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Effects of Rosuvastatin on the expression of the genes involved in cholesterol metabolism in rats: adaptive responses by extrahepatic tissues
Accepted Manuscript Effects of Rosuvastatin on the expression of the genes involved in cholesterol metabolism in rat: Adaptive responses by extrahepatic tissues
Yasin Ahmadi, Amir Ghorbani Haghjoo, Siavoush Dastmalchi, Mahboob Nemati, Nasrin Bargahi PII: DOI: Reference:
S0378-1119(18)30343-3 doi:10.1016/j.gene.2018.03.092 GENE 42714
To appear in:
Gene
Received date: Revised date: Accepted date:
14 February 2018 25 March 2018 28 March 2018
Please cite this article as: Yasin Ahmadi, Amir Ghorbani Haghjoo, Siavoush Dastmalchi, Mahboob Nemati, Nasrin Bargahi , Effects of Rosuvastatin on the expression of the genes involved in cholesterol metabolism in rat: Adaptive responses by extrahepatic tissues. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2018.03.092
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ACCEPTED MANUSCRIPT Effects of Rosuvastatin on the Expression of the Genes Involved in Cholesterol Metabolism in Rat: Adaptive Responses by Extrahepatic Tissues
Yasin Ahmadi* a, b, Amir Ghorbani Haghjoo* b, Siavoush Dastmalchi b, Mahboob Nemati c,
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Nasrin Bargahi a
Tabriz University of Medical Sciences, Student Research Committee, Tabriz, Iran
b
Tabriz University of Medical Sciences, Biotechnology Research Center, Tabriz, Iran
c
Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
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a
*
Co-corresponding authors. Phone:
00984134426078;
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[email protected];
Fax:
00984133364666
[email protected], [email protected]; phone: 00989187227695;
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Fax: 00984133363666
ACCEPTED MANUSCRIPT Abstract Background: Statins mostly target the liver; therefore, increase in the synthesis of cholesterol by extra-hepatic tissues and then transferring this cholesterol to the liver can be regarded as adaptive responses by these tissues. In addition to cholesterol, these adaptive responses can increase isoprenoid units as byproducts of the cholesterol
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biosynthesis pathway; isoprenoids play a key role in regulating cell signaling pathways
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and cancer development. Thus, there is a primary need for in vivo investigation of the
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effects of statins on the cholesterol metabolism in the extra-hepatic tissues. Materials: Eighteen male Sprague-Dawley rats were randomly divided into control (n=9)
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and treatment (n=9) groups. The treatment group was orally given 10mg/kg/day of Rosuvastatin for 6 weeks. Then, serum lipid profile, expression levels of 3-hydroxy-3methyl-glutaryl-coenzyme A reductase (HMGCR), ABCA1, ABCG1 and ApoA1, and
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activity of HMGCR were measured in the liver, intestine and adipose tissues. Results: Rosuvastatin significantly reduced total cholesterol and LDL-C. The
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expression levels of ABCA1, ABCG1, and ApoA1 in the liver and HMGCR in both liver
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and intestine were significantly increased in the Rosuvastatin treated-group. However, in the intestine, there were no significant differences in the expression levels of ABCA1
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and ABCG1 between the study groups. Rosuvastatin had no effect on the adipose tissue.
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The HMGCR activity was significantly increased in the liver and intestine of the Rosuvastatin-treated group.
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Conclusions: In spite of the adipose tissue, the intestine efficiently responses to the reduced levels of cholesterol and increases its cholesterogenesis capacity. However, adipose tissue seems to play a small role in correcting cholesterol deficiency during the course of statin therapy. Keywords: Adipose tissue; Intestine; Isoprenoids; Liver; Statin
ACCEPTED MANUSCRIPT 1. Introduction: Not only dietary cholesterol is directly transported to the liver, but also hepatocytes synthesize approximately 60-70% of the whole body cholesterol [1, 2]; hence, the liver is the primary target of the cholesterol-lowering drugs, statins [2, 3]. Statins via competitive inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) -
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the main enzyme of the cholesterol biosynthesis pathway - reduce the whole body
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cholesterol [3, 4].
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Regarding the vital roles of cholesterol in the liver, correcting of the cholesterol deficiency seems to be vital for maintaining the physiological function of the liver
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during the course of statin treatment.
The induction of reverse cholesterol transport (RCT) process, responsible for
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transferring excess cholesterol from the extrahepatic tissues to the liver, in addition to the increase in the cholesterol absorption in the intestine can be regarded as the main
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mechanism underlying cholesterol providing during the course of statin therapy. Peripheral cells (apart from steroidogenic and skin cells) cannot degrade cholesterol;
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thus, the excess amount of cholesterol must be exported to the liver [5]. Most studies have shown that statins in the liver induce different components of the RCT process,
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including ATP binding cassette subfamily A member 1 (ABCA1), ATP binding cassette
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subfamily G member 1 (ABCG1) and apolipoprotein A1 (ApoA1) [6-8]. ApoA1 is the major apolipoprotein of the high-density lipoprotein (HDL) particles which
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is produced by the liver and intestine. ABCA1 plays the major role in cholesterol loading of the newly-synthesized ApoA1 or nascent-HDL. Nascent-HDLs take further cholesterol by other transporters such as ABCG1 and aqueous diffusion and finally transfer it to the liver [8]. Only after the liver, the intestine produces the highest amount of cholesterol in the body [9]. Adipose tissue is the major pool of free cholesterol in obesity [10]. Thus, these tissues can play an outstanding role in supplying cholesterol.
ACCEPTED MANUSCRIPT Generally, statins are classified into hydrophilic and lipophilic groups. Hydrophilic statins are mostly taken up by hepatocytes through organic anion-transporting polypeptides (OATPs), particularly, OATP1B1 which is exclusively expressed in the hepatocytes [11]. Other types of OATPs such as OATP1B3 and OATP2B1 are responsible for transporting different statins into the extrahepatic cells [12].
[13].
Most
of
these
effects
arise
from
blocking
of
the
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pleiotropic effects
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Statins induce several other effects beyond their lipid-lowering effect known as
mevalonate/isoprenoid pathway, which is vital for prenylation and then tethering various
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intracellular signaling proteins to the cell membrane, which is essential for their biological activity [4, 13]. Farnesyl pyrophosphate (FPP) and geranylgeranyl
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pyrophosphate (GGPP) are the major isoprenoids involved in the prenylation of different proteins [14].
