, Hubei, China), and protein concen-
trations were determined using a Bio-Rad protein
assay reagent.
Statistical analysis
All results are expressed as mean SEM. Statistical sig-
nificance was determined with analysis of variance
(ANOVA) tests and post-hoc Dunnett’s t-tests; p-values
<.05 were considered to indicate statistical significance.
Results and discussion
Measurement of phosphorylated AMPK
The present study aimed to determine the in vitro cyto-
protective activity of piceatannol against damage
induced by MG in MC3T3-E1 osteoblastic cells.
Previously, our research group observed a dose-
dependent loss in the viability of MC3T3-E1 osteoblastic
cells exposed to varying concentrations of MG
(100–400 mM) [39]. Based on the results of these cyto-
toxicity studies, the 400 mM dose of MG was selected for
the biochemical assays in the present study. To deter-
mine whether piceatannol had a protective effect
against MG-induced cytotoxicity, the cells were preincu-
bated with piceatannol for 1 h and then cultured with
MG for 48 h (Figure 1(A)). Concentrations of piceatannol
ꢄ1 mM had no effect on the viability of MC3T3-E1 cells.
The cells were lysed and homogenised in PBS and then
the homogenates were centrifuged at 13,000 ꢃ g for
15 min at 4 ꢂC. The supernatants were collected and
used for ELISA and protein content measurements.
Phosphorylated AMPK levels were measured with an
ELISA kit (MyBioSource, San Diego, CA, USA).
Measurement of NO generation
4-Amino-5-methylamino-20,70-difluorofluorescein (DAF-
FM) diacetate is a sensitive fluorescent indicator used
for the detection of NO because it is a cell-permeable
derivative of DAF-FM. Upon entry into the cell, DAF-FM

716
K. S. SUH ET AL.
Treatment with 400 mM MG resulted in MC3T3-E1 cell
death of nearly 50% compared with untreated control
cells. However, piceatannol (0.01–1 mM) inhibited MG-
induced cytotoxicity. LDH is localised in the cytoplasm
of cells and, when extruded into the medium, indicates
that cells are damaged or necrotic [40]. Therefore, the
leakage of LDH into the culture medium was measured
in the present study to determine the cytoprotective
effects of piceatannol. MG (400 mM) was cytotoxic to
MC3T3-E1 cells but piceatannol (0.1 and 1 mM) reduced
these effects (Figure 1(B)). Aminoguanidine (AG;
300 mM), which is a carbonyl scavenger, also inhibited
MG-induced cytotoxicity.
The abnormal cellular accumulation of MG, a dicar-
bonyl metabolite, is associated with increased ROS
formation [41] and thus the present study examined
whether piceatannol would attenuate MG-induced ROS
production. The exposure of MC3T3-E1 cells to 400 mM
MG for 48 h increased H2DCFDA fluorescence
(Figure 2(A)), but the preincubation of the cells with
0.01–1 mM piceatannol or AG for 1 h prior to MG treat-
ment prevented the intracellular oxidation of the
fluorescent probe. MitoSOXTM Red localises to the mito-
chondria and serves as a fluoroprobe for the selective
detection of superoxides in organelles [42]. In the pre-
sent study, MG (400 mM) significantly increased mito-
chondrial superoxide production compared with that of
untreated cells but pretreatment with piceatannol
(0.01–1 mM) or AG decreased this production
(Figure 2(B)). These findings demonstrate that piceatan-
nol reduced MG-induced ROS generation.
Because MG-mediated damage can be countered by
GSH-dependent metabolism via glyoxalase I [43], the
effects of piceatannol on glyoxalase I activity and GSH
levels were examined in osteoblast MC3T3-E1 cells.
There was a significant decrease in glyoxalase I activity
(A)
150
125
100
75
(A)
#
150
100
50
Without MG
With MG
*
*
*
*
*
*
*
*
#
50
MG
-
-
-
+
-
-
+
0.01
-
+
+
+
-
+
0
Pic (μM)
AG
0.1
1
0
0.010.1
1
AG
0
0.010.1
1
AG
-
-
Piceatannol (μM)
(B)
(B)
150
250
200
150
100
50
#
#
125
100
75
*
*
*
*
*
*
*
50
0
MG
-
-
-
+
-
-
+
+
+
+
-
+
MG
-
-
-
+
-
-
+
0.01
-
+
+
+
-
Pic (μM)
AG
0.01
0.1
1
Pic (μM)
0.1
1
-
-
-
AG
-
-
+
Figure 2. Inhibitory effects of piceatannol on MG-induced oxi-
dative stress in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells
were preincubated with piceatannol (Pic) or AG (300 mM) prior
to treatment with MG (400 mM) for 48 h. Data for (A) ROS and
(B) mitochondrial superoxide levels are expressed as mean
relative percentages of the fluorescence. #p < .05, compared
Figure 1. Protective effects of piceatannol against MG-induced
cytotoxicity in osteoblastic MC3T3-E1 cells. Osteoblastic
MC3T3-E1 cells were treated with piceatannol (Pic) or AG
(300 mM) in the absence or presence of MG (400 mM) for 48 h
and then (A) cell viability and (B) lactate LDH release were
ꢀ
ꢀ
measured. #p < .05, compared to untreated cells; p < .05,
compared to cells treated with MG alone.
to untreated cells; p < .05, compared to cells treated with
MG alone.

