Resveratrol alters the lipid composition, metabolism and peroxide level in senescent rat hepatocytes

Full text

Resveratrol alters the lipid composition, metabolism and peroxide level
in senescent rat hepatocytes
Albena Momchilova
a,
⇑
, Diana Petkova
a
, Galya Staneva
a
, Tania Markovska
a
, Roumen Pankov
b
,
Ralica Skrobanska
b
, Mariana Nikolova-Karakashian
c
, Kamen Koumanov
a
a
Department of Lipid-Protein Interactions in Biomembranes, Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. Bl. 21,
1113 Sofia, Bulgaria
b
Department of Cytology, Histology and Embryology, Biological Faculty, Sofia University, 14, Dragan Cankov Str, 1164 Sofia, Bulgaria
c
Department of Physiology, University of Kentucky, College of Medicine, Lexington, KY 40536, USA
article info
Article history:
Received 12 August 2013
Received in revised form 20 September 2013
Accepted 18 October 2013
Available online 30 October 2013
Keywords:
Resveratrol
Hepatocytes
Oxidative stress
Membrane lipids
Aging
abstract
Investigations were performed on the influence of resveratrol on the lipid composition, metabolism, fatty
acid and peroxide level in plasma membranes of hepatocytes, isolated from aged rats. Hepatocytes were
chosen due to the central role of the liver in lipid metabolism and homeostasis. The obtained results
showed that the level of sphingomyelin (SM) and phosphatidylserine (PS) was augmented in plasma
membranes of resveratrol-treated senescent hepatocytes. The saturated/unsaturated fatty acids ratio of
the two most abundant membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanol-
amine (PE), was decreased as a result of resveratrol treatment. The neutral sphingomyelinase was found
to be responsible for the increase of SM and the decrease of ceramide in plasma membranes of resvera-
trol-treated senescent hepatocytes. Using labeled acetate as a precursor of lipid synthesis we demon-
strated, that resveratrol treatment resulted in inhibition mainly of phospholipid synthesis, followed by
fatty acids synthesis. Resveratrol induced reduction of specific membrane-associated markers of apopto-
sis such as localization of PS in the external plasma membrane monolayer and ceramide level. Finally, the
content of lipid peroxides was investigated, because the unsaturated fatty acids, which were augmented
as a result of resveratrol treatment, are an excellent target of oxidative attack. The results showed that the
lipid peroxide level was significantly lower, ROS were slightly reduced and GSH was almost unchanged in
resveratrol-treated hepatocytes. We suggest, that one possible biochemical mechanism, underlying the
reported resveratrol-induced changes, is the partial inactivation of neutral sphingomyelinase, leading
to increase of SM, the latter acting as a native membrane antioxidant.
In conclusion, our studies indicate that resveratrol treatment induces beneficial alterations in the
phospholipid and fatty acid composition, as well as in the ceramide and peroxide content in plasma
membranes of senescent hepatocytes. Thus, the presented results imply that resveratrol could improve
the functional activity of the membrane lipids in the aged liver by influencing specific membrane param-
eters, associated with the aging process.
Ó2013 Published by Elsevier Ireland Ltd.
1. Introduction
Oxidative damage of cellular components has been postulated
to be among the factors underlying the genesis of a wide range
of patho-physiological events, which accompany the aging pro-
cesses [1]. Thus, the ability of cells to resist or prevent oxidative at-
tack could possibly slow down the development of age-related
changes, and so the therapies aimed at reduction of oxidative
stress could as well induce anti-aging effects [2].
One of the widely studied antioxidants, which has also been
associated with anti-aging effects, is the naturally occurring phyto-
alexin resveratrol (3,4
0
,5
0
-trihydroxystilbene) [3]. It can be found in
different plants, mainly grapes, peanuts, berries, as well as in many
types of red wines [4]. Resveratrol has been reported to exhibit
various beneficial effects such as antioxidant, anti-inflammatory
and anti-aging, among others. [4,5]. In addition, there is evidence
that resveratrol has lipid-lowering effect on serum and liver lipids
and also inhibits hyperlipidemia and atherosclerosis in diabetic
LDL receptor-deficient mice [6].
The aim of the present study was to investigate the effect of res-
veratrol treatment of hepatocytes isolated form aged rats on plas-
ma membrane lipid composition, metabolism, fatty acids and
0009-2797/$ - see front matter Ó2013 Published by Elsevier Ireland Ltd.
http://dx.doi.org/10.1016/j.cbi.2013.10.016
⇑
Corresponding author. Tel.: +359 2 9792686; fax: +359 898 238971.