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The compensatory induction of Hydroxy-Methyl-Glutaryl-CoA-Reductase (HMGCR) not only increases the cholesterol synthesis but also upregulates mevalonate/isoprenoid
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pathway, which in turn initiate the cell signaling cascades in the extrahepatic tissues.
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Rosuvastatin (Crestor) is the most recent produced statin and the first statin approved by the regulatory authorities since the withdrawal of cerivastatin (2001) [15].
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Regarding the high hydrophobicity, Rosuvastatin is mostly taken up by hepatocytes,
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and therefore partially diffuses into extrahepatic tissues [16]. It should be mentioned that most studies have been conducted in cell cultures and liver
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as the major target of statins have not existed. Thus, there is a primary need for in vivo investigation of the effects of statins on the extra-hepatic tissues. In this study, we aimed to investigate the effects of Rosuvastatin on the HMGCR expression as well as its enzymatic activity in the liver, intestine and adipose tissues -as the major sites for cholesterol biosynthesis and cholesterol storage- in rats with the normal diet. The normal diet was used to prevent correcting of the cholesterol deficiency by diet cholesterol, and so the roles of the different tissues in this regard be investigated.
ACCEPTED MANUSCRIPT Moreover, we measured the expression levels of ABCA1 and ABCG1 (as the major transporters responsible for cholesterol efflux), and ApoA1 (as the key apolipoprotein of HDL particles which are the major regulators of the RCT). 2. Material and Methods: 2.1. Animals
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Eighteen male Sprague-Dawley rats weighing 220 to 280 g were purchased from
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Pasteur Institute (Tehran, Iran). All procedures were followed according to the National Institute of Health guide for the care and use of Laboratory Animals (NIH Publications
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No. 8023, revised 1978) and local guidelines for compassionate use of animals in research; two rats were kept per cage with free access to tap water and compact
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standard chow. The animals were kept in the same laboratory conditions (18°C to 23°C room temperature and controlled humidity) with alternating 12-h light and dark cycles.
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Animals were randomly divided into control (n-9) and Rosuvastatin (n-9) groups. Rosuvastatin group was treated with 10mg/kg/day Rosuvastatin (calcium salt,
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AstraZeneca) freshly prepared in 0.5% carboxymethyl cellulose as the vehicle [17, 18],
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and the control group only received the vehicle. The administration period was six weeks. The conditions of the administrations were based on previous studies [19, 20].
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At the end of the treatment course, all the rats were anesthetized by a single dose (100 mg/kg) intraperitoneal injection of ketamine. Blood samples were collected by cardiac
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puncture. Serum samples were prepared and kept frozen at −20 °C until analysis. Then, using the Ketamine lethal dose (200 mg/kg), all the rats were killed. Liver of the
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rats was excised, weighed and plunged into liquid nitrogen and stored at -80°C. The small intestine was excised and the luminal contents were washed out with cold phosphate buffered saline (PBS). The ileal segments (20 cm proximal from the ileocecal valve) were dissected, the mucosa was scraped off, immediately plunged into liquid nitrogen and stored at -80°C. The adipose tissue samples (from the left inguinal fat pad) were dissected and washed with PBS, plunged into liquid nitrogen and then stored at 80°C. 2.2. Biochemical Parameters
ACCEPTED MANUSCRIPT The serum lipid profile, including total cholesterol, triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) and very low-density lipoprotein (VLDL) was measured by a colorimetric method using commercial reagents in an automated chemical analyzer (Roche Cobas Mira, Basel, Switzerland).
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2.3. Gene Expression analyses Total RNA of the liver, intestine and adipose tissues was extracted by RNeasy Mini Kit Hilden,
Germany)
according to
the
manufacturer’s
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(Qiagen,
protocol. Then,
complementary DNA (cDNA) was synthesized using 1 μg of the total RNA with Oligo
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(dT) primers and the RevertAid first-strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The quantitative real-time polymerase chain
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reaction (QPCR) was performed in duplicate using SYBR Premix Ex Taq II (Takara Bio Inc., Japan) on the Rotor-Gene 6000 real-time PCR detection system (Corbett
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Research, Sydney, Australia). Results were normalized according to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. Table 1
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shows the used specific primer-sequences.
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Table 1. the primer sequences Primer Forward Reverse HMGCR 5’-ACCGTGGGTGGTGGGAC-3’ 5’-GCCCCTTGAACACCTAGCATC-3’ ABCA1 5’-ACGAGATTGATGACCGCCTC-3’ 5’-GCATCCACCCCACTCTCTTC-3’ ABCG1 5’-AGGTCTCAGTCTAAAGTTCCTC-3’ 5’-TCTCTCGAAGTGAATGAAATTTATCG-3’ ApoA1 5’-CACCGAGCTTCACAAAAA CG-3’ 5’-TGATCGCTGTAGAGCCCAAA-3’ GAPDH 5’-GTCTCCTGCGACTTCAACA-3’ 5’-TCATTGTCATACCAGGAAATGAGC-3’ HMGCR: 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; ABCA1: ATP-binding cassette transporter subfamily A member 1; ABCG1: ATP-binding cassette transporter subfamily G member 1; ApoA1: apolipoprotein A1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase
PCR program for HMGCR was run as a preincubation step at 94°C for 10 min, followed by 40 cycles of denaturation (95°C, 10 s), annealing (62°C, 30 s) and extension (72°C, 30 s) steps with the final extension step at 72˚C for 10 min; the ApoA1 and ABCA1 run program included a preincubation step at 94°C for 10 min, followed by 40 cycles of denaturation (95°C, 10 s), annealing (64°C, 30 s) and extension (72°C, 30 s) steps with the final extension step at 72˚C for 10 min; the ABCG1 run program was as a preincubation step at 94°C for 10 min, followed by 40 cycles of denaturation (95°C, 10
ACCEPTED MANUSCRIPT s), annealing (63°C, 22 s), and extension (72°C, 15 s) steps with the final extension step at 72˚C for 10 min. The 2−ΔΔCt method was used to calculate the relative changes in the gene expression. HMGCR Activity Assay
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Microsomes were isolated using Microsome Isolation Kit (Biovision Inc., Milpitas, CA,
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USA) according to the manufacturer’s instructions. Then, the activity of HMGCR for isolated microsomes was measured according to the instructions of the HMGCR Activity
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Assay Kit (K588-100; BioVison Inc.).
