STZ on expression of genes involved in lipid, cholesterol and glucose metabolism in rats

    Effect of HFD/STZ on expression of genes involved in lipid, cholesterol and glucose metabolism in rats Luisa Pozzo, Andrea Vornoli, Ilaria Coppola, Clara Maria Della Croce, Lucia Giorgetti, Pier Giovanni Gervasi, Vincenzo Longo PII: DOI: Reference:

S0024-3205(16)30579-3 doi: 10.1016/j.lfs.2016.09.022 LFS 15032

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

14 April 2016 13 September 2016 26 September 2016

Please cite this article as: Pozzo Luisa, Vornoli Andrea, Coppola Ilaria, Croce Clara Maria Della, Giorgetti Lucia, Gervasi Pier Giovanni, Longo Vincenzo, Effect of HFD/STZ on expression of genes involved in lipid, cholesterol and glucose metabolism in rats, Life Sciences (2016), doi: 10.1016/j.lfs.2016.09.022

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ACCEPTED MANUSCRIPT Effect of HFD/STZ on expression of genes involved in lipid, cholesterol and glucose

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metabolism in rats

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Luisa Pozzoa, Andrea Vornolia,b, Ilaria Coppolaa, Clara Maria Della Crocea, Lucia Giorgettia, Pier

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Giovanni Gervasia, Vincenzo Longoa,*.

Institute of Agricultural Biology and Biotechnology, CNR, ViaMoruzzi 1, 56124, Pisa, Italy.

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[email protected]. Phone: +390503152690

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*Correspondence to: Dr. Vincenzo Longo, IBBA-CNR, Via Moruzzi 1, 56124 Pisa, Italy. E-mail:

Ph.D. candidate of the Pharmacological and Toxicological Science School of the University of

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Bologna.

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ACCEPTED MANUSCRIPT Abstract Aims: The aim of the study was to evaluate lipid, cholesterol and glucose metabolism in a novel rat

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model of non-alcoholic fatty liver disease (NAFLD).

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Main methods: Rats (Wistar) were fed high fat/cholesterol diet (HFD) and a single low dose (35

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mg/kg) of streptozotocin (STZ). Collagen and glycogen content, oxidative stress and glucokinase activity were measured using biochemical assays. Other metabolic pathway were assessed by qRT-

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PCR.

Key findings: HFD/STZ treated rats, compared to control ones, showed an increase in expression of

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biomarkers of inflammation (TNFα, IL6), fibrosis (TGFβ), mitochondrial stress (UCP2) and oxidative stress (GSH and carbonylated proteins) but not of ER stress (CHOP, XBP1).

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Additionally, HFD/STZ treatment caused a reduction in glycogen content, glucokinase activity (a

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limiting step in glycolysis) and expression of ChREBP gene (a de novo lipogenesis regulator),

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suggesting a modified glycolytic pathway. The cholesterol biosynthesis in HFD/STZ treated rats was inhibited (reduced expression of SREBP-

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2−regulated HMGCoA red and LDLr), instead the cholesterol catabolism was increased, as shown by the mRNA induction of the CYP7A1 and CYP8B1 (key genes for BA acid). A reduced gene expression of FXR-dependent SHP (a key gene for feedback inhibition of CYP7A1 and CYP8B1) and of bile acids (NTCP, OATP1A1, BSEP) and cholesterol (ABCA1) transporters was found. Significance: These results widely extend the characterization of HFD/STZ rat model, which might mimic the NAFLD/NASH in diabetic humans.

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ACCEPTED MANUSCRIPT Keywords: HFD/STZ rat model, lipid, cholesterol and glucose metabolism, bile acid transporters,

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inflammation, mitochondrial and endoplasmic reticulum oxidative stress.

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ACCEPTED MANUSCRIPT Introduction Non-alcoholic fatty liver disease (NAFLD) is an increasing public health problem and its

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prevalence in the general population ranges from 5 to 20%, but in diabetic patients the rates reach

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up to 75% [1-3]. The advanced stage of NAFLD, non-alcoholic steatohepatitis (NASH), features

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liver inflammation and fibrosis, and has a strong association with the metabolic syndrome [4-6]. The complexity and chronology of pathophysiological events leading to the development of

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NAFLD/NASH are not fully understood.