FREE RADICAL RESEARCH
717
110
100
90
80
70
60
50
40
30
20
10
0
diabetic neuropathy compared with patients with pain-
less diabetic neuropathy [46]. These authors also found
that increased glyoxalase I activity is significantly corre-
lated with reductions in the occurrence of painful dia-
betic neuropathies. In contrast to the stimulating effects
of piceatannol in osteoblast MC3T3-E1 cells noted in
this study, piceatannol has been shown to inhibit glyox-
alase I activity and the glyoxalase I -dependent prolifer-
ation of cancer cells [47]. Thus, piceatannol appears to
have different biological effects in different cell types.
The mechanisms through which piceatannol exerts
multidirectional action remain to be clarified.
(A)
*
*
*
*
#
MG
Pic (μM)
-
-
-
+
-
-
+
0.01
-
+
+
+
-
+
0.1
1
Methylglyoxal-modified proteins are particularly
potent inducers of TNF-a expression [48]. Another cyto-
kine, IL-6, is regulated by the nuclear factor (NF)-jb and
c-Jun N-terminal kinase (JNK) pathways [49]. Onishi
et al. [50] reported that MG augments IL-6 and IL-1b
levels, enhances the expression of toll-like receptor
(TLR)-4 and TLR9, and increases the production of mito-
gen-activated protein kinases (MAPKs). Because cyto-
kine production induced by MG-modified proteins is
enhanced by increasing concentrations of these pro-
teins [51], the development of pathological changes
may be elevated in poorly controlled diabetic patients.
Thus, the induction of proinflammatory cytokines may
represent a mechanism by which hyperglycemia and its
derived products manifest complications in diabetes.
Wang et al. [52] demonstrated that MG significantly
increases the secretion of proinflammatory cytokines,
including IL-6, IL-8, and TNF-a, and induces apoptosis in
neutrophils. However, when antioxidants are added to
MG-treated cells, there is a marked reduction in all of
these proinflammatory cytokines. The present study
also investigated whether piceatannol would modulate
MG-induced increases in TNF-a and IL-6 (Figure 4).
When 400 mM MG was added to cells, the production
levels of TNF-a and IL-6 increased significantly.
However, these levels were significantly inhibited by
treatment with piceatannol (0.01–1 mM) or AG. Because
accumulated MG causes significant damage due to oxi-
dative stress, there is also an inflammatory response in
osteoblastic cells. Thus, the anti-inflammatory efficacy
of piceatannol may have an important counter-effect to
prevent impairments associated with diabetic
bone disease.
AG
-
-
(B)
200
*
*
*
150
100
50
0
MG
-
-
-
+
-
-
+
0.01
-
+
+
+
-
+
Pic (μM)
AG
0.1
1
-
-
Figure 3. Effects of piceatannol on glyoxalase I activity and
GSH levels in MG-treated MC3T3-E1 cells. MC3T3-E1 cells were
preincubated with piceatannol (Pic) or AG (300 mM) prior to
treatment with MG (400 mM) for 48 h. The control values for
(A) glyoxalase I activity and (B) GSH were 0.29 0.001 DOD/
min/mg and 72.03 6.91 mg/mg, respectively. #p < .05, com-
ꢀ
pared to untreated cells; p < .05, compared to cells treated
with MG alone.
in MC3T3-E1 cells treated with MG (400 mM; Figure 3(A)),
but treatment with piceatannol (0.01–1 mM) or AG
increased the glyoxalase I activity inhibited by MG.
Additionally, piceatannol (0.01–1 mM) increased GSH
levels in MG-treated MC3T3-E1 osteoblastic cells
(Figure 3(B)). Under conditions of aging and oxidative
stress, decreased glyoxalase
I activity results in
increased levels of glycation and tissue injury [43]. In
obese diabetic (ob/ob) mice and streptozotocin-treated
lean MF1 mice, hyperglycemia is accompanied by sig-
nificant decreases in erythrocyte glyoxalase I activity as
well as a marked increase in MG concentrations [44].
