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Differences in hepatic expression of genes involved in lipid homeostasis between hereditary hypertriglyceridemic rats and healthy Wistar rats and in their response to dietary cholesterol
Food and Chemical Toxicology 47 (2009) 2624–2630
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Differences in hepatic expression of genes involved in lipid homeostasis between hereditary hypertriglyceridemic rats and healthy Wistar rats and in their response to dietary cholesterol J. Orolin a, R. Vecera a,*, I. Markova b, A. Zacharova a, P. Anzenbacher a a b
Institute of Pharmacology, Faculty of Medicine, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic Institute for Clinical and Experimental Medicine, Videnska 1958/9, 140 21, Praha 4, Czech Republic
a r t i c l e
i n f o
Article history: Received 19 May 2009 Accepted 21 July 2009
Keywords: Hereditary hypertriglyceridemic rats Metabolic syndrome Wistar rats Dietary cholesterol Cytochromes p450
a b s t r a c t Differences in expression of mRNA of genes regulating lipid and drug metabolism between hereditary hypertriglyceridemic rats (HHTg, accepted model of metabolic syndrome) and healthy Wistar–Kyoto (WKY) rats were studied. Also, differences in expression due to intake of high cholesterol diet (1% w/w) were determined to investigate possible differences in response of the WKY and HHTg rats to increased intake of dietary cholesterol. Levels of ATP-binding cassette transporters (ABCG5, ABCG8), fatty acid synthase (FAS) and cytochrome P450 (CYP2C11) mRNA were significantly lower in HHTg rats on standard laboratory diet; in contrary, CYP7A1, CYP2C6 and CYP2B2 gene expression was significantly higher. The WKY rats responded to high cholesterol diet by an increase in expression of mRNAs for sterol regulatory element binding protein (SREBP1c), CYP2B2 and CYP7A1; lower expression was found in the FAS, ABCG5, ABCG8, CYP4A1, CYP4A2 and acyl-CoA oxidase. HHTg rats responded to cholesterol intake in a similar manner, however, differences were found in expression of the FAS and CYP4A1 mRNA (decrease was not observed), CYP2B2 (decrease instead of an increase). Conclusions: (i) dietary cholesterol significantly influences expression of genes involved in lipid homeostasis and drug metabolism, and (ii) the HHTg rats responded to dietary cholesterol in a different way. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Hypertriglyceridemia is an independent risk factor for coronary heart disease as it participates in development of atherosclerosis and hypertension. Moreover, hypertriglyceridemia is associated with several other metabolic disorders, such as insulin resistance, decreased level of HDL-cholesterol, hypertension, abdominal obesity and hyperinsulinemia. Reaven (1988) designated this cluster of related abnormalities as syndrome X, which was later named the metabolic syndrome or insulin resistance syndrome (Sarafidis and Nilsson, 2006).
Abbreviations: ABCA1, G5, G8, ATP-binding cassette transporters; ACO, acyl-CoA oxidase; CYP7A1, CYP2C6, CYP2C11, CYP2B2, CYP4A1, CYP4A2, types of cytochromes P450; FAS, fatty acid synthase; HCD, high cholesterol diet; HHTg, hereditary hypertriglyceridemic rats; HLR, spontaneously hyperlipidemic rats; LXRa, liver X receptor alpha; PPARa, peroxisome proliferator-activated receptor alpha; SD, Sprague–Dawley rats; SHR, spontaneously hypertensive rats; SPF, specific pathogen free; SREBP1c, sterol regulatory element binding protein 1c; STD, standard laboratory diet; WKY, Wistar–Kyoto rats. * Corresponding author. Tel.: +420 585 632 553; fax: +420 585 632 966. E-mail address: [email protected] (R. Vecera). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.07.022
Some interrelated symptoms of the metabolic syndrome such as insulin resistance, hypertriglyceridemia, and hypertension are known important cardiovascular risk factors, which are hence studied intensively. Several experimental models of insulin resistance and related abnormalities including hypertension have been developed in the spontaneously hypertensive rats (SHR) with hypertension, fasting hyperglycemia, impaired oral glucose tolerance, hyperinsulinemia and hypertriglyceridemia in comparison with healthy Wistar–Kyoto rats (WKY) (Yamori et al., 1978; Reaven and Chang, 1991). The second model of lipid dysregulation is represented by spontaneously hyperlipidemic rats (HLR) originated from Sprague–Dawley rats (SD), which show a higher plasma cholesterol level than control SD rats (Watanabe et al., 1996). Other genetically defined models of lipid disturbances are obese Zucker rats (Bray, 1977; Reaven et al., 1991), Dahl salt-sensitive rats (Dahl, 1961), also used for studies on insulin resistance (Reaven et al., 1991) or the hereditary hypertriglyceridemic (HHTg) rats (originally Wistar) modeling human hypertriglyceridemia (Vrána and Kazdová, 1990). Hypertension, hypertriglyceridemia, insulin resistance, hyperinsulinemia, signs of oxidative stress and other metabolic disturbances have been demonstrated in HHTg rats in comparison with controls (Klimeš et al., 1995).
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This work is focused on relative differences between the HHTg and healthy Wistar–Kyoto (WKY) rats in hepatic expression of mRNA of 14 genes important in regulation of lipid metabolism. In parallel, the effect of dietary cholesterol on differences in expression of the same genes was determined. The following genes expressing the following proteins known to be involved in regulation of lipid homeostasis were chosen: peroxisome proliferatoractivated receptor a (PPARa), liver X receptor a (LXRa), acyl-CoA oxidase (ACO), fatty acid synthase (FAS), ATP-binding cassette transporters (ABCA1, ABCG5 and ABCG8) and sterol regulatory element binding protein (SREBP1c) together with six enzymes of the cytochrome P450 family (CYP7A1, CYP2C6, CYP2C11, CYP4A1, CYP4A2, CYP2B2) which take part in biotransformation of lipids and are also important in drug–drug interactions (warfarin, diclofenac, diazepam, omeprazole and others) in therapy of wide range of diseases. 2. Materials and methods 2.1. Chemicals Cholesterol from Sigma (New York, USA), fentanyl was from Torrex Pharma (Vienna, Austria), medetomidin (Domitor) from Pfizer Animal Health (Brussels, Belgium), diazepam (Apaurin) from Krka (Slovenia), the Bio-La-Test Cholesterol 250 E and Bio-La-Test Triacylglycerol T 500 from Lachema (Brno, Czech Republic), TRIzol from Invitrogen Carlsbad (CA, USA), DNase I treatment from Roche (Maneheim, Germany), SuperScript II from Invitrogen Carlsbad (CA, USA), SYBR Green PCR Master Mix from Applied Biosystems (Weiterstadt, Germany). Other chemicals were of analytical grade. 2.2. Animals Male hereditary hypertriglyceridemic (HHTg, kindly provided by Dr. Kazdová of IKEM, Prague, Czech Republic) rats (body weight 180–240 g) and male Wistar–Kyoto SPF (specific pathogen free, BioTest, Konárovice, Czech Republic) rats (body weight 160–220 g) were maintained under standard laboratory conditions with free access to water. The animals (seven animals per group in each strain) were fed on a standard laboratory diet (STD; KrmiMo Mohelsky, Brno, Czech Republic) or on a high cholesterol diet (HCD) prepared by adding 1% (w/w) cholesterol to the STD. No additional fat was used when the diet was prepared. Rats were fed their respective diets ad libitum for a period of 20 days and the amounts of diet consumed were checked daily per cage of three rats. After this period, the rats were fasted overnight (16 h), anesthetized by intramuscular (i.m.) administration of fentanyl (40 lg kg1 of body weight) in combination with medetomidin (200 lg kg1 of body weight), followed by an i.m. administration of diazepam (5 mg kg1 of body weight). The liver was removed and rinsed in ice-cold sucrose solution (pH 7.4; 0.25 M sucrose, 1 mM Na2EDTA, 0.025 M TRIS), weighed, divided into several portions, and frozen in liquid nitrogen. Blood samples were taken from the aortic bifurcation and collected into Na2EDTA (1 mg ml1) and NaN3 (0.1 mg ml1). All experiments with animals were approved by the Ethics Committee of the Ministry of Education, Czech Republic. 2.3. Lipid determination Distribution of lipids was determined by established procedures (Havel et al., 1955; Haug and Hostmark, 1987). Plasma was separated by centrifugation (2500g, 20 min, 10 °C) and divided into two aliquots. The first aliquot of plasma was used for determination of cholesterol and triacylglycerols (TAG). The second aliquot of plasma was used for isolation of plasma lipoproteins (d < 1.006 g ml1 containing VLDL, d < 1.063 g ml1 containing LDL and d < 1.125 g ml1 containing HDL), which were isolated by sequential density gradient ultracentrifugation (Havel et al., 1955). Plasma, VLDL, LDL, HDL, and liver lipids were extracted according to Haug and Hostmark (1987). Cholesterol and TAG in plasma, lipoprotein fractions, and the liver extracts were measured enzymatically using commercial kits (BioLa-Test). 2.4. RNA isolation and RT-PCR procedures A 30–50-mg piece of liver tissue was homogenized (FastPrep, Qbiogene, Illkirch, France) and the RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Basel, Switzerland). Contaminating genomic DNA was removed by DNase I treatment (Roche, Rotkreuz, Switzerland). One microgram of RNA was reverse-transcribed with SuperScript II (Invitrogen, Carlsbad, CA, USA) using random primers. Generated cDNA was used for real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems, Weiterstadt, Germany) in an