Hepatic lipid metabolism is a complex, multicomponent process. Many studies have focused on the

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biological roles of the nuclear receptors liver X receptor α (LXRα) and peroxisome proliferatoractivated receptor α (PPARα), which serve as lipid sensors [7,8]. Through the oxysterols activation

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[8], LXRα up-regulates the sterol regulatory element binding protein 1c (SREBP-1c), which

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controls the de novo lipogenesis [9-11]. LXRα down-regulates the sterol regulatory element binding

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protein 2 (SREBP-2), a transcriptional factor which controls de novo cholesterol synthesis through the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA red), and the low-density lipoprotein receptor (LDLr) [10]. Furthermore, LXRα regulates the first step of the conversion of

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cholesterol to bile acids (BAs) by cholesterol 7α-hydroxylase (CYP7A1) [12]. The bile acids are ligands of the farnesoid X receptor (FXR), which is implied in the regulation of lipoprotein metabolism, glucose homeostasis, BAs synthesis, BAs uptake, and BAs secretion by various hepatic transporters (BSEP, MDR2, MRP2, NTCP and OATPs) [13]. Another well studied major regulator of lipid metabolism is the transcription factor PPARα. When activated by fatty acids (FA), PPARα regulates genes involved in the microsomal and mitochondrial oxidation [14]. FA may be subjected eitherto β-oxidation in mitochondria to generate ATP or to esterification as well as conversion to triglycerides (TG) [15].

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ACCEPTED MANUSCRIPT Insulin enhances glycogen synthesis through induction of glycogen synthase, and inhibits the expression of phosphoenolpyruvatecarboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase),

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two rate-limiting genes in gluconeogenesis [16,17]. In the presence of insulin resistance, the

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enhanced glucose synthesis, reduced glycolysis and glycogen stored in the liver, together with a

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reduced glucose deposition in extra-hepatic tissues, contribute to hyperglycemia. The importance of hepatic inflammation and fibrosis in the pathogenesis of NAFLD/NASH has been long proven [18,19]. It has been shown that the consumption of high fat diet induces

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inflammation of adipose tissue, which releases the pro-inflammatory tumor necrosis factor α

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(TNFα), leading to activation of an inflammatory response by the liver and in particular interleukin6 (IL6) secretion and activation of fibrogenic stellate cells [20]. In order to recapitulate the metabolic characteristics of NAFLD/NASH in diabetic human, a

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protocol has been developed in rats combining a high fat diet (HFD), causing insulin resistance, and a low dose (35 mg/kg) of streptozotocin (STZ), which reduces the β-cell function and promotes

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hyperglycemia [21]. This differs from the simple HFD-fed rat model, which does not take into account the diabetes-related high glucose levels and rarely proceeds to NASH [22].

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In the present study, we have used the above-mentioned HFD/STZ rat model to examine the expression of several genes related to inflammatory stress, lipids, cholesterol, glucose metabolism, and bile salt transport.

Materials and methods Chemicals All chemicals and solvents were obtained from common commercial sources and were of the highest grade available.

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ACCEPTED MANUSCRIPT Animal procedure This study was performed using Wistar male rats (180-200 g/bw) as previously reported [23]. The

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animals received food and drinking water ad libitum and were maintained on a 12 h light/dark cycle

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in cages at room temperature with 55% of relative humidity. Animals were divided into two groups:

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the control (CTR) group (n=5) fed a standard diet containing 19% of protein, 6% of fiber, 7% of minerals and vitamins, 64% of carbohydrates and 4% of fats (the latter percentage corresponding to the 11% of the diet-deriving energy); the HFD/STZ group (n=5) fed a high fat/cholesterol diet,

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containing 13,8% of protein, 4,4% of fiber, 5,1% of minerals and vitamins, 48,7% of carbohydrates

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and 28% of fat including 2% cholesterol, (this 28% of fats corresponds to the 55% of diet-deriving energy). After 14 days, the rats were treated with a single i.p. injection of STZ (35 mg/kg) (Sigma, St Louis, MO). The treated rats were scored as diabetic once the blood glucose levels reached >350

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mg/dl. The animals remained on the HFD diet for an additional 8 weeks. The weight of the animals was measured at the beginning and at the end of the experiment in order to calculate the weight

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increase. At the end of the experiment, the two groups of rats were sacrificed. Blood samples were obtained by cardiac puncture under general anesthesia prior to rat sacrifice. Serum samples were

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obtained by centrifugation at 2,000 x g for 15 min and used for laboratory analysis. After liver weight recording, samples were stored at -80 °C for extraction and quantification of hepatic lipids, glycogen, collagen, and carbonylated proteins, and for total RNA extraction. Other liver samples were collected and stored in formalin at 4 °C until the histological analysis. All animal procedures were performed with the approval of the Local Ethical Committee and in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Biochemical Analysis Plasma analysis for aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), glucose and total cholesterol (TC) had data very similar to those previously 6

ACCEPTED MANUSCRIPT reported [23] and they are not shown. The hepatic GSH content was determined as previously

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Extraction and quantification of hepatic lipids, glycogen and collagen

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described [24], whereas the glucokinase activity was performed according to Castro et al. [25].