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2.4. Statistical Analysis
Data are shown as mean ± SD. Statistical comparisons of the groups were performed
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by the non-parametric Mann-Whitney test. A p value of less than 0.05 was considered
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statistically significant. The analyses were performed in SPSS 16.0 software. 3. Results:
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3.1. Rosuvastatin Effect on the Serum Lipid Profile Rosuvastatin significantly (p<0.05) reduced the serum levels of total cholesterol and
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LDL-C by 20.07% and 17.47% respectively. The serum levels of HDL-C, TG and VLDL were insignificantly (p>0.05) decreased in the Rosuvastatin-treated group (Fig.1). Fig. 1. The effect of Rosuvastatin on the serum lipid profile. In the Rosuvastatin-treated group, the serum concentrations of total cholesterol (52.6 ± 3.43 µg/dl) and LDL-C (16.72 ± 1.28 µg/dl) were significantly (p<0.05) reduced compared to those in the control group (total cholesterol (65.81 ± 3.45 µg/dl) and LDL-C (20.26 ±0.91 µg/dl). There were not seen significant differences (p>0.05) in the serum concentrations of TG (18.8 ± 2.14 µg/dl), HDL-C (30.80 ± 1.68 µg/dl), and VLDL (3.97±0.50) in the Rosuvastatin-treated group versus those in the control group; TG (21.09 ± 2.66 µg/dl), HDL-C (28.45 ± 3.17 µg/dl), VLDL (4.19±0.89).
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3.2. Results of the Gene Expression Analyses 3.2.1. Relative Expression of HMGCR, ABCA1, ABCG1, and ApoA1 in the Liver The results of the QPCR in the liver revealed that the expression of HMGCR, ABCA1,
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ABCG1, and ApoA1 was significantly (p<0.05) upregulated in the Rosuvastatin group
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compared to the control group (Fig. 2).
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Fig. 2. Relative expression of the genes in the liver. There was observed a significant (p<0.05) elevation in the expression levels of ABCG1 (2.85±1.21 fold),
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ABCA1 (3.96±1.81 fold), ApoA1 (8.87±4.68 fold) and HMGCR (8.09±3.83 fold) in the Rosuvastatin-treated group compared to those in the control group including
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ABCG1 (1.14±0.50 fold), ABCA1 (1.06±0.49 fold),
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ApoA1 (1.07±0.46 fold), and HMGCR (1.27±0.62 fold).
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3.2.2. Relative Expression of HMGCR, ABCA1, and ABCG1 in the Intestine The results of the gene expression assay in the intestine indicated that the expression
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of HMGCR was significantly (p<0.05) higher in the Rosuvastatin-treated group;
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however, there were not significant (p>0.05) differences in the expression level of ABCA1
and
ABCG1
between
the
Rosuvastatin and control groups (Fig. 3). Fig. 3. Relative expression of the genes in the intestine. There was shown a significant (p<0.05) increase
in
the
expression
level
of
HMGCR
(3.10±1.89 fold) in the Rosuvastatin group versus that in the control group (0.94±0.52 fold). However, there were not observed significant (p>0.05) differences in the expression levels of ABCA1 (1.18±0.93 fold) and ABCG1 (1.37±0.81) in the treatment group compared
ACCEPTED MANUSCRIPT to the control group (ABCA1 (0.93±0.41 fold) and ABCG1 (1.09±0.48 fold).
3.2.3. Relative Expression of HMGCR, ABCA1, and ABCG1 in the Adipose Tissue The results of the QPCR in the adipose tissue showed insignificant (p>0.05) differences
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in the expression levels of ABCA1, ABCG1 and HMGCR between the study groups
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(Fig. 4).
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Fig. 4. Relative expression of the genes in the adipose tissue. The QPCR results showed an
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insignificant (p>0.05) change in the expression levels of ABCG1 (0.91±0.55 fold), ABCA1 (0.98±0.67 fold), and HMGCR (0.89±0.33 fold) in the Rosuvastatin-
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treated group compared to those in the control group; ABCG1 (1.48±1.39 fold), ABCA1 (1.21±0.84 fold) and
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HMGCR (1.00±0.53 fold).
3.3. The HMGCR activity in the liver, intestine and adipose tissues The results of the HMGCR activity assay in the Rosuvastatin-treated group showed a
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significant (p<0.05) increase in the activity of HMGCR in the liver and intestine by
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57.14% and 30.76% respectively. However, in the adipose tissue, there was not observed a significant (p>0.05) difference between the study groups (see Fig. 5). Fig. 5. The HMGCR activity in different tissues. The HMGCR activity in the liver and intestine of the Rosuvastatin-treated group was significantly (p<0.05) higher (0.022±0.003 and 0.017±.003 units/mg protein respectively)
than
those
(0.014±0.002
and
respectively).
However,
in
the
0.013±.003 there
control
units/mg was
not
group protein
seen
a
significant (p>0.05) difference in the HMGCR activity between (.0085±.003 and .0081±0.002 units/mg protein respectively).
the
Rosuvastatin
and
control
groups
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4. Discussion: Different transcription factors, including liver X receptors (LXRs), peroxisome proliferative activated receptors (PPARs) and sterol regulatory element binding transcription (SREBPs) are responsible for regulating the expression of the genes
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involved in the lipid metabolism [21]. LXRα is known as the whole-body cholesterol
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sensor, regulating expression of the genes involved in the cholesterol efflux such as
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ABCA1 and ABCG1 [22]. High level of cholesterol causes an increase in the generation of the oxidized forms of cholesterol (oxysterols ) which serve as the agonist ligands of
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LXR [11].
Statins through a biphasic mechanism regulate the transcriptional activity of LXR in the
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liver: first, suppression of LXR activity via reducing the oxysterols; then, induction of the transcriptional activity of LXR through reducing GGpp [23]; statins by inhibiting prenylation and then activating RhoA (a small GTPase protein) prevent the inhibitory
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phosphorylation of PPAR-γ; PPAR- γ upregulates the expression of LXR. Also, GGPP
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can directly antagonize activity of LXR [6].