Additionally, the overexpression of glyoxalase I prevents
the formation of AGEs in endothelial cells cultured in
high levels of glucose [45]. Consistent with its anti-gly-
cation and detoxification properties, glyoxalase I activity
is significantly reduced in diabetic patients with painful
Mitochondrial dysfunction is thought to play an
important role in the pathogenesis of age-associated
diseases. High concentrations of MG induce toxicity in
cells via the secondary depletion of ATP and modula-
tion of the MMP [53]. The exposure of MC3T3-E1 cells to
MG (400 mM) induced marked disruption in MMP
(Figure 5(A)), but treatment with piceatannol (0.1 and
1 mM) or AG reduced this disruption. Additionally,

718
K. S. SUH ET AL.
(A)
is regulated by several different key factors involved in
mitochondrial biogenesis, including AMPK, which acts
as an energy sensor of the cell and a key regulator of
mitochondrial biogenesis. Piceatannol (0.1 and 1 mM)
significantly stimulated the activation of AMPK under
MG-treated conditions (Figure 5(E)), which indicates
that piceatannol is a potent inducer of AMPK. These
findings suggest that piceatannol enhanced cell func-
tion due to the activation of AMPK and protection of
the mitochondria.
300
#
200
100
0
*
*
*
*
It has also been reported that the dysregulation of
mitochondrial biogenesis is related to impairments in
NO bioavailability [54]. NO regulates mitochondrial
biogenesis through the transcriptional activation of
PGC-1a16 and thus the present study evaluated NO gen-
eration using DAF-FM, which is a specific probe used to
quantify low concentrations of NO. MG (400 mM)
decreased the production of NO in MC3T3-E1 cells, but
this was prevented by pretreatment with piceatannol
(0.01–1 mM) or AG (Figure 5(F)). The present data indi-
cate that piceatannol is a powerful means of enhance-
ment for these mitochondrial biogenesis-related factors
that can improve cell function by protecting mitochon-
dria in MG-treated osteoblasts.
MG
-
-
-
+
-
+
0.01
-
+
+
+
-
Pic (μM)
AG
0.1
1
-
-
-
+
500
400
300
200
100
0
(B)
#
*
*
*
*
The endoplasmic reticulum stress is generally
assessed using ATF6 or IRE1 levels. In the present study,
the effects of piceatannol on the activation of ER-stress
sensors elicited by MG stimulation were investigated.
The levels of ATF6 in MC3T3-E1 cells stimulated with
MG were significantly inhibited by piceatannol
(0.1–1 mM) or AG (Figure 6(A)). Additionally, piceatannol
(0.1 mM) or AG significantly reduced IRE1 levels that
were increased by MG (Figure 6(B)). These findings indi-
cate that piceatannol inhibited the activation of ER-
stress sensors induced by MG. Excessive or prolonged
ER stress may result in apoptosis via mitochondrial-
dependent or mitochondrial-independent mechanisms
[55,56]. Guo et al. [57] found that the MC3T3-E1 osteo-
blastic cells exhibit decreased cellular viability in
response to ER stress. When unfolded proteins accumu-
late in the ER, binding immunoglobulin protein (BiP)
dissociates from IRE1a, which leads to its homodimer-
isation and trans-autophosphorylation, and activates its
kinase and endoribonuclease (RNase) domains. IRE1
RNase then cleaves an intron from X-box-binding pro-
tein 1 (XBP1) mRNA, which induces a translational
frameshift [58]. ATF6 is translocated to the Golgi appar-
atus where it is cleaved by two proteases, which
releases a fragment containing a basic region and leu-
cine zipper (bZIP) domain that migrates to the nucleus
and induces the transcription of UPR-inducible genes
[58]. The ER stress-induced apoptosis of osteoblasts is
implicated in the pathogenesis of osteoporosis [59,60].
MG
-
-
-
+
-
-
+
0.01
-
+
+
+
-
+
Pic (μM)
AG
0.1
1
-
-
Figure 4. Effects of piceatannol on MG-induced changes in
TNF-a and IL-6 production in MC3T3-E1 cells. MC3T3-E1 cells
were preincubated with piceatannol (Pic) or AG (300 mM) prior
to treatment with MG (400 mM) for 48 h. The control values
for TNF-a and IL-6 were 18.09 2.713 pg/mg and
0.655 0.033 ng/mg, respectively. #p < .05, compared to
ꢀ
untreated cells; p < .05, compared to cells treated with
MG alone.
