Chemistry and Physics of Lipids 204 (2017) 85–93

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Chemistry and Physics of Lipids

journal homepage: www.elsevier.com/locate/chemphyslip

The effect of cannabinoid receptor 1 blockade on hepatic free fatty acid

profile in mice with nonalcoholic fatty liver disease
Bojan Jorga9cevi cevi
ca , Danijela Vu9 cb , Slapana Šobaji
ca , Ivana Ðuri9ci cb ,
Dušan Mladenovi c , Milena Veskovi
a
c , Rada Ješi
a
c Vuki cevi
c , Tatjana Radosavljevi
c
ca,*
a
Institute of Pathophysiology “Ljubodrag Buba Mihailovic”, Faculty of Medicine, University of Belgrade, Serbia
b
Department for Bromatology, Faculty of Farmacy, University of Belgrade, Serbia
c
Institute of Digestive Diseases, Clinical Centre of Serbia, Belgrade, Serbia

A R T I C L E I N F O A B S T R A C T

Article history:
Received 14 January 2017 We used rimonabant to investigate the role of CB1 receptor on hepatic FFAs profile during NAFLD. Male
Received in revised form 6 March 2017 mice C57BL/6 were divided into: control group fed with control diet 20 weeks (C; n = 6); group fed with
Accepted 14 March 2017 HFD 20 weeks (HF; n = 6); group fed with control diet and treated with rimonabant after 18 weeks (R;
Available online 29 March 2017 n = 9); group fed with HFD and treated with rimonabant after 18 weeks (HFR; n = 10). Rimonabant (10 mg/
kg) was administered daily to HFR and R group by oral gavage. Rimonabant decreased liver palmitic acid
Keywords: proportion in HFR group compared to HF group (p < 0.05). Liver stearic and oleic acid proportions were
NAFLD decreased in R group compared to control (p < 0.01 respectively). Rimonabant increased liver linoleic and
Endocannabinoid system
arachidonic acid proportions in HFR group compared to HF group (p < 0.01 respectively). CB1 blockade
Rimonabant
may be useful in the treatment of HFD-induced NAFLD due to modulation of plasma lipid and hepatic FFA
CB1 receptor blockade
Free fatty acid profile profile.
Mice © 2017 Elsevier B.V. All rights reserved.

1. Introduction
Nonalcoholic fatty liver disease (NAFLD) includes wide spec-
trum of liver diseases ranging from steatosis (triglyceride/TG/
accumulation within hepatocytes) to steatohepatitis, associated
with hepatocellular injury and activation of fibrogenic pathways
(fibrosis and cirrhosis), that may progess to hepatocellular
carcinoma (Mallat et al., 2013; Pardo et al., 2015; Friedman,
2016). Although, pathogenesis of NAFLD is still incompletely
understood, two hit mechanisms have been identified. Firstly,
insulin resistance (IR) in the adipose tissue increases lipolysis and
the hepatic entry of free fatty acids (FFAs), as well as de novo
synthesis of fatty acids and TG. Secondly, oxidative/nitrosative
stress, mitochondrial dysfunction, lipotoxicity and overproduction
of proinflammatory cytokines have been involved in progression of
liver steatosis to steatohepatitis and fibrosis (Fraulob et al., 2010;
Aubert et al., 2011; Leamy et al., 2013; Stankovi c et al., 2014;
Jorga9cevi
c et al., 2015). However, this “two hit hypothesis” is
increasingly replaced by the concept that benign steatosis and
nonalcoholic steatohepatitis (NASH) could be two different entities
* Corresponding author at: Institute of Pathophysiology, Faculty of Medicine,
University of Belgrade, Dr Suboti
ca 9, 11000 Belgrade, Serbia.
E-mail address: tanjamm@med.bg.ac.rs (T. Radosavljevi c).
with divergent pathogenic pictures (Tilg and Moschen, 2010; Fuchs
et al., 2014). Thus, “multiple hits” may occur in parallel rather than
in strict sequence (Leamy et al., 2013). In this context lipid
accumulation may be considered as a “bystander phenomenon”(-
Fuchs et al., 2014). Constant exposure of hepatocytes to FFAs,
phosphatidic acid, lysophosphatidic acid, ceramides and diacyl-
glycerols (DAG) may result in lipotoxic insults (Trauner et al., 2010),
characterized by endoplasmatic reticulum (ER) stress, inflamma-
tion, apoptosis and necrosis of hepatocytes (Neuschwander-Tetri,
2010; Fuchs et al., 2014). In addition to potential lipotoxic TG
degradation products, cholesterol may also be an important trigger
for NAFLD development (Simonen et al., 2011; Enjoji et al., 2012).
There are evidence that increased dietary cholesterol uptake does
not only act as disease initiator but can also promote its
progression beyond the more severe stages (Subramanian et al.,
2011; Fuchs et al., 2014). Many studies have demonstrated that
saturated fatty acids (SFAs) are more toxic than their unsaturated
counterparts, resulting in a progressive lipotoxic cascade, both in
experimental models and in NAFLD patients (El-Badry et al., 2007;
Puri et al., 2007; Malhi and Gores, 2008; Leamy et al., 2013; Fuchs
et al., 2014; Stankovic et al., 2014).
The cannabinoid system (CS) refers to the cannabinoids,
cannabinoid receptors (cannabinoid receptor type 1/CB1/and type
2/CB2/) and enzymes involved in endocannabinoids (ECs)

http://dx.doi.org/10.1016/j.chemphyslip.2017.03.009
0009-3084/© 2017 Elsevier B.V. All rights reserved.
