Differential effects of gemfibrozil and fenofibrate on reverse cholesterol transport from macrophages to feces in vivo

Biochimica et Biophysica Acta 1811 (2011) 104–110

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Differential effects of gemfibrozil and fenofibrate on reverse cholesterol transport from macrophages to feces in vivo Noemí Rotllan a,b,1, Gemma Llaverías a,b,1, Josep Julve a,b, Matti Jauhiainen c, Laura Calpe-Berdiel a, Cristina Hernández b,d, Rafael Simó b,d, Francisco Blanco–Vaca a,b,e,⁎, Joan Carles Escolà-Gil a,b,⁎ a

IIB Sant Pau. 08025 Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas. CIBERDEM, 08036 Barcelona, Spain National Institute for Health and Welfare, and FIMM Institute for Molecular Medicine Finland, Biomedicum, Helsinki, Finland d Diabetes and Metabolism Research Unit. Institut de Recerca Hospital Universitari Vall d'Hebron. Universitat Autònoma de Barcelona. 08035 Barcelona, Spain e Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08025 Barcelona, Spain b c

a r t i c l e

i n f o

Article history: Received 29 September 2010 Received in revised form 4 November 2010 Accepted 19 November 2010 Available online 30 November 2010 Keywords: Fibrate PPAR-alpha HDL apoA-I Atherosclerosis Cardiovascular disease

a b s t r a c t Gemfibrozil and fenofibrate, two of the fibrates most used in clinical practice, raise HDL cholesterol (HDLc) and are thought to reduce the risk of atherosclerotic cardiovascular disease. These drugs act as PPARα agonists and upregulate the expression of genes crucial in reverse cholesterol transport (RCT). In the present study, we determined the effects of these two fibrates on RCT from macrophages to feces in vivo in human apoA-I transgenic (hApoA-ITg) mice. [3H]cholesterol-labeled mouse macrophages were injected intraperitoneally into hApoA-ITg mice treated with intragastric doses of fenofibrate, gemfibrozil or a vehicle solution for 17 days, and radioactivity was determined in plasma, liver and feces. Fenofibrate, but not gemfibrozil, enhanced [3H]cholesterol flux to plasma and feces of female hApoA-ITg mice. Fenofibrate significantly increased plasma HDLc, HDL phospholipids, hApoA-I levels and phospholipid transfer protein activity, whereas these parameters were not altered by gemfibrozil treatment. Unlike gemfibrozil, fenofibrate also induced the generation of larger HDL particles, which were more enriched in cholesteryl esters, together with higher potential to generate preβ-HDL formation and caused a significant increase in [3H]cholesterol efflux to plasma. Our findings demonstrate that fenofibrate promotes RCT from macrophages to feces in vivo and, thus, highlight a differential action of this fibrate on HDL. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Clinical and epidemiologic studies have demonstrated an inverse correlation between the concentration of plasma high-density lipoprotein cholesterol (HDLc) and apolipoprotein (apo) A-I and the incidence of atherosclerotic cardiovascular disease [1]. The most widely accepted mechanistic explanation for the HDL-mediated cardioprotective effects is that HDL promotes cholesterol efflux from lipid-laden macrophages located in the artery wall and delivers that cholesterol to the liver, from where it will be partly eliminated through bile and feces in a process termed reverse cholesterol transport (RCT) [1]. The flux of cholesterol through the entire macrophage-specific RCT pathway is crucial for antiatherogenic HDL