SREBP-2 is another nuclear receptor involved in the metabolism of sterol. Cholesterol
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deficiency causes the proteolytic activation of SREBP-2 [24] that upregulates genes
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involved in the cholesterol biosynthesis and cholesterol efflux [8, 25] In addition to the nuclear receptors, miR-33 which is located within introns of the
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SREBP-2 gene downregulates the expression of ABCA1 and ABCG1 [26, 27]. Therefore, the effects of statins on the genes responsible for cholesterol effluxing are dependent on the balance between the inducing mechanisms (LXR activation) and inhibitory mechanisms (miR-33). The results of the present study showed that Rosuvastatin in the liver increased the activity of HMGCR and upregulated the expression of HMGCR, ABCA1, ABCG1, and ApoA1. These findings may indicate the adaptive responses by the liver. Consistent with these findings, various studies demonstrated that statins could increase the expression level of HMGCR [28], ApoA1 [29], ABCA1 [30] and ABCG1[31] in the liver or
ACCEPTED MANUSCRIPT HepG2 cells. However, several other studies showed that statins through upregulating SREBP-2 and then miR-33 downregulate the expression of ABCA1 and ABCG1 in the liver [27, 32]. In the intestine, Rosuvastatin increased both the mRNA level and activity of HMGCR. Rosuvastatin through direct inhibiting cholesterol synthesis in the liver may reduce the
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cholesterol levels in the intestine and then induce the compensatory upregulation of
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HMGCR. In spite of our findings, Harshman et al. showed that atorvastatin did not affect
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the expression of HMGCR in the intestine of C57Bl6 Male Mice [33].
The results of this study did not show a significant difference in the intestinal expression
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of ABCA1 and ABCG1 between the study groups.
In the extrahepatic tissues, ABCA1 has been shown to be differently regulated;
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Tamehiro et al. showed that Pravastatin reduced both mRNA and protein levels of ABCA1 in the intestine; however, increased them in the liver [34]. Moreover, it was
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shown that statins could reduce the mRNA levels of ABCA1 and ABCG1 in Caco-2 cells (heterogeneous human epithelial colorectal adenocarcinoma), respectively [35]. These
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findings may indicate that statins cannot induce the intestine cholesterol efflux via these protein transporters.
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Adipocytes are unique for their large capacity of the cholesterol storage, these cells include 25% of the whole-body cholesterol. However, adipocytes have a low capacity of
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cholesterogenesis, less than 10% of the systemic cholesterol production [36].
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There is a dynamic equilibrium between the serum lipoprotein-cholesterol and adipocyte free-cholesterol (FC) pools. Thus, the adipose tissue has been suggested to play a “buffer” role for the serum-cholesterol [37]. In spite of the essential role of the adipose tissue in the cholesterol metabolism, the effects of statins on the adipose tissue have not been widely disclosed. The results of this study showed that Rosuvastatin had no effects on the cholesterol metabolism in the adipose tissue. Regarding the role of the cellular cholesterol load in regulating the transcriptional activity of different factors such as LXR and SREBP-2, the
ACCEPTED MANUSCRIPT high amount of cholesterol in the adipocytes would keep the constant expression levels of the genes involved in the cholesterol metabolism during the course of the statin treatment. Overall, our findings revealed that in response to the reduced level of cholesterol by Rosuvastatin, liver, and intestine increase their cholesterol synthesis capacity. The
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adaptive response by the liver was expected because it is the primary target of statins.
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However, the intestinal response to the reduced level of cholesterol indicates the vital role of this organ in maintaining cholesterol homeostasis. It was shown that the
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activation of intestinal SREBP2 alone seems to be sufficient to increase plasma cholesterol, highlighting the essential role of the intestine in cholesterol homeostasis in
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the body [38].
Statins, in addition to the metabolism of cholesterol, play an important role in the
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regulation of the cellular signaling pathways and it depends on the effect of statins on the synthesis of isoprenoids. Theatrically, the adaptive response by the intestine
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increases isoprenoids including GGpp and FPP, and then initiates the cell signaling
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cascades, and increases the risk of cancer development. However, most of the population-based studies showed that statins had no effects on the risk of
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gastrointestinal cancers [39, 40].
The small sample size and the use of laboratory animals were limitations of this study.
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On the other hand, measuring cholesterol synthesis in-vivo condition is a more reliable method to evaluate the adaptive responses by different tissues to the reduced level of
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cholesterol after administration statins. Also, measuring of the isoprenoids in the extrahepatic tissues can exactly clarify the effects of statins on the cellular signaling pathways and cancer development. 5. Conclusion: The intestine, in response to the reduced level of the cholesterol synthesis in the liver, enhances its cholesterogenesis capacity. However, Rosuvastatin does not affect the cholesterol efflux through protein transporters of ABCA1 and ABCG1. Therefore, other cholesterol transmission pathways such as aqueous diffusion are likely to be
ACCEPTED MANUSCRIPT responsible for effluxing the newly-synthesized cholesterol from the extra-hepatic tissues to the HDL particles. Thus, the intestine may play the major role in compensating for the reduced level of cholesterol during the statin treatment course. Rosuvastatin does not affect the adipose tissue. Thus, adipose tissue is not likely to play an important role in compensating for the reduced level of cholesterol in the body.
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6. Declarations of Interest: none
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7. Acknowledgments: we would like to express my special thanks to everyone who
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supported us during the course of this project.
8. Funding: This work was supported by Biotechnology Research Center at Tabriz
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University of Medical Sciences (grant number: 5/126558).
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Genvigir, F.D., A.C. Rodrigues, A. Cerda, M.H. Hirata, R. Curi, and R.D. Hirata, ABCA1 and ABCG1 expressions are regulated by statins and ezetimibe in Caco-2 cells. Drug metabolism and drug interactions, 2011. 26(1): p. 33-36.
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40.
ACCEPTED MANUSCRIPT Abbreviations list: ABCA1: ATP binding cassette subfamily A member 1 ABCG1: ATP binding cassette subfamily G member 1 ApoA1: apolipoprotein A1 HMGCR: 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase
PPAR: peroxisome proliferator activated receptor
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SREBP: sterol regulatory element binding transcription
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miR-33: microRNA 33
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LXR: liver X receptors
ACCEPTED MANUSCRIPT Highlights:
The intestine, efficiently responses to the reduced level of cholesterol by statin (Rosuvastatin) and upregulates its expression of HMGCR to compensate for cholesterol deficiency in the liver.
The adipose tissue as the main cholesterol storage in the body, does not play a significant role in correcting cholesterol deficiency in the liver of the rats treated with Rosuvastatin.