piceatannol (0.1 and 1 mM) or AG increased ATP synthe-
sis that was inhibited by MG (Figure 5(B)). Taken
together, these results suggest that piceatannol amelio-
rated MG-induced mitochondrial dysfunction. To deter-
mine whether the increased mitochondrial function in
piceatannol-treated cells was a consequence of the
induction of mitochondrial biogenesis, the levels of
mitochondrial biogenesis-related factors were exam-
ined. PGC-1a is a cotranscriptional regulation factor that
induces mitochondrial biogenesis by activating different
transcription factors, including nuclear respiratory factor
(NRF), which subsequently activates mitochondrial tran-
scription factor A. The latter drives the transcription and
replication of mitochondrial DNA [14]. In the present
study, the levels of PGC-1a and NRF-1 significantly
increased following piceatannol treatment (0.01–1 mM
and 0.01 mM, respectively; Figures 5(C,D)). PGC-1a itself

FREE RADICAL RESEARCH
719
(A)
(B)
120
100
80
60
40
20
0
175
150
125
100
75
*
*
*
*
*
*
#
50
#
25
0
MG
MG
-
-
-
-
-
-
+
-
-
+
0.01
-
+
+
+
-
+
+
-
-
+
0.01
-
+
+
+
-
+
Pic (μM)
AG
0.1
1
Pic (μM)
AG
0.1
1
-
-
-
-
(C)
(D)
150
100
50
300
200
100
0
*
*
*
*
*
0
MG
MG
-
-
-
-
-
-
+
-
-
+
0.01
-
+
+
+
-
+
+
-
-
+
0.01
-
+
+
+
-
+
Pic (μM)
AG
0.1
1
Pic (μM)
AG
0.1
1
-
-
-
-
(E)
(F)
150
100
50
250
200
150
100
50
*
*
*
*
*
#
*
*
#
0
0
MG
MG
Pic (μM)
AG
-
-
-
-
-
-
+
-
-
+
0.01
-
+
+
+
-
+
+
-
-
+
0.01
-
+
+
+
-
+
0.1
1
Pic (μM)
AG
0.1
1
-
-
-
-
Figure 5. Effects of piceatannol on mitochondrial function and mitochondrial biogenesis-related factors in MG-treated MC3T3-E1
cells. MC3T3-E1 cells were preincubated with piceatannol (Pic) or AG (300 mM) prior to treatment with MG (400 mM) for 48 h.
Data for (A) MMP and (F) NO are expressed as mean relative percentages of the fluorescence. The control values for (B) ATP lev-
els, (C) PGC-1a levels, (D) NRF-1 levels, and (E) AMPK activity were 1.496 0.193 nmol/mg, 387.98 5.39 ng/mg, 2.44 0.17 ng/mg,
ꢀ
and 15.27 0.886 units/mg, respectively. #p < .05, compared to untreated cells; p < .05, compared to cells treated with
MG alone.
Thus, the modulation of ER stress signalling pathways
by piceatannol is an important issue regarding protec-
tion against cellular damage induced by MG.
The present study evaluated the autophagy activity
of MC3T3-E1 osteoblastic cells pretreated with piceatan-
nol in the presence of MG. The autophagy activity of
MG-treated cells exhibited an increase compared with
that in the control group (Figure 7). When incubated
with piceatannol (0.1 mM) or AG, MG-induced autophagy
activation in MC3T3-E1 cells significantly decreased.
Notably, the autophagy-reducing effects of piceatannol
were abolished after the addition of Ex527 (1 mM), a
selective inhibitor of SIRT1, which suggests that the
piceatannol-induced reduction in autophagy was

720
K. S. SUH ET AL.
400
300
200
100
0
(A) 400
#
**
300
200
100
0
#
*
*
*
*
*
MG
AG
MG
-
-
-
+
-
+
-
+
-
+
+
+
-
-
+
0.01
-
+
+
1
-
+
-
+
-
-
-
-
+
+
-
Pic (μM)
AG
0.1
Pic
-
+
-
-
Ex527
-
-
Figure 7. Autophagy activity in MC3T3-E1 cells exposed to
piceatannol in the presence of MG. MC3T3-E1 cells were prein-
cubated with piceatannol (0.1 mM Pic) or AG (300 mM) prior to
treatment with MG (400 mM) for 24 h. The cells were also
treated with Pic in the presence or absence of 1 mM Ex527, a
selective inhibitor of SIRT1. Data are expressed as mean rela-
tive percentages of the fluorescence. #p < .05, compared to
300
200
100
0
(B)
#
*
ꢀ
untreated cells; p < .05, compared to cells treated with MG
ꢀꢀ
alone; p < .05, compared to cells treated with Pic and MG.
*
regulated kinase (ERK) and JNK pathways to inhibit
mechanistic target of rapamycin (mTOR) signalling via
the activation of AMPK [68,69]. There is also evidence
showing that ROS mediate the strong upregulation of
Beclin 1 by activating NF-jB, which is responsible for
ROS-induced autophagy [70,71]. Taken together, these
results indicate that MG-induced ROS generation might
be the primary mechanism underlying MG-induced
autophagy. In the present study, ROS levels significantly
increased when the MC3T3-E1 osteoblastic cells were
incubated with MG but treatment with piceatannol
decreased intracellular ROS levels. Therefore, it can be
speculated that MG-induced autophagy by elevating
ROS levels in MC3T3-E1 cells and that piceatannol pro-
tected the cells by reducing the generation of ROS,
which weakened MG-induced autophagy.