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ABI PRISM 7700 Sequence Detection system with the following thermal cycling conditions: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min for denaturation, annealing and elongation. Cycle-to-cycle fluorescence emission was monitored and quantified using the SDS 1.1.9 software (Applied Biosystems, Weiterstadt, Germany). All samples were performed in duplicate. Rat primers were synthesized by Operon (Cologne, Germany). Data were normalized to hypoxanthine–guanine phosphoribosyltransferase (HPRT) and quantified using the comparative Ct method. The following primer sequences were used: PPARa Fw 50 -TGGAGTCCACGCATGTGAAG-30 , PPARa Rev 50 -CGCCAGCTTTAGCCGAATAG-30 , ACO Fw 50 -ATTGGCACCTACGCCCAG-30 , ACO Rev 50 -GTGGTTTCCAAGCCTCGAAG-30 , CYP4A1 Fw 50 -TGACATTCTGAAACCCTATGTAAAAAA-30 , CYP4A1 Rev 50 -CCTGCCAGCTGTTCCCATT-30 , CYP4A2 Fw 50 - GTTATGAAGTGTGCTTTCAGCCA-30 , CYP4A2 Rev 50 -CAACAGCCTTGGTGTAGGACC-30 , HPRT Fw 50 -TCGACCCTCAGTCCCAGC-30 , HPRT Rev 50 -CAGCATAATGATTAGGTATGCAAAATAAA-30 , LXRa Fw 50 -CCGGGAAGACTTTGCCAAA, LXRa.Rev 50 -CATGGATCTGGAGAACTCAAAGATG, FAS Fw 50 -GACATGGTCACAGACGATGACA, FAS Rev 50 -TTTCCAGACCGCTTAGGCA, SREBP-1c Fw 50 -GGAGCCATGGATTGCACATT, SREBP-1c Rev 50 -GCTTCCAGAGAGGAGCCCAG, ABCA1 Fw 50 -TACACCTGACACACCAGCTACAAG, ABCA1 Rev 50 -GGAACAAAGCCAGCTCCTGA, ABCG5 Fw 50 -GGCCAGACCATGTGCATCTT, ABCG5 Rev 50 -CCAGAGATGGCGTCCAGC, ABCG8 Fw 50 -GATGCTGGCTATCATAGGGAGC, ABCG8 Rev 50 -CTCTGCCTGTGATAACGTCGAG, CYP7A1 Fw 50 -TGGATCAAGTGCAACTGAATGAC, CYP7A1 Rev 50 -GCACTGGAAAGCCTCAGAGC, CYP2C11 Fw 50 -AGGAGGCTCAGTGCCTTGTG, CYP2C11 Rev 50 -CCAGGATAAAGGTGGGATCAAA, CYP2C6 Fw 50 -GCCTTGTGGAGGAACTGAGG, CYP2C6 Rev 50 -GCACAGCCCAGGATAAACGT, CYP2B2 Fw 50 -GGACACTGAAAAAGAGTGAAGCTTT, CYP2B2 Rev 50 -AATGCCTTCGCCAAGACAAA.
2.5. Statistical analysis All data are reported as the means ± SEM (n = 7). Differences between groups were analyzed using analysis of variance (ANOVA) followed by the appropriate post hoc test at the overall significance threshold of p < 0.05.
3. Results 3.1. Comparison of mRNA expression in HHTg and WKY rats fed with standard diet At first, the healthy (WKY) and hypertriglyceridemic (HHTg) rats were compared in their ability to express the 14 genes selected, when only a standard laboratory diet was given to both groups of animals. The results yielded a direct comparison of animals modeling human hypertriglyceridemia with the healthy ones under ‘‘normal” conditions, i.e. without the influence of elevated cholesterol intake. The data (Fig. 1) have shown important differences between healthy WKY and HHTg rats in basal expression of genes involved either in regulation of lipid homeostasis or in drug metabolism. Cytochrome P450 7A1 (CYP7A1) mRNA was expressed 3.4-fold more in HHTg rats than in the WKY ones. Basal expression of SREBP-1c was also higher (to 185%) in HHTg rats, this difference almost reached statistical significance (p = 0.07). Both CYP2C6 and CYP2B2 mRNA levels were significantly higher in HHTg rats than in WKY rats fed with STD (to 156% and 181%, respectively). In contrast to the genes mentioned above, hepatic expression of mRNA of ABCG5 and ABCG8 transporters was significantly lower in HHTg rats fed with STD as it decreased to 56 % and 41%. FAS mRNA levels were also significantly lower (decreased to 69%) in HHTg rats
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on STD when compared with healthy WKY rats. Of the selected enzymes of cytochrome P450 system only the level of CYP2C11 mRNA was lower in HHTg rats (decreased to 56%). (Levels of CYP2C6 and CYP2B2 mRNA increased as mentioned in the preceding paragraph.) The rest of the genes studied in this work (PPARa, LXRa, ACO, ABCA1, CYP4A1 and CYP4A2) did not significantly differ in their expression between the WKY and HHTg rats fed with STD. 3.2. Influence of high cholesterol diet on mRNA expression in WKY rats Dietary cholesterol caused significant increase of expression of SREBP-1c mRNA as well as of the CYP2B2 (to 280% and 159%, respectively); CYP7A1 mRNA increased to 194 % however without statistical significance. Genes which were expressed to a lower extent, were both corresponding to mRNAs of ABC transporters, ABCG5 and ABCG8 (to 38% and 21% of values obtained with standard diet). A similar finding was found for the CYP4A mRNAs (CYP4A1, to 69%, CYP4A2, to 57%); also, levels of mRNA of FAS
and ACO decreased significantly (to 63% and 77%, respectively). Results are displayed in Fig. 2. 3.3. Effect of high cholesterol diet on mRNA expression in HHTg rats In the HHTg rats, high cholesterol diet again caused (Fig. 3) a marked and significant increase of mRNA levels of SREBP-1c (to 514%) and of CYP7A1 (to 233%); lower expression of CYP4A2 as well as of ABCG5 and ABCG8 mRNAs was also found to be 50%, 50% and 24%, respectively. Interestingly, no other major or significant changes were observed resulting from dietary intake of cholesterol in this strain of rats. 3.4. Differences in response of HHTg and WKY rats to high cholesterol diet Changes in gene expression described above illustrated the differences between WKY and HHTg rats, either on the STD or HCD
Fig. 1. Differences of relative expression of genes involved in lipid homeostasis or drug metabolism between WKY and HHTg rats on STD. Expression in WKY rats on STD was set to 100%. Means ± SEM from groups of seven animals are shown (+p < 0.05, ++p < 0.02, +++p < 0.01, ++++p < 0.001 vs. healthy WKY rats on the STD diet).
Fig. 2. Differences of relative expression of genes involved in lipid homeostasis or drug metabolism between WKY rats on STD or HCD. Expression in WKY rats on STD was set to 100%. Means ± SEM from groups of seven animals are shown (xp < 0.05, xxp < 0.02, xxxp < 0.01, xxxxp < 0.001 vs. healthy WKY rats on the STD diet).
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diet (Fig. 4). While the majority of genes reacted to the cholesterol excess in the same way in WKY rats as in HHTg rats, three genes form the studied groups were expressed in a completely different manner. While the expression of CYP4A1 mRNA of WKY rats on HCD decreased to 69% compared to control WKY group fed STD, the HHTg rats showed no such reaction. In other words, no significant change in expression of CYP4A1 was observed in HHTg rats fed HCD (Fig. 4). The mRNA expression of another member of P450 family, CYP2B2, increased to 159% in WKY rats on HCD compared to WKY rats fed on STD. In contrast, in HHTg rats fed with HCD, no up-regulation was observed compared to HHTg rats on STD as the expression of CYP2B2 mRNA in HHTg rats on HCD (which was relatively high when these rats were on the STD, 181% of control WKY rats on STD, cf. Fig. 4) decreased (nonsignificantly) to 154%. Hepatic expression of FAS mRNA in WKY rats on
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HCD decreased to 63% when compared to the control group (WKY rats on STD). The HHTg rats again responded to the excessive amounts of cholesterol in a different way: instead of a significant decrease as in WKY rats, there was only a non-significant increase (from 69% to 80%).
3.5. Lipid parameters To confirm the hyperlipidemic effect of dietary cholesterol excess and to demonstrate its absorption, cholesterol and TAG levels were determined in plasma, lipoprotein fractions, and the liver. Parameters of lipid and lipoprotein metabolism are shown in Tables 1–3. High cholesterol diet (HCD) caused a significant accumulation of cholesterol and TAG in the liver as demonstrated in Table
Fig. 3. Differences of relative expression of genes involved in lipid homeostasis or drug metabolism between HHTg rats on STD or HCD. Expression in HHTg rats on STD was set to 100%. Means ± SEM from groups of seven animals are shown (op < 0.05, ooop < 0.01, oooop < 0.001 vs. HHTg rats on the STD diet).