86 cevic et al. / Chemistry and Physics of Lipids 204 (2017) 85–93
B. Jorga9
synthesis and degradation (Howlett, 2002; Alswat, 2013; Mallat
et al., 2013; Romero-Zerbo and Bermúdez-Silva, 2014). Cannabi-
noids are a class of diverse chemical compounds that activate
cannabinoid receptors, including phytocannabinoids (found in
cannabis and some other plants), the ECs (produced in the body,
e.g. noladine, N-arachidonoyl ethanolamine, also known as
anandamide/ANA/, 2-arachidonoyl glycerol/2-AG/and virodh-
amine) and synthetic cannabinoids (Pacher et al., 2006; Alswat,
2013; Romero-Zerbo and Bermúdez-Silva, 2014).
In the normal liver, the activity of endocannabinoid system
(ECS) is modest (Alswat, 2013). However, during liver pathology
ECS is overactivated and CB1 receptors undergo marked up-
regulation, most particularly in hepatocytes and stellate cells
(Jeong et al., 2008; Alswat, 2013). The liver is identified as a
primary site for endocannabinoid-mediated modulation of lipo-
genesis (Osei-Hyiaman et al., 2005). In fact, the activation of the
CB1 receptors increase the expression of lipogenic genes in the
liver (sterol regulatory element binding protein-1/SREBP-1/, etc.)
and its target enzymes, which is the main source of de novo FA
synthesis in the body (Osei-Hyiaman et al., 2005; Jeong et al., 2008;
Westerbacka et al., 2010; Shi et al., 2014). Additionally, animal
studies showed that high fat diet (HFD) increases hepatic levels of
anandamide, CB1 density and basal rates of FA synthesis (Alswat,
2013). In relation to, it is suggested that deletion of CB1 receptors in
the liver decreases hepatic lipogenesis and ameliorates hypercho-
lesterolemia, hypertriglyceridemia and hepatocellular damage
(Osei Hyiaman et al., 2008; Després et al., 2009; Vettor and
Pagano, 2009; Bartelt et al., 2011; Quarta et al., 2011), in this
context, modulation of hepatic ECS may represent a potential novel
target for NAFLD treatment (Alswat, 2013; Mallat et al., 2013).
Current study attempted for the first time to explore FFA profile in
high saturated fat diet model of NAFLD. Although the brain-
penetrant CB1 antagonist rimonabant was withdrawn because of
an alarming adverse effect on mood in humans (Despres et al.,
2005, 2009), we used this centrally acting weight loss drug having
in mind its efficacy in treating obesity and obesity-related MS
components, including dyslipidemia and diabetes (Despres et al.,
2005, 2009; Scheen et al., 2006). In relation to, it is known that
many of metabolic benefits of rimonabant may be explained by
actions on brain CB1 receptors influencing autonomic output to the
gastrointestinal tract, adipose tissue and liver. Experimentally
distinguishing centrally mediated peripheral effects from direct
effects on peripherally located CB1 receptors (e.g., in adipose tissue
or hepatic and pancreatic islet cells) is difficult and not yet
conclusive (Sam et al., 2011). Therefore, in this study we decided to
investigate the effect of rimonabant, on hepatic FFAs profile in mice
with NAFLD. Besides, since the development of the second
generation of neutral and inverse peripherally-restricted CB1
antagonists show promissing effects in preclinical studies (Mallat
et al., 2013), we will concentrate our further research attention on
these molecules. Based on this background, the aim of the present
study was to investigate the effects of CB1 receptor blockade on
hepatic FFAs profile in mice with NAFLD.
2. Materials and methods
2.1. Animals
The experiment was performed on male mice C57BL/6 wt 21–
25 g, raised at Military Medical Academy, Belgrade. Animals were
kept in individual cages under standard laboratory conditions
(temperature 22  2  C, relative humidity 50  10%, 12/12 light-
dark cycle with lights turned on at 9.00 A.M.) and had free access to
tap water and standard chow diet before experiment. All
experimantal procedures were in full compliance with Directive
of the European Parliament and of the Council (2010/63 EU) and
Table 1
High saturated fat diet composition (MP Biochemical, CA, USA).
Nutrient %
Casein Purified 20
DL-Methionine 0.30
Sucrose 30.58
Corn Starch 20
Coconut Oil 20
Alphacel, Non Nutritive Bulk 5
DL a-Tocopherol Powder (250 IU/gr) 0.12
AIN 76 Mineral Mix 4
Total 100
approved by the Ethical Commitee of the University of Belgrade
(695/2).
2.2. Experimental design
Before experiment all animals (n = 31) were fed with control
diet. At the age of 8 weeks they were randomly divided into
following groups: 1. control group fed with control chow diet 20
weeks (C; n = 6) 2. group fed with high saturated fat diet 20 weeks
(HF; n = 6); 3. group fed with standard chow diet and treated with
rimonabant after 18 weeks (R; n = 9); 4. group fed with high
saturated fat diet and treated with rimonabant after 18 weeks
(HFR; n = 10). Composition of high saturated fat diet (MP
Biochemicals, CA, USA) is shown in Table 1. Daily dose of
rimonabant (10 mg/kg) was administered to HFR and R group by
oral gavage for a two weeks. Simultaneously, C and HF group
received vehicle (saline, 0.9% NaCl) in the same manner. Before
administration, rimonabant was dissolved into 0.1% Tween 80 in
distilled water and for 20 s sonificated on ice using a digital
Branson sonificator.