In conclusion, piceatannol offered significant cyto-
protection against oxidative damage induced by MG in
MC3T3-E1 osteoblastic cells as revealed by reductions in
LDH release, a remarkable decrease in intracellular ROS
levels, and elevations in glyoxalase I activity and GSH
levels. Additionally, the present results revealed that
piceatannol prevented the release of inflammatory cyto-
kines and significantly upregulated markers of mito-
chondrial biogenesis in MC3T3-E1 cells. These results
suggest that piceatannol might aid in prevention of the
development of diabetic osteopathy by activating the
glyoxalase system and enhancing mitochondrial bio-
genesis. Furthermore, piceatannol treatment resulted in
MG
Pic (μM)
AG
-
-
-
+
-
-
+
0.01
-
+
+
1
-
+
-
0.1
-
+
Figure 6. Effects of piceatannol on ATF-6 and IRE1 levels in
MG-treated MC3T3-E1 cells. MC3T3-E1 cells were preincubated
with piceatannol (Pic) or AG (300 mM) prior to treatment with
MG (400 mM) for 24 h. The control values for (A) ATF-6 and (B)
IRE1 were 54.72 0.497 ng/mg and 3.095 0.179 ng/mg,
ꢀ
respectively. #p < .05, compared to untreated cells; p < .05,
compared to cells treated with MG alone.
mediated by SIRT1. The effects of autophagy on cell
death are complicated as the cytoprotective and cyto-
toxic functions of autophagy have both been reported
[61]. When autophagy levels surpass a certain threshold,
it may cause autophagic cell death [62,63]. In some dis-
eases, endogenous ROS levels are increased, which then
activate mitochondrial autophagy [64,65]. Increased
intracellular ROS levels can cause destabilisation of the
lysosomal compartment, which, in turn, results in mito-
chondrial damage that is similar to the damage induced
by apoptotic pathways [66]. Autophagy is primarily a
survival mechanism in response to ROS, but the
molecular targets of ROS in autophagy remain unclear
[67]. In the present study, MG treatment led to early
increases in ROS levels, which indicates that the cells
experienced oxidative stress. Autophagy occurs to deal
with this cellular stress response [67]. ROS can induce
autophagy by activating the extracellular-signal-

FREE RADICAL RESEARCH
721
[13] Spiegelman BM. Transcriptional control of mitochon-
drial energy metabolism through the PGC1 coactiva-
tors. Novartis Found Symp. 2007;287:60–3; discussion
63.
[14] Scarpulla RC. Transcriptional paradigms in mammalian
mitochondrial biogenesis and function. Physiol Rev.
2008;88(2):611–638.
significant reductions in the levels of MG-induced ER
stress and autophagy. These findings could have
important implications for the pathophysiological states
of patients with diabetes and other degenerative disor-
ders that are associated with systemic or elevated car-
bonyl stress.
[15] Kelly DP, Scarpulla RC. Transcriptional regulatory cir-
cuits controlling mitochondrial biogenesis and func-
tion. Genes Dev. 2004;18(4):357–368.
Disclosure statement
[16] Nisoli E, Clementi E, Paolucci C, et al. Mitochondrial
biogenesis in mammals: the role of endogenous nitric
oxide. Science. 2003;299(5608):896–899.
No potential conflict of interest was reported by the authors.
Funding
[17] Nishino Y, Miura T, Miki T, et al. Ischemic precondi-
tioning activates AMPK in a PKC-dependent manner
and induces GLUT4 up-regulation in the late phase of
cardioprotection. Cardiovasc Res. 2004;61(3):610–619.
[18] Gugliucci A. “Blinding” of AMP-dependent kinase by
methylglyoxal: a mechanism that allows perpetuation
of hepatic insulin resistance? Med Hypotheses.
2009;73(6):921–924.
[19] Jiang B, Le L, Pan H, et al. Dihydromyricetin amelio-
rates the oxidative stress response induced by methyl-
glyoxal via the AMPK/GLUT4 signaling pathway in
PC12 cells. Brain Res Bull. 2014;109:117–126.
[20] Yung HW, Korolchuk S, Tolkovsky AM, et al.
Endoplasmic reticulum stress exacerbates ischemia-
reperfusion-induced apoptosis through attenuation of
Akt protein synthesis in human choriocarcinoma cells.
FASEB J. 2007;21(3):872–884.
[21] Boot-Handford RP, Briggs MD. The unfolded protein
response and its relevance to connective tissue dis-
eases. Cell Tissue Res. 2010;339(1):197–211.
This research was supported by the Basic Science Research
Program of the National Research Foundation of Korea
(NRF), funded by the Ministry of Education [NRF-
2016R1D1A1B03930082].
References
[1] Brownlee M. Negative consequences of glycation.
Metab Clin Exp. 2000;49(2 Suppl 1):9–13.