⁎ Corresponding author. Hospital de la Santa Creu i Sant Pau, Servei de Bioquímica, C/Antoni M Claret 167, 08025 Barcelona, Spain. Tel.: +34 93 2919451; fax: +34 93 2919196. E-mail addresses: [email protected] (F. Blanco–Vaca), [email protected] (J.C. Escolà-Gil). 1 Contributed equally to this work. 1388-1981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2010.11.006

function (reviewed in references 2,3). Therefore, the study of how these antiatherogenic functions are influenced by different treatments may provide a preclinical insight into their antiatherogenic potential [2,3]. Peroxisome proliferator-activated receptors (PPARs), which form a subfamily of the nuclear receptor gene family, are transcriptional factors activated by ligands [4]. Fibrates have long been known to be relatively weak PPARα agonists [4]. PPARα heterodimerizes with the retinoid X receptor (RXR) to modulate the expression of a number of target genes critical for lipid and lipoprotein metabolism [4,5]. Several clinical trials have demonstrated a benefit of fibrate treatment in cardiovascular risk reduction and improvement in the lipid profile [5]. The clinical effects of these drugs are attributed to their improving of the lipoprotein profile by reducing triglyceride and increasing HDLc levels [5]. Bezafibrate, gemfibrozil and fenofibrate have all shown atheroprotective effects in imaging studies. However, their effects in cardiovascular end-point prevention have yielded more complex results [5]. While several randomized controlled clinical trials with bezafibrate showed no significant protective effects in terms of hard outcomes (myocardial infarction, sudden death), several post hoc

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analyses showed significant event-rate reduction in patients with elevated triglyceride levels or the metabolic syndrome [6]. Gemfibrozil treatment has been associated with significant reductions in coronary events [5]. Treatment with fenofibrate failed to significantly prevent coronary death and non-fatal myocardial infarction events in patients with well-controlled type 2 diabetes mellitus in a recent clinical trial [5]. However, there was a significant reduction in total cardiovascular events, particularly in patients with high triglycerides and/or low HDLc [6]. The effects of fenofibrate in cardiovascular disease prevention are particularly important since current guidelines recommend its use for high-risk statin-treated patients with atherogenic dyslipidemia owing to its lower risk of causing severe myopathy and rhabdomyolysis [7]. Studies in vitro and in transgenic mice showed that fibrates increased liver human apoA-I production [8,9]. However, fenofibrate and gemfibrozil appeared to exert distinct effects on human apoA-I levels (reviewed in references 9,10). Fenofibrate also increased phospholipid transfer protein (PLTP) activity in mice [11,12], although fenofibrate and other fibrates seemed not to consistently increase human PLTP in human beings [12–16]. Furthermore, fibrates and other selective PPARα agonists increased macrophage cholesterol efflux in vitro by upregulating ATP-binding cassette transporter A1 (ABCA1) and limited macrophage foam cell formation and atherosclerosis [17–19]. However, the effects of fibrates on the entire macrophage-dependent RCT pathway are unknown. The present study tested the ability of fenofibrate and gemfibrozil to induce the macrophage-specific RCT pathway in vivo using a validated mouse assay in which radiolabeled cholesterol from macrophages is traced in plasma, liver and feces [2,3]. 2. Methods 2.1. Mice and diet All animal procedures were reviewed and approved by the Institutional Animal Care Committee of the Hospital de la Santa Creu i Sant Pau. Human (h) ApoA-I transgenic (Tg) mice were used in this study since human and mouse apoA-I genes are regulated by fibrates in an opposite manner [9]. Male and female 14- to 16-week-old hApoA-ITg mice in C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME; #003904). At the beginning of the study, male and female hApoA-ITg mice were matched for body weight, HDLc and hApoA-I levels and randomized into three groups treated respectively with daily intragastric doses of fenofibrate (250 mg/kg) (Sigma Diagnostics, St. Louis, MO, USA), gemfibrozil (625 mg/kg) (Sigma) or a vehicle solution (1.0% wt./vol. carboxymethylcellulose medium viscosity) for 17 days, a time at which a steady-state response is reached (see Online Figure 1). All mice were maintained on a regular chow diet (Safe, Scientific Animal Food & Engineering, Augy, France) containing 0.02% cholesterol. All animal manipulations began at 12 p.m. with the mice fed ad libitum. 2.2. In vivo macrophage-specific reverse cholesterol transport Macrophage-like cell line P388D1 derived from DBA/2 mice (ATCC; Manassas, VA) was cultured in 75-cm tissue culture plates at 5 million cells per plate and grown to 90% confluence in RPMI 1640 supplemented with 10% fetal bovine serum [20]. Mouse macrophages were incubated for 48 h in the presence of 5 μCi/mL of [1α,2α(n)-3H] cholesterol (GE Healthcare), 100 μg/mL of acetylated LDL and 10% lipoprotein-depleted serum. These cells were washed, equilibrated, detached by gently pipeting and resuspended in 0.9% (wt./vol.) saline and pooled before being intraperitoneally injected into the mice [20,21]. Two independent experiments were performed: one in hApoA-ITg males and the other in females. In both experiments, mice treated with a vehicle solution, fenofibrate and gemfibrozil,