On the day prior to sacrifice mice were fasted overnight. After
the treatment, animals were sacrificed by exsanguination in
ketamine (100 mg/kg intraperitoneally/i.p./) anesthesia. Blood
samples were taken from right side of the heart by cardiac
puncture for determination of lipid status and activity of amino-
transferases. Liver samples were taken for pathohistology and FFA
level measurement. Epididymal and inguinal fat were surgically
removed as representatives of visceral and subcutaneous fat,
respectively (Kim et al., 2011).
2.3. Serum biochemical analysis
Liver injury was confirmed biochemically by determination of
serum alanine aminotransferase (ALT) and aspartate aminotrans-
ferase (AST) activity. Activities of these enzymes were measured
spectrophotometrically at photometer BTS-330 according to the
manufacturer‘s instruction using special kits containing 2-oxo-
glutarate (Sigma Aldrich, St.Louis, MO).
Triglyceride (TG) and high density lipoprotein (HDL) concen-
tration in plasma is measured by enzymatic colorimetric method.
According to this method, modified cholesterol-oxidase reagent by
inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA) was
used to measure plasma TG concentration. Cholesterol and TG
assay kits were purchased from Abcam (Cambridge, UK, Choles-
terol Assay Kit, ab65390; TG Quantification Kit, ab65336).
Total cholesterol (TC) concentration in plasma was determined
by measuring hydrogen peroxide generated by cholesterol oxidase
in oxidative coupling reaction with 4-aminoantipyrine and phenol
at 500 nm (Allain et al., 1974). Plasma low density lipoprotein (LDL)
concentration was calculated by a combination of ultracentrifuga-
tion and precipitation procedure by Friedewaldet al. (Friedwald
et al., 1972). Plasma HDL concentration was determined by using
enzymatic colorimetric method (Roche Diagnostics, Basel,
 cevic et al. / Chemistry and Physics of Lipids 204 (2017) 85–93
B. Jorga9
Switzerland). In the presence of double positively charged anions,
the HDL fraction was measured after selective LDL precipitation by
polyanion sulfates. In our experimental protocol, dextran sulfate
and magnesium sulfate were used in accordance with the Center of
Disease Control and prevention (Atlanta, GA: Lipid Reference
Section). After centrifugation, HDL fraction was determined
directly in the supernatant by the PAP method (Trinder, 1983).
2.4. Free fatty acids analysis
Lipids for further fatty acid analysis were extracted according to
the method of Bligh and Dyer (Biligh and Dyer, 1959). The methyl
esters of the fatty acids (FAMEs) from the lipid extract were
transesterified with hydrochloric acid (HCl) in methanol according
to the method described by Ichihara and Fukubayashi (Ichihara and
Fukubayashi, 2010). FAMEs were quantified using Agilent Tech-
noligies (7890A Gas Chromatograph, Santa Clara, CA) with a flame
ionization detector. Separation of the FAMEs was performed on a
112-88A7, HP-88 capillary column (100 m  0.25 mm  0.2 mm)
using helium (He) as a carrier gas at a flow rate of 105 ml/min. The
samples were injected at a starting oven temperature of 175  C,
injector temperature was 250  C and detector temperature was
280  C. The oven temperature was programmed to increase from
175  C to 220  C at 5  C/min. Fatty acids were identified by their
retention time with reference fatty acid standards (Supelco, FAME
Mix, Sigma Aldrich) and were expressed as a percentage of total
fatty acids in the liver.
2.5. Pathohistological analysis
Liver tissue was incubated in 10% formalin solution at room
temperature. After the fixation, the liver samples were processed
by the standard method. Tissues were incorporated in parafin
sectioned at 5 mm and then stained with hematoxylin-eosin (HE)
and prepared for light microscopy analysis. All samples were
evaluated by an expirienced pathohistologist who was blinded to
the experiment. Preparations were analyzed and photographed
using a combined photobinocular light microscope Olympus BX51
equipped Artcore 500MI artray, Co. Ltd. Japan Camera.
2.6. Statistical analysis
All results are expressed as means  SD. As the normal
distribution of parameters was confirmed by Kolmogorov-Smirnov
test, one-way analysis of variance (ANOVA) with Tukey‘s post hoc
test was used for testing the difference among groups. The
difference was considered statistically significant if p < 0.05.
Computer software SPSS 15.0 was used for the statistical analysis.
Table 2
Effect of rimonabant on liver/body weight ratio.
Group
C 4.58  0.22
R 4.75  0.20
HF 4.86  0.17*
HFR 4.67  0.14#
C-control group fed with control chow diet for 20 weeks; R-group fed with standard
chow diet and treated with rimonabant after 18 weeks; HF-group fed with high
saturated fet diet for 20 weeks; HFR-group fed with high saturated fet diet and
treated with rimonabant after 18 weeks. Significance of the difference was
estimated by using one-way analysis of variance (ANOVA) with Tukey’s post hoc
test (*p < 0.05 vs. C, #p < 0.05 vs. HF).
87
3. Results
3.1. Effect of rimonabant on liver/body weight ratio
There were no statistically significant difference in liver/body
weight ratio between R group (4.75  0.20%) and C group
(4.58  0.22%) (p > 0.05). HFD induced a significant increase in
liver/body ratio in HF group (4.86  0.17%) in comparison with C
group (4.58  0.22%) (p < 0.05), as well as in HF group compared to
HFR (4.67  0.14%) (p < 0.05 respectively). (Table 2).
