Long-chain saturated fatty acids, such as palmitate, induce apoptosis in liver cells.. These data suggest that adiponectin is an important determinant of saturated fatty acid-induced apo
Trang 1palmitate-induced apoptosis via inhibition of c-Jun NH2 terminal kinase
Tae W Jung1, Yong J Lee2, Myung W Lee3,4, Seon M Kim3and Tae W Jung1
1 Samsung Biomedical Institute, Seoul, Korea
2 Division of Clinical Research, Seoul Medical Center Research Institute, Korea
3 Department of Family Medicine, Brain Korea 21 Project Medical Science, College of Medicine, Korea University, Seoul, Korea
4 Department of Anatomy, College of Medicine, Korea University, Seoul, Korea
Non-alcoholic fatty liver disease (NAFLD) is a chronic
disease that is initially characterized by steatosis, with
progression in some individuals to non-alcoholic
ste-atohepatitis (NASH) and last-stage hepatic disease
[1,2] NAFLD is a common cause of chronic liver enzyme elevation and cryptogenic cirrhosis It has been proposed that the trigger for a progression into the more processed stages of NAFLD involves damage to
Keywords
adiponectin; AMPK; apoptosis; JNK;
palmitate
Correspondence
T W Jung, Samsung Biomedical Institute,
Seoul, Korea, Annex B235, 50 Ilwon-Dong,
Kangnam-Ku, PO Box 135-710, Seoul, Korea
Fax: +82 2 873 8071
Tel: +82 2 873 8071
E-mail: ohayo2030@hanmail.net
(Received 24 December 2008, revised 31
January 2009, accepted 10 February 2009)
doi:10.1111/j.1742-4658.2009.06955.x
Hepatic apoptosis is elevated in patients with non-alcoholic steatohepatitis and is correlated with the severity of the disease Long-chain saturated fatty acids, such as palmitate, induce apoptosis in liver cells The present study examined adiponectin-mediated protection against saturated fatty acid-induced apoptosis in the human hepatoma cell line, HepG2 Cells were cultured in a control media (i.e without fatty acids) or the same media containing 250 lmolÆL)1 of albumin-bound oleate or palmitate for 24 h The adiponectin concentrations used were: 0, 1, 10 or 100 lgÆmL)1 (n = 4–6 per treatment) Palmitate and thapsigargin, but not oleate, acti-vated caspase-3 and decreased cell viability in the absence of adiponectin Adiponectin reduced palmitate- and thapsigargin-induced activation of cas-pase-3 and cell death in a dose-dependent manner Phosphatidylinositol 3-kinase and AMP-activated protein kinase inhibitors abolished the effects
of adiponectin Adiponectin-induced inhibition of palmitate- and thapsigar-gin-induced apoptosis was not the result of an augmentation in the unfolded protein response or the increased expression of genes encoding the inhibitor of apoptosis proteins, inhibitor of apoptosis protein-2 and X-linked mammalian inhibitor of apoptosis protein Palmitate and thapsi-gargin, but not oleate, increased c-Jun NH2 terminal kinase phosphoryla-tion in the absence of adiponectin Adiponectin blocked palmitate- and thapsigargin-induced activation of c-Jun NH2 terminal kinase and reduced apoptosis These data suggest that adiponectin is an important determinant
of saturated fatty acid-induced apoptosis in liver cells and may have impli-cations for fatty acid-mediated liver cell injury in adiponectin-deficient individuals
Abbreviations
AMPK, AMP-activated protein kinase; CHOP, CCAAT ⁄ enhancer-binding protein homologous protein; ER, endoplasmic reticulum; IAP, inhibitor of apoptosis protein; JNK, c-Jun NH2terminal kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PI3 kinase, phosphatidylinositol 3-kinase; UPR, unfolded protein response; XIAP, X-linked mammalian inhibitor of apoptosis protein.