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respectively, were injected intraperitoneally with [3H]cholesterollabeled P388D1 mouse macrophages (3.7 × 106 cells containing 1.36 × 106 cpm in 0.5 mL of saline for each mouse; cell viability was 77% measured by trypan blue staining). Mice were then individuallyhoused in metabolic cages and stools were collected over the next two days [20,21]. Plasma radioactivity was determined at 24 and 48 h by liquid scintillation counting. HDL-associated [3H]cholesterol was measured after precipitation of β-lipoproteins with phosphotungstic acid and magnesium ions (Roche Diagnostics). At that point, mice were euthanized and livers removed. Liver and fecal lipids were extracted with isopropyl alcohol–hexane. The lipid layer was collected, evaporated and [3H]cholesterol radioactivity measured by liquid scintillation counting [20,21]. The [3H]tracer detected in fecal bile acids was determined in the remaining aqueous portion of fecal material extracts. A known amount of [1α,2α(n)-3H]cholesterol (GE Healthcare) and [3H(G)]-taurocholic acid (PerkinElmer LAS, Boston, USA) was used as internal control. The amount of [3H] tracer was also expressed as a fraction of the injected dose. 2.3. Lipid analyses The methods used for plasma lipid analyses have been described in detail elsewhere [20–22]. HApoA-I levels were determined using nephelometric commercial kits (Kamiya, Biomedical Company, Seattle, WA) adapted to a BM/HITACHI 917 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland). Mouse apoA-I was quantified by an ELISA assay as previously reported [23]. Lipoprotein fractions, including HDL, were separated by an FPLC system using a Superose 6® column (GE Healthcare, Buckinghamshire, UK). Cholesterol and phospholipids were measured in each eluted fraction using the commercial kits for plasma. A crossed immunoelectrophoresis was carried out and the areas of α-HDL and preβ-HDL determined as previously reported [23]. 2.4. Liver and fecal analyses Stools from individually-housed mice were collected over 2 days (from the 15th to 17th days of treatment). Mice were euthanized and exsanguinated by cardiac puncture at the end of the study and livers removed after being perfused extensively with saline. Lipids were extracted from 1 g of feces and 100 mg of liver with isopropyl alcohol– hexane (2:3, vol./vol.) and cholesterol was determined using a commercial kit adapted to a BM/HITACHI 917 autoanalyzer [20,21]. Bile acids of 1 g of feces were extracted in ethanol and used to determine total bile acid content by the 3α-hydroxysteroid dehydrogenase method [20,21]. Net intestinal cholesterol absorption was measured in mice at the end of the study by a fecal dual-isotope ratio method as previously described [20]. 2.5. Enzyme activities and cholesterol efflux assay LCAT activity towards endogenous lipoproteins labeled with radioactive cholesterol was measured as previously reported [24]. PLTP radiometric assay was carried out as described [25]. Cellular cholesterol efflux to plasma was determined using [3H]cholesterollabeled P388D1 mouse macrophages as described [26]. 2.6. Metabolism of [3H]-cholesteryl oleate HDL [3H]cholesteryl oleate-labeled HDL (containing 1 million cpm) derived from hApoA-ITg female mice (specific activity of 4.5 × 103 cpm/pmol) were prepared and injected intravenously into each mouse as described [27]. Blood was collected into tubes at 1, 3, 6, 24 and 48 h and the radioactivity contained in 20 μL of plasma aliquots determined. This analysis was used to fit an exponential curve to each set of plasma-decay data and fractional