E-mail address: albena_momchilova@abv.bg (A. Momchilova).
Chemico-Biological Interactions 207 (2014) 74–80
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oxidative status. Liver cells were chosen due to the central role of
the liver in lipid metabolism and lipid homeostasis. Isolated hepa-
tocytes represent a convenient model for investigation of liver
functions, which is very close to in vivo conditions. Studies were
performed on the ability of resveratrol to reverse some parameters
of the membrane lipids, which change in the process of aging, such
as phospholipid and fatty acid composition, lipid synthesis, sphin-
gomyelin metabolism, accumulation of sphingolipid metabolites,
cholesterol level, phospholipid asymmetry etc.
The obtained results showed that resveratrol treatment induced
beneficial alterations of the membrane lipids and peroxide content
in plasma membranes of hepatocytes isolated from old rats, thus
implying that this lipophilic antioxidant could partially improve
the functional activity of the membrane lipids in the aged liver.
2. Materials and methods
2.1. Animals
Male Wistar rats (purchased from the Department for Labora-
tory Animals, Bulgarian Academy of Sciences) were kept for
20 months in laboratory conditions (in a ventilated room at ambi-
ent temperature 22 ± 2 °C) and had free access to food and water.
All experiments with animals were performed in strict accordance
with the national and institutional rules for use of animals for
experimental purposes. The performed experiments with animals
have been approved by the Ethical commission of Bulgarian Acad-
emy of Sciences.
2.2. Reagents
Trans-resveratrol (more than 99% pure) was purchased from
Sigma–Aldrich. C
6
-NBD-Cer{6-[N-(7-nitro-2,1,3-benzoxadiazol-4-
yl) amino] hexanoylceramide}, C
6
NBD-SM {6-[N-(7-nitro-2,1,3
benzoxadiazol-4-yl)amino] hexanoylsphingosyl phosphocholine},
C
17:0
ceramide (N-heptadecanoyl – D-sphingosine), palmitoyl-
(NBD-hexanoyl)-phosphatidylserine (NBD-PS) and cis-parinaric
acid were obtained from Avanti Polar Lipids. [1-
14
C] acetate
(58.9 mCi/mmol) was from Amersham Int.
2.3. Isolation of hepatocytes from old rats (senescent hepatocytes) and
incubation with resveratrol
Hepatocytes were isolated by liver perfusion with collagenase
[7]. The hepatocytes thus obtained were suspended in Krebs–
Henseleit buffer, pH 7.4, supplemented with 10 mM glucose and
1% (w/v) defatted bovine serum albumin [8]. The viability of
the isolated cells was monitored by the trypan blue test. In our
experiments we used preparations, which exhibited at least 90%
viability. After isolation, the hepatocytes were incubated in condi-
tioned shaker under 95% air and 5% CO
2
in Erlenmeyer flasks, con-
taining 10 mg of cellular protein per ml for 1 h in the presence or
absence of resveratrol (50
l
M). This concentration of resveratrol
was chosen because the measured alterations of sphingomyelin
content were linear up to 70
l
M. Resveratrol was delivered from
a stock solution in dimethyl sulfoxide. Control cells were incubated
only with dimethyl sulfoxide.
2.4. Cell viability assay after incubation with resveratrol and [1-
14
C]
acetate incorporation
After incubation with resveratrol, cell viability was determined
by tetrazolum salt measurement (MTT assay), involving assess-
ment of succinate dehydrogenase-induced conversion of
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolum bromide
into formazan crystals [9]. Formation of formazan was measured
at 570 nm. The viability of the cells after incubations was esti-
mated as percentage of the absorbance of resveratrol-treated cells
compared to controls. In the experiments involving incubation
with labeled acetate the incubation medium contained 10 mM glu-
cose, 1% defatted albumin and labeled acetate at a final concentra-
tion of 20
l
M in accordance with the procedure described by
Gnoni and Paglialonga [10]. The reactions were stopped with 10
N NaOH after 1 h of incubation and the hepatocytes were used
immediately for isolation of plasma membranes or for analysis of
reactive oxygen species (ROS) and glutathione (GSH) as explained
below.
2.5. Isolation of liver plasma membranes
Plasma membranes from hepatocytes were isolated according
to the procedure described by Pankov et al. [11] with modifica-
tions, involving differential centrifugation. Briefly, the post-nuclear
supernatant was loaded on a discontinuous sucrose gradient and
centrifuged at 100,000gfor 2.5 h. The plasma membrane fraction
was obtained at a density of 8% (w/v), suspended in ice-cold
10 mM Tris buffer, pH 7.4 and used immediately for lipid analysis.