Fig. 4. Different response of three genes to dietary cholesterol excess (HCD) between WKY and HHTg rats. Expression in WKY rats on STD was set to 100%. Means ± SEM from groups of seven animals are shown (+p < 0.05, xxp < 0.02, xxx,+++p < 0.01. = STD vs. HCD (WKY rats only), + = WKY vs. HHTg (STD only).
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Table 1 Cholesterol and TAG (lmol g1) in liver in WKY and HHTg rats. WKY rats
Cholesterol TAG
HHTg rats
STD
HCD
STD
HCD
10.13 ± 0.62 12.91 ± 1.22
15.23 ± 0.89 21.48 ± 2.24
9.60 ± 0.34 11.49 ± 1.29
19.59 ± 1.61*,ssss 23.07 ± 1.03ssss
STD, standard diet; HCD, high cholesterol diet. Means ± SEM from groups of seven animals are shown: *p < 0.05; ,ssssp < 0.001. = STD vs. HCD (WKY rats only), s = STD vs. HCD (HHTg rats only), = WKY vs. HHTg (HCD diet only).
Table 2 Cholesterol levels (mmol l1) in plasma and lipoprotein fractions. WKY rats
Plasma VLDL LDL HDL
HHTg rats
STD
HCD
STD
HCD
1.59 ± 0.11 0.07 ± 0.01 0.26 ± 0.03 0.74 ± 0.03
1.71 ± 0.10 0.21 ± 0.02 0.20 ± 0.02 0.56 ± 0.04
1.57 ± 0.08 0.11 ± 0.03 0.38 ± 0.04+ 0.81 ± 0.06
1.70 ± 0.08 0.30 ± 0.04ss 0.31 ± 0.04* 0.76 ± 0.05
STD, standard diet; HCD, high cholesterol diet. Means ± SEM from groups of seven animals are shown: *,+p < 0.05; ssp < 0.02; p < 0.01; p < 0.001. = STD vs. HCD (WKY rats only), s = STD vs. HCD (HHTg rats only), + = WKY vs. HHTg (STD only), = WKY vs. HHTg (HCD only).
Table 3 TAG levels (mmol l1) in plasma and VLDL fraction. WKY rats
Plasma VLDL
HHTg rats
STD
HCD
STD
HCD
0.48 ± 0.02 0.24 ± 0.05
0.67 ± 0.04 0.44 ± 0.04
0.83 ± 0.07++++ 0.46 ± 0.09+
0.74 ± 0.10 0.52 ± 0.07
STD, standard diet; HCD, high cholesterol diet. Means ± SEM from groups of seven animals are shown: +p < 0.05; p < 0.01; ++++p < 0.001. = STD vs. HCD (WKY rats only), + = WKY vs. HHTg (STD only).
1. Increase of cholesterol content in the liver was significantly higher in HHTg compared to WKY rats (Table 1). Distribution of cholesterol in lipoprotein fractions was significantly changed after administration of HCD (Table 2). Hypercholesterolemia was demonstrated by an increase of VLDL cholesterol levels, and associated with a decrease in HDL cholesterol levels in WKY rats. In HHTg rats fed HCD, an increase of VLDL cholesterol levels was the main change in the distribution of cholesterol in lipoprotein fractions. Focusing on the difference between in LDL levels between the rat strains, elevated level of LDL cholesterol in HHTg rats compared to WKY rats both on STD and HCD was detected. The HCD significantly increased levels of TAG in plasma in WKY rats, as shown in Table 3. The HCD also caused an apparent increase of TAG in the VLDL fraction in WKY rats. Although similar changes were not observed in HHTg rats, this rat strain exhibited a dyslipidemia manifesting by higher levels of TAG in plasma and VLDL fraction compared to WKY rats. 4. Discussion HHTg rats are a suitable model for phenotyping and genotyping such complex diseases as hypertension, hypertriglyceridemia, insulin resistance, and others, which represent components of the metabolic syndrome (Štolba et al., 1992; Zicha et al., 2006). However, even though the HHTg rats are a well established model
for metabolic syndrome, not many studies were performed to clarify the differences of this rat strain at molecular level. This work focuses (i) on differences in reference (i.e. with standard laboratory diet – STD) mRNA expression of several genes in liver samples of WKY and HHTg rats (as an accepted model of metabolic syndrome) (ii) on differences in the expression due to intake of high cholesterol diet – HCD. Genes were chosen with their respective role in lipid homeostasis and/or in drug metabolism. In parallel, the levels of cholesterol and TAG in plasma, lipoprotein fractions and in the liver were determined during this experiment. The results obtained here clearly indicate that HHTg rats have lower levels of ABCG5 and ABCG8 mRNA in liver compared to WKY rats (Fig. 1). ABCG5 and ABCG8 transporters are connected in a functional heterodimer and its function is to excrete sterols to intestinal lumen (in the intestine) and to the bile (in the liver). Higher hepatic expression of ABCG5/8 lowered the risk of atherosclerosis in mice on a ‘‘western” diet (Basso et al., 2007). The data also confirms the suitability of HHTg rats as a model of diabetes mellitus or insulin resistance as it is known that levels of ABCG5/ 8 mRNA in intestine and in liver are down-regulated in rats with streptozotocin induced diabetes (Bloks et al., 2004). This downregulation was also shown to coincide with a reduction of hepatobiliary cholesterol secretion and with an increased absorption of cholesterol together with increased plasma plant sterol concentrations (Bloks et al., 2004). When similar events occur in human diabetic patients, as suggested by increased plasma levels of plant sterols in subjects with poorly controlled Type I diabetes (Kojima et al., 1999), this fact could contribute to an enhanced risk for development of atherosclerosis (Sudhop et al., 2002). Feeding rats with HCD caused repression of ABCG5 and ABCG8 mRNA levels in the liver of both, WKY and HHTg rats (Figs. 2 and 3). This is in agreement with work of Dieter et al. (2004), who used the same diet (1% w/w of cholesterol) in Sprague–Dawley rats. (It is interesting to note that mice and rats exhibit differences regarding this parameter as it was published by Dieter et al. (2004) indicating interspecies differences in regulation of these genes controlled by the LXRa). Hydroxylation of cholesterol by CYP7A1 is the main step in bile acid synthesis; transformation into bile acids is necessary for elimination of cholesterol excess (Myant and Mitropoulos, 1977). Levels of CYP7A1 mRNA were increased in HHTg rats (Figs. 1 and 2). An interpretation of this fact might be based on a concomitant decrease of the sterol transport (decreased ABCG5/8 mRNA expression discussed previously) leading to a greater supply of cholesterol and hence in an increased need to convert this cholesterol surplus to bile acids. In other words, higher content of liver cholesterol may stimulate a regulation cascade resulting in up-regulation of CYP7A1 and conversion of abundant cholesterol into bile acids (Lehmann et al., 1997; Zhang et al., 2001). Hence, higher expression of CYP7A1 in HHTg rats can contribute to the fact that the plasma cholesterol levels are not elevated in this strain. Comparison with two other models of dyslipidemia, spontaneously hypertensive rats (SHR) and spontaneously hyperlipidemic rats (HLR), points to differences in regulation of lipid homeostasis in the experimental models. For example, no difference in expression of liver CYP7A1 mRNA was found in the literature in SHR rats in comparison to WKY (Kumai et al., 2003), however, expression of ABCG5/8 was not determined in this model (SHR) which makes a clear comparison of these two models (SHR and HHTg rats) impossible. An opposite effect, decreased level of CYP7A1 at the mRNA (Kumai et al., 2003) and protein (Brassil et al., 1998) level, was found in another model, i.e. in HLR derived from Sprague–Dawley rats. However, this model suffers with inherent hypercholesterolemia, and, hence, it is very likely that the decreased CYP7A1 level may be one of the mechanisms behind the increase in circulating cholesterol levels in HLR rats. This fact again documents that a
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more direct comparison of results with one parameter obtained with different experimental models may be often biased by individual characteristics of the model used. SREBP-1c (Sterol Regulatory Element Binding Protein 1c) belongs to important genes in lipid metabolism as it is crucial in de novo synthesis of fatty acids and triglycerides (Repa et al., 2000). The results presented in this work show that SREBP-1c mRNA level in liver is up-regulated in HHTg rats (Fig. 1). This fact corresponds with hypertriglyceridemia in HHTg rats (Klimeš et al., 1995). As it is well known that cholesterol excess induce expression of SREBP1c (Repa et al., 2000), feeding HHTg rats with HCD even accented the up-regulation of SREBP-1c mRNA (Fig. 3). This is in agreement with suggestion of Liu et al. (1995) and our previous results (Orolin et al., 2007) that increased cholesterol levels contribute to enhanced fatty acid synthesis and decreased fatty acid utilization in order to keep fatty acid (triglycerides)/cholesterol balance. Although rat models of metabolic syndrome are just an approximation of human disease, these models can provide invaluable data. Therefore, better characterization of these models is necessary to fully evaluate data from experimental models. The results presented here also show significant changes in the expression of mRNA of several enzymes of the cytochrome P450 family in response to the hypertriglyceridemia as well as on the diet. CYP2C11 is strongly down-regulated in HHTg rats compared to the WKY ones, both at the STD and HCD diet. The effects of dyslipidemia on expression of CYP2C11 mRNA in HHTg rats were in agreement with another model – Zucker rats – which exhibited a decrease of CYP2C11 mRNA compared to SD rats (Kim et al., 2004). Moreover, decrease of CYP2C11 was observed on the protein level in the SD rats with ‘‘poloxamer 407” – induced hyperlipidemia (Shayeganpour et al., 2008). Interestingly, rat CYP2C11 exhibits high (85%) sequence identity with human CYP2C9 (Pons et al., 1991) which is known to play an important role in metabolism of wide range of drugs such as S-warfarin or diclofenac (Rettie et al., 1992; Tang, 2003). These findings may be hence important with regard to possible drug–drug interactions as the decreased levels of this protein will be most probably reflected in elevated levels of drugs metabolized by this (down-regulated) enzyme. Expression of CYP2C6 mRNA was also changed significantly compared to controls as it was up-regulated in HHTg rats (Fig. 1). Different expression of CYP2C6 and CYP2C11 in HHTg rats should be explained by differences in regulation of these enzymes as suggested in earlier studies (Nadin et al., 1995). Not many studies were focused on CYP2C6 expression in different rat models of dyslipidemia or metabolic syndrome. Rat CYP2C6 shares high sequence similarity with another member of the CYP2C subfamily, human CYP2C19, which is reflected in the fact that both enzymes (rat CYP2C6 and human 2C19) metabolize similar group of xenobiotics (Scollon et al., 2009). CYP2C19 in humans is involved in metabolism of e.g. omeprazole or diazepam (Meyer, 1996). Changes in cytochrome P450 expression and activity therefore represent an important factor which must be considered when the response on the therapy is studied in experimental models of diseases such as metabolic syndrome as well in the light of possible consequences for human pharmacotherapy. The results of this study have clearly shown that there are genes with different response to dietary cholesterol excess in HHTg rats compared to WKY rats (Fig. 4). This finding is presented here for the first time as there is a lack of data in other models of dyslipidemia or metabolic syndrome in the literature. The results point at differences not only in the status of metabolic regulation caused by the disease itself, but also at possible important changes in the function of these genes due to abundant dietary cholesterol. The reason for the changes in expression of genes described may be an adjustment of organism to pathologic metabolic state; however, this hypothesis has to be verified by further experimental data.