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Lipids were extracted from hepatic tissue following the method described by Folch et al. [26]. Glycogen content was determined in liver, as described by Roe and Dailey [27]. Hepatic collagen levels were quantified using a commercial kit (Sircol™ Collagen Assay,SCA; Biocolor Ltd.,

Carbonylated proteins quantification

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Northern Ireland), and performed according to manufacturer’s recommendations.

The protein oxidation level was determined by the carbonyl protein assay according to Mercier et

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al. [28] and Terevinto et al. [29], with slight modifications. Liver samples from rats were homogenized and incubated with 2,4-dinitrophenylhydrazine (DNPH) 0.02M in HCl 2M. After

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TCA 20% addition and subsequent centrifugation, the pellets were washed three times with ethanol:ethyl acetate (1:1). The pellets were dissolved inguanidine-HCl 6M in KH2PO4 0.02 M (pH

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6.5) and centrifuged. The absorbance of the supernatant was measured at 370 nm.

Histopathological analysis Livers (n=5) were collected for histological examination and fixed in 10% neutral buffered formalin. Tissues were then divided in two parts. One part was routinely embedded in paraffin wax blocks, sectioned at a thickness of 5 µm and stained with haematoxylin and eosin. The other part was stored until use at -20°C, processed for cryosections 10 μm thick, dried for 1 hour, rinsed in absolute propylene glycolfor 3 minutes, stained with Oil Red O (Sigma-Aldrich, Milan, Italy) for 10 minutes at 60°C, rinsed in 85% propylene glycolfor 3 minutes, washed with bidistilled water twice

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ACCEPTED MANUSCRIPT and stained with haematoxylin for 30 seconds at room temperature. All the slide sections were

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examined under a light microscope [30].

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Isolation of total RNA and cDNA synthesis

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Total RNA was isolated from frozen rat liver tissues (about 20 mg of tissue) with the RNeasy Mini Kit (Qiagen, Valencia, CA), following the supplied protocol. Genomic DNA elimination and reverse transcription of total RNA were performed using the QuantiTech Reverse Transcription Kit

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(Qiagen, Valencia, CA). RNA was quantified using NanoDrop (Celbio, Milan, Italy); its purity and integrity were evaluated by checking the absorbance ratio at 260–280 nm and assessing the

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sharpness of 18S and 28S ribosomal RNA bands on GelRed™-stained 1% agarose gel and

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visualized by transillumination with ultra-violet light.

RT-PCR and sequence analysis

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Real-time qPCR was performed according to the manufacturer’s instructions. Briefly, for each reaction, cDNA (100 ng) was added to SsoFast™ EvaGreen Supermix (BioRad, Segrate, Italy) plus

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300 M of forward–reverse primers specific for genes reported in Table 1 and was run in triplicates using conditions that have been previously described [31]. In order to evaluate the quality, the extracted RNA was separated on GelRed™-stained 1% agarose gel and visualized by transillumination with ultra-violet light. Primer pairs were designed with Beacon Designer™ 5 (Premier Biosoft, International,Palo Alto, CA)using the rat cDNA sequences available in GenBank.

Table 1.Primer pairs and annealing temperatures for real time PCR experiments. Target gene

Direction

Primer sequence (5’-3’)

Temp. (°C)