2.6. Lipid extraction and analysis
Lipid extraction was performed with chloroform/methanol
according to the method of Bligh and Dyer [12]. The organic phase
obtained after extraction was concentrated and analyzed by thin
layer chromatography. The phospholipid fractions were separated
on silica gel G 60 plates in a solvent system containing chloro-
form/methanol/2-propanol/triethylamine/0.25% KCl (30:9:25:18:
6 v/v) [13]. The location of the separate fractions was determined
by spraying the plates with 2
0
,7
0
-dichlorofluorescein. The spots
were scraped and quantified by determination of the inorganic
phosphorus [14]. Neutral lipids were analyzed by thin-layer chro-
matography in a solvent system containing hexan:dietyl ether:ace-
tic acid (90:30:1v/v).
The incorporation of labeled acetate into the separate lipid frac-
tions was assayed by measuring the radioactivity of the spots
which were scraped and eluted.
Cholesterol content was assayed by gas chromatography using a
medium polarity RTX-65 capillary column (0.32 mm internal
diameter, length 30 m, thickness 0.25
l
m). Calibration was
achieved by a weighted standard of cholestane.
2.7. Fatty acid and ceramide analysis
The phospholipid extracts were saponified with 0.5 N methano-
lic KOH and methylated with boron trifluoride-methanol complex
(Merck) [15]. The fatty acid methyl esters were extracted with hex-
ane and separated by gas chromatography on a capillary column
coated with Supelcowax 10-bound phase 9 (i.d. 0.32 mm, length
30 m, film thickness 0.25
l
m; (Supelco, Bellafonte, PA) fitted in a
Perichrom gas chromatograph. Quantification was referred to an
internal standard of heptadecanoic methyl ester. The level of cera-
mide was determined by the fatty acid content in its molecules
after separation from the total phospholipids in developing system
containing diethyl ether:methanol (99:1 v/v).
2.8. Sphingomyelinase activity assay
Sphingomyelinase activity was determined by the method of
Nikolova-Karakashian et al. [16] with minor modifications. Briefly,
aliquots of the cell suspensions were lysed in 0.2% Triton X-100 in
100 mM Tris pH 7.4 buffer supplemented with 25
l
M genestein for
10 min on ice. The samples were homogenized with three passes
A. Momchilova et al. / Chemico-Biological Interactions 207 (2014) 74–80 75

through a 25-gauge needle and 10
l
l aliquots were taken for
protein assay. NBD-sphingomyelin was added to the lysates to a fi-
nal concentration of 20
l
M and incubations were performed for
10 min at 4 °C. Aliquots of this mixture containing 0.1 mg protein
and 3
l
M substrate were added to 5 mM MgCl
2
, 10 mM Tris pH
7.4 to a final volume of 0.3 ml. All buffers contained 0.2% Triton
X-100. After incubation for 1 h at 37 °C the reaction was stopped
by addition of 2 ml chloroform-methanol 2:1 (v/v). The samples
were evaporated and separated in a solvent system containing
diethyl ether:methanol (99:1v/v) and the spots corresponding to
ceramide were scraped and eluted. After addition of hexane the
fluorescence of the samples was measured at 455 nm (excitation)
and 530 nm (emission).
2.9. Sphingomyelin synthase assay
Sphingomyelin synthase was determined by the procedure de-
scribed by Tefesse et al. [17] with modifications. The incubation
mixture contained 20 mM Tris pH 7.4, 0.3 mg membrane protein,
50
l
g NBD-ceramide and 5
l
g PC to final volume of 500
l
l. After
incubation for 2 h at 37 °C the reaction was stopped with 1 ml
chloroform-methanol 2:1 (v/v). The lipids were separated in a sys-
tem containing diethyl ether:methanol (99:1v/v) and the spots
corresponding to sphingomyelin was scraped and eluted. Hexane
was added to the samples and fluorescence was measured at
455 nm (excitation) and 530 nm (emission).
2.10. Ceramidase assay
Ceramidase activity was determined by the method of Nikol-
ova-Karakashian et al. [16] with modifications. Briefly, the cells
were removed from the dishes and centrifuged at 300gfor 5 min.