Trang 2liver by oxidative stress, or a second effect, in addition
to hepatic steatosis and abnormal apoptosis [3]
Hepatic apoptosis is present in patients with high
calorie-induced hepatic steatosis and correlates with
the severity of the disease [4,5] Excess circulating and
non-adipose tissue lipids, in particular long-chain
satu-rated fatty acids, induce apoptosis in a number of cell
types, including hepatocytes [6–11] Obesity and insulin
resistance, which are both conditions associated with
and determined by excess lipids, play important roles
in the development and progression of NAFLD
[12,13] Notably, insulin and several growth factors
inhibit apoptosis and promote cell survival via
phos-phatidylinositol 3-kinase (PI3 kinase)- and
Akt-depen-dent mechanisms [14–17] Adiponectin is a known
adipokine in which plasma levels are decreased in
hyperlipidemic conditions such as obesity and type 2
diabetes [18] It has been reported that intravenous
injection of adiponectin normalizes decreased insulin
signaling and sensitivity [19] The effect of adiponectin,
an antidiabetic adipokine, has also been suggested to
involve the PI3 kinase⁄ Akt signaling pathway, and the
ability of PI3 kinase⁄ Akt to suppress the c-Jun NH2
terminal kinase (JNK) pathway has been studied in a
variety of cell types [20] Therefore, excess lipid
deliv-ery together with the role of reduced adiponectin may
comprise an environment that promotes apoptosis and
the development and⁄ or severity of NASH The
pres-ent study aimed to determine whether adiponectin
restricts lipid-mediated apoptosis in hepatocytes and, if
so, whether this involved: (a) augmentation of the
unfolded protein response (UPR); (b) up-regulation of
members of the inhibitor of apoptosis protein (IAP)
family; and⁄ or (c) inhibition of JNK activity [8,11,17]
Results and Discussion
Adiponectin reduces endoplasmic reticulum (ER)
stress-mediated apoptosis
Hyperlipidemia has been reported to induce ER stress,
which may phosphorylate JNK and contribute to the
development of insulin resistance and cell death [21]
Therefore, we treated HepG2 cells with thapsigargin
and palmitate to confirm the inhibitory effect of
adipo-nectin in chemically induced- or palmitate-induced ER
stress In the absence of adiponectin, elevated
caspase-3 activity (Fig 1A) and decreased cell viability in the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) assay (Fig 1B) were observed in
HepG2 cells incubated with thapsigargin or palmitate
Adiponectin inhibits thapsigargin- and
palmitate-induced caspase-3 activity (Fig 1A) and recovered cell
viability in the MTT assay (Fig 1B) in a dose-depen-dent manner
AMP-activated protein kinase (AMPK) inhibitor and PI3 kinase inhibitor inhibit adiponectin-mediated inhibition of apoptosis Adiponectin has been reported to be an AMPK activa-tor [18] and there is a known connection between AMPK and the PI3 kinase⁄ Akt signaling pathway [20] Therefore, we verified the signaling pathway of AMPK-ER stress-induced cell death using compound
c, as an AMPK inhibitor, and wortmannin, as a PI3 kinase inhibitor In the absence of adiponectin, thapsi-gargin and palmitate elevated caspase-3 activity in HepG2 cells (Fig 2) Wortmannin (Fig 2A) or com-pound c (Fig 2B) interrupted the protective effects of
7
*
*
!
!
!
!
!
!
!
*
0 µg·mL –1
10 µg·mL –1
100 µg·mL –1
Adiponectin
A
B
0 µg·mL –1
10 µg·mL –1
1 µg·mL –1
100 µg·mL –1
Adiponectin
5 4 3
Caspase 3 activity (f
2 1 0
120 100 80 60 40 20 0
Fig 1 Adiponectin inhibits thapsigargin- and palmitate-induced apoptosis in a dose-dependent manner (A) Caspase-3 activity is presented as the mean ± SD (n = 5) (B) Cell death was measured
by the MTT assay from a total of three independent experiments Treatments were carried out for 24 h Con, not treated; TG, 250 n M
thapsigargin; O300, 300 l M oleate; P300, 300 l M palmitate *Signif-icantly different from Con and O250 Signif*Signif-icantly different from the same treatment in the absence of adiponectin.