ABCG5

Forward

GGCAACAGCGTCAGAATCC

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CYP7A1

CYP7B1

CATCTTTTCACGGTTGGCCTTA

Forward

CGGGTGGCAGCGACAGAG

Reverse

CAGGTGTGGTGGTGTATGAAGATG

Forward

AGGTCTGGCTCTACCACGAT

Reverse

CACTCTGTCTGCAGCAGTGA

Forward

CACCATTCCTGCAACCTTTT

Reverse

GTACCGGCAGGTCATTCAGT

Forward

ACTACAGAGCCGCCAGAG

Reverse

GGTGACCGCAAAGAGAAGG

GLUT2

HMGCoA red

IL-6

60

58

58

58

58

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CCAGCAGGGAGTAGACAAACC TCACCGATGGCTGAGGAAGAG

Reverse

CCCAGGCAGGACCGAACC

Forward

CACGGCGGCAGCAGGAACAG

Reverse

AGCACTCTCAGACAGGCACTCAG

Forward

CCACCATCCCAGAAGCACAT

Reverse

ACACGGCGTTTTTGGTGATG

Forward

GCTGGAGTCTTGTCAGGCAT

Reverse

GCCCCTTTAGCAGCAGGTAA

Forward

TCACACCAGCACATACGACACC

Reverse

GGACACAGACAGAGACCAGAGC

Forward

CGACAGCACGAGCAGATTTG

Reverse

TGGACTGGAGACGGATGTAGAG

Forward

TCCTACCCCAACTTCCAATGCTC

Reverse

TTGGATGGTCTTGGTCCTTAGCC

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G6Pase

Forward

CCAAGGACAAGGAGCAGGAC

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CYP27A1

FXR

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Forward Reverse

FAS

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Reverse

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CYP8B1

CCCCATTGAACACGGATT

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CPT1α

Forward

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CHOP

ACTGCGTCTCCTACCTCCTG

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β-actin

Reverse

58

62

58

64

58

60

62

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MRP4

NTCP

OATP1A1

Forward

TCCGCCGCAGTGTCATCAAGG

Reverse

TTCCGCCGCATGTAGGTGTCC

Forward

CCAATGTGCCAGTGCTTCAG

Reverse

TGGACCACGGTGCTCTTC

Forward

GGTCAGAAGCAGCGAGTCAGC

Reverse

GAGCATCCACAGCCGACAGG

Forward

TTCAGAGGCTTGGTTCTTGTTCC

Reverse

CCGAAGGCAACGACGATGAC

Forward

GGCAAGGAAGGACAGCAGCAGAG

AGGCGGCAGGGAGAAATTGAAAGG

Forward

GGCTTTTTGGTCTGTGCAGG

SHP

SREBP-1c

SREBP-2

SULT2A1

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AC

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58

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58

58

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CACCTTGTGTTGCAGTCAGC

Forward

CAGCTGTGGAAAACGTGGTG

Reverse

TGTCCATCCTTGCCCCATTC

Forward

CCATCTGTCCTCTCTCCCCA

Reverse

CCTCTCCGAGGGACTGAGAA

Forward

GCTCACCTCTGGCCAAGATT

Reverse

AGGCCCAGTTGTTGACCAAA

Forward

GGCACTATCCTCTTCAACCCA

Reverse

TCCAGGACTTCACACAATGCC

Forward

CTGTCGTCTACCATAAGCTGCAC

Reverse

ATAGCATCTCCTGCACACTCAGC

Forward

GCAACAACAGCAGTGGCAGAG

Reverse

TGAGGGAGAGAAGGTAGACAATGG

Forward

GTCCATGCGAGAATGGGACAA

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OATP1B2

PEPCK

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Reverse

Reverse

PPARα

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CATGGATCCAGTCTACCGCC

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MRP2

Reverse

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MDR2

AGATCTACAGCGCCGTGATG

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LXRα

Forward

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LDLr

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60

62

62

60

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CCTGTATTCCGTCTCCTTGG

Forward

AAAGCATGATCCGAGATGTG

Reverse

AGCAGGAATGAGAAGAGGCT

Forward

CGTCTGCACTCCTGTGTTCT

Reverse

GCTATCATGGCCTGATCCCC

Forward

TCCGCAGCACTCAGACTAC

Reverse

CCGTGTATCTCAGCGTCTCC

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Reverse

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56

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XBP1

ATTCCTGGCGTTACCTTGG

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UCP2

Forward

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TNFα

TCATGAGGCCAATTCCAGTAAA

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TGFβ

Reverse

Statistical analysis

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Significant differences between the means of the two rat groups (CTR vs HFD/STZ) were evaluated

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Results

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(San Diego, CA, USA).

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using the Student’s t-test. All statistical analyses were carried out using Prism, GraphPad Software

Effect of HFD/STZ on body weight, food intake and liver weight The rats of the HFD/STZ group did not differ from the CTR group in final body weight (354.9±13.5 vs 322.0±36.9 g/rat, respectively) and food intake (23.5±0.3 vs 21.8±1.9 g/rat/day, respectively). However, rats of HFD/STZ group showed a statistically significant increase in liver weight when compared to CTR group (17.1±3.2 vs 10.9±0.3 g, respectively) (p<0.01) and relative liver weight (5.3±0.4 vs 3.1±0.1 g liver/100 g bw) (p<0.001).