They were lysed in 0.2% Triton X-100 in 100 mM Tris pH 7.4 buffer
supplemented with 25
l
M sodium vanadate for 10 min on ice. The
lysed cells were homogenized with three passes through a 25-
gauge needle and 10
l
l aliquots were taken for protein assay.
NBD-ceramide was added to the lysates to a final concentration
20
l
M and incubations were performed for 10 min at 4 °C. Aliquots
of this mixture containing 0.1 mg protein and 3
l
M substrate were
added to 0.5 M acetate buffer pH 4.5 to a final volume of 0.3 ml. All
buffers contained 0.2% Triton X-100. After incubation for 1 h at
37 °C the reaction was stopped by addition of 5 ml hexane and
4 ml 10% citric acid. The samples were evaporated, dissolved in
hexane and NBD-labeled fatty acid was quantified after addition
of hexane and determination of the fluorescence intensity (excita-
tion 455 nm and emission 530 nm).
2.11. Measurement of reactive oxygen species (ROS)
The generation of ROS was assessed by spectrofuorimetric anal-
ysis, using 2’-7’dichlorodihydrofluorescin diacetate, a non-polar
compound which reacts with ROS to produce the highly fluores-
cent dihydrofluorescein. Control and resveratrol-treated hepato-
cytes were incubated with 10
l
M dichlorodihydrofluorescein at
37 °C for 30 min. The cells were washed 3 times with warm PBS
to remove the unincorporated dye, placed in 2 ml PBS and fluores-
cence was measured at 485 nm (excitation beam) and 525 nm
(emission beam) [18]. The level of ROS is presented as measured
fluorescence intensity per mg protein.
2.12. Measurement of the reduced glutathione (GSH)
The content of GSH was determined according to Ellman [19]
using cell lysate obtained from control and resveratrol-treated
hepatocytes in 0.25 M Tris, 20 mM EDTA, pH 8.2). The obtained val-
ues are expressed as nmol GSH per mg protein.
2.13. Determination of lipid peroxidation
Lipid peroxidation was determined by the procedure described
by Kuypers et al [20] and Carini et al. [21]. The lipid peroxidation
was assessed by measuring the loss of fluorescence.of cis-parinaric
acid (PNA) (Molecular Probes, Invitrogen, UK) The hepatocytes
plasma membranes were incubated with 10
l
M PNA at 37 °C for
30 min in the dark. The incubation buffer was immediately re-
moved and the plasma membranes suspensions were washed 3
times with warm PBS to remove the unincorporated dye. The
membranes were transferred to fluorescence cuvettes in 2 ml
10 mM Tris pH 7.4. The emission fluorescence of wavelength
455 nm (slit width 5 nm) was measured using an excitation wave-
length of 312 nm (slit width 5 nm). A blank sample containing
unlabeled cells was also used and the measured values of the la-
beled cells were accordingly corrected by subtraction. The level
of lipid peroxidation was calculated as fluorescence intensity per
mg membrane protein.
2.14. Back exchange assay
The internalization of the lipid fluorescent analogs was assessed
by back exchange to serum albumin as described by Pomorski et al.
[22] with modifications. Exchangeable fluorescence lipids residing
in the outer membrane leaflet can be removed, allowing quantita-
tive determination of the lipid internalized into the inner mono-
layer. In short, cells were incubated with fatty acid-free BSA on
ice for 10 min, followed by washing with HBS and centrifugation
at 12,000g. The pellets were solubilized in 2% Triton X-100 and
the amount of internalized lipid was determined by comparing
the fluorescent intensity before and after back exchange to
albumin.
2.15. Protein determination
The content of protein was determined according to Bradford
[23].
2.16. Statistical analysis
Statistical processing of the data was made by one-way analysis
of variance (ANOVA), using InStat software.
3. Results
The influence of resveratrol on the lipid composition of plasma
membranes, isolated from hepatocytes of aged rats (referred to as
senescent hepatocytes or aged hepatocytes), is presented in Table 1.
The mol% of sphingomyelin (SM) and phosphatidylserine (PS) was
elevated by 39% and 21% respectively as a result of resveratrol
treatment. Phosphatidylcholine (PC) was decreased by about 12%,
Table 1
Phospholipid composition of plasma membranes isolated from resveratrol-treated
senescent hepatocytes (mol%).
Lipids Control Resveratrol
Sphingomyelin 8.1 11.4
*
Phosphatidylcholine 37.4 33.1
*
Phosphatidylserine 8.2 10.1
⁄
Phosphatidylinositol 10.6 9.9
Phosphatidylethanolamine 25.3 25.8
Phosphatidylglycerol 10.4 9.7
Cholesterol/phospholipids 0.345 0.359
Results are means of three separate experiments.