3.2. Effect of rimonabant on subcutaneous and visceral adipose tissue
weight
Subcutaneous adipose tissue (SAT) weight was significantly
higher in HFD fed mice (0.05  0.03 g) compared to group fed with
control chow diet (0.03  0.015 g) (p < 0.05). Treatment with
rimonabant induced a significant decrease of SAT in R group
(0.01  0.03 g) compared to C group (p < 0.05). Similarly, significant
decrease of SAT was observed in HFR group (0.03  0.02 g) in
comparison with HF (p < 0.05) (Fig. 1).
Visceral adipose tissue (VAT) weight was significantly increased
in HF group (0.73  0.09 g) compared to control (0.35  0.07 g)
(p < 0.01). On the other hand, rimonabant treatment induced a
significant decrease of VAT in R group (0.23  0.04 g) compared to C
group, as well as in HFR group (0.48  0.06 g) in comparison with
HF group (p < 0.05 and p < 0.01 respectively) (Fig. 1).
3.3. Effect of rimonabant on serum AST and ALT activity
Our study has shown that serum AST activity was significantly
increased in HF group (258.95  19.99 U/L) in comparison with
control group (191.91 7.65 U/L) (p < 0.01). Significant decrease in
AST activity was evident between R group (155.60  10.27 U/L) and
control, as well as in HFR group (125.46  8.70 U/L) compared to HF
group (p < 0.01 respectively) (Fig. 2).
HF diet caused an increase in serum ALT activity
(60.98  0.96 U/L) compared to control (49.25  3.38 U/L) (p
< 0.01). On the other hand, a significant decrease in ALT activity
was observed in R group (30.77  1.07 U/L) compared to C
(p < 0.01). Rimonabant treatment induced the significant decrease
in serum ALT activity in HFR group (34.29  2.89 U/L) when
compared with HF group (p < 0.01) (Fig. 2).
3.4. Effect of rimonabant on plasma TG concentration
Plasma TG concentration was significantly lower in R group
(0.85  0.04 mmol/L) in comparison with control (1.06  0.09
mmol/L), as well as in HFR group (1.14  0.06 mmol/L) compared
to HF group (1.56  0.08 mmol/L) (p < 0.01 respectively). However,
TG concentration was significantly higher in HFD fed mice
compared to group fed with control chow diet (p < 0.01) (Fig. 3).
3.5. Effect of rimonabant on plasma cholesterol level
Plasma TC level was significantly increased in HF group
% (3,29  0.03 mmol/L) compared to control (2,41  0.05 mmol/L)
(p < 0.01). On the other hand, rimonabant treatment caused
decrease in plasma TC level in R group (1.98  0.08 mmol/L)
compared to C group, as well as in HFR group (2.82  0.09 mmol/L)
compared to HFD fed mice (p < 0.01 respectively) (Fig. 3).
Analysis of plasma cholesterol fractions has shown that HFD
induced a significant decline in HDL level in HF group (2.33  0.04
mmol/L) in comparison with control (3.48  0.05 mmol/L) (p
< 0.01). Similarly, plasma HDL level was significantly elevated in
R group (3.94  0.08 mmol/L) compared to control, as well as in
88 cevic et al. / Chemistry and Physics of Lipids 204 (2017) 85–93
B. Jorga9
Fig. 1. The effect of rimonabant on on subcutaneous and visceral fat tissue weight after 20 weeks feeding with high fat and control chow diet C-control group fed with control
chow diet for 20 weeks; R-group fed with standard chow diet and treated with rimonabant after 18 weeks; HF-group fed with high saturated fet diet for 20 weeks; HFR-group
fed with high saturated fet diet and treated with rimonabant after 18 weeks. Statistical significance of the difference was estimated by using two-way analysis of variance
(ANOVA) with Tukey’s post hoc test (**p < 0.01 vs. C, *p < 0.05 vs. C, ## p < 0.01 vs. HF).
Fig. 2. The effect of rimonabant on serum AST and ALT activity after 20 weeks
feeding with high fat and control chow diet (C, R, HF, HFR)*. Statistical significance
of the difference was estimated by using two-way analysis of variance (ANOVA)
with Tukey’s post hoc test (**p < 0.01 vs. C, ## p < 0.01 vs. HF). *Abbreviations as in
Fig. 1.
Fig. 3. The effect of rimonabant on on serum triacylglycerol, total cholesterol and
HDL concentration after 20 weeks feeding with high fat and control chow diet (C, R,
HF, HFR)*. Statistical significance of the difference was estimated by using two-way
analysis of variance (ANOVA) with Tukey’s post hoc test (**p < 0.01 vs. C, ## p < 0.01
vs. HF, # p < 0.05 vs. HF).*Abbreviations as in Fig. 1.
HFR group (4.07  0.04 mmol/L) in comparison with HF group
(p < 0.01 respectively) (Fig. 3).
Plasma LDL level was significantly increased in HF group
(0.29  0.02 mmol/L) compared to control (0.07  0.03 mmol/L)
(p < 0.01). Besides, rimonabant treatment caused nonsignificant
increase in plasma LDL level in R group (0.08  0.01 mmol/L)
compared to C group (p > 0.05). On the other hand, plasma LDL
concentration was significantly lower in HFR group (0.17  0.03
mmol/L) in comparison with HF group (p < 0.01) (Fig. 4).