The liver, as the main target of statins, efficiently responses to Rosuvastatin and upregulates expression of the genes involved in the cholesterol synthesis and cholesterol effluxing.
Regarding the targeting of the liver by Rosuvastatin, it is possible that the increased-cholesterogenesis in the intestine also increases isoprenoids which are the byproducts of the cholesterol synthesis pathways
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Yasin Ahmadi, Amir Ghorbani Haghjoo, Siavoush Dastmalchi, Mahboob Nemati, Nasrin Bargahi PII: DOI: Reference:
S0378-1119(18)30343-3 doi:10.1016/j.gene.2018.03.092 GENE 42714
To appear in:
Gene
Received date: Revised date: Accepted date:
14 February 2018 25 March 2018 28 March 2018
Please cite this article as: Yasin Ahmadi, Amir Ghorbani Haghjoo, Siavoush Dastmalchi, Mahboob Nemati, Nasrin Bargahi , Effects of Rosuvastatin on the expression of the genes involved in cholesterol metabolism in rat: Adaptive responses by extrahepatic tissues. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2018.03.092
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ACCEPTED MANUSCRIPT Effects of Rosuvastatin on the Expression of the Genes Involved in Cholesterol Metabolism in Rat: Adaptive Responses by Extrahepatic Tissues
Yasin Ahmadi* a, b, Amir Ghorbani Haghjoo* b, Siavoush Dastmalchi b, Mahboob Nemati c,
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Nasrin Bargahi a
Tabriz University of Medical Sciences, Student Research Committee, Tabriz, Iran
b
Tabriz University of Medical Sciences, Biotechnology Research Center, Tabriz, Iran
c
Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
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a
*
Co-corresponding authors. Phone:
00984134426078;
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[email protected];
Fax:
00984133364666
[email protected], [email protected]; phone: 00989187227695;
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Fax: 00984133363666
ACCEPTED MANUSCRIPT Abstract Background: Statins mostly target the liver; therefore, increase in the synthesis of cholesterol by extra-hepatic tissues and then transferring this cholesterol to the liver can be regarded as adaptive responses by these tissues. In addition to cholesterol, these adaptive responses can increase isoprenoid units as byproducts of the cholesterol
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biosynthesis pathway; isoprenoids play a key role in regulating cell signaling pathways
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and cancer development. Thus, there is a primary need for in vivo investigation of the
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effects of statins on the cholesterol metabolism in the extra-hepatic tissues. Materials: Eighteen male Sprague-Dawley rats were randomly divided into control (n=9)
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and treatment (n=9) groups. The treatment group was orally given 10mg/kg/day of Rosuvastatin for 6 weeks. Then, serum lipid profile, expression levels of 3-hydroxy-3methyl-glutaryl-coenzyme A reductase (HMGCR), ABCA1, ABCG1 and ApoA1, and
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activity of HMGCR were measured in the liver, intestine and adipose tissues. Results: Rosuvastatin significantly reduced total cholesterol and LDL-C. The
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expression levels of ABCA1, ABCG1, and ApoA1 in the liver and HMGCR in both liver
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and intestine were significantly increased in the Rosuvastatin treated-group. However, in the intestine, there were no significant differences in the expression levels of ABCA1
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and ABCG1 between the study groups. Rosuvastatin had no effect on the adipose tissue.
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The HMGCR activity was significantly increased in the liver and intestine of the Rosuvastatin-treated group.
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Conclusions: In spite of the adipose tissue, the intestine efficiently responses to the reduced levels of cholesterol and increases its cholesterogenesis capacity. However, adipose tissue seems to play a small role in correcting cholesterol deficiency during the course of statin therapy. Keywords: Adipose tissue; Intestine; Isoprenoids; Liver; Statin
ACCEPTED MANUSCRIPT 1. Introduction: Not only dietary cholesterol is directly transported to the liver, but also hepatocytes synthesize approximately 60-70% of the whole body cholesterol [1, 2]; hence, the liver is the primary target of the cholesterol-lowering drugs, statins [2, 3]. Statins via competitive inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) -
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the main enzyme of the cholesterol biosynthesis pathway - reduce the whole body
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cholesterol [3, 4].
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Regarding the vital roles of cholesterol in the liver, correcting of the cholesterol deficiency seems to be vital for maintaining the physiological function of the liver
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during the course of statin treatment.
The induction of reverse cholesterol transport (RCT) process, responsible for
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transferring excess cholesterol from the extrahepatic tissues to the liver, in addition to the increase in the cholesterol absorption in the intestine can be regarded as the main
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mechanism underlying cholesterol providing during the course of statin therapy. Peripheral cells (apart from steroidogenic and skin cells) cannot degrade cholesterol;
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thus, the excess amount of cholesterol must be exported to the liver [5]. Most studies have shown that statins in the liver induce different components of the RCT process,
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including ATP binding cassette subfamily A member 1 (ABCA1), ATP binding cassette
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subfamily G member 1 (ABCG1) and apolipoprotein A1 (ApoA1) [6-8]. ApoA1 is the major apolipoprotein of the high-density lipoprotein (HDL) particles which
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is produced by the liver and intestine. ABCA1 plays the major role in cholesterol loading of the newly-synthesized ApoA1 or nascent-HDL. Nascent-HDLs take further cholesterol by other transporters such as ABCG1 and aqueous diffusion and finally transfer it to the liver [8]. Only after the liver, the intestine produces the highest amount of cholesterol in the body [9]. Adipose tissue is the major pool of free cholesterol in obesity [10]. Thus, these tissues can play an outstanding role in supplying cholesterol.
ACCEPTED MANUSCRIPT Generally, statins are classified into hydrophilic and lipophilic groups. Hydrophilic statins are mostly taken up by hepatocytes through organic anion-transporting polypeptides (OATPs), particularly, OATP1B1 which is exclusively expressed in the hepatocytes [11]. Other types of OATPs such as OATP1B3 and OATP2B1 are responsible for transporting different statins into the extrahepatic cells [12].
[13].
Most
of
these
effects
arise
from
blocking
of
the
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pleiotropic effects
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Statins induce several other effects beyond their lipid-lowering effect known as
mevalonate/isoprenoid pathway, which is vital for prenylation and then tethering various
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intracellular signaling proteins to the cell membrane, which is essential for their biological activity [4, 13]. Farnesyl pyrophosphate (FPP) and geranylgeranyl
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pyrophosphate (GGPP) are the major isoprenoids involved in the prenylation of different proteins [14].