[2] Casazza JP, Felver ME, Veech RL. The metabolism of
acetone in rat. J Biol Chem. 1984;259(1):231–236.
[3] Thornalley PJ. The glyoxalase system: new develop-
ments towards functional characterization of a meta-
bolic pathway fundamental to biological life. Biochem
J. 1990;269(1):1–11.
[4] Liu YW, Zhu X, Zhang L, et al. Up-regulation of glyoxa-
lase 1 by mangiferin prevents diabetic nephropathy
progression in streptozotocin-induced diabetic rats.
Eur J Pharmacol. 2013;721(1–3):355–364.
[5] Morcos M, Du X, Pfisterer F, et al. Glyoxalase-1 pre-
vents mitochondrial protein modification and enhan-
ces life span in Caenorhabditis elegans. Aging Cell.
2008;7(2):260–269.
[6] Weinstein RS. Clinical practice. Glucocorticoid-induced
bone disease. N Engl J Med. 2011;365(1):62–70.
[7] Boyce BF, Schwarz EM, Xing L. Osteoclast precursors:
cytokine-stimulated immunomodulators of inflamma-
tory bone disease. Curr Opin Rheumatol. 2006;18(4):
427–432.
€€
€
[22] Høyer-Hansen M, Jaattela M. Connecting endoplasmic
reticulum stress to autophagy by unfolded protein
response and calcium. Cell Death Differ. 2007;14(9):
1576–1582.
ꢀ
[23] Ułamek-Kozioł M, Furmaga-Jabłonska W, Januszewski
S, et al. Neuronal autophagy: selfeating or selfcanni-
balism in Alzheimer’s disease. Neurochem Res. 2013;
38(9):1769–1773.
[24] Banerjee R, Starkov AA, Beal MF, et al. Mitochondrial
dysfunction in the limelight of Parkinson’s disease
pathogenesis. Biochim Biophys Acta. 2009;1792(7):
651–663.
[25] Rimando AM, Kalt W, Magee JB, et al. Resveratrol,
pterostilbene, and piceatannol in Vaccinium berries.
J Agric Food Chem. 2004;52(15):4713–4719.
[26] Piotrowska H, Kucinska M, Murias M. Biological activity
of piceatannol: leaving the shadow of resveratrol.
Mutat Res. 2012;750(1):60–82.
[27] Lee SK, Mbwambo ZH, Chung H, et al. Evaluation of
the antioxidant potential of natural products. Comb
Chem High Throughput Screen. 1998;1(1):35–46.
[28] Kimura Y, Okuda H, Arichi S. Effects of stilbenes on
arachidonate metabolism in leukocytes. Biochim
Biophys Acta. 1985;834(2):275–278.
[8] Kastelan D, Kastelan M, Massari LP, et al. Possible asso-
ciation of psoriasis and reduced bone mineral density
due to increased TNF-alpha and IL-6 concentrations.
Med Hypotheses. 2006;67(6):1403–1405.
[9] Gilbert L, He X, Farmer P, et al. Inhibition of osteoblast
differentiation by tumor necrosis factor-alpha.
Endocrinology. 2000;141(11):3956–3964.
[10] Byun CH, Koh JM, Kim DK, et al. Alpha-lipoic acid
inhibits TNF-alpha-induced apoptosis in human bone
marrow stromal cells.
20(7):1125–1135.
[11] Newsholme P, Gaudel C, Krause M. Mitochondria and
diabetes: an intriguing pathogenetic role. Adv Exp
Med Biol. 2012;942:235–247.
J Bone Miner Res. 2005;
[29] Kawakami S, Kinoshita Y, Maruki-Uchida H, et al.
Piceatannol and its metabolite, isorhapontigenin,
induce SIRT1 expression in THP-1 human monocytic
cell line. Nutrients. 2014;6(11):4794–4804.
[12] Rosca MG, Monnier VM, Szweda LI, et al. Alterations in
renal mitochondrial respiration in response to the
reactive oxoaldehyde methylglyoxal. Am J Physiol
Renal Physiol. 2002;283(1):F52–F59.

722
K. S. SUH ET AL.
[30] Sano S, Sugiyama K, Ito T, et al. Identification of the
strong vasorelaxing substance scirpusin B, a dimer of
piceatannol, from passion fruit (Passiflora edulis) seeds.
J Agric Food Chem. 2011;59(11):6209–6213.
[31] Kinoshita Y, Kawakami S, Yanae K, et al. Effect of long-
term piceatannol treatment on eNOS levels in cultured
endothelial cells. Biochem Biophys Res Commun.
2013;430(3):1164–1168.
[32] Matsui Y, Sugiyama K, Kamei M, et al. Extract of pas-
sion fruit (Passiflora edulis) seed containing high
amounts of piceatannol inhibits melanogenesis and
promotes collagen synthesis. J Agric Food Chem.