3.6. Effect of rimonabant on hepatic FFA profile
Palmitic acid (C16:0) proportion was significantly higher in HFD
fed mice (23.32  0.69%) compared to group fed with control chow
diet (22.66  0.58%) (p < 0.05). Additionally, rimonabant induced a
significant decrease in liver palmitic acid proportion in R group
(21.22  1.2%) compared to control, as well as in HFR group
(22.53  1.00%) in comparison with HF group (p < 0.05 respective-
ly) (Fig. 5).
Stearic acid (C18:0) proportion was significantly higher in HF
group (13.22  1.13%) compared to group fed with control chow
diet (10.44  0.63%) (p < 0.01). In contrast, liver stearic acid
proportion was significantly decreased in R group (7.22  1.2%)
compared to control, as well as in HFR group (11.68  0.62%) in
comparison with HF group (p < 0.01 respectively) (Fig. 5).
Oleic acid (C18:1) proportion was higher in HF group
(31.14  0.62%) when compared to control (13.26  0.67%) (p
< 0.01). However, rimonabant caused a significant decrease in
liver oleic acid proportion in R group (10.24  1.1%) compared to
control, as well as in HFR group (24.33  1.3%) in comparison with
HF group (p < 0.01 respectively) (Fig. 5).
Fig. 4. The effect of rimonabant on on serum LDL concentration after 20 weeks
feeding with high fat and control chow diet (C, R, HF, HFR)*. Statistical significance
of the difference was estimated by using two-way analysis of variance (ANOVA)
with Tukey’s post hoc test (**p < 0.01 vs. C, ## p < 0.01 vs. HF). *Abbreviations as in
Fig. 1.
 cevic et al. / Chemistry and Physics of Lipids 204 (2017) 85–93
B. Jorga9 89

Fig. 5. The effect of rimonabant on liver free fatty acid profile after 20 weeks feeding with high fat and control chow diet (C, R, HF, HFR)*. Statistical significance of the
difference was estimated by using two-way analysis of variance (ANOVA) with Tukey’s post hoc test (**p < 0.01 vs. C, *p < 0.05 vs. C, # p < 0.05 vs. HF). *Abbreviations as in
Fig. 1.

Linoleic acid (C18:2 n6) proportion was significantly higher in
HFD fed mice (4.78  0.64%) compared to group fed with control
chow diet (3.78  0.64%) (p < 0.05). Additionally, rimonabant
induced a significant increase in liver linoleic acid proportion in
R group (18.6  1.27%) compared to control, as well as in HFR group
(20.98  1.59%) in comparison with HF group (p < 0.01 respective-
ly) (Fig. 5).
Arachidonic acid (C20:4 n6) proportion was significantly lower
in HF group (8.16  1.34%) when compared to control
(11.88  0.39%) (p < 0.01). However, rimonabant did not cause
any changes in arachidonic acid proportion in R (12.02  0.1%)
group compared to C group (p > 0.05). In contrast, rimonabant
treatment induced a significant increase in arachidonic acid
proportion in HFR group (11.67  0.9%) in comparison with HF
group (p < 0.01) (Fig. 5).

Fig. 6. The effect of HF diet on histopathological changes in the liver. HE-stained preparations were sectioned with blade thickness of 5 mm and analyzed and photographed
using a combined photobinocular light microscope Olympus BX51. (A) Control with normal liver histology/10x/; (B) rimonabant treated control chow diet fed group with
normal liver histology/10x/; (C) liver histology in HF* group shows mild steatosis with portal inflammatory infiltrate and focal necrotic changes in parenchyma/20x/; (D) liver
histology in HFR* group shows mild steatosis/10x/. *Abbreviations as in Fig. 1.
90 cevic et al. / Chemistry and Physics of Lipids 204 (2017) 85–93
B. Jorga9
3.7. Pathohistological findings
There were no pathohistological changes in the liver in C and R
group (Fig. 6A and B). HFD caused mild hepatic steatosis with
portal inflammatory infiltrate and focal necrotic changes in
parenchyme (Fig. 6C). In contrast to HF group, mild steatosis
and no inflammatory infiltrate in portal space and focal necrotic
changes in the liver parenchyma were found in HFR group (Fig. 6D).
4. Discussion
NAFLD is a major cause of liver morbidity and mortality with no
proven effective therapy as of yet (Alswat, 2013). For studying
NAFLD/NASH animal models are essential tools. By using a HFD
model of NAFLD in C57BL/6 mice in our study, the results have
shown evident fatty changes in the mice liver after 18 weeks of
HFD (Fig. 6C). Since mild hepatic steatosis with portal inflamma-
tory infiltrate and focal necrotic changes was found in liver
parenchyme of HFD fed mice (Fig. 6C), it may be suggested that
FFAs are involved in this pathohistological finding. In our
experimental protocol, a significant increase in liver/body ratio
was evident in HFD fed mice compared to group fed with control
chow diet (Table 2). This finding is in agreement with reports
demonstrating a link between HFD and hepatocellular damage
(Osei-Hyiaman et al., 2005; Osei Hyiaman et al., 2008; Fraulob
et al., 2010; Trauner et al., 2010; Bartelt et al., 2011; Jorga9cevi
c et al.,
2015). It is known that an increased dietary supply of fat to the liver
may promote steatosis by increasing hepatic lipid uptake (Fig. 6C).
In this pathway, lipogenic gene expression and FA synthesis
induced by HFD appear to play an important role in the
pathogenesis of NASH (Osei-Hyiaman et al., 2005; Osei Hyiaman
cevi
et al., 2008; Enjoji et al., 2012; Jorga9 c et al., 2015). In our study
HFD caused a significant increase in serum AST and ALT activity
compared to control values (Fig. 2). These data also indicate that
increased dietary fat uptake leads to hepatocellular damage
(Fig. 6C).