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catabolic rates and secretion rates of HDLc were determined [27]. At the end of the experiment, liver and fecal [3H]cholesterol and the [3H]tracer detected in fecal bile acids were determined as described above. 2.7. Quantitative real-time RT–PCR analyses The whole small intestine was cut into three segments with a length ratio of 1:3:2 (duodenum–jejunum–ileum). From the middle of each intestinal segment, 1.5 cm of the duodenal, jejunal and ileal tissues were extracted and pooled for each mouse. Liver and small intestine RNA were isolated using the trizol RNA isolation method (Gibco/BRL, Grand Island, NY, USA). Total RNA samples were repurified, checked for integrity by agarose gel electrophoresis and reverse-transcribed with Oligo(dT)15 using M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant to generate cDNA. Predesigned validated primers (Assays-on-Demand, Applied Biosystems, Foster City, CA) were used with Taqman probes. PCR assays were performed on an Applied Biosystems Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) as described [21]. All analyses were performed in duplicate and relative RNA levels were determined using GAPDH as internal control. Fibrate treatment did not affect the expression of the housekeeping gene. 2.8. Statistical methods One-way ANOVA with a Dunnett post-test was used to compare differences among groups. GraphPad Prism 4.0 software (GraphPad, San Diego, CA) was used to perform all statistical analyses. A P value b 0.05 was considered statistically significant.

Fecal [3H]cholesterol and bile acid excretions from radiolabeled P388D1 mouse macrophages (0.21 ± 0.06% and 0.22 ± 0.12% of the injected dose) were similar to those obtained with peritoneal endogenous murine macrophages in female hApoA-ITg mice (0.17 ± 0.03% and 0.16 ± 0.05% of the injected dose, respectively). To ascertain whether fibrates promote macrophage-dependent RCT pathway in vivo, radiolabeled P388D1 macrophages were injected into female hApoA-ITg mice treated with fenofibrate, gemfibrozil or a vehicle solution for 17 days. Fenofibrate enhanced [3H]cholesterol flux to plasma in female hApoA-ITg mice at both 24 and 48 h (Fig. 1A). The plasma [3H]cholesterol increase resulted mainly from the increase in radiolabeled HDL-bound [3H]cholesterol (Fig. 1A). 3 The [ H]tracer recovery was also measured in liver at 48 h and feces collected over 48 h. Liver [3H]cholesterol was slightly increased in fenofibrate-treated female hApoA-ITg mice (1.2-fold), but did not reach statistical significance compared to the vehicle group (Fig. 1B). Fecal [3H]cholesterol excretion in female hApoA-ITg mice given fenofibrate was 1.7-fold higher than that of mice given the vehicle solution (Fig. 1C). No significant changes were found in fecal [3H]bile acid excretion (Fig. 1D). Overall, fenofibrate increased the net [3H] cholesterol + bile acid excretion 1.5-fold compared with mice given the vehicle solution (P b 0.05). 3.2. Effects of fibrates on plasma lipids and liver and fecal parameters Mouse weight and dietary cholesterol intake were similar in the control and treated groups (Online Figure 1 and Table 1). After

11.00

VLDL+LDL cholesterol

* 5.50

50000 2.75

60000

4.4

30000

2.2

0.0 Fenofibrate

Gemfibrozilil

fib

ro zi

l

V hi l Vehicle

G

em

e

ra te

hi cl Ve

Fe n

of ib

ro zi

ra te

fib

G

em

e hi cl

of ib

Ve

Fe n

6.6

0

0.00 l

0

90000

% injeccted dose

8.25 100000

Liver [3H] cholesterol (cpm)

HDL cholesterol

*

150000

% injeccted dose

48h

24h

C

D 8000

0.590

0.295

2000 0

0.000 Vehicle

Fenofibrate

Gemfibrozil

8000

0.590

6000 4000

0.295

2000 0

% injeccted dose

4000

% injeccted dose

*

6000

Fecal [3H] bile acid output (cpm)