Effect of HFD/STZ on hepatic lipids and collagen content

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ACCEPTED MANUSCRIPT In order to assess the effectiveness of the HFD/STZ treatment to induce hepatic steatosis and fibrosis, we measured the lipid and collagen content in the liver. Both the hepatic lipids and

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collagen contents were significantly higher in the HFD/STZ rats than in the controls (213.8±31.5 vs

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32.7±3.6 mg of lipids/g of liver, respectively) (p<0.001) and (549.9±28.8 vs 343.2±53.8 mg of

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collagen/g liver, respectively) (p<0.001).

Liver GSH and oxidative stress by carbonylated proteins quantification

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The hepatic GSH content was significantly decreased in the HFD/STZ treated group than in the

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CTR group (0.59±0.02 vs 0.75±0.05 mg of GSH/g of liver, respectively) (p<0.01). Since it has been observed that the malondialdehyde assay could fail to detect lipid peroxidation [32,33], we determined the content of the carbonylated proteins as more stable products of oxidative stress.

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Indeed, the carbonylated proteins were significantly increased in the HFD/STZ group, compared to

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CTR group (405.2±17.6 vs 215.7±13.3 nmol/g of liver, respectively) (p<0.001).

Liver glycogen content and glucokinase activity

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In order to assess the effect of HFD/STZ treatment on glucose metabolism, we measured the stored hepatic glycogen content and the glucokinase activity. The content of glycogen was significantly decreased in HFD/STZ group compared to the CTR group (211.0±18.8 vs 671.0±145.0 mg/100 g of tissue, respectively) (p<0.001). The glucokinase activity was significantly lower in the rats of HFD/STZ group than in the CTR group (7.3±1.9 vs 17.3±4.9 nmol/mg of protein min, respectively) (p<0.01).

Histopathological analysis

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ACCEPTED MANUSCRIPT The histology of liver sections from the CTR group (Figure1a, b, e and f) exhibited normal liver architecture with well-preserved cytoplasm and prominent nucleus, whereas extensive

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macrovescicularsteatosis was observed in the livers of the HFD/STZ rats (Figure 1c, d, g and h).

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Effect of HFD/STZ on inflammatory and ER stress genes expression

In order to study the pathogenetic mechanisms involved in the steatosis, some inflammatory and ER stress genes were evaluated by quantitative RT-PCR. As shown in Figure 2, the HFD/STZ group

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showed a significant increase of the hepatic transforming growth factor β (TGFß) (p<0.05), TNF

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(p<0.01) and IL6 (p<0.05) gene expression compared to CTR group. Furthermore, the HFD/STZ group showed a significant increase of the mitochondrial uncoupling protein-2 (UCP2) gene

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expression compared to CTR group (p<0.05), whereas no significant difference was found for

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C/EBP homologous protein (CHOP) and X-box binding protein-1 (XBP1) gene expression between

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the two groups.

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BP 1 X

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P2 C U

IL -6

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TG

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Fold mRNA levels

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Figure 2. Relative expression of TGFβ, TNFα, IL6, UCP2, CHOP, and XBP1, by real-time PCR in liver from CTR (○) and HFD/STZ (●) rats. Data represent the mean ± SD (bar) of 5 rats. Results are normalized for the levels of β-actin housekeeping gene and referred to the mean of the controls, to which a value of 1 was assigned. Significantly different from control value by Student’s t-test, **p < 0.01; ***p< 0.001

Effect of HFD/STZ on expression of genes involved in lipid, cholesterol and glucose metabolism To investigate the molecular mechanisms involved in the hypercholesterolaemia and steatosis, some cholesterol and fatty acid homeostasis genes were evaluated by quantitative RT-PCR including 13

ACCEPTED MANUSCRIPT LXR, SREBP-1c, carbohydrate responsive element-binding protein (ChREBP), acetyl-CoA carboxylase (ACC1), fatty acid synthase (FAS), PEPCK, G6Pase, SREBP2, HMGCoA red, LDLr,

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PPAR, carnitinepalmitoyl-transferase1α (CPT1α), and long-chain acyl-CoA dehydrogenase

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(LCAD). As shown in Figure 3, the HFD/STZ group showed a significant induction of the SREBP-

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1c (p<0.01), LXRα (p<0.05), PPAR (p<0.05), and CPT1α (p<0.001), and a significant reduction of SREBP2 (p<0.005), HMGCoA red (p<0.05), LDLr (p<0.001), ChREBP (p<0.01), and ACC1 (p<0.001) genes expression compared to CTR group. On the other hand, the gene expression of

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FAS and LCAD were not affected. In addition, the HFD/STZ treatment did not affect the

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expression of PEPCK and G6Pase genes involved in the gluconeogenesis.