*
P< 0.001.
76 A. Momchilova et al. / Chemico-Biological Interactions 207 (2014) 74–80

whereas the changes in the rest of the membrane lipids were insig-
nificant. The cholesterol/total phospholipids (CH/TPL) molar ratio
remained almost unchanged in plasma membranes of resvera-
trol-treated aged hepatocytes (Table 1).
Since SM was affected most significantly by resveratrol
treatment, we analyzed the possible reasons for its increase by
measuring the activities of two enzymes involved in SM metabo-
lism – neutral sphingomyelinase (nSMase) and SM synthase
(Fig. 1). The results showed that the activity of nSMase was mark-
edly lower in plasma membranes from resveratrol-treated hepato-
cytes, whereas the activity of SM synthase was insignificantly
augmented, thus suggesting that the decreased SMase activity
was likely responsible for the elevated SM content after resveratrol
treatement.
In addition, the level of ceramides was altered from 236 to
174 nmol/mol phospholipid in control and resveratrol-treated cells
respectively. This change was also attributed to the inhibition of
nSMase activity since, as evident from Fig. 1, the activity of the
membrane bound ceramidase, which catalyses the conversion of
ceramide to sphingosine remained unchanged after resveratrol
treatment.
Besides SM, the other plasma membrane lipid which was in-
creased as a result of resveratrol treatment was PS (Table 1). This
aminophospholipid is important in physiological aspect, because
its appearance in the outer membrane monolayer is associated
with apoptosis initiation [24] and aging is known to alter the
asymmetrical distribution of membrane phospholipids [25]. Since
under normal conditions PS is localized predominantly in the inner
membrane monolayer, we analyzed its distribution between the
two membrane leaflets before and after resveratrol treatment of
aged hepatocytes. The obtained results indicated that resveratrol
treatment affected the intramembrane distribution of PS by reduc-
ing its content in the outer plasma membrane leaflet (Fig. 2).
Since the fatty acid composition of biological membranes
changes in the course of aging and is also closely related to lipid sus-
ceptibility to oxidative damage, we analyzed the acyl chain compo-
sition of the two most abundant membrane phospholipids,
phosphatidylcholine (PC) and phosphatidylethanolamine (PE), be-
fore and after treatment of senescent hepatocytes with resveratrol
(Table 2). The results showed that one major saturated fatty acid,
palmitic acid (C16:0), was reduced in both tested phospholipids,
whereas some of the polyunsaturated fatty acids (C18:3 and
C22:6) were increased in membranes of resveratrol-treated hepato-
cytes (Table 2). The relative content of the polyunsaturated long
chain C22:5 was elevated about two fold in PC, but not in PE. It
should also be noted that the ratio between the saturated (SAT)
and unsaturated (UNSAT) fatty acids was reduced as a result of res-
veratrol treatment (0.802 vs. 0.763 for PC and 0.597 vs. 0.524 for PE).
To analyze the mechanisms underlying the influence of resvera-
trol on liver membrane lipids, we investigated the changes in the
lipid synthesis by incubating control and resveratrol-treated hepa-
tocytes with radiolabeled acetate. We followed the distribution of
the labeled precursor into the major membrane lipid fractions –
fatty acids, phospholipids, diacylglycerols, triacylglycerols and
cholesterol (Fig. 3). Apparently, resveratrol reduced the incorpora-
tion of acetate mainly in the total phospholipid fraction (by 21%),
followed by the fatty acid fraction (by 13%). The decrease of acetate
incorporation in diacylglycerols and triacylglycerols was statisti-
cally insignificant. There was no change at all in the precursor
incorporation into cholesterol after resveratrol treatment. Since
the reduction of acetate incorporation was most prominent in
the phospholipid fraction, further studies were carried out on the
label distribution among the separate phospholipid classes
(Fig. 4). The results showed a lower incorporation of acetate mainly
in PC (by 20%) followed by phosphatidylinositol (PI). The observed
reduction of the label incorporation into PS and PE was statistically
insignificant. No difference was found in acetate incorporation into
SM after resveratrol treatment (data not shown).