Plasma [3H] cholesterol (cpm/ml)

3.1. Effects of fibrates on in vivo macrophage-specific RCT pathway in female hApoA-ITg mice

B

A

Fecal [3H] cholesterol output (cpm)

3. Results

0.000 Vehicle

Fenofibrate

Gemfibrozil

Fig. 1. In vivo reverse cholesterol transport from macrophages to feces in female hApoA-ITg mice treated with a vehicle (n = 8), fenofibrate (n = 9) or gemfibrozil (n = 9) solution for 17 days. Individually-housed mice were injected intraperitoneally with [3H]cholesterol-labeled P388D1 mouse macrophages (3.7 × 106 cells containing 1.36 × 106 cpm in 0.5 mL of saline in each mouse). (A) Plasma total [3H]cholesterol and HDL-associated [3H]cholesterol at 24 and 48 h. (B) Liver [3H] cholesterol. (C and D) Fecal [3H]cholesterol and [3H]tracer from fecal bile acids over 48 h. Values are mean ± SEM. The amount of [3H]tracer was also expressed as a fraction of the injected dose. *P b 0.05 vs. vehicle mice.

N. Rotllan et al. / Biochimica et Biophysica Acta 1811 (2011) 104–110 Table 1 Plasma lipids in female hApoA-ITg mice treated for 17 days with vehicle, fenofibrate and gemfibrozil.

Weight (g) Total cholesterol (mmol/L) Free cholesterol (%) Phospholipids (mmol/L) Triglycerides (mmol/L) HDL cholesterol (mmol/L) HDL phospholipids (mmol/L)

Vehicle (n = 8)

Fenofibrate (n = 9)

Gemfibrozil (n = 9)

26.3 ± 7.7 4.1 ± 0.6 36.9 ± 3.7 4.1 ± 0.3 0.9 ± 0.2 3.2 ± 0.4 3.4 ± 0.3

26.4 ± 3.6 9.5 ± 0.9⁎ 31.2 ± 0.4 5.9 ± 0.2⁎ 0.4 ± 0.1⁎ 7.3 ± 0.7⁎ 5.3 ± 0.6⁎

22.8 ± 4.2 7.7 ± 1.4⁎ 33.0 ± 0.8 3.4 ± 0.4 0.5 ± 0.2⁎ 5.0 ± 0.8 2.6 ± 0.3

Values are mean ± SEM. ⁎ P b 0.05 vs. vehicle mice.

17 days of fenofibrate treatment, plasma total cholesterol and phospholipids and HDLc levels in female hApoA-ITg mice were 2.3-, 1.4-and 2.3-fold higher, respectively, than those of vehicle-treated mice (Table 1). Gemfibrozil treatment resulted in a significant increase in plasma total cholesterol (1.9-fold), with no significant effect on phospholipids and HDLc levels (Table 1). Both fibrates reduced triglyceride levels to a similar extent. FPLC analyses (Fig. 2), consistent with the results of Table 1, revealed that the fenofibrate treatment resulted in a large increase in cholesterol levels in HDL fractions together with an increased HDL particle size. Composition analyses of pooled HDL peak fractions showed that HDL of fenofibrate-treated female hApoA-ITg mice were enriched in esterified cholesterol (26.7% vs. 13.3% in vehicle mice), whereas phospholipid and protein % were reduced (38.0 vs. 46.8 in phospholipids and 24.1 vs. 32.0 in protein % of vehicle mice). HDL composition in gemfibrozil-treated mice was similar to that of vehicle-treated mice (data not shown). On the other hand, fenofibrate increased liver

HDL cholesterol (mM)