2010;58(20):11112–11118.
[33] Maruki-Uchida H, Kurita I, Sugiyama K, et al. The pro-
tective effects of piceatannol from passion fruit
(Passiflora edulis) seeds in UVB-irradiated keratinocytes.
Biol Pharm Bull. 2013;36(5):845–849.
[34] Son Y, Byun SJ, Pae HO. Involvement of heme oxygen-
ase-1 expression in neuroprotection by piceatannol, a
natural analog and a metabolite of resveratrol, against
glutamate-mediated oxidative injury in HT22 neuronal
cells. Amino Acids. 2013;45(2):393–401.
[35] Ashikawa K, Majumdar S, Banerjee S, et al. Piceatannol
inhibits TNF-induced NF-kappaB activation and NF-
kappaB-mediated gene expression through suppres-
sion of IkappaBalpha kinase and p65 phosphorylation.
J Immunol. 2002;169(11):6490–6497.
[36] Chang JK, Hsu YL, Teng IC, et al. Piceatannol stimu-
lates osteoblast differentiation that may be mediated
by increased bone morphogenetic protein-2 produc-
tion. Eur J Pharmacol. 2006;551(1–3):1–9.
[37] Thornalley PJ, Tisdale MJ. Inhibition of proliferation
of human promyelocytic leukaemia HL60 cells by S-d-
lactoylglutathione in vitro. Leuk Res. 1988;12(11–12):
897–904.
[38] Wang H, Meng QH, Chang T, et al. Fructose-induced
peroxynitrite production is mediated by methylglyoxal
in vascular smooth muscle cells. Life Sci.
2006;79(26):2448–2454.
[39] Suh KS, Choi EM, Rhee SY, et al. Methylglyoxal induces
oxidative stress and mitochondrial dysfunction in
osteoblastic MC3T3-E1 cells. Free Radic Res.
2014;48(2):206–217.
[40] Kim KA, Lee WK, Kim JK, et al. Mechanism of refractory
ceramic fiber- and rock wool-induced cytotoxicity in
alveolar macrophages. Int Arch Occup Environ Health.
2001;74(1):9–15.
[41] Xue M, Rabbani N, Momiji H, et al. Transcriptional con-
trol of glyoxalase 1 by Nrf2 provides a stress-respon-
sive defence against dicarbonyl glycation. Biochem J.
2012;443(1):213–222.
[42] Mukhopadhyay P, Rajesh M, Yoshihiro K, et al. Simple
quantitative detection of mitochondrial superoxide
production in live cells. Biochem Biophys Res
Commun. 2007;358(1):203–208.
[43] Thornalley PJ. Glyoxalase I – structure, function and a
critical role in the enzymatic defence against glyca-
tion. Biochem Soc Trans. 2003;31(6):1343–1348.
[44] Atkins TW, Thornally PJ. Erythrocyte glyoxalase activity
in genetically obese (ob/ob) and streptozotocin dia-
betic mice. Diabetes Res. 1989;11(3):125–129.
[45] Shinohara M, Thornalley PJ, Giardino I, et al.
Overexpression of glyoxalase-I in bovine endothelial
cells inhibits intracellular advanced glycation endprod-
uct formation and prevents hyperglycemia-induced
increases in macromolecular endocytosis. J Clin Invest.
1998;101(5):1142–1147.
[46] Skapare E, Konrade I, Liepinsh E, et al. Association of
reduced glyoxalase 1 activity and painful peripheral
diabetic neuropathy in type 1 and 2 diabetes mellitus
patients.
J
Diabetes Complications. 2013;27(3):
262–267.
[47] Takasawa R, Akahane H, Tanaka H, et al. Piceatannol, a
natural trans-stilbene compound, inhibits human
glyoxalase I. Bioorg Med Chem Lett. 2017;27(5):
1169–1174.
[48] Webster L, Abordo EA, Thornalley PJ, et al. Induction
of TNF alpha and IL-1 beta mRNA in monocytes by
methylglyoxal- and advanced glycated end product-
modified human serum albumin. Biochem Soc Trans.
1997;25(2):250S.
[49] Zhu J, Krishnegowda G, Gowda DC. Induction of
proinflammatory responses in macrophages by the
glycosylphosphatidylinositols of Plasmodium falcip-
arum: the requirement of extracellular signal-regulated
kinase, p38, c-Jun N-terminal kinase and NF-kappaB
pathways for the expression of proinflammatory cyto-
kines and nitric oxide. J Biol Chem. 2005;280(9):
8617–8627.
[50] Onishi A, Akimoto T, Urabe M, et al. Attenuation of
methylglyoxal-induced peritoneal fibrosis: immunomo-
dulation by interleukin-10. Lab Invest. 2015;95(12):
1353–1362.