Our study suggests that ECS contribute to the development of
NASH through CB1 receptors. In fact, it highlights the importance
of CB1 receptor blockade in protection against the pathological
consequences of a HFD in the liver (Fig. 6D). We used rimonabant,
the first CB receptor blocker with high potency and selectivity for
the CB1 receptor (Muccioli, 2007), since this centrally acting
weight loss drug is used therapeutically for the treatment of
obesity-related MS components, including dyslipidemia and
diabetes (Despres et al., 2005, 2009; Scheen et al., 2006;
Mechoulam et al., 2014). However, the drug was withdrawn from
the market, because of the alarming incidence of anxiety, sleeping
disorders and depression (Mallat et al., 2013). Nevertheless, the
development of rimonabant by Sanofi-Aventis can be considered to
be a major contributor to our understanding that CB1 receptors is
present and functional in tissues, such as adipose tissue, liver and
pancreas under pathological conditions of HFD or obesity (Di
Marzo and Despres, 2009; Mechoulam et al., 2014). This new
understanding of the role of CB1 receptors in metabolic regulation
has inspired the search for novel possible drugs that fail to gain
access to the brain (Mallat et al., 2013; Mechoulam et al., 2014;
Kaur et al., 2016; Krentz et al., 2016; Lipina et al., 2016).
It is known that effect of rimonabant is associated with
reduction of inflammation. Previous preclinical and clinical
nephropathy and cardiomiopathy studies have shown antiinflam-
matory and cytoprotective effects of CB1 antagonists, due to
reduction of tumor necrosis factor-alpha (TNF-a) production and
inactivation of nuclear factor-kappa B (NF-kB) (Despres et al.,
2005; Di Marzo and Matias, 2005; Van Gaal et al., 2005; Pi-Sunyer
et al., 2006; Scheen et al., 2006; Hudson et al., 2010; Mukho-
padhyay et al., 2010a, 2010b; Cheung et al., 2013). Moreover, our
recent experimental NAFLD study clearly suggested that antiin-
flammatory effect of rimonabant may be additionally mediated by
decline in lipid peroxidation (Jorga9 cevic et al., 2015). Besides,
hepatoprotective effect of rimonabant in this study is in accor-
dance with higher level of ANA and upregulation of CB1 receptor in
HFD-induced NAFLD (Osei-Hyiaman et al., 2005, 2008; West-
erbacka et al., 2010; Alswat, 2013; Shi et al., 2014).
In present work HFD caused lipid accumulation not only in liver,
but also in SAT and VAT (Fig. 1). These findings is in agreement with
reports demonstrating a link between HFD and distribution of
body fat (Osei Hyiaman et al., 2008; Park et al., 2008; Starowicz
et al., 2008; Van der Poorten et al., 2008; Jourdan et al., 2010;
Verrijken et al., 2010; Hashimoto and Tokushige, 2011; Ioannou,
2016). Visceral obesity is recognized as an important risk factor for
development of hepatic steatosis (Park et al., 2008; Starowicz et al.,
2008; Van der Poorten et al., 2008; Jourdan et al., 2010; Verrijken
et al., 2010; Hashimoto and Tokushige, 2011; Ioannou, 2016). Thus,
Eguchi et al. have found positive correlation between severity of
steatosis and amount of VAT (Eguchi et al., 2006). Similar finding
regarding positive correlation between VAT, inflammation and
fibrosis in NASH patients is published by Van der Poorten and his
colleagues (Van der Poorten et al., 2008). Results of our study
showed that treatment with rimonabant induced a significant
decrease of SAT and VAT (Fig. 1). Additionally, the role of ECS and
CB1 receptors in the accumulation of neutral lipids in fat cells was
confirmed by Matias et al. Namely, it is noteworthy that adipose
cell differentiation is preceded by a rise in 2AG and ANA production
by adipocytes, thus supporting the role of ECS in driving adipose
conversion of preadipocytes (Matias et al., 2006). Consistent with
previous reports (Osei-Hyiaman et al., 2005; Matias et al., 2006;
Pacher et al., 2006; Vettor and Pagano, 2009; Bartelt et al., 2011; Shi
et al., 2014), our findings also indicate that selective CB1 receptor
antagonist rimonabant persistently reduces body weight in obese
animals, suggesting that CB1 receptor blockade stimulates energy
metabolism, leading to a decreased fat content. Besides, there are
similar data strongly suggesting that there is an increased EC tone
in obese humans. In fact, blood levels of ANA only, or both 2-AG and
ANA were increased in obese women (Engeli et al., 2005;
Monteleone et al., 2005), and increased VAT 2-AG concentrations
were observed in obese individuals (Matias et al., 2006). These
results support the crucial role of VAT activation of the ECS in the
pathophysiology of obesity and its complications (Matias et al.,
2006; Di Marzo, 2008; Starowicz et al., 2008; Jourdan et al., 2010).
Present and above mentioned studies suggest that CB1 receptors in
adipose tissue could be selectively targeted for the treatment of
obesity and related metabolic disorders.
The liver has a critical role in the production and processing of
circulating lipoproteins (Enjoji et al., 2012). Extensive dysregula-
tion of cholesterol homeostasis has been documented in NAFLD
(Musso et al., 2009; Yasutake et al., 2009; Neuschwander-Tetri,
2010; Wouters et al., 2010; Pinzani, 2011; Min et al., 2012; Souza
et al., 2012; Chaurasia and Summers, 2015; Ioannou, 2016). Excess
dietary cholesterol also has been shown consistently to cause the
development of experimental NASH in different animal models,
especially in the setting of concurrent HFD, obesity or hyperphagia
(Wouters et al., 2010; Ioannou et al., 2013, 2015; Savard et al., 2013;
Hendrikx et al., 2014). Similarly, in our experimental procedure,
dysregulation of cholesterol homeostasis has been observed.