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PT 1 C

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PP A

Lr

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*

M G C

EB P2 SR

6P as e G

K PE PC

S

1

C

C

A

EB P hR C

SR

EB P1 c

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FA

2

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3

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Fold mRNA levels

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Figure 3. Relative expression of LXRα, SREBP-1c, ChREBP, ACC1, PEPCK, G6Pase, SREBP-2, LDLr, PPARα, CPT1, and LCAD by real-time PCR in liver from CTR (○) and HFD/STZ (●) rats. Data represent the mean ± SD (bar) of 5 rats. Results are normalized for the levels of β-actin housekeeping gene and referred to the mean of the controls, to which a value of 1 was assigned. Significantly different from control value by Student’s t-test, *p < 0.05; **p <0.01; ***p < 0.001.

Effect of HFD/STZ on the expression of genes involved in bile acids biosynthesis and transport In order to investigate the combined effect of steatosis and diabetes on bile acids metabolism, some of the major genes such as CYP7A1, sterol 27-hydroxylase (CYP27A1), sterol 12 α-hydroxylase (CYP8B1), oxysterol 7α-hydroxylase (CYP7B1), FXR, small heterodimer partner (SHP), multidrug resistance protein 2 (MDR2), multidrug resistance associated protein 2 (MRP2), multidrug resistance associated protein 4 (MRP4), ATP binding casette A1 (ABCA1), ATP binding casette G5 14

ACCEPTED MANUSCRIPT (ABCG5), Na-taurocholate cotransporting polypeptide (NTCP), organic anion transporting polypeptide 1A1 (OATP1A1), organic anion transporting polypeptide 1B2 (OATP1B2), bile salt

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export pump (BSEP) and sulfotransferase 2A1 (SULT2A1), involved in bile acids formation and

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transport, were analyzed by quantitative RT-PCR. As reported in Figure 4, the HFD/STZ group

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showed, with respect to the control group, a strong induction of CYP7A1 (p<0.001), a significant enhancement of CYP8B1 (p<0.05) and ABCA1 (p<0.05) genes, whereas the expression of SHP (p<0.05), NTCP (p<0.05), OATP1A1 (p<0.01), BSEP (p<0.01), and SULT2A1 (p<0.05) genes was

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significantly reduced. On the other hand, the expression of CYP27A1, CYP7B1, FXR, MDR2,

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MRP2, MRP4, ABCG5 and OATP1B2 genesdid not change.

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TP 1B 2

BS EP

SU

A

O

TC N O

A

1

P

5

1

BC G A

BC A A

P4 M R

P2 M R

2 R M D

P SH

R

FX

P8 B1

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P7 B1

*

**

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C Y

Y C

C

Y

P7 A

P2 7A

1

1

0

**

*

*

TP 1A

*

2

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10

D

15

C Y

Fold mRNA levels

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Figure 4. Relative expression of CYP7A1, CYP27A1, CYP7B1, CYP8B1, FXR, SHP, MDR3, MRP2, MRP4, ABCA1, ABCG5, NTCP, OATP1A1, OATP1B2, BSEP and SULT2A1 by real-time PCR in liver from CTR (○) and HFD/STZ (●) rats. Data represent the mean ± SD (bar) of 5 rats. Results are normalized for the levels of β-actin housekeeping gene and referred to the mean of the controls, to which a value of 1 was assigned. Significantly different from control value by Student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001.

Discussion Several NAFLD/NASH models were developed and explored during the recent years [34]. Among these models, HFD fed rats injected with a low dose of STZ has gained a particular interest. After 14 days of HFD, the STZ injection induces diabetes by a selective destruction of pancreatic β-cells, 15

ACCEPTED MANUSCRIPT resulting in reduced insulin secretion and giving rise to impaired glucose tolerance [21,35]. This situation seems to reproduce the same pathological conditions of human NAFLD patients

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characterized by insulin resistance and limited insulin production by β-cells.

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In the current study, rats from HFD/STZ group displayed hyperglycemia, hypercholesterolemia,

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hepatic lipid deposition, pronounced steatosis and high blood levels of ALT and AST similar to the results previously reported [23]. In addition, we observed a significant depletion of GSH and oxidative stress, as indicated by the formation of carbonylated proteins, in line with the findings

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previously described [21, 36-39].