[51] Westwood ME, Thornalley PJ. Induction of synthesis
and secretion of interleukin 1 beta in the human
monocytic THP-1 cells by human serum albumins
modified with methylglyoxal and advanced glycation
endproducts. Immunol Lett. 1996;50(1–2):17–21.
[52] Wang H, Meng QH, Gordon JR, et al. Proinflammatory
and proapoptotic effects of methylglyoxal on neutro-
phils from patients with type 2 diabetes mellitus. Clin
Biochem. 2007;40(16–17):1232–1239.
[53] de Arriba SG, Stuchbury G, Yarin J, et al. Methylglyoxal
impairs glucose metabolism and leads to energy
depletion in neuronal cells – protection by carbonyl
scavengers. Neurobiol Aging. 2007;28(7):1044–1050.
[54] Addabbo F, Ratliff B, Park HC, et al. The Krebs cycle
and mitochondrial mass are early victims of endothe-
lial dysfunction: proteomic approach. Am J Pathol.
2009;174(1):34–43.
€
[55] Schroder M. Endoplasmic reticulum stress responses.
Cell Mol Life Sci. 2008;65(6):862–894.
[56] Boyce M, Yuan J. Cellular response to endoplasmic
reticulum stress: a matter of life or death. Cell Death
Differ. 2006;13(3):363–373.
[57] Guo YS, Sun Z, Ma J, et al. 17b-estradiol inhibits ER
stress-induced apoptosis through promotion of TFII-I-
dependent Grp78 induction in osteoblasts. Lab Invest.
2014;94(8):906–916.
[58] Zhang K, Kaufman RJ. From endoplasmic-reticulum
stress to the inflammatory response. Nature.
2008;454(7203):455–462.

FREE RADICAL RESEARCH
723
[59] Park SJ, Kim KJ, Kim WU, et al. Involvement of endo-
plasmic reticulum stress in homocysteine-induced
apoptosis of osteoblastic cells. J Bone Miner Metab.
2012;30(4):474–484.
[60] He L, Lee J, Jang JH, et al. Osteoporosis regulation by
salubrinal through eIF2a mediated differentiation of
osteoclast and osteoblast. Cell Signal. 2013;25(2):
552–560.
during necrotizing enterocolitis. Oxid Med Cell Longev.
2009;2(5):297–306.
[66] Carlo-Stella C, Di Nicola M, Turco MC, et al. The anti-
human leukocyte antigen-DR monoclonal antibody
1D09C3 activates the mitochondrial cell death pathway
and exerts a potent antitumor activity in lymphoma-
bearing non obese diabetic/severe combined immuno-
deficient mice. Cancer Res. 2006;66(3):1799–1808.
[67] Scherz-Shouval R, Elazar Z. Regulation of autophagy
by ROS: physiology and pathology. Trends Biochem
Sci. 2011;36(1):30–38.
€
[61] Fulda S, Kogel D. Cell death by autophagy: emerging
molecular mechanisms and implications for cancer
therapy. Oncogene. 2015;34(40):5105–5113.
[62] Lockshin RA, Zakeri Z. Programmed cell death and
apoptosis: origins of the theory. Nat Rev Mol Cell Biol.
2001;2(7):545–550.
[63] Shimizu S, Kanaseki T, Mizushima N, et al. Role of Bcl-
2 family proteins in a non-apoptotic programmed cell
death dependent on autophagy genes. Nat Cell Biol.
2004;6(12):1221–1228.
[68] Cheng P, Ni Z, Dai X, et al. The novel BH-3 mimetic
apogossypolone induces Beclin-1- and ROS-mediated
autophagy in human hepatocellular carcinoma cells.
Cell Death Dis. 2013;4:e489.
[69] Kang R, Livesey KM, Zeh HJ, et al. HMGB1 as an
autophagy sensor in oxidative stress. Autophagy.
2011;7(8):904–906.
[64] Xu YN, Cui XS, Sun SC, et al. Mitochondrial dysfunc-
tion influences apoptosis and autophagy in porcine
[70] Haar L, Ren X, Liu Y, et al. Acute consumption of a
high-fat diet prior to ischemia-reperfusion results in
cardioprotection through NF-jB-dependent regulation
of autophagic pathways. Am J Physiol Heart Circ
Physiol. 2014;307(12):H1705–H1713.
[71] Zeng M, Wei X, Wu Z, et al. NF-jB-mediated induction
of autophagy in cardiac ischemia/reperfusion injury.
Biochem Biophys Res Commun. 2013;436(2):180–185.
parthenotes developing in vitro.
J Reprod Dev.
2011;57(1):143–150.
[65] Baregamian N, Song J, Bailey CE, et al. Tumor necrosis
factor-a and apoptosis signal-regulating kinase 1 control
reactive oxygen species release, mitochondrial autoph-
agy and c-Jun N-terminal kinase/p38 phosphorylation