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The results presented in this paper demonstrate accumulation of both cholesterol and TAG in the liver of rats (both WKY and HHTg) fed with HCD (Table 1). As expected, increased levels of TAG in plasma and VLDL in HHTg rats fed with STD (compared to WKY rats on the same diet) were observed here (Table 3). A positive correlation between blood pressure and plasma TAG concentration is known from the literature (Štolba et al., 1992). Hence, this, pathologically increased, TAG level may explain the absence of increase of TAG levels in plasma and VLDL fraction of HHTg rats on HCD. Increased TAG content in the liver of HHTg rats fed with HCD (Table 1), however, indicates up-regulation of de novo synthesis of fatty acids and TAG according to previously published suggestions (Liu et al., 1995; Orolin et al., 2007). 5. Conclusion In summary, this work documents significant differences in regulation of lipid homeostasis between healthy WKY rats and hypertriglyceridemic HHTg rats at the molecular level. The quantitative as well as qualitative differences are important not only with regard to regulation of lipid homeostasis (changes in expression of CYP7A1, ABC transporters or SREBP-1c) but, as well, due to possible consequences in metabolism of drugs and other xenobiotics (as a result of significant differences in expression of drug metabolizing enzymes) in organism facing high cholesterol diet. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This project was covered by Czech Ministry of Education, Youth and Sports grants OC09004 (COST B25), MSM 6198959216 and Grant Agency of the Czech Republic project 305/09/1177. The authors wish to thank Prof. Meyer (University of Basel) and Dr. Kazdová (IKEM Prague) for help in provision of experiments and for valuable discussions. References Basso, F., Freeman, L.A., Ko, C., Joyce, C., Amar, M.J., Shamburek, R.D., Tansey, T., Thomas, F., Wu, J., Paigen, B., Remaley, A.T., Santamarina-Fojo, S., Brewer Jr, H.B., 2007. Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited. J. Lipid Res. 48, 114– 126. Bloks, V.W., Bakker-van Waardel, W.M., Verkadel, H.J., Kema, I.P., Wolters, H., Vink, E., Groen, A.K., Kuipers, F., 2004. Down-regulation of hepatic and intestinal Abcg5 and Abcg8 expression associated with altered sterol fluxes in rats with streptozotocin-induced diabetes. Diabetologia 47, 104–112. Brassil, P.J., Debri, K., Nakura, H., Kobayashi, S., Davis, D.S., Edwards, R.J., 1998. Reduced hepatic expression of CYP7A1 and CYP2C13 in rats with spontaneous hyperlipidemia. Biochem. Pharmacol. 56, 253–257. Bray, G.A., 1977. The Zucker-fatty rat: a review. Fed. Proc. 36, 148–153. Dahl, L.K., 1961. Effects of chronic excess salt feeding. Introduction of selfsustaining hypertension in rats. J. Exp. Med. 114, 231–236. Dieter, M.Z., Maher, J.M., Cheng, X., Klaassen, C.D., 2004. Expression and regulation of the sterol half-transporter genes ABCG5 and ABCG8 in rats. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 4, 209–218. Haug, A., Hostmark, A.T., 1987. Lipoprotein lipases, lipoproteins and tissue lipids in rats fed fish oil or coconut oil. J. Nutr. 117, 1011–1017. Havel, R.J., Eder, H.A., Bragdon, J.H., 1955. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34, 1345–1353. Kim, M.S., Wang, S., Shen, Z., Kochansky, C.J., Strauss, J.R., Franklin, R.B., Vincent, S.H., 2004. Differences in the pharmacokinetics of peroxisome proliferatoractivated receptor agonists in genetically obese Zucker and Sprague–Dawley rats: implications of decreased glucuronidation in obese Zucker rats. Drug Metab. Dispos. 32, 909–914. Klimeš, I., Vrána, A., Kuneš, J., Šeböková, E., Dobešová, Z., Štolba, P., Zicha, J., 1995. Hereditary hypertriglyceridemic rat: a new animal model of metabolic alterations in hypertension. Blood Press. 4, 137–142.
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Kojima, H., Hidaka, H., Matsumura, K., Fujita, Y., Yamada, S., Haneda, M., Yasuda, H., Kikkawa, R., Kashiwagi, A., 1999. Effect of glycemic control on plasma plant sterol levels and post-heparin diamine oxidase activity in type 1 diabetic patients. Atherosclerosis 145, 389–397. Kumai, T., Oonuma, S., Kitaoka, Y., Tadokoro, M., Kobayashi, S., 2003. Biochemical and morphological characterization of spontaneously hypertensive hyperlipidaemic rats. Clin. Exp. Pharmacol. Physiol. 30, 537–544. Lehmann, J.M., Kliewer, S.A., Moore, L.B., Smith-Oliver, T.A., Oliver, B.B., Su, J.L., Sundseth, S.S., Winegar, D.A., Blanchard, D.E., Spencer, T.A., Willson, T.M., 1997. Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway. J. Biol. Chem. 272, 3137–3140. Liu, C.H., Huang, M.T., Huang, P.C., 1995. Sources of triacylglycerol accumulation in livers of rats fed a cholesterol supplemented diet. Lipids 30, 527–531. Meyer, U.A., 1996. Interaction of proton pump inhibitors with cytochromes P450: consequences for drug interactions. Yale J. Biol. Med. 69, 203–209. Myant, N.B., Mitropoulos, K.A., 1977. Cholesterol 7a-hydroxylase. J. Lipid Res. 18, 135–153. Nadin, L., Butler, A.M., Farrell, G.C., Murray, M., 1995. Pretranslational downregulation of cytochromes P450 2C11 and 3A2 in male rat liver by tumor necrosis factor alpha. Gastroenterology 109, 198–205. Orolin, J., Vecˇerˇa, R., Jung, D., Meyer, U.A., Škottová, N., Anzenbacher, P., 2007. Hypolipidemic effects of silymarin are not mediated by the peroxisome proliferator-activated receptor alpha. Xenobiotica 37, 725–735. Pons, C., Dansette, P.M., Amar, C., Jaouen, M., Wolf, C.R., Gregeois, J., Homberg, J.C., Mansuy, D., 1991. Detection of human hepatitis anti-liver kidney microsomes (LKM2) autoantibodies on rat liver sections is redominantly due to reactivity with rat liver P-450 IIC11. J. Pharmacol. Exp. Ther. 259, 1328–1334. Reaven, G.M., 1988. Role of insulin resistance in human disease. Diabetes 37, 1595– 1607. Reaven, G.M., Chang, H., 1991. Relationship between blood pressure, plasma insulin and triglyceride concentration, and insulin action in spontaneous hypertensive and Wistar–Kyoto rats. Am. J. Hypertens. 4, 34–38. Reaven, G.M., Twersky, J., Chang, H., 1991. Abnormalities of carbohydrate and lipid metabolism in Dahl rats. Hypertension 18, 630–635. Repa, J.J., Liang, G., Ou, J., Bashmakov, Y., Lobaccaro, J.M., Shimomura, I., Shan, B., Brown, M.S., Goldstein, J.L., Mangelsdorf, D.J., 2000. Regulation of mouse sterol
regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta. Genes Dev. 14, 2819–2830. Rettie, A.E., Korzekwa, K.R., Kunze, K.L., Lawrence, R.F., Eddy, A.C., Aoyama, T., Gelboin, H.V., Gonzalez, F.J., Trager, W.F., 1992. Hydroxylation of warfarin by human cDNA-expressed cytochrome P-450: a role for P-4502C9 in the etiology of (S)-warfarin-drug interactions. Chem. Res. Toxicol. 5, 54–59. Sarafidis, P.A., Nilsson, P.M., 2006. The metabolic syndrome: a glance at its history. J. Hypertens. 24, 621–626. Scollon, E.S., Starr, J.M., Godin, S.J., Devito, M.J., Hughes, M.F., 2009. In vitro metabolism of pyrethroid pesticides by rat and human hepatic microsomes and cytochrome P450 isoforms. Drug Metab. Dispos. 37, 221–228. Shayeganpour, A., Korashy, H., Patel, J.P., El-Kadi, A.O., Brocks, D.R., 2008. The impact of experimental hyperlipidemia on the distribution and metabolism of amiodarone in rat. Int. J. Pharm. 361, 78–86. Štolba, P., Dobešová, Z., Hušek, P., Opltová, H., Zicha, J., Vrána, A., Kuneš, J., 1992. The hypertriglyceridemic rat as a genetic model of hypertension and diabetes. Life Sci. 51, 733–740. Sudhop, T., Gottwald, B.M., von Bergman, K., 2002. Serum plant sterols as a potential risk factor for coronary heart disease. Metabolism 51, 1519–1521. Tang, W., 2003. The metabolism of diclofenac-enzymology and toxicology perspectives. Curr. Drug Metab. 4, 319–329. Vrána, A., Kazdová, L., 1990. The hereditary hypertriglyceridemic nonobese rat: an experimental model of human hypertriglyceridemia. Transpl. Proc. 22, 2579. Watanabe, M., Nakura, H., Tateishi, T., Tanaka, M., Kumai, T., Kobayashi, S., 1996. Reduced hepatic-drug metabolising enzyme activities in spontaneously hyperlipidemic rat as an animal model of hyper-cholesterolaemia. J. Med. Sci. Res. 24, 257–259. Yamori, Y., Ohta, M., Ueshima, H., Nara, Y., Horie, R., Shimamoto, T., Komachi, Y., 1978. Glucose tolerance in spontaneously hypertensive rats. Jpn. Circ. 42, 841– 847. Zhang, Z., Li, D.S., Blanchard, D.E., Lear, S.R., Erickson, S.K., Spencer, T.A., 2001. Key regulatory oxysterols in liver: analysis as D4-3-ketone derivatives by HPLC and response to physiological perturbations. J. Lipid Res. 42, 649–658. ˇ acˇányiová, S., Cebová, M., Kristek, F., Török, J., Šimko, F., Zicha, J., Pechánˇová, O., C Dobešová, Z., Kuneš, J., 2006. Hereditary hypertriglyceridemic rat: a suitable model of cardiovascular disease and metabolic syndrome? Physiol. Res. 55 (Suppl. 1), 49–63.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Differences in hepatic expression of genes involved in lipid homeostasis between hereditary hypertriglyceridemic rats and healthy Wistar rats and in their response to dietary cholesterol J. Orolin a, R. Vecera a,*, I. Markova b, A. Zacharova a, P. Anzenbacher a a b
Institute of Pharmacology, Faculty of Medicine, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic Institute for Clinical and Experimental Medicine, Videnska 1958/9, 140 21, Praha 4, Czech Republic
a r t i c l e
i n f o
Article history: Received 19 May 2009 Accepted 21 July 2009
Keywords: Hereditary hypertriglyceridemic rats Metabolic syndrome Wistar rats Dietary cholesterol Cytochromes p450
a b s t r a c t Differences in expression of mRNA of genes regulating lipid and drug metabolism between hereditary hypertriglyceridemic rats (HHTg, accepted model of metabolic syndrome) and healthy Wistar–Kyoto (WKY) rats were studied. Also, differences in expression due to intake of high cholesterol diet (1% w/w) were determined to investigate possible differences in response of the WKY and HHTg rats to increased intake of dietary cholesterol. Levels of ATP-binding cassette transporters (ABCG5, ABCG8), fatty acid synthase (FAS) and cytochrome P450 (CYP2C11) mRNA were significantly lower in HHTg rats on standard laboratory diet; in contrary, CYP7A1, CYP2C6 and CYP2B2 gene expression was significantly higher. The WKY rats responded to high cholesterol diet by an increase in expression of mRNAs for sterol regulatory element binding protein (SREBP1c), CYP2B2 and CYP7A1; lower expression was found in the FAS, ABCG5, ABCG8, CYP4A1, CYP4A2 and acyl-CoA oxidase. HHTg rats responded to cholesterol intake in a similar manner, however, differences were found in expression of the FAS and CYP4A1 mRNA (decrease was not observed), CYP2B2 (decrease instead of an increase). Conclusions: (i) dietary cholesterol significantly influences expression of genes involved in lipid homeostasis and drug metabolism, and (ii) the HHTg rats responded to dietary cholesterol in a different way. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Hypertriglyceridemia is an independent risk factor for coronary heart disease as it participates in development of atherosclerosis and hypertension. Moreover, hypertriglyceridemia is associated with several other metabolic disorders, such as insulin resistance, decreased level of HDL-cholesterol, hypertension, abdominal obesity and hyperinsulinemia. Reaven (1988) designated this cluster of related abnormalities as syndrome X, which was later named the metabolic syndrome or insulin resistance syndrome (Sarafidis and Nilsson, 2006).
Abbreviations: ABCA1, G5, G8, ATP-binding cassette transporters; ACO, acyl-CoA oxidase; CYP7A1, CYP2C6, CYP2C11, CYP2B2, CYP4A1, CYP4A2, types of cytochromes P450; FAS, fatty acid synthase; HCD, high cholesterol diet; HHTg, hereditary hypertriglyceridemic rats; HLR, spontaneously hyperlipidemic rats; LXRa, liver X receptor alpha; PPARa, peroxisome proliferator-activated receptor alpha; SD, Sprague–Dawley rats; SHR, spontaneously hypertensive rats; SPF, specific pathogen free; SREBP1c, sterol regulatory element binding protein 1c; STD, standard laboratory diet; WKY, Wistar–Kyoto rats. * Corresponding author. Tel.: +420 585 632 553; fax: +420 585 632 966. E-mail address: [email protected] (R. Vecera). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.07.022
Some interrelated symptoms of the metabolic syndrome such as insulin resistance, hypertriglyceridemia, and hypertension are known important cardiovascular risk factors, which are hence studied intensively. Several experimental models of insulin resistance and related abnormalities including hypertension have been developed in the spontaneously hypertensive rats (SHR) with hypertension, fasting hyperglycemia, impaired oral glucose tolerance, hyperinsulinemia and hypertriglyceridemia in comparison with healthy Wistar–Kyoto rats (WKY) (Yamori et al., 1978; Reaven and Chang, 1991). The second model of lipid dysregulation is represented by spontaneously hyperlipidemic rats (HLR) originated from Sprague–Dawley rats (SD), which show a higher plasma cholesterol level than control SD rats (Watanabe et al., 1996). Other genetically defined models of lipid disturbances are obese Zucker rats (Bray, 1977; Reaven et al., 1991), Dahl salt-sensitive rats (Dahl, 1961), also used for studies on insulin resistance (Reaven et al., 1991) or the hereditary hypertriglyceridemic (HHTg) rats (originally Wistar) modeling human hypertriglyceridemia (Vrána and Kazdová, 1990). Hypertension, hypertriglyceridemia, insulin resistance, hyperinsulinemia, signs of oxidative stress and other metabolic disturbances have been demonstrated in HHTg rats in comparison with controls (Klimeš et al., 1995).