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The compensatory induction of Hydroxy-Methyl-Glutaryl-CoA-Reductase (HMGCR) not only increases the cholesterol synthesis but also upregulates mevalonate/isoprenoid
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pathway, which in turn initiate the cell signaling cascades in the extrahepatic tissues.
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Rosuvastatin (Crestor) is the most recent produced statin and the first statin approved by the regulatory authorities since the withdrawal of cerivastatin (2001) [15].
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Regarding the high hydrophobicity, Rosuvastatin is mostly taken up by hepatocytes,
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and therefore partially diffuses into extrahepatic tissues [16]. It should be mentioned that most studies have been conducted in cell cultures and liver
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as the major target of statins have not existed. Thus, there is a primary need for in vivo investigation of the effects of statins on the extra-hepatic tissues. In this study, we aimed to investigate the effects of Rosuvastatin on the HMGCR expression as well as its enzymatic activity in the liver, intestine and adipose tissues -as the major sites for cholesterol biosynthesis and cholesterol storage- in rats with the normal diet. The normal diet was used to prevent correcting of the cholesterol deficiency by diet cholesterol, and so the roles of the different tissues in this regard be investigated.
ACCEPTED MANUSCRIPT Moreover, we measured the expression levels of ABCA1 and ABCG1 (as the major transporters responsible for cholesterol efflux), and ApoA1 (as the key apolipoprotein of HDL particles which are the major regulators of the RCT). 2. Material and Methods: 2.1. Animals
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Eighteen male Sprague-Dawley rats weighing 220 to 280 g were purchased from
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Pasteur Institute (Tehran, Iran). All procedures were followed according to the National Institute of Health guide for the care and use of Laboratory Animals (NIH Publications
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No. 8023, revised 1978) and local guidelines for compassionate use of animals in research; two rats were kept per cage with free access to tap water and compact
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standard chow. The animals were kept in the same laboratory conditions (18°C to 23°C room temperature and controlled humidity) with alternating 12-h light and dark cycles.
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Animals were randomly divided into control (n-9) and Rosuvastatin (n-9) groups. Rosuvastatin group was treated with 10mg/kg/day Rosuvastatin (calcium salt,
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AstraZeneca) freshly prepared in 0.5% carboxymethyl cellulose as the vehicle [17, 18],
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and the control group only received the vehicle. The administration period was six weeks. The conditions of the administrations were based on previous studies [19, 20].
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At the end of the treatment course, all the rats were anesthetized by a single dose (100 mg/kg) intraperitoneal injection of ketamine. Blood samples were collected by cardiac
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puncture. Serum samples were prepared and kept frozen at −20 °C until analysis. Then, using the Ketamine lethal dose (200 mg/kg), all the rats were killed. Liver of the
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rats was excised, weighed and plunged into liquid nitrogen and stored at -80°C. The small intestine was excised and the luminal contents were washed out with cold phosphate buffered saline (PBS). The ileal segments (20 cm proximal from the ileocecal valve) were dissected, the mucosa was scraped off, immediately plunged into liquid nitrogen and stored at -80°C. The adipose tissue samples (from the left inguinal fat pad) were dissected and washed with PBS, plunged into liquid nitrogen and then stored at 80°C. 2.2. Biochemical Parameters
ACCEPTED MANUSCRIPT The serum lipid profile, including total cholesterol, triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) and very low-density lipoprotein (VLDL) was measured by a colorimetric method using commercial reagents in an automated chemical analyzer (Roche Cobas Mira, Basel, Switzerland).
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2.3. Gene Expression analyses Total RNA of the liver, intestine and adipose tissues was extracted by RNeasy Mini Kit Hilden,
Germany)
according to
the
manufacturer’s
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(Qiagen,
protocol. Then,
complementary DNA (cDNA) was synthesized using 1 μg of the total RNA with Oligo
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(dT) primers and the RevertAid first-strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The quantitative real-time polymerase chain
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reaction (QPCR) was performed in duplicate using SYBR Premix Ex Taq II (Takara Bio Inc., Japan) on the Rotor-Gene 6000 real-time PCR detection system (Corbett
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Research, Sydney, Australia). Results were normalized according to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. Table 1
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shows the used specific primer-sequences.
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Table 1. the primer sequences Primer Forward Reverse HMGCR 5’-ACCGTGGGTGGTGGGAC-3’ 5’-GCCCCTTGAACACCTAGCATC-3’ ABCA1 5’-ACGAGATTGATGACCGCCTC-3’ 5’-GCATCCACCCCACTCTCTTC-3’ ABCG1 5’-AGGTCTCAGTCTAAAGTTCCTC-3’ 5’-TCTCTCGAAGTGAATGAAATTTATCG-3’ ApoA1 5’-CACCGAGCTTCACAAAAA CG-3’ 5’-TGATCGCTGTAGAGCCCAAA-3’ GAPDH 5’-GTCTCCTGCGACTTCAACA-3’ 5’-TCATTGTCATACCAGGAAATGAGC-3’ HMGCR: 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; ABCA1: ATP-binding cassette transporter subfamily A member 1; ABCG1: ATP-binding cassette transporter subfamily G member 1; ApoA1: apolipoprotein A1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase
PCR program for HMGCR was run as a preincubation step at 94°C for 10 min, followed by 40 cycles of denaturation (95°C, 10 s), annealing (62°C, 30 s) and extension (72°C, 30 s) steps with the final extension step at 72˚C for 10 min; the ApoA1 and ABCA1 run program included a preincubation step at 94°C for 10 min, followed by 40 cycles of denaturation (95°C, 10 s), annealing (64°C, 30 s) and extension (72°C, 30 s) steps with the final extension step at 72˚C for 10 min; the ABCG1 run program was as a preincubation step at 94°C for 10 min, followed by 40 cycles of denaturation (95°C, 10
ACCEPTED MANUSCRIPT s), annealing (63°C, 22 s), and extension (72°C, 15 s) steps with the final extension step at 72˚C for 10 min. The 2−ΔΔCt method was used to calculate the relative changes in the gene expression. HMGCR Activity Assay
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Microsomes were isolated using Microsome Isolation Kit (Biovision Inc., Milpitas, CA,
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USA) according to the manufacturer’s instructions. Then, the activity of HMGCR for isolated microsomes was measured according to the instructions of the HMGCR Activity
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Assay Kit (K588-100; BioVison Inc.).