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To further examine the extent of the oxidative stress, we measured CHOP and XBP1 gene expression, as markers of ER stress due to the unfolded protein response (UPR) activation, an important process of animal and human NASH pathogenesis [40-41]. The HFD/STZ treatment in

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this study did not lead to the up-regulation of CHOP and XBP1, suggesting the absence of ER stress and highlighting an advanced stage of NAFLD, unlike the rat model induced by a saturated fats diet

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[42]. Although, the model, described here, shows all the mechanistic characteristics of human NAFLD, the exact timing of the onset of the pathology remains unknown. The onset cannot be

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extrapolated based on previously reported data due to the alterations in the experimental setup (STZ concentration and HFD composition), making straight comparisons with other models difficult. PPARs control the expression of genes related to lipid and glucose homeostasis and inflammatory responses [43,44]. In the present study, we detected a significant increase in mRNA levels of the transcription factor PPARα and CPT1α enzyme, responsible for the fatty acid transport into mitochondria, an observation that has previously been reported by a number of studies [22,42]. In the liver, FA may either undergo the mitochondrial β-oxidation for energy production or be esterified to triglycerides, which are incorporated into VLDL particles to be transported and used by the peripheral tissues. The up-regulation of PPARα mRNA could be linked to FA overload, which are known endogenous ligands of PPARα receptor. The increase in PPARα expression lead to the 16

ACCEPTED MANUSCRIPT subsequent up-regulation of hepatic CYP4A, a protein involved FA (ω)-oxidation in ER [23]. However, in this study an up-regulation of LCAD, a key gene of FA β-oxidation in mitochondria

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that was expected to be increased by the up-regulation of PPARα, was not observed. [45].

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Accordingly, wefound an over-expression of UCP2 mRNA, suggesting a spillover of ROS

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production, which is also caused by the up-regulation of CYP2E1 [23]. Indeed, UCP2 is an anion transporter which negatively regulates mitochondrial membrane potential and ATP synthesis, decreasing the superoxide anion emission [46]. Moreover, the CYP4As induction [23] suggests an

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over production of dicarboxylic acids that, together with ROS, might cause mitochondrial

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dysfunction, as shown by over expression of UCP2 mRNA.

It is well known that the hepatic inflammation, resulting from proinflammatory cytokines, plays an important role in the development of NAFLD and progression of the fibrogenic process [18,47,48].

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In the present study, the HFD/STZ treatment induced gene expression of the inflammatory cytokines TNFα and IL6, in agreement with an earlier study in HFD/STZ rats [49]. We also

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observed an increase in collagen deposition and an up-regulation of TGFβ gene. The TGFβ cytokine, which is produced by Kupffer cells and is considered the most powerful mediator of

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hepatic stellate cells activation, leads to the formation of collagen, subsequently promoting fibrosis and NASH [50].

Underphysiological conditions, the excessof glucose leads to de novo lipogenesis via glycolysis, which is regulated by the step limiting activity of the insulin-inducible glucokinase. Consequently, an increase in the glycolysis-intermediate fructose 2-6 diphosphate up-regulates the expression of lipogenic genes such as ACC1 and FAS, via ChREBP [51-52]. In the present study, the reduction of hepatic glycogen content, glucokinase activity and ChREBP, ACC1 and FAS genes expression in rats of HFD/STZ group suggests that the conversion of glucose into FA by this pathway was restricted.

17

ACCEPTED MANUSCRIPT In addition to glycolysis via ChREBP, another source of hepatic lipid accumulation is de novo FA synthesis activated by LXRα-SREBP1c axis [53]. An up-regulation of SREBP-1c mRNA

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expression, along with its lipogenic target genes, was reported in human and rat steatotic liver [54-

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56]. The present study showed that, although an over-expression of SREBP1c and ABCA1 (a target

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gene) was observed, neither ACC1 nor FAS were up-regulated, suggesting that the HFD/STZ treatment induced an advanced stage of NAFLD, in agreement with Xia et al. in the same rat model [17].

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As previously reported [57], the observed elevated intracellular cholesterol levels were correlated

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with a down-regulation of SREBP-2 and its target genes: LDLr, responsible of plasma cholesterol uptake, and HMGCoA red, a rate-limiting enzyme for cholesterol biosynthesis. BAs production plays a key role for the biotransformation of cholesterol in the liver. This pathway

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is mainly accomplished via transcriptional regulation of CYP7A1, the rate-limiting enzyme of neutral BAs biosynthesis, which leads to cholic acid (CA) formation via CYP8B1 [58]. An alternate

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or acidic minor BAs route, which involves CYP27A1 and CYP7B1 producing chenodeoxycholic acid (CDCA), is also present in the liver [58].