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This work is focused on relative differences between the HHTg and healthy Wistar–Kyoto (WKY) rats in hepatic expression of mRNA of 14 genes important in regulation of lipid metabolism. In parallel, the effect of dietary cholesterol on differences in expression of the same genes was determined. The following genes expressing the following proteins known to be involved in regulation of lipid homeostasis were chosen: peroxisome proliferatoractivated receptor a (PPARa), liver X receptor a (LXRa), acyl-CoA oxidase (ACO), fatty acid synthase (FAS), ATP-binding cassette transporters (ABCA1, ABCG5 and ABCG8) and sterol regulatory element binding protein (SREBP1c) together with six enzymes of the cytochrome P450 family (CYP7A1, CYP2C6, CYP2C11, CYP4A1, CYP4A2, CYP2B2) which take part in biotransformation of lipids and are also important in drug–drug interactions (warfarin, diclofenac, diazepam, omeprazole and others) in therapy of wide range of diseases. 2. Materials and methods 2.1. Chemicals Cholesterol from Sigma (New York, USA), fentanyl was from Torrex Pharma (Vienna, Austria), medetomidin (Domitor) from Pfizer Animal Health (Brussels, Belgium), diazepam (Apaurin) from Krka (Slovenia), the Bio-La-Test Cholesterol 250 E and Bio-La-Test Triacylglycerol T 500 from Lachema (Brno, Czech Republic), TRIzol from Invitrogen Carlsbad (CA, USA), DNase I treatment from Roche (Maneheim, Germany), SuperScript II from Invitrogen Carlsbad (CA, USA), SYBR Green PCR Master Mix from Applied Biosystems (Weiterstadt, Germany). Other chemicals were of analytical grade. 2.2. Animals Male hereditary hypertriglyceridemic (HHTg, kindly provided by Dr. Kazdová of IKEM, Prague, Czech Republic) rats (body weight 180–240 g) and male Wistar–Kyoto SPF (specific pathogen free, BioTest, Konárovice, Czech Republic) rats (body weight 160–220 g) were maintained under standard laboratory conditions with free access to water. The animals (seven animals per group in each strain) were fed on a standard laboratory diet (STD; KrmiMo Mohelsky, Brno, Czech Republic) or on a high cholesterol diet (HCD) prepared by adding 1% (w/w) cholesterol to the STD. No additional fat was used when the diet was prepared. Rats were fed their respective diets ad libitum for a period of 20 days and the amounts of diet consumed were checked daily per cage of three rats. After this period, the rats were fasted overnight (16 h), anesthetized by intramuscular (i.m.) administration of fentanyl (40 lg kg1 of body weight) in combination with medetomidin (200 lg kg1 of body weight), followed by an i.m. administration of diazepam (5 mg kg1 of body weight). The liver was removed and rinsed in ice-cold sucrose solution (pH 7.4; 0.25 M sucrose, 1 mM Na2EDTA, 0.025 M TRIS), weighed, divided into several portions, and frozen in liquid nitrogen. Blood samples were taken from the aortic bifurcation and collected into Na2EDTA (1 mg ml1) and NaN3 (0.1 mg ml1). All experiments with animals were approved by the Ethics Committee of the Ministry of Education, Czech Republic. 2.3. Lipid determination Distribution of lipids was determined by established procedures (Havel et al., 1955; Haug and Hostmark, 1987). Plasma was separated by centrifugation (2500g, 20 min, 10 °C) and divided into two aliquots. The first aliquot of plasma was used for determination of cholesterol and triacylglycerols (TAG). The second aliquot of plasma was used for isolation of plasma lipoproteins (d < 1.006 g ml1 containing VLDL, d < 1.063 g ml1 containing LDL and d < 1.125 g ml1 containing HDL), which were isolated by sequential density gradient ultracentrifugation (Havel et al., 1955). Plasma, VLDL, LDL, HDL, and liver lipids were extracted according to Haug and Hostmark (1987). Cholesterol and TAG in plasma, lipoprotein fractions, and the liver extracts were measured enzymatically using commercial kits (BioLa-Test). 2.4. RNA isolation and RT-PCR procedures A 30–50-mg piece of liver tissue was homogenized (FastPrep, Qbiogene, Illkirch, France) and the RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Basel, Switzerland). Contaminating genomic DNA was removed by DNase I treatment (Roche, Rotkreuz, Switzerland). One microgram of RNA was reverse-transcribed with SuperScript II (Invitrogen, Carlsbad, CA, USA) using random primers. Generated cDNA was used for real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems, Weiterstadt, Germany) in an
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ABI PRISM 7700 Sequence Detection system with the following thermal cycling conditions: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min for denaturation, annealing and elongation. Cycle-to-cycle fluorescence emission was monitored and quantified using the SDS 1.1.9 software (Applied Biosystems, Weiterstadt, Germany). All samples were performed in duplicate. Rat primers were synthesized by Operon (Cologne, Germany). Data were normalized to hypoxanthine–guanine phosphoribosyltransferase (HPRT) and quantified using the comparative Ct method. The following primer sequences were used: PPARa Fw 50 -TGGAGTCCACGCATGTGAAG-30 , PPARa Rev 50 -CGCCAGCTTTAGCCGAATAG-30 , ACO Fw 50 -ATTGGCACCTACGCCCAG-30 , ACO Rev 50 -GTGGTTTCCAAGCCTCGAAG-30 , CYP4A1 Fw 50 -TGACATTCTGAAACCCTATGTAAAAAA-30 , CYP4A1 Rev 50 -CCTGCCAGCTGTTCCCATT-30 , CYP4A2 Fw 50 - GTTATGAAGTGTGCTTTCAGCCA-30 , CYP4A2 Rev 50 -CAACAGCCTTGGTGTAGGACC-30 , HPRT Fw 50 -TCGACCCTCAGTCCCAGC-30 , HPRT Rev 50 -CAGCATAATGATTAGGTATGCAAAATAAA-30 , LXRa Fw 50 -CCGGGAAGACTTTGCCAAA, LXRa.Rev 50 -CATGGATCTGGAGAACTCAAAGATG, FAS Fw 50 -GACATGGTCACAGACGATGACA, FAS Rev 50 -TTTCCAGACCGCTTAGGCA, SREBP-1c Fw 50 -GGAGCCATGGATTGCACATT, SREBP-1c Rev 50 -GCTTCCAGAGAGGAGCCCAG, ABCA1 Fw 50 -TACACCTGACACACCAGCTACAAG, ABCA1 Rev 50 -GGAACAAAGCCAGCTCCTGA, ABCG5 Fw 50 -GGCCAGACCATGTGCATCTT, ABCG5 Rev 50 -CCAGAGATGGCGTCCAGC, ABCG8 Fw 50 -GATGCTGGCTATCATAGGGAGC, ABCG8 Rev 50 -CTCTGCCTGTGATAACGTCGAG, CYP7A1 Fw 50 -TGGATCAAGTGCAACTGAATGAC, CYP7A1 Rev 50 -GCACTGGAAAGCCTCAGAGC, CYP2C11 Fw 50 -AGGAGGCTCAGTGCCTTGTG, CYP2C11 Rev 50 -CCAGGATAAAGGTGGGATCAAA, CYP2C6 Fw 50 -GCCTTGTGGAGGAACTGAGG, CYP2C6 Rev 50 -GCACAGCCCAGGATAAACGT, CYP2B2 Fw 50 -GGACACTGAAAAAGAGTGAAGCTTT, CYP2B2 Rev 50 -AATGCCTTCGCCAAGACAAA.
2.5. Statistical analysis All data are reported as the means ± SEM (n = 7). Differences between groups were analyzed using analysis of variance (ANOVA) followed by the appropriate post hoc test at the overall significance threshold of p < 0.05.
3. Results 3.1. Comparison of mRNA expression in HHTg and WKY rats fed with standard diet At first, the healthy (WKY) and hypertriglyceridemic (HHTg) rats were compared in their ability to express the 14 genes selected, when only a standard laboratory diet was given to both groups of animals. The results yielded a direct comparison of animals modeling human hypertriglyceridemia with the healthy ones under ‘‘normal” conditions, i.e. without the influence of elevated cholesterol intake. The data (Fig. 1) have shown important differences between healthy WKY and HHTg rats in basal expression of genes involved either in regulation of lipid homeostasis or in drug metabolism. Cytochrome P450 7A1 (CYP7A1) mRNA was expressed 3.4-fold more in HHTg rats than in the WKY ones. Basal expression of SREBP-1c was also higher (to 185%) in HHTg rats, this difference almost reached statistical significance (p = 0.07). Both CYP2C6 and CYP2B2 mRNA levels were significantly higher in HHTg rats than in WKY rats fed with STD (to 156% and 181%, respectively). In contrast to the genes mentioned above, hepatic expression of mRNA of ABCG5 and ABCG8 transporters was significantly lower in HHTg rats fed with STD as it decreased to 56 % and 41%. FAS mRNA levels were also significantly lower (decreased to 69%) in HHTg rats
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on STD when compared with healthy WKY rats. Of the selected enzymes of cytochrome P450 system only the level of CYP2C11 mRNA was lower in HHTg rats (decreased to 56%). (Levels of CYP2C6 and CYP2B2 mRNA increased as mentioned in the preceding paragraph.) The rest of the genes studied in this work (PPARa, LXRa, ACO, ABCA1, CYP4A1 and CYP4A2) did not significantly differ in their expression between the WKY and HHTg rats fed with STD. 3.2. Influence of high cholesterol diet on mRNA expression in WKY rats Dietary cholesterol caused significant increase of expression of SREBP-1c mRNA as well as of the CYP2B2 (to 280% and 159%, respectively); CYP7A1 mRNA increased to 194 % however without statistical significance. Genes which were expressed to a lower extent, were both corresponding to mRNAs of ABC transporters, ABCG5 and ABCG8 (to 38% and 21% of values obtained with standard diet). A similar finding was found for the CYP4A mRNAs (CYP4A1, to 69%, CYP4A2, to 57%); also, levels of mRNA of FAS
and ACO decreased significantly (to 63% and 77%, respectively). Results are displayed in Fig. 2. 3.3. Effect of high cholesterol diet on mRNA expression in HHTg rats In the HHTg rats, high cholesterol diet again caused (Fig. 3) a marked and significant increase of mRNA levels of SREBP-1c (to 514%) and of CYP7A1 (to 233%); lower expression of CYP4A2 as well as of ABCG5 and ABCG8 mRNAs was also found to be 50%, 50% and 24%, respectively. Interestingly, no other major or significant changes were observed resulting from dietary intake of cholesterol in this strain of rats. 3.4. Differences in response of HHTg and WKY rats to high cholesterol diet Changes in gene expression described above illustrated the differences between WKY and HHTg rats, either on the STD or HCD
Fig. 1. Differences of relative expression of genes involved in lipid homeostasis or drug metabolism between WKY and HHTg rats on STD. Expression in WKY rats on STD was set to 100%. Means ± SEM from groups of seven animals are shown (+p < 0.05, ++p < 0.02, +++p < 0.01, ++++p < 0.001 vs. healthy WKY rats on the STD diet).