Taking into consideration the reported resveratrol-induced aug-
mentation of the fatty acid degree of unsaturation, we further
analyzed the accompanying changes in the level of lipid peroxides,
0
20
40
60
80
100
120
n SMase SM synthase ceramidase
Arbitrary units %
Fig. 1. Alterations of enzyme activities (as indicated in the figure), participating in
sphingolipid metabolism in control (black bars) and resveratrol-treated (white
bars) hepatocytes isolated from aged rats. Results are given as means ± SD of three
separate experiments. Statistically significant differences were observed only
between the values obtained for neutral sphingomyelinase activity in control and
resveratrol treated senescent hepatocytes (P< 0.001).
0
10
20
30
40
50
60
70
80
90
020406080
NBD-P S in the inner membrane
leafl et (%)
Time (min)
Fig. 2. Inward translocation of NBD-phosphatidylserine in membranes of control
(squares) and resveratrol-treated (circles) senescent hepatocytes. Cells were labeled
as described under Materials and methods. The fraction of fluorescent phospholipid
in the inner membrane leaflet was determined by back exchange to albumin. The
data represent means ± SD of four separate determinations.
Table 2
Fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in
plasma membranes of resveratrol-treated senescent hepatocytes (mol%).
Fatty acids Phosphatidylcholine Phosphatidylethanolamine
Control Resveratrol Control Resveratrol
C16:0 26.7 23.8
*
24.8 22.3
*
C16:1 2.1 2.2 2.2 2.5
C18:0 19.8 19.5 12.6 12.1
C18:1 10.7 9.8 9.6 10.2
C18:2 10.9 11.1 15.9 15.2
C18:3 5.6 7.2
*
6.5 7.4
⁄⁄
C20:4 21.8 22.4 24.7 25.1
C22:5 0.5 1.2
*
1.1 1.3
C22:6 1.9 2.8
*
2.6 3.9
*
SAT/UNSAT 0.802 0.763
**
0.597 0.524
**
SAT-saturated fatty acids; UNSAT-unsaturated fatty acids.
Values are means of four separate experiments.
*
P< 0.001.
**
P< 0.01.
A. Momchilova et al. / Chemico-Biological Interactions 207 (2014) 74–80 77

because unsaturated acyl chains are an excellent target of oxida-
tive attack and resveratrol is known to act as a potent natural anti-
oxidant [4]. Thus, the alterations of the membrane content of lipid
peroxides, together with the level of ROS and GSH in whole hepa-
tocytes, would be indicative of the degree of age-induced oxidative
stress and the impact of resveratrol on the cellular oxidative status.
Peroxide formation was assessed by the decrease of cis-parinaric
acid fluorescence. Due to its conjugated tetraene structure cis-
parinaric acid becomes fluorescent only when incorporated in lipid
environment [21]. Oxidative destruction of the double bonds is di-
rectly translated into irreversible loss of fluorescence, and this is
the basis for the use of parinaric acid as a sensitive indicator of
the oxidation of conjugated double bonds of membrane lipids
[21]. As evident from Fig. 5 resveratrol treatment induced a
marked reduction in the level of lipid peroxides. The content of
ROS was also reduced in resveratrol-treated aged hepatocytes,
whereas GSH remained almost unchanged (Table 3).
4. Discussion
Resveratrol is a naturally occurring phytoalexin, that has been
reported to exhibit antioxidant, anti-inflammatory and anti-aging
effect on cells [3]. In the present studies we used as experimental
model resveratrol-treated hepatocytes isolated from old rats. Plas-
ma membranes were isolated from control and resveratrol-treated
hepatocytes, and were used for analysis of the lipid composition
and metabolism, fatty acids and lipid peroxides.
The analysis of the plasma membrane lipid composition re-
vealed augmentation of sphingomyelin and phosphatidylserine,
as well as reduction of phosphatidylcholine. The effect of resvera-
trol on sphingomyelin content is a finding of particular interest. On
one hand, SM is a major component of the membrane raft domains,
which are recognized as cellular signaling platforms [26]. On the
other hand, SM is the main source of ceramide, a bioactive lipid
second messenger, which is reported to increase in the course of
aging and is also considered as a marker of senescence [27]. The
accumulation of ceramide has been correlated with the onset of
aging-associated inflammation, cellular senescence, growth arrest
and many aging-associated diseases [27]. Our results showed that
in vitro resveratrol treatment significantly reduced ceramide con-
tent in plasma membranes of senescent hepatocytes. This observa-
tion is in contrast with the results of Scarlatti et al. [28], who
reported resveratrol-induced increase of ceramide in breast cancer
cells. It is possible that this discrepancy is due to the differences in
the type of cells under investigation – we used normal hepatocytes,
isolated from healthy aged animals, whereas Scarlatti et al. [28]
used cancer cells. It is quite likely, that resveratrol exhibits diverse
effects on ceramide accumulation, depending on the degree of
patho-physiological changes, occurring in the corresponding cell
model. One could further speculate, that in cancer cells the in-
crease of ceramide and the consequential initiation of apoptosis
0
20
40
60
80
100
120
Incorporaon of labeled acetate into
separate lipid fracons (% of controls)
FA PL DG TG CH
Fig. 3. Incorporation of labeled acetate into the separate lipid fractions in plasma
membranes of control (black bars) and resveratrol-treated (white bars) hepatocytes
isolated from aged rats (nmol [1-
14
C] acetate.h
1
.mg protein
1
). The values
obtained for control hepatocytes were taken for 100% for each separate lipid
fraction. PL-phospholipids; FA-fatty acids; DG-diglycerides; TG-triglycerides; CH-
cholesterol. The data represent means ± SD of three separate determinations. Only
the differences between control and resveratrol-treated hepatocytes, obtained for
PL (P< 0.001) and FA (P< 0.01) were statistically significant.