Trang 3adiponectin on thapsigargin- and palmitate-mediated
apoptosis
Adiponectin is unable to augment the UPR
The UPR is a signaling pathway that serves to reduce
the protein load degradative ability of the ER in
response to the accumulation of mis- and unfolded
proteins [22] An inappropriate response to these
stres-sors results in apoptotic cell death [22] We
hypothe-sized that adiponectin might inhibit thapsigargin- and
palmitate-mediated apoptosis via augmentation of the
UPR However, in the absence of adiponectin,
thasi-gargin and palmitate elevated the expression of several
genes involved in the UPR in HepG2 cells (Fig 3) In
the presence of thapsigargin or palmitate, adiponectin was unable to decrease the expression of these genes (Fig 3)
Thapasigargin, palmitate and adiponectin are unable to influence the expression of Bcl-2, cIAP2 and the IAP family
Bcl-2 proteins play important roles in caspase-depen-dent apoptosis, and the IAP family, cIAP2 and X-linked mammalian inhibitor of apoptosis protein (XIAP) all play a protective role in ER stress-induced apoptosis in human breast cancer cells [23] Therefore,
we evaluated the expression levels of Bcl-2, cIAP2and XIAP in HepG2 cells in the presence and absence of adiponectin Thapsigargin and palmitate, as well as adiponectin, were unable to affect the expression of Bcl-2, cIAP2and XIAP (Fig 4)
Adiponectin inhibits thapsigargin- and palmitate-induced JNK phosphorylation Palmitate has been reported to induce JNK dependent apoptosis in liver cells [8] Thus, we evaluated the
1.2
A
B
Control
Wortmannin
Adiponectin
Adipo
+ Wort
Control
Compound c
Adiponectin
Adipo
+ Com c
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
!
**
!
!
!
!
*
*
*
!
!
!
*
*
Fig 2 Adiponectin inhibits thapsigargin- and palmitate-induced
cas-pase-3 activity via PI3 kinase and AMPK in HepG2 cells (A) The
effects of 10 lgÆmL)1of adiponectin and 1 l M of wortmannin, or
both, on caspase-3 activity (B) The effects of 10 ugÆmL)1of
adipo-nectin and 10 l M of compound c, or both, on caspase-3 activity.
Treatments were carried out for 24 h These data were obtained
from a total of three independent experiments or represent the
mean ± SD (n = 3) Con, not treated; TG, 250 n M thapsigargin;
O300, 300 l M oleate; P300, 300 l M palmitate *Significantly
differ-ent from Con and O250 Significantly differdiffer-ent from same
treat-ment in the absence of adiponectin.
A
B
Con GRP78
CHOP
GRP78 CHOP
8
6
4
2
0
Beta actin
TG TG + A P P + A A
Con TG TG + A P P + A A
Fig 3 Adiponectin is unable to inhibit thapsigargin- and palmitate-induced ER stress markers (GRP78 and CHOP) in HepG2 cells The effects of adiponectin on the expression of GRP78 and CHOP mRNA were measured by semiquantitative RT-PCR Treatments were car-ried out for 24 h These data were obtained from a total of three independent experiments or represent the mean ± SD (n = 3) Con, not treated; TG, 250 n M thapsigargin; P, 300 l M palmitate; A,
10 lgÆmL)1adiponectin *Significantly different from Con and A.