Fig. 2. Differences of relative expression of genes involved in lipid homeostasis or drug metabolism between WKY rats on STD or HCD. Expression in WKY rats on STD was set to 100%. Means ± SEM from groups of seven animals are shown (xp < 0.05, xxp < 0.02, xxxp < 0.01, xxxxp < 0.001 vs. healthy WKY rats on the STD diet).
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diet (Fig. 4). While the majority of genes reacted to the cholesterol excess in the same way in WKY rats as in HHTg rats, three genes form the studied groups were expressed in a completely different manner. While the expression of CYP4A1 mRNA of WKY rats on HCD decreased to 69% compared to control WKY group fed STD, the HHTg rats showed no such reaction. In other words, no significant change in expression of CYP4A1 was observed in HHTg rats fed HCD (Fig. 4). The mRNA expression of another member of P450 family, CYP2B2, increased to 159% in WKY rats on HCD compared to WKY rats fed on STD. In contrast, in HHTg rats fed with HCD, no up-regulation was observed compared to HHTg rats on STD as the expression of CYP2B2 mRNA in HHTg rats on HCD (which was relatively high when these rats were on the STD, 181% of control WKY rats on STD, cf. Fig. 4) decreased (nonsignificantly) to 154%. Hepatic expression of FAS mRNA in WKY rats on
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HCD decreased to 63% when compared to the control group (WKY rats on STD). The HHTg rats again responded to the excessive amounts of cholesterol in a different way: instead of a significant decrease as in WKY rats, there was only a non-significant increase (from 69% to 80%).
3.5. Lipid parameters To confirm the hyperlipidemic effect of dietary cholesterol excess and to demonstrate its absorption, cholesterol and TAG levels were determined in plasma, lipoprotein fractions, and the liver. Parameters of lipid and lipoprotein metabolism are shown in Tables 1–3. High cholesterol diet (HCD) caused a significant accumulation of cholesterol and TAG in the liver as demonstrated in Table
Fig. 3. Differences of relative expression of genes involved in lipid homeostasis or drug metabolism between HHTg rats on STD or HCD. Expression in HHTg rats on STD was set to 100%. Means ± SEM from groups of seven animals are shown (op < 0.05, ooop < 0.01, oooop < 0.001 vs. HHTg rats on the STD diet).
Fig. 4. Different response of three genes to dietary cholesterol excess (HCD) between WKY and HHTg rats. Expression in WKY rats on STD was set to 100%. Means ± SEM from groups of seven animals are shown (+p < 0.05, xxp < 0.02, xxx,+++p < 0.01. = STD vs. HCD (WKY rats only), + = WKY vs. HHTg (STD only).
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Table 1 Cholesterol and TAG (lmol g1) in liver in WKY and HHTg rats. WKY rats
Cholesterol TAG
HHTg rats
STD
HCD
STD
HCD
10.13 ± 0.62 12.91 ± 1.22
15.23 ± 0.89 21.48 ± 2.24
9.60 ± 0.34 11.49 ± 1.29
19.59 ± 1.61*,ssss 23.07 ± 1.03ssss
STD, standard diet; HCD, high cholesterol diet. Means ± SEM from groups of seven animals are shown: *p < 0.05; ,ssssp < 0.001. = STD vs. HCD (WKY rats only), s = STD vs. HCD (HHTg rats only), = WKY vs. HHTg (HCD diet only).
Table 2 Cholesterol levels (mmol l1) in plasma and lipoprotein fractions. WKY rats
Plasma VLDL LDL HDL
HHTg rats
STD
HCD
STD
HCD
1.59 ± 0.11 0.07 ± 0.01 0.26 ± 0.03 0.74 ± 0.03
1.71 ± 0.10 0.21 ± 0.02 0.20 ± 0.02 0.56 ± 0.04
1.57 ± 0.08 0.11 ± 0.03 0.38 ± 0.04+ 0.81 ± 0.06
1.70 ± 0.08 0.30 ± 0.04ss 0.31 ± 0.04* 0.76 ± 0.05
STD, standard diet; HCD, high cholesterol diet. Means ± SEM from groups of seven animals are shown: *,+p < 0.05; ssp < 0.02; p < 0.01; p < 0.001. = STD vs. HCD (WKY rats only), s = STD vs. HCD (HHTg rats only), + = WKY vs. HHTg (STD only), = WKY vs. HHTg (HCD only).
Table 3 TAG levels (mmol l1) in plasma and VLDL fraction. WKY rats
Plasma VLDL
HHTg rats
STD
HCD
STD
HCD
0.48 ± 0.02 0.24 ± 0.05
0.67 ± 0.04 0.44 ± 0.04
0.83 ± 0.07++++ 0.46 ± 0.09+
0.74 ± 0.10 0.52 ± 0.07
STD, standard diet; HCD, high cholesterol diet. Means ± SEM from groups of seven animals are shown: +p < 0.05; p < 0.01; ++++p < 0.001. = STD vs. HCD (WKY rats only), + = WKY vs. HHTg (STD only).
1. Increase of cholesterol content in the liver was significantly higher in HHTg compared to WKY rats (Table 1). Distribution of cholesterol in lipoprotein fractions was significantly changed after administration of HCD (Table 2). Hypercholesterolemia was demonstrated by an increase of VLDL cholesterol levels, and associated with a decrease in HDL cholesterol levels in WKY rats. In HHTg rats fed HCD, an increase of VLDL cholesterol levels was the main change in the distribution of cholesterol in lipoprotein fractions. Focusing on the difference between in LDL levels between the rat strains, elevated level of LDL cholesterol in HHTg rats compared to WKY rats both on STD and HCD was detected. The HCD significantly increased levels of TAG in plasma in WKY rats, as shown in Table 3. The HCD also caused an apparent increase of TAG in the VLDL fraction in WKY rats. Although similar changes were not observed in HHTg rats, this rat strain exhibited a dyslipidemia manifesting by higher levels of TAG in plasma and VLDL fraction compared to WKY rats. 4. Discussion HHTg rats are a suitable model for phenotyping and genotyping such complex diseases as hypertension, hypertriglyceridemia, insulin resistance, and others, which represent components of the metabolic syndrome (Štolba et al., 1992; Zicha et al., 2006). However, even though the HHTg rats are a well established model
for metabolic syndrome, not many studies were performed to clarify the differences of this rat strain at molecular level. This work focuses (i) on differences in reference (i.e. with standard laboratory diet – STD) mRNA expression of several genes in liver samples of WKY and HHTg rats (as an accepted model of metabolic syndrome) (ii) on differences in the expression due to intake of high cholesterol diet – HCD. Genes were chosen with their respective role in lipid homeostasis and/or in drug metabolism. In parallel, the levels of cholesterol and TAG in plasma, lipoprotein fractions and in the liver were determined during this experiment. The results obtained here clearly indicate that HHTg rats have lower levels of ABCG5 and ABCG8 mRNA in liver compared to WKY rats (Fig. 1). ABCG5 and ABCG8 transporters are connected in a functional heterodimer and its function is to excrete sterols to intestinal lumen (in the intestine) and to the bile (in the liver). Higher hepatic expression of ABCG5/8 lowered the risk of atherosclerosis in mice on a ‘‘western” diet (Basso et al., 2007). The data also confirms the suitability of HHTg rats as a model of diabetes mellitus or insulin resistance as it is known that levels of ABCG5/ 8 mRNA in intestine and in liver are down-regulated in rats with streptozotocin induced diabetes (Bloks et al., 2004). This downregulation was also shown to coincide with a reduction of hepatobiliary cholesterol secretion and with an increased absorption of cholesterol together with increased plasma plant sterol concentrations (Bloks et al., 2004). When similar events occur in human diabetic patients, as suggested by increased plasma levels of plant sterols in subjects with poorly controlled Type I diabetes (Kojima et al., 1999), this fact could contribute to an enhanced risk for development of atherosclerosis (Sudhop et al., 2002). Feeding rats with HCD caused repression of ABCG5 and ABCG8 mRNA levels in the liver of both, WKY and HHTg rats (Figs. 2 and 3). This is in agreement with work of Dieter et al. (2004), who used the same diet (1% w/w of cholesterol) in Sprague–Dawley rats. (It is interesting to note that mice and rats exhibit differences regarding this parameter as it was published by Dieter et al. (2004) indicating interspecies differences in regulation of these genes controlled by the LXRa). Hydroxylation of cholesterol by CYP7A1 is the main step in bile acid synthesis; transformation into bile acids is necessary for elimination of cholesterol excess (Myant and Mitropoulos, 1977). Levels of CYP7A1 mRNA were increased in HHTg rats (Figs. 1 and 2). An interpretation of this fact might be based on a concomitant decrease of the sterol transport (decreased ABCG5/8 mRNA expression discussed previously) leading to a greater supply of cholesterol and hence in an increased need to convert this cholesterol surplus to bile acids. In other words, higher content of liver cholesterol may stimulate a regulation cascade resulting in up-regulation of CYP7A1 and conversion of abundant cholesterol into bile acids (Lehmann et al., 1997; Zhang et al., 2001). Hence, higher expression of CYP7A1 in HHTg rats can contribute to the fact that the plasma cholesterol levels are not elevated in this strain. Comparison with two other models of dyslipidemia, spontaneously hypertensive rats (SHR) and spontaneously hyperlipidemic rats (HLR), points to differences in regulation of lipid homeostasis in the experimental models. For example, no difference in expression of liver CYP7A1 mRNA was found in the literature in SHR rats in comparison to WKY (Kumai et al., 2003), however, expression of ABCG5/8 was not determined in this model (SHR) which makes a clear comparison of these two models (SHR and HHTg rats) impossible. An opposite effect, decreased level of CYP7A1 at the mRNA (Kumai et al., 2003) and protein (Brassil et al., 1998) level, was found in another model, i.e. in HLR derived from Sprague–Dawley rats. However, this model suffers with inherent hypercholesterolemia, and, hence, it is very likely that the decreased CYP7A1 level may be one of the mechanisms behind the increase in circulating cholesterol levels in HLR rats. This fact again documents that a