0
20
40
60
80
100
120
Incorporaon of labeled acetate
into separate phospholipids
(% of controls)
PC PE PS PI
Fig. 4. Incorporation of labeled acetate into the separate phospholipid fractions in
plasma membranes of control (black bars) and resveratrol-treated (white bars)
hepatocytes isolated from aged rats (nmol [1-
14
C] acetate.h
1
.mg protein
1
). The
values obtained for control hepatocytes were taken for 100% for each separate
phospholipid fraction. PC-phosphatidylcholine; PE-phosphatidylethanolamine; PS-
phosphatidylserine; PI-phosphatidylinositol. The data represent means ± SD of
three separate determinations. Only the differences between control and resvera-
trol-treated senescent hepatocytes, obtained for PC and PI were statistically
significant (P< 0.001).
0
20
40
60
80
100
120
Lipid peroxidation (%)
Control Resveratrol
Fig. 5. Alerations in the lipid peroxides assessed by the decrease of fluorescence of
cis-parinaric acid in plasma membranes of control (black bars) and resveratrol-
treated (white bars) senescent hepatocytes. Parinaric acid fluorescence was
measured as described in Materials and methods. The obtained results were
calculated as fluorescence intensity per mg membrane protein. The control values
were taken as 100%. Values are means ± SD of four determinations. Statistical
significance is calculated by comparison of control and resveratrol-treated cells
(P< 0.01).
Table 3
Levels of ROS and GSH in control and resveratrol-treated senescent hepatocytes.
Control Resveratrol
ROS 387 295
*
GSH 456 471
ROS – reactive oxygen species.
GSH – reduced glutathione.
ROS are expressed as fluorescence intensity per mg protein.
GSH is expressed as nmol per mg protein.
Values are means of three separate determinations.
*
P< 0.01.
78 A. Momchilova et al. / Chemico-Biological Interactions 207 (2014) 74–80

is a rational way for elimination of such pathological cells. How-
ever, in hepatocytes of old animals, resveratrol reduced the level
of ceramide by inhibiting nSMase activity, which is most probably
beneficial for the senescent cells.
To elucidate the biochemical mechanism, underlying the alter-
ations of ceramide, we analyzed the activities of specific sphingo-
lipid-metabolizing enzymes, that are related to SM hydrolysis
and ceramide accumulation (Fig. 1). As mentioned above, the activ-
ity of neutral SMase was markedly lower in resveratrol-treated
hepatocytes. What is more, membrane-bound ceramidase, which
hydrolyzes ceramide to sphingosine, was not changed as a result
of resveratrol treatment (Fig. 1). Thus, resveratrol-induced reduc-
tion of ceramide in aged hepatocytes was due mainly to the de-
crease of nSMase activity and not to stimulation of ceramide
hydrolysis. In addition, the accumulation of SM, a lipid acting as
a natural membrane antioxidant, was also a result mainly of the re-
duced SM hydrolysis, performed by nSMase. So it seems likely, that
nSMase is the key enzyme responsible for ceramide reduction and
SM accumulation in resveratrol-treated aged cells. The observed
effect seems to be specific for senescent cells, because similar stud-
ies, performed on hepatocytes of three month old rats did not show
any statistically significant differences neither in SM content, nor
in nSMase activity (data not shown). Since sphingomyelin pathway
plays an important role in age-related changes occurring in liver
plasma membranes [27,29], we presume, that one of the major
mechanisms, underlying effect of resveratrol on membranes of
aged hepatocytes, is its impact on the sphingolipid-metabolizing
enzymes and more specifically on nSMase and its products.