Trang 4effect of adiponectin on thapsigargin- and
palmitate-induced phosphorylation of JNK in HepG2 cells
Thapsigargin and palmitate elevated JNK
phosphory-lation and activity As expected, adiponectin inhibited
these inductions (Fig 5)
An elevation of plasma free fatty acids and fat
accu-mulation in the liver are the cause of hepatic insulin
resistance and liver disease [24–26] Adiponectin
induces fatty acid oxidation and insulin sensitivity [27]
Therefore, an adequate adiponectin signaling pathway
in the liver may prove to be important in provoking
apoptosis, which is a cause of hepatic inflammation
and fibrosis In the present study, we evaluated the
ability of adiponectin to prevent palmitate-induced
apoptosis in HepG2 cells The results obtained
demon-strate that adiponectin partially inhibits both
palmi-tate- and thapsigargin-induced apoptosis via JNK
phosphorylation
In the present study, adiponectin inhibits caspase-3
and cell death induced by thapsigargin and palmitate
(Fig 1) Interestingly, the addition of either
wortman-nin or compound c in the presence of adiponectin
pre-vented the effects of adiponectin (Fig 2) These results
suggest that adiponectin inhibits apoptotic cell death via AMPK and PI3 kinase activation
Adiponectin reduced palmitate- and thapsigargin-induced apoptosis, although it did not inhibit elevated
ER stress markers, suggesting that adiponectin-medi-ated protection involves a pathway independent of ER stress markers (Fig 3)
A
B
Con Bcl-2
clAP2
XIAP
Bcl-2
clAP2
XIAP
Beta actin
2.0
1.5
1.0
0.0
0.5
TG TG + A P P + A A
Con TG TG + A P P + A A
Fig 4 Thapsigargin, palmitate and adiponectin are unable to affect
the expression of Bcl-2 and inhibitor of apoptosis family members
in HepG2 cells The effects of adiponectin on the expression of
Bcl-2, cIAP2and XIAP mRNA were measured by semiquantitative
RT-PCR Treatments were carried out for 24 h These data were
obtained from a total of three independent experiments or
repre-sent the mean ± SD (n = 3) Con, not treated; TG, 250 n M
thapsi-gargin; P, 300 l M palmitate; A, 10 lgÆmL)1adiponectin.
A
B
Con
54 kDa
46 kDa
42 kDa T-JNK
Beta actin
6
5
4
3
2
1
0
5
4
3
2
1
0
TG TG + A P P + A
Con TG TG + A P
*
*
#
#
*
*
!
!
P + A
Con TG TG + A P P + A
Fig 5 Adiponectin inhibits thapsigargin- and palmitate-induced JNK phosphorylation in HepG2 cells (A) The effects of adiponectin on JNK phosphorylation were measured by western blot analysis (B) The effects of adiponectin on enzymatic JNK activity were mea-sured using the JNK activity assay kit Treatments were carried out for 24 h These data were obtained from a total of three indepen-dent experiments or represent the mean ± SD (n = 4) There was
no effect of sole adiponectin on JNK phosphorylation Con, not treated; TG, 250 n M thapsigargin; P, 300 l M palmitate; A,
10 lgÆmL)1adiponectin *Significantly different from Con Signifi-cantly different from TG #SignifiSignifi-cantly different from P.
Trang 5Bcl-2, XIAP and the IAP family are related to
caspase-dependent cell death [17] However, thapsigargin and
palmitate did not induce Bcl-2, XIAP and the IAP
fam-ily Moreover, adiponectin was also unable to influence
their expression (Fig 4) These results suggest that the
adiponectin-mediated protective effects of
thapsigargin-and palmitate-induced apoptosis occur independently of
the expression of Bcl-2, XIAP and the IAP family
The mitogen-activated protein kinase family responds
to a variety of stressors [28] Especially, JNK is a
criti-cal metabolic regulator and plays a role in lipoapoptosis
in a variety of cell types, including hepatocytes [29] In
the present study, palmitate and thapsigargin induced
JNK phosphorylation Adiponectin inhibited
palmitate-and thapsigargin-mediated JNK phosphorylation
These results coincide with the findings of a study
per-formed in mouse hepatocyte and HepG2 cells in which