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The presence of 2% cholesterol in the diet of rats from HFD/STZ group caused a significant transcriptional increase of CYP7A1 and CYP8B1, but not of CYP27A1 and CYP7B1 genes. In the absence of STZ, HFD and cholesterol diet resulted in CYP7A1 up-regulation only, whereas CYP8B1 espression was reported to be down-regulated [59]. On the other hand, an enhancement of CYP8B1 expression was observed in STZ-treated diabetic rats and in diabetic patients, where CYP8B1-dependent 12-α-hydroxylated BAs were found to be increased in their serum samples and correlated with insulin resistance [60,61]. Other authors found that CYP8B1 activity was regulated at the transcriptional level and the insulin decreased its mRNA levels [62]. Thus, the increased CYP8B1 mRNA level observed in HFD/STZ rats of this study may be associated with insulin dysfunction. 18

ACCEPTED MANUSCRIPT CYP8B1 regulates CA/CDCA ratio, therefore, an up-regulation of CYP8B1 gene expression might indicate an increase in CA at the expense of CDCA and an increase in its derived deoxycholic acid

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(DCA) production. This CYP controls the production of CA, which was found to be more efficient

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than CDCA in the intestinal cholesterol re-absorption and its storage in the liver [63,64].

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On the other hand, CDCA and DCA are the most potent agonists of FXR [65] which, upon activation, protects the liver from BAs toxicity and from the gallstone formation by up-regulation of export transporter genes of BAs (BSEP and MRP2/4), phosphatidylcholine (MDR2), cholesterol

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(ABCG5), and by down-regulation of NTCP [66]. In addition, activated FXR potently induces the

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transcription of SHP, which is able to produce a feedback reaction on the BAs synthesis by repressing CYP7A1 and CYP8B1 transcription [67]. In the current study, this protective feedback loop appears to be absent. We hypothesize that the increased CYP8B1 gene expression, whose

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increased protein expression [63], in the HFD/STZ group may lead to decreased levels of CDCA and DCA, which would consequently prevent the FXR activation.

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In fact, FXR did not appear to be activated, and the expression of its target genes SHP, BSEP and OATP1A1 appeared to be reduced. Furthermore, this hypothesis of lack of activation is also

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associated with the decreasing trend in FXR expression, in line with the findings by Lulu et al. [59], in spite of the reported inhibition of CYP7A1 and CYP8B1 gene expression. On the contrary, in HFD fed rats, the mRNA expression of BSEP and OATP1A1 was found to be induced [68-70]. Previously, it was demonstrated that TNFα administration to rodents suppresses BSEP and OATP1A1 mRNA levels [71]. Thus, the reduced gene expression of BAs transporters and SULT2A1 found in the HFD/STZ rats might be due to the observed high levels of proinflammatory cytokines. Since the hepatobiliary transporters regulate the flux and concentration of BAs, their alterations may lead to a different BAs pool composition, which characterizes some hepatic disease [13]. Indeed,

a

reduced

expression

of

canalicular

transporters

for

cholesterol,

BAs

and 19

ACCEPTED MANUSCRIPT phosphatidylcholine is relevant for gallstone formation and/or a cholestatic condition, as demonstrated in knockout mice lacking FXR [72] and in patients with severe NAFLD [73].

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In the current study, the reduced mRNA expression profile of BAs transporters, along with an

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elevated CA/CDCA ratio, a lack of FXR expression and a cholesterol accumulation in liver, might

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indicate the presence of a gallstone/cholestatic condition in our HFD/STZ rats model of NAFLD/NASH.

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Conclusion

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In the present work, we widely extended the characterization of important genes involved in the lipid, cholesterol and glucose metabolism and bile acids transport in rats treated with a high fat/cholesterol diet combined with a low dose of streptozotocin. It is important to note that the

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current model remains artificially induced and although the final outcome recapitulates human pathology of NAFLD, the progression of the disease in rodents may not be fully representative of

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what is commonly observed in human patients. That being said, we still believe that the HFD/STZ model assessed in this study is suitable for pharmacological and nutraceutical screening. Moreover,

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future assessment of protein levels could further extend our understanding of NAFLD etiology.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgment The authors gratefully thank M. Maltinti (Fondazione Toscana Gabriele Monasterio, Pisa, Italy) for plasma analysis, and Dr. C. Kusmic (Institute of Clinical Physiology, CNR, Pisa, Italy) for the assistance during glucose measurement.

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ACCEPTED MANUSCRIPT Figure 1. Hematoxylin eosin staining of liver tissue from CTR (a and b) and HFD/STZ (c and d).

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Oil Red O staining of liver tissue from CTR (e and f) and HFD/STZ (g and h).

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Figure 1

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