As mentioned above PS, which was augmented in resveratrol-
treated membranes, is important in physiological and patho-phys-
iological aspect, because its translocation to the outer membrane
leaflet is a marker of apoptosis and serves as a signal for macro-
phage attack. The observed slight, but statistically significant
reduction of PS exposure in the external membrane monolayer, ta-
ken together with the decrease of ceramide implies a reduction of
specific apoptotic markers in the senescent cells, which could be
considered as a favorable event for the hepatocytes functional
activity.
The fatty acid analysis of the most abundant membrane phos-
pholipids, PC and PE, showed an increase of the polyunsaturated
acids such as linolenic and docosahexaenoic, as well as a reduction
of the saturated palmitic acid. The latter observation is in accor-
dance with the finding of Gnoni and Paglialonga [10], who reported
reservatrol-induced short-term inhibition of palmitic acid synthe-
sis in hepatocytes of young rats. Thus, it is possible that resveratrol
exerts a similar effect on this saturated fatty acid in hepatocytes of
both young and old rats. It is also possible that resveratrol stimu-
lates the activities of elongase and desaturase enzymes, because
we observed an elevation in the mol% of the long-chain polyunsat-
urated docosahexaenoic (in PC and PE), and docosapentaenoic
(only in PE) acids.
In our previous paper we reported that SM acts as a natural
membrane antioxidant, which could protect the polyunsaturated
fatty acid chains against oxidative destruction [30]. Since the level
of SM was higher in resveratrol-treated hepatocytes, we followed
the accompanying changes in the content of lipid peroxides, ROS
and GSH (Fig. 5 and Table 3). We did not observe significant
changes in the GSH level, which was rather unexpected, because
resveratrol is known to act as a potent antioxidant, and has been
reported to increase the expression of glutathione peroxidase
[31]. It is possible, that such effect takes place in young cells and
is not valid for cells of aged animals, where the synthesis of GSH
is hindered. However, the content of ROS was lower in resvera-
trol-treated cells (Table 3). Nevertheless, it should be noted that
free radicals do not only cause molecular damage to cells, but could
also participate in cell signaling and thus act as mediators of
physiological processes [32]. Also, there are reports which associ-
ate increased ROS production with higher longevity. So our finding
that ROS have been reduced by resveratrol treatment could be a
subject of a more complex interpretation. Based on the observed
decrease of lipid peroxides in plasma membranes from resvera-
trol-treated cells (Fig. 5) we presume, that the influence of resvera-
trol was most prominent on the membranes of senescent
hepatocytes, possibly due to the lipophillic nature of this
polyphenol.
The finding that lipid peroxidation was lower and the degree of
fatty acid unsaturation was elevated in membranes of resveratrol-
treated senescent hepatocytes is of particular interest. This implies
that certain intrinsic antioxidant factors are responsible for the
reduction of lipid peroxides in the membranes of resveratrol-trea-
ted aged hepatocytes. One possible explanation is the antioxidant
effect of SM [30,33] which was elevated due to resveratrol treat-
ment. Subbaiah et al. reported that the sphingosine backbone of
SM has unique structure, which not only makes SM less susceptible
to free radical reactions, but also inhibits the oxidation of the
neighboring unsaturated lipid molecules [33]. Another mechanism
could be related to the reported lower level of ROS (Table 3). Of
course, other cellular mechanisms which could be activated by res-
veratrol treatment should not be ruled out. In addition, the relative
increase of omega-3 fatty acids, represented by docosahexaenoic
acid, should also be emphasized, because the abundance of these
essential fatty acids in the membrane phospholipids is considered
favorable for the membrane functional activity and structural orga-
nization [34].
In conclusion, the presented results demonstrate that resvera-
trol treatment of hepatocytes obtained from old rats induces sig-
nificant changes especially on membrane level. Particular
attention require the alterations in the fatty acid composition,
the ceramide level, the activity of nSMase as well as the lipid per-
oxide level in plasma membranes of resveratrol-treated senescent
hepatocytes. Although at this point the exact mechanisms underly-
ing the reported changes could not be specified, it is clear that res-
veratrol induces specific alterations in the membrane lipids which
can be estimated as beneficial for the plasma membrane and for
the whole senescent liver cell.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgement
This work was financially supported by the National Science
Fund – Bulgarian Ministry of Education, Grant #DFNI-B01-5/2012.
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