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2.4. Statistical Analysis
Data are shown as mean ± SD. Statistical comparisons of the groups were performed
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by the non-parametric Mann-Whitney test. A p value of less than 0.05 was considered
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statistically significant. The analyses were performed in SPSS 16.0 software. 3. Results:
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3.1. Rosuvastatin Effect on the Serum Lipid Profile Rosuvastatin significantly (p<0.05) reduced the serum levels of total cholesterol and
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LDL-C by 20.07% and 17.47% respectively. The serum levels of HDL-C, TG and VLDL were insignificantly (p>0.05) decreased in the Rosuvastatin-treated group (Fig.1). Fig. 1. The effect of Rosuvastatin on the serum lipid profile. In the Rosuvastatin-treated group, the serum concentrations of total cholesterol (52.6 ± 3.43 µg/dl) and LDL-C (16.72 ± 1.28 µg/dl) were significantly (p<0.05) reduced compared to those in the control group (total cholesterol (65.81 ± 3.45 µg/dl) and LDL-C (20.26 ±0.91 µg/dl). There were not seen significant differences (p>0.05) in the serum concentrations of TG (18.8 ± 2.14 µg/dl), HDL-C (30.80 ± 1.68 µg/dl), and VLDL (3.97±0.50) in the Rosuvastatin-treated group versus those in the control group; TG (21.09 ± 2.66 µg/dl), HDL-C (28.45 ± 3.17 µg/dl), VLDL (4.19±0.89).
ACCEPTED MANUSCRIPT
3.2. Results of the Gene Expression Analyses 3.2.1. Relative Expression of HMGCR, ABCA1, ABCG1, and ApoA1 in the Liver The results of the QPCR in the liver revealed that the expression of HMGCR, ABCA1,
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ABCG1, and ApoA1 was significantly (p<0.05) upregulated in the Rosuvastatin group
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compared to the control group (Fig. 2).
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Fig. 2. Relative expression of the genes in the liver. There was observed a significant (p<0.05) elevation in the expression levels of ABCG1 (2.85±1.21 fold),
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ABCA1 (3.96±1.81 fold), ApoA1 (8.87±4.68 fold) and HMGCR (8.09±3.83 fold) in the Rosuvastatin-treated group compared to those in the control group including
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ABCG1 (1.14±0.50 fold), ABCA1 (1.06±0.49 fold),
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ApoA1 (1.07±0.46 fold), and HMGCR (1.27±0.62 fold).
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3.2.2. Relative Expression of HMGCR, ABCA1, and ABCG1 in the Intestine The results of the gene expression assay in the intestine indicated that the expression
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of HMGCR was significantly (p<0.05) higher in the Rosuvastatin-treated group;
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however, there were not significant (p>0.05) differences in the expression level of ABCA1
and
ABCG1
between
the
Rosuvastatin and control groups (Fig. 3). Fig. 3. Relative expression of the genes in the intestine. There was shown a significant (p<0.05) increase
in
the
expression
level
of
HMGCR
(3.10±1.89 fold) in the Rosuvastatin group versus that in the control group (0.94±0.52 fold). However, there were not observed significant (p>0.05) differences in the expression levels of ABCA1 (1.18±0.93 fold) and ABCG1 (1.37±0.81) in the treatment group compared
ACCEPTED MANUSCRIPT to the control group (ABCA1 (0.93±0.41 fold) and ABCG1 (1.09±0.48 fold).
3.2.3. Relative Expression of HMGCR, ABCA1, and ABCG1 in the Adipose Tissue The results of the QPCR in the adipose tissue showed insignificant (p>0.05) differences
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in the expression levels of ABCA1, ABCG1 and HMGCR between the study groups
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(Fig. 4).
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Fig. 4. Relative expression of the genes in the adipose tissue. The QPCR results showed an
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insignificant (p>0.05) change in the expression levels of ABCG1 (0.91±0.55 fold), ABCA1 (0.98±0.67 fold), and HMGCR (0.89±0.33 fold) in the Rosuvastatin-
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treated group compared to those in the control group; ABCG1 (1.48±1.39 fold), ABCA1 (1.21±0.84 fold) and
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HMGCR (1.00±0.53 fold).
3.3. The HMGCR activity in the liver, intestine and adipose tissues The results of the HMGCR activity assay in the Rosuvastatin-treated group showed a
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significant (p<0.05) increase in the activity of HMGCR in the liver and intestine by
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57.14% and 30.76% respectively. However, in the adipose tissue, there was not observed a significant (p>0.05) difference between the study groups (see Fig. 5). Fig. 5. The HMGCR activity in different tissues. The HMGCR activity in the liver and intestine of the Rosuvastatin-treated group was significantly (p<0.05) higher (0.022±0.003 and 0.017±.003 units/mg protein respectively)
than
those
(0.014±0.002
and
respectively).
However,
in
the
0.013±.003 there
control
units/mg was
not
group protein
seen
a
significant (p>0.05) difference in the HMGCR activity between (.0085±.003 and .0081±0.002 units/mg protein respectively).
the
Rosuvastatin
and
control
groups
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4. Discussion: Different transcription factors, including liver X receptors (LXRs), peroxisome proliferative activated receptors (PPARs) and sterol regulatory element binding transcription (SREBPs) are responsible for regulating the expression of the genes
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involved in the lipid metabolism [21]. LXRα is known as the whole-body cholesterol
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sensor, regulating expression of the genes involved in the cholesterol efflux such as
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ABCA1 and ABCG1 [22]. High level of cholesterol causes an increase in the generation of the oxidized forms of cholesterol (oxysterols ) which serve as the agonist ligands of
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LXR [11].
Statins through a biphasic mechanism regulate the transcriptional activity of LXR in the
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liver: first, suppression of LXR activity via reducing the oxysterols; then, induction of the transcriptional activity of LXR through reducing GGpp [23]; statins by inhibiting prenylation and then activating RhoA (a small GTPase protein) prevent the inhibitory
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phosphorylation of PPAR-γ; PPAR- γ upregulates the expression of LXR. Also, GGPP
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can directly antagonize activity of LXR [6].