A

1.0

Vehicle Fenofibrate

0.8

Gemfibrozil 0.6 0.4

107

weight in female hApoA-ITg mice with no significant effects on liver cholesterol levels (Table 2). Fecal cholesterol output in fenofibratetreated mice was unchanged compared with gemfibrozil and vehicle groups (Table 2). However, a significant reduction in fecal bile acid output was observed in female mice given fenofibrate or gemfibrozil. None of the drugs affected net intestinal cholesterol absorption (Table 2). 3.3. Effects of fibrates on plasma hApoA-I, preβ-HDL and enzyme activities After 17 days of fenofibrate treatment, total plasma hApoA-I levels in female hApoA-ITg mice were 1.8-fold higher than those of vehicletreated mice (Fig. 3A). Fenofibrate treatment also resulted in an increase in the preβ-HDL fraction of total HDL hApoA-I compared with that of hApoA-ITg females given vehicle solution or gemfibrozil (Fig. 3B). Murine apoA-I levels were very low in hApoA-ITg mice given vehicle (0.52 ± 0.01 g/L) and did not differ from those of mice given fenofibrate or gemfibrozil (0.51 ± 0.01 and 0.53 ± 0.01 g/L, respectively). To ascertain whether the modifications in HDL composition induced by fenofibrate were associated with those of HDL-converting plasma enzyme activities, we determined murine LCAT and PLTP activities. Fenofibrate induced an increase in plasma PLTP activity (1.9-fold), with no significant effect on LCAT activity (Fig. 3C and D). 3.4. Effects of fibrates on in vitro macrophage cholesterol efflux and [3H]-cholesteryl oleate HDL catabolism The ability of plasma from each group to induce cholesterol efflux from [3H]cholesterol-labeled macrophages was also studied. The efflux to plasma of mice treated with fenofibrate was significantly increased compared with the vehicle and gemfibrozil groups (Fig. 4). In order to determine the fate of [3H]cholesterol from the HDL core, mice from each group were injected intravenously with HDL[3H]cholesteryl oleate (Table 3). None of the drugs affected the fractional catabolic rate of intravenously-injected [3H]HDL or the recovery of HDL-derived [3H]cholesterol in the liver or feces (Table 3). The secretion rate of HDLc in female hApoA-ITg mice given fenofibrate was 2.0-fold higher than that of mice given the vehicle solution (Table 3). 3.5. Effects of fibrates on liver and small intestine lipid-related gene expression

0.2 0.0 25

30

35

40

Both fibrates increased mRNA levels of liver PPARα, ABCG5, ABCG8 and hApoA-I (Fig. 5A and B). Fenofibrate strongly upregulated PLTP gene expression. ABCG1 gene expression was found to be significantly

Time (min)

HDL phospholipids (mM)

B

1.0

Table 2 Liver and fecal parameters in female hApoA-ITg mice treated for 17 days with vehicle, fenofibrate and gemfibrozil.

0.8 0.6 0.4 0.2 0.0 25

30

35

40

Time (min) Fig. 2. Representative fast protein liquid chromatography distribution profiles of HDL cholesterol (A) and HDL phospholipids (B) in pooled plasma from female hApoA-I-Tg mice treated with a vehicle, fenofibrate or gemfibrozil for 17 days.

Liver weight (g) Liver cholesterol (μmol/total liver) Dietary cholesterol intake (μmol/day/100 g bw) Fecal cholesterol output (μmol/day/100 g bw) Fecal bile acid output (μmol/day/100 g bw) Intestinal cholesterol absorption (%)⁎⁎

Vehicle (n = 8)

Fenofibrate (n = 9)

Gemfibrozil (n = 9)

1.0 ± 0.1 1.3 ± 0.1 21.2 ± 1.2

1.6 ± 0.1⁎ 1.5 ± 0.2 19.6 ± 0.7

1.1 ± 0.1 1.2 ± 0.1 17.8 ± 2.3

15.0 ± 2.8

15.7 ± 1.3

14.1 ± 2.0

6.3 ± 0.3 74.7 ± 4.4

4.0 ± 0.4⁎ 73.3 ± 4.0

4.5 ± 0.7⁎ 81.4 ± 2.2

Values are mean ± SEM. Bw, body weight. ⁎ P b 0.05 vs. vehicle mice. ⁎⁎ Intestinal cholesterol absorption was determined by a fecal dual-isotope ratio method in 5 animals per group.