Namely, a significant increase in plasma TG, TC and LDL
concentration was registered in HFD fed mice compared to group
fed with control chow diet (Figs. 3 and 4). In addition, HFD in
present study induced a significant decline in plasma HDL level in
HF group in comparison with control (Fig. 3). These findings is in
agreement with numerous studies demonstrating a link between
dyslipidemia and NAFLD (Trauner et al., 2010; Westerbacka et al.,
2010; Pinzani, 2011; Souza et al., 2012; Fuchs et al., 2014). It is well
 cevic et al. / Chemistry and Physics of Lipids 204 (2017) 85–93
B. Jorga9
documented that NASH develops when the liver is exposed to
lipotoxic lipid species, while simple steatosis develops in response
to the storage of lipids that do not cause lipotoxicity (Neuschwan-
der-Tetri, 2010; Wouters et al., 2010; Chaurasia and Summers,
2015; Ioannou, 2016). Thus, hepatic free cholesterol is now
considered a critical lipotoxic molecule in NASH, due to activation
of Kupffer cells and hepatic stellate cells and induction of
necroinflammation and fibrosis (Ioannou, 2016).
In current study, rimonabant caused a significant decrease in
plasma TG and TC concentration compared to control (Fig. 3). Our
study also has shown that administration of rimonabant to HFD-
fed mice significantly reduced plasma TG and TC concentration in
comparison with mice fed with HFD (Fig. 3). Besides, results of our
study were found a significant decline in plasma LDL concentration
in HFD-fed mice treated with rimonabant compared to mice fed
with HFD (Fig. 4). On the other hand, our study demonstrated that
plasma HDL concentration was significantly elevated in mice
treated with rimonabant compared with control values (Fig. 3), as
well as in mice fed a HFD after rimonabant treatment compared to
mice fed on HFD (Fig. 3). Judging from these findings, it seems that
CB1 receptor blockade may improve lipid profile abnormalities
associated with HFD-induced NAFLD. More importantly, these
findings suggest that CB1 antagonists may be effective not only as
antiobesity agents, but also in preventing/reversing the develop-
ment of fatty liver (Fig. 6D). Our results may be related to a great
number of experimental reports that describe beneficial effects of
CB1 antagonism on fatty liver in mice (Matias et al., 2006; Pacher
et al., 2006; Vettor and Pagano, 2009; Jourdan et al., 2010; Quarta
et al., 2011; Alswat, 2013; Mallat et al., 2013) and humans (Howlett,
2002; Engeli et al., 2005; Di Marzo, 2008; Mallat et al., 2013;
Romero-Zerbo and Bermúdez-Silva, 2014). Furthermore, these
published data strongly indicate that ECS overactivity has a major
role in the regulation of lipid metabolism not only at the central but
also at the peripheral level (Howlett, 2002; Vettor and Pagano,
2009; Romero-Zerbo and Bermúdez-Silva, 2014). This overactivity
occurs at the level of both the hypothalamus and peripheral
tissues, including the liver, pancreas and epididymal adipose tissue
in animals fed with a HFD and in the visceral fat and blood of obese
patients (Howlett, 2002; Monteleone et al., 2005; Osei-Hyiaman
et al., 2005; Kim et al., 2011; Alswat, 2013). In relation to, more and
more evidences show that HFD-induced steatosis resulting from
increased FA synthesis is mediated via ANA-induced CB1 receptor
activation (Starowicz et al., 2008; Westerbacka et al., 2010; Mallat
et al., 2013; Romero-Zerbo and Bermúdez-Silva, 2014; Shi et al.,
2014; Jorga9 cevi
c et al., 2015). These effects are blocked or
prevented by CB1 antagonist (Osei-Hyiaman et al., 2005; Osei
Hyiaman et al., 2008; Vettor and Pagano, 2009; Shi et al., 2014).
Similar to our findings, evidence from in vivo studies with the
CB1 antagonist rimonabant in obese, overweight and diabetic
patients indicates that blocking CB1 receptor favourably modifies
the lipid profile (Van Gaal et al., 2005; Pi-Sunyer et al., 2006;
Scheen et al., 2006; Després et al., 2009; Vettor and Pagano, 2009).
Since CB1 antagonist rimonabant is involved in improving
dyslipidemia and reducing liver fat deposits, it has been hypothe-
sized recently that peripheral CB1 receptors could be selectively
targeted for the treatment of fatty liver and dyslipidemia (Cheung
et al., 2013).