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more direct comparison of results with one parameter obtained with different experimental models may be often biased by individual characteristics of the model used. SREBP-1c (Sterol Regulatory Element Binding Protein 1c) belongs to important genes in lipid metabolism as it is crucial in de novo synthesis of fatty acids and triglycerides (Repa et al., 2000). The results presented in this work show that SREBP-1c mRNA level in liver is up-regulated in HHTg rats (Fig. 1). This fact corresponds with hypertriglyceridemia in HHTg rats (Klimeš et al., 1995). As it is well known that cholesterol excess induce expression of SREBP1c (Repa et al., 2000), feeding HHTg rats with HCD even accented the up-regulation of SREBP-1c mRNA (Fig. 3). This is in agreement with suggestion of Liu et al. (1995) and our previous results (Orolin et al., 2007) that increased cholesterol levels contribute to enhanced fatty acid synthesis and decreased fatty acid utilization in order to keep fatty acid (triglycerides)/cholesterol balance. Although rat models of metabolic syndrome are just an approximation of human disease, these models can provide invaluable data. Therefore, better characterization of these models is necessary to fully evaluate data from experimental models. The results presented here also show significant changes in the expression of mRNA of several enzymes of the cytochrome P450 family in response to the hypertriglyceridemia as well as on the diet. CYP2C11 is strongly down-regulated in HHTg rats compared to the WKY ones, both at the STD and HCD diet. The effects of dyslipidemia on expression of CYP2C11 mRNA in HHTg rats were in agreement with another model – Zucker rats – which exhibited a decrease of CYP2C11 mRNA compared to SD rats (Kim et al., 2004). Moreover, decrease of CYP2C11 was observed on the protein level in the SD rats with ‘‘poloxamer 407” – induced hyperlipidemia (Shayeganpour et al., 2008). Interestingly, rat CYP2C11 exhibits high (85%) sequence identity with human CYP2C9 (Pons et al., 1991) which is known to play an important role in metabolism of wide range of drugs such as S-warfarin or diclofenac (Rettie et al., 1992; Tang, 2003). These findings may be hence important with regard to possible drug–drug interactions as the decreased levels of this protein will be most probably reflected in elevated levels of drugs metabolized by this (down-regulated) enzyme. Expression of CYP2C6 mRNA was also changed significantly compared to controls as it was up-regulated in HHTg rats (Fig. 1). Different expression of CYP2C6 and CYP2C11 in HHTg rats should be explained by differences in regulation of these enzymes as suggested in earlier studies (Nadin et al., 1995). Not many studies were focused on CYP2C6 expression in different rat models of dyslipidemia or metabolic syndrome. Rat CYP2C6 shares high sequence similarity with another member of the CYP2C subfamily, human CYP2C19, which is reflected in the fact that both enzymes (rat CYP2C6 and human 2C19) metabolize similar group of xenobiotics (Scollon et al., 2009). CYP2C19 in humans is involved in metabolism of e.g. omeprazole or diazepam (Meyer, 1996). Changes in cytochrome P450 expression and activity therefore represent an important factor which must be considered when the response on the therapy is studied in experimental models of diseases such as metabolic syndrome as well in the light of possible consequences for human pharmacotherapy. The results of this study have clearly shown that there are genes with different response to dietary cholesterol excess in HHTg rats compared to WKY rats (Fig. 4). This finding is presented here for the first time as there is a lack of data in other models of dyslipidemia or metabolic syndrome in the literature. The results point at differences not only in the status of metabolic regulation caused by the disease itself, but also at possible important changes in the function of these genes due to abundant dietary cholesterol. The reason for the changes in expression of genes described may be an adjustment of organism to pathologic metabolic state; however, this hypothesis has to be verified by further experimental data.
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The results presented in this paper demonstrate accumulation of both cholesterol and TAG in the liver of rats (both WKY and HHTg) fed with HCD (Table 1). As expected, increased levels of TAG in plasma and VLDL in HHTg rats fed with STD (compared to WKY rats on the same diet) were observed here (Table 3). A positive correlation between blood pressure and plasma TAG concentration is known from the literature (Štolba et al., 1992). Hence, this, pathologically increased, TAG level may explain the absence of increase of TAG levels in plasma and VLDL fraction of HHTg rats on HCD. Increased TAG content in the liver of HHTg rats fed with HCD (Table 1), however, indicates up-regulation of de novo synthesis of fatty acids and TAG according to previously published suggestions (Liu et al., 1995; Orolin et al., 2007). 5. Conclusion In summary, this work documents significant differences in regulation of lipid homeostasis between healthy WKY rats and hypertriglyceridemic HHTg rats at the molecular level. The quantitative as well as qualitative differences are important not only with regard to regulation of lipid homeostasis (changes in expression of CYP7A1, ABC transporters or SREBP-1c) but, as well, due to possible consequences in metabolism of drugs and other xenobiotics (as a result of significant differences in expression of drug metabolizing enzymes) in organism facing high cholesterol diet. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This project was covered by Czech Ministry of Education, Youth and Sports grants OC09004 (COST B25), MSM 6198959216 and Grant Agency of the Czech Republic project 305/09/1177. The authors wish to thank Prof. Meyer (University of Basel) and Dr. Kazdová (IKEM Prague) for help in provision of experiments and for valuable discussions. References Basso, F., Freeman, L.A., Ko, C., Joyce, C., Amar, M.J., Shamburek, R.D., Tansey, T., Thomas, F., Wu, J., Paigen, B., Remaley, A.T., Santamarina-Fojo, S., Brewer Jr, H.B., 2007. Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited. J. Lipid Res. 48, 114– 126. Bloks, V.W., Bakker-van Waardel, W.M., Verkadel, H.J., Kema, I.P., Wolters, H., Vink, E., Groen, A.K., Kuipers, F., 2004. Down-regulation of hepatic and intestinal Abcg5 and Abcg8 expression associated with altered sterol fluxes in rats with streptozotocin-induced diabetes. Diabetologia 47, 104–112. Brassil, P.J., Debri, K., Nakura, H., Kobayashi, S., Davis, D.S., Edwards, R.J., 1998. Reduced hepatic expression of CYP7A1 and CYP2C13 in rats with spontaneous hyperlipidemia. Biochem. Pharmacol. 56, 253–257. Bray, G.A., 1977. The Zucker-fatty rat: a review. Fed. Proc. 36, 148–153. Dahl, L.K., 1961. Effects of chronic excess salt feeding. Introduction of selfsustaining hypertension in rats. J. Exp. Med. 114, 231–236. Dieter, M.Z., Maher, J.M., Cheng, X., Klaassen, C.D., 2004. Expression and regulation of the sterol half-transporter genes ABCG5 and ABCG8 in rats. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 4, 209–218. Haug, A., Hostmark, A.T., 1987. Lipoprotein lipases, lipoproteins and tissue lipids in rats fed fish oil or coconut oil. J. Nutr. 117, 1011–1017. Havel, R.J., Eder, H.A., Bragdon, J.H., 1955. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34, 1345–1353. Kim, M.S., Wang, S., Shen, Z., Kochansky, C.J., Strauss, J.R., Franklin, R.B., Vincent, S.H., 2004. Differences in the pharmacokinetics of peroxisome proliferatoractivated receptor agonists in genetically obese Zucker and Sprague–Dawley rats: implications of decreased glucuronidation in obese Zucker rats. Drug Metab. Dispos. 32, 909–914. Klimeš, I., Vrána, A., Kuneš, J., Šeböková, E., Dobešová, Z., Štolba, P., Zicha, J., 1995. Hereditary hypertriglyceridemic rat: a new animal model of metabolic alterations in hypertension. Blood Press. 4, 137–142.
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