SREBP-2 is another nuclear receptor involved in the metabolism of sterol. Cholesterol
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deficiency causes the proteolytic activation of SREBP-2 [24] that upregulates genes
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involved in the cholesterol biosynthesis and cholesterol efflux [8, 25] In addition to the nuclear receptors, miR-33 which is located within introns of the
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SREBP-2 gene downregulates the expression of ABCA1 and ABCG1 [26, 27]. Therefore, the effects of statins on the genes responsible for cholesterol effluxing are dependent on the balance between the inducing mechanisms (LXR activation) and inhibitory mechanisms (miR-33). The results of the present study showed that Rosuvastatin in the liver increased the activity of HMGCR and upregulated the expression of HMGCR, ABCA1, ABCG1, and ApoA1. These findings may indicate the adaptive responses by the liver. Consistent with these findings, various studies demonstrated that statins could increase the expression level of HMGCR [28], ApoA1 [29], ABCA1 [30] and ABCG1[31] in the liver or
ACCEPTED MANUSCRIPT HepG2 cells. However, several other studies showed that statins through upregulating SREBP-2 and then miR-33 downregulate the expression of ABCA1 and ABCG1 in the liver [27, 32]. In the intestine, Rosuvastatin increased both the mRNA level and activity of HMGCR. Rosuvastatin through direct inhibiting cholesterol synthesis in the liver may reduce the
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cholesterol levels in the intestine and then induce the compensatory upregulation of
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HMGCR. In spite of our findings, Harshman et al. showed that atorvastatin did not affect
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the expression of HMGCR in the intestine of C57Bl6 Male Mice [33].
The results of this study did not show a significant difference in the intestinal expression
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of ABCA1 and ABCG1 between the study groups.
In the extrahepatic tissues, ABCA1 has been shown to be differently regulated;
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Tamehiro et al. showed that Pravastatin reduced both mRNA and protein levels of ABCA1 in the intestine; however, increased them in the liver [34]. Moreover, it was
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shown that statins could reduce the mRNA levels of ABCA1 and ABCG1 in Caco-2 cells (heterogeneous human epithelial colorectal adenocarcinoma), respectively [35]. These
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findings may indicate that statins cannot induce the intestine cholesterol efflux via these protein transporters.
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Adipocytes are unique for their large capacity of the cholesterol storage, these cells include 25% of the whole-body cholesterol. However, adipocytes have a low capacity of
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cholesterogenesis, less than 10% of the systemic cholesterol production [36].
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There is a dynamic equilibrium between the serum lipoprotein-cholesterol and adipocyte free-cholesterol (FC) pools. Thus, the adipose tissue has been suggested to play a “buffer” role for the serum-cholesterol [37]. In spite of the essential role of the adipose tissue in the cholesterol metabolism, the effects of statins on the adipose tissue have not been widely disclosed. The results of this study showed that Rosuvastatin had no effects on the cholesterol metabolism in the adipose tissue. Regarding the role of the cellular cholesterol load in regulating the transcriptional activity of different factors such as LXR and SREBP-2, the
ACCEPTED MANUSCRIPT high amount of cholesterol in the adipocytes would keep the constant expression levels of the genes involved in the cholesterol metabolism during the course of the statin treatment. Overall, our findings revealed that in response to the reduced level of cholesterol by Rosuvastatin, liver, and intestine increase their cholesterol synthesis capacity. The
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adaptive response by the liver was expected because it is the primary target of statins.
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However, the intestinal response to the reduced level of cholesterol indicates the vital role of this organ in maintaining cholesterol homeostasis. It was shown that the
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activation of intestinal SREBP2 alone seems to be sufficient to increase plasma cholesterol, highlighting the essential role of the intestine in cholesterol homeostasis in
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the body [38].
Statins, in addition to the metabolism of cholesterol, play an important role in the
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regulation of the cellular signaling pathways and it depends on the effect of statins on the synthesis of isoprenoids. Theatrically, the adaptive response by the intestine
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increases isoprenoids including GGpp and FPP, and then initiates the cell signaling
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cascades, and increases the risk of cancer development. However, most of the population-based studies showed that statins had no effects on the risk of
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gastrointestinal cancers [39, 40].
The small sample size and the use of laboratory animals were limitations of this study.
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On the other hand, measuring cholesterol synthesis in-vivo condition is a more reliable method to evaluate the adaptive responses by different tissues to the reduced level of
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cholesterol after administration statins. Also, measuring of the isoprenoids in the extrahepatic tissues can exactly clarify the effects of statins on the cellular signaling pathways and cancer development. 5. Conclusion: The intestine, in response to the reduced level of the cholesterol synthesis in the liver, enhances its cholesterogenesis capacity. However, Rosuvastatin does not affect the cholesterol efflux through protein transporters of ABCA1 and ABCG1. Therefore, other cholesterol transmission pathways such as aqueous diffusion are likely to be
ACCEPTED MANUSCRIPT responsible for effluxing the newly-synthesized cholesterol from the extra-hepatic tissues to the HDL particles. Thus, the intestine may play the major role in compensating for the reduced level of cholesterol during the statin treatment course. Rosuvastatin does not affect the adipose tissue. Thus, adipose tissue is not likely to play an important role in compensating for the reduced level of cholesterol in the body.
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6. Declarations of Interest: none
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7. Acknowledgments: we would like to express my special thanks to everyone who
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supported us during the course of this project.
8. Funding: This work was supported by Biotechnology Research Center at Tabriz
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University of Medical Sciences (grant number: 5/126558).
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ACCEPTED MANUSCRIPT Abbreviations list: ABCA1: ATP binding cassette subfamily A member 1 ABCG1: ATP binding cassette subfamily G member 1 ApoA1: apolipoprotein A1 HMGCR: 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase
PPAR: peroxisome proliferator activated receptor
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SREBP: sterol regulatory element binding transcription
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miR-33: microRNA 33
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LXR: liver X receptors
ACCEPTED MANUSCRIPT Highlights:
The intestine, efficiently responses to the reduced level of cholesterol by statin (Rosuvastatin) and upregulates its expression of HMGCR to compensate for cholesterol deficiency in the liver.
The adipose tissue as the main cholesterol storage in the body, does not play a significant role in correcting cholesterol deficiency in the liver of the rats treated with Rosuvastatin.
The liver, as the main target of statins, efficiently responses to Rosuvastatin and upregulates expression of the genes involved in the cholesterol synthesis and cholesterol effluxing.
Regarding the targeting of the liver by Rosuvastatin, it is possible that the increased-cholesterogenesis in the intestine also increases isoprenoids which are the byproducts of the cholesterol synthesis pathways
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