Keeping in mind that elevated plasma levels of FFA can result in
increased intracellular TG synthesis and formation of lipid droplets
(Musso et al., 2009; Yasutake et al., 2009; Min et al., 2012; Leamy
et al., 2013; Savard et al., 2013; Fuchs et al., 2014; Stankovic et al.,
2014), our study is the first that determined FFA profile in high
saturated fat diet model of NAFLD. The results of present
investigation found a significant increase in hepatic saturated
acid proportion (palmitic and stearic acid) in HFD fed mice
compared to control group (Fig. 5). These findings correspond to
91
the well known hepatotoxic effect of SFAs and may aggravate liver
injury caused by HFD (Fig. 6C) (Puri et al., 2007; Leamy et al., 2013;
Fuchs et al., 2014; Stankovic et al., 2014; Pardo et al., 2015). Studies
on patients with NAFLD suggested that increase in plasma FFA
concentration is the primary source for TG synthesis in hepato-
cytes (Puri et al., 2007; Stankovic et al., 2014). Within TG and DAG,
the saturated palmitic acid was elevated, reflecting, perhaps, its
greater availability in the circulating FFA pool (Puri et al., 2007;
Trauner et al., 2010; Leamy et al., 2013). Additionally, several
putative mechanisms including the accumulation of reactive
oxygen species (ROS), ER stress and increased ceramide synthesis
have been hypothesized to explain how SFAs initiate hepatocellu-
lar injury (Jung et al., 2016; Povero and Feldstein, 2016; Ress and
Kaser, 2016).
The result of the present study was found a significant decline in
liver palmitic and stearic acid proportion in both group treated
with rimonabant (Fig. 5). This effect of rimonabant was associated
with reduction of inflammation (Fig. 6D). Besides, this finding is in
accordance with higher level of ANA and upregulation of CB1
receptor in HFD-induced NAFLD (Vettor and Pagano, 2009).In
present study, HFD caused a significant increase in oleic acid
proportion compared to control (Fig. 5). This finding is in
agreement with reports demonstrating a link between abnormal
plasma accumulation of oleic acid and pathohistological severity of
NASH (Malhi and Gores, 2008; Stankovi c et al., 2014; Povero and
Feldstein, 2016). However, it is not completely clear how elevated
concentration of oleic acid in liver with steatohepatitis interacts to
expand hepatic lipid stores, causes hepatocellular injury and
recruits inflammation. Our study indicates that rimonabant caused
a significant decrease in liver oleic acid proportion in R group
compared to control, as well as in HFR group in comparison with
HF group (Fig. 5). Possible explanation for this result refers to
antiinflammatory and cytoprotective effects of CB1 pharmacolog-
ical inhibition or genetic deletion observed in the numerous
preclinical and clinical reports (Di Marzo, 2008; Vettor and Pagano,
2009; Jourdan et al., 2010; Kim et al., 2011; Alswat, 2013; Mallat
et al., 2013).
It is important to note that the inflammatory response induced
in the liver of NAFLD patients may also be related to LCPUFA
depletion (El-Badry et al., 2007; Neuschwander-Tetri, 2010).
Besides, low plasma levels of LCPUFA is reported in patients with
end-stage liver disease or in cirrhotic patients with hepatitis B and
C (El-Badry et al., 2007). Contrary to these reports, in current study
linoleic acid proportion was significantly higher in HFD fed mice
compared to group fed with control chow diet (Fig. 5). This
discrepancy between our study and other similar investigations in
humans may be a consequence of HFD model that requires a longer
time period to develop depletion of linoleic acid concentration in
liver. Additionally, in current research liver linoleic acid proportion
in both rimonabant treated groups of mice was significantly
increased compared to control values and mice fed on HFD (Fig. 5).
This finding suggests that the pharmacological CB1 inactivation in
hepatocytes in some manner may contribute to protective effects
of linoleic acid.
Arachidonic acid are biologically active even in small quantities
(Puri et al., 2007; Fuchs et al., 2014). This essential FA is an
important component of all membranes and influences membrane
fluidity and the behaviour of membrane-bound enzymes and
receptors (El-Badry et al., 2007). However, the arachidonic acid
may be utilized for the formation of various eicosanoids, which
then in high concentrations contribute to development of fatty
liver, inflammatory disorders, blood viscosity, vasospasm and
vasoconstriction (Malhi and Gores, 2008; Neuschwander-Tetri,
2010; Pinzani, 2011; Ress and Kaser, 2016). In our study significant
decrease in arachidonic acid proportion was observed in liver of
HFD fed mice in comparison with control group (Fig. 5). This
92 cevic et al. / Chemistry and Physics of Lipids 204 (2017) 85–93
B. Jorga9
decline is in agreement with published data that indicate the
ability of the arachidonic acid-derived eicosanoids to impair
hepatic lipid homeostasis (El-Badry et al., 2007; Malhi and Gores,
2008; Neuschwander-Tetri, 2010; Westerbacka et al., 2010;
Pinzani, 2011; Ress and Kaser, 2016). In contrast, result in our
study showed that rimonabant treatment induced a significant
increase in arachidonic acid proportion in HFR group in compari-
son with HF group (Fig. 5). This finding put into attention CB1
receptor blockade as a potential contributor to the amelioration of
hepatic lipid metabolism disturbances.
5. Conclusions
To date, no effective therapy has been proposed for patients
with NAFLD. To our knowledge, this is the first study that
investigates FFA profile in high saturated fat diet model of NAFLD.
Based on differing mechanisms of NAFLD/NASH development in
humans and rodents, HFD model can not completely reflect all
relevant histopathological and pathophysiological characteristics
associated with human NAFLD/NASH. According to our results, it
can be noted the potential usefulness of CB1 blockade in the
treatment of HFD-induced NAFLD, particularly due to modulation
of plasma lipid and hepatic FFA profile and improvement of liver
histology. However, further investigations regarding precise
mechanisms by which HFD stimulate ECS production in the liver
are required before moving toward clinical phase of investigation.
Funding
This research was financially supported by the Ministry of
Education, Science and Technological Development of Republic of
Serbia, Grant no. 175015.
Conflict of interest
The authors declare no conflicts of interest.
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