free fatty acid-induced apoptosis was reported to be
partially dependent on JNK [30] The present data
sug-gest that adiponectin-mediated protection from
apopto-sis may involve a JNK-dependent pathway
In conclusion, the results obtained in the present
study demonstrate that both the AMPK and PI3
kinase signaling pathways are critical factors for the
protective effects of adiponectin with respect to
palmi-tate- and thapsigargin-induced apoptosis via JNK
phosphorylation in HepG2 cells These data may be
valuable for identifying adiponectin as a candidate for
the treatment of NASH, which is characterized by
abnormal hepatic apoptosis
Experimental procedures
Culture media and reagents
HepG2 cells were plated at a density of 2· 105
cellsÆmL)1 and grown in DMEM medium supplemented with heat
inactivated 10% (v⁄ v) fetal bovine serum, 100 UÆmL)1
pen-icillin and 100 lgÆmL)1 streptomycin Palmitate was
pur-chased from Sigma (St Louis, MO, USA) Palmitate was
conjugated to BSA at a 2 : 1 molar ratio [11] Thapsigargin,
which was used to chemically induce the misfolded or
unfolded protein response and apoptosis, and wortmannin,
a PI3 kinase inhibitor, were purchased from Sigma AMPK
inhibitor compound c was purchased from Calbiochem
(San Diego, CA, USA) Human full-length adiponectin was
purchased from BioVision (Mountain View, CA, USA)
RNA extraction and analysis
Total RNA was isolated using TRIzol according to the
USA) Primer sequences and their respective PCR fragment
homolo-gous protein (CHOP), forward: 5¢-ATGAGGACCTGC AAGAGGTCC-3¢, reverse: 5¢-TCCTCCTCAGTCAGCCA AGC-3¢ (137 bp); glucose regulated protein 78, forward: 5¢-GTTCTTGCCGTTCAAGGTGG-3¢, reverse: 5¢-TGGTA CAGTAACAACTGCATG-3¢ (182 bp); b-actin, forward: 5¢-GAGACCTTCAACACCCCAGCC-3¢, reverse: 5¢-GGA TCTTCATGAGGTAGTCAG-3¢ (206 bp); Bcl-2, forward: 5¢-TTTTAGGAGACCGAAGTCCG-3¢, reverse: 5¢-AGCC
5¢-TTTATCCTAATTTGGTTTCC-3¢, reverse: 5¢-AATTCT TAAAGGTTAACTC-3¢ (253 bp); and XIAP, forward: 5¢-GAAGACCCTTGGGAACAGCA-3¢, reverse: 5¢-CGCC TTAGCTGCTCTTCAGT-3¢ (383 bp)
Immunoblot analysis
Cells were washed with NaCl⁄ Pi and harvested using lysis buffer contatining 20 mm Hepes (pH 7.4), 1% Triton X-100, 15% glycerol, 2 mm EGTA, 1 mm sodium vanadate, 2 mm dithiothreitol, 10 lm leupeptin and 5 lm pepstatin Equiva-lent amounts of total extracts (20–30 lg) were loaded onto
(Amer-sham Pharmacia Biotech, Piscataway, NJ, USA) and the membranes were incubated with antibodies against phosphor-ylated JNK (Cell Signaling Technology, Beverly, MA, USA), total JNK (Cell Signaling Technology) and b-actin (Sigma) Proteins were detected using horseradish peroxidase conju-gated secondary antibodies and reacted with ECL solution (Amersham Pharmacia Biotech) Signals were detected using horseradish peroxidase conjugated secondary antibodies and
a chemoluminescence reagent (Pierce, Rockford, IL, USA)
Determination of JNK activity
Cell lysates were assayed for JNK phosphorylation using the Phospho-JNK DuoSet IC ELISA kit (R&D Systems, Minneapolis, MN, USA)
Determination of caspase-3 activity and cell death
Activity of the caspase-3 class of cysteine protease was deter-mined with the colorimetric activity assay (R&D Systems) Caspase-3 activity was normalized to the total extracted pro-tein concentration After treatment, culture medium was removed and cells were incubated in NaCl⁄ Pi containing
HepG2 cells were solubilized with dimethyl sulfoxyde
Statistical analysis
Statistical comparisons were calculated using analysis of variance P < 0.05 was considered statistically significant All data are reported as the mean ± SD
Trang 6This study was supported by the Brain Korea 21
pro-gram of Korea University We thank Dr Bong Soo
Cha and Dr Myung Shik Lee for their critical
sugges-tions and for providing facilities to conduct this
research
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