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A

B Amount of HDL paarticles (%)

Human ApoA-I (g/L)

10 8

*

6 4 2 0

Vehicle

Fenofibrate

Gemfibrozil

80

*

60

*

40 20 0

Vehicle

Feno

Gemfibrozil

D 250

PLTP activity (μmol/L/h)

LCAT activity (μmol/L/h)

C

preβ-HDL α-HDL

100

200 150 100 50 0

Vehicle

Fenofibrate

Gemfibrozil

*

30000

20000

10000

0

Vehicle

Fenofibrate

Gemfibrozil

Fig. 3. Effects of fibrates on HDL hApoA-I distribution and plasma enzyme activities. (A) Plasma hApoA-I levels. (B) Amounts of α-mobile and preβ-mobile HDL particles quantified by two-dimensional crossed immunoelectrophoresis. (C and D) Plasma lecithin:cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP) activities. Results are expressed as mean ± SEM of 5 individual animals per group. *P b 0.05 vs. vehicle mice.

upregulated in the fenofibrate group, whereas LCAT and SR-BI expression was only upregulated in gemfibrozil group (Fig. 5A and B). No significant changes in small intestine ABCG5, ABCG8 and NPC1L1 mRNA expression were found among groups (Fig. 5C). 4. Discussion This study demonstrates for the first time, to our knowledge, the ability of fenofibrate to increase the macrophage-specific RCT pathway in vivo in female hApoA-ITg mice, a mouse model that elicits a humanized response to fibrates [9]. In contrast, gemfibrozil did not change the rate of macrophage-specific RCT in the same animal model. It is of note that both fibrates were given at periods able to reach maximum effectiveness in mice in vivo (see Online Figure 1) and, therefore, differences in the RCT rate did not appear to be related to the different fibrate doses administered (fenofibrate, 250 mg/kg and gemfibrozil, 625 mg/kg). In support of this, the increased macrophage-specific RCT in female fenofibrate-treated mice was observed despite their having a similar hypotriglyceridemic profile to those treated with gemfibrozil. As previously reported, fenofibrate increased plasma mouse PLTP activity [11,12] and HDLc and hApoA-I levels [9,10], whereas

gemfibrozil appeared to exert no effects on these parameters. The effect of fenofibrate on mouse PLTP activity is apparently murinespecific since fenofibrate did not increase the transcriptional activity of human PLTP promoter [13]. Nevertheless, systemic PLTP overexpression in transgenic mice induced HDL deficiency and impaired macrophage-specific RCT in vivo [28]. In contrast, PLTP expression resulted in a major increase in HDLc, apoA-I and preβ-HDL particles in hapoA-ITg mice [29]. This point together with an increased liver ABCG1 expression in fenofibrate-treated hapoA-ITg mice could emphasize the PLTP-mediated HDL changes in these Tg mice. Our results are also consistent with the recognized ability of PLTP to remodel HDL, thereby inducing both the formation of large fused HDL particles and preβ-HDL [30]. Therefore, the effects of fenofibrate on RCT in vivo might be attributed, at least in part, to an increased efflux from macrophages to these two efficient cholesterol acceptors in plasma [30]. Our results also rule out the possibility that fenofibrate differentially affected HDL cholesterol catabolism or inhibited intestinal cholesterol absorption and that this resulted in increased RCT in vivo in our hApoA-ITg mice, as occurred with liver SR-BI overexpression or ezetimibe treatment in mice [31,32]. This was rather surprising since fenofibrate has been reported to increase fecal cholesterol output in wild-type mice, probably by reducing

Macrophage cholesterol efflux to plasma (%/4h)

40

*

Table 3 In vivo clearance of [3H]cholesteryl oleate-radiolabeled HDL and excretion of tracer in fecal cholesterol and bile acids in female hApoA-ITg mice.

30

20

10

Vehicle

Fenofibrate

Gemfibrozil

Fig. 4. Effects of fibrates on in vitro macrophage cholesterol efflux. Cholesterol efflux from [3H]cholesterol-labeled P388D1 mouse macrophages to plasma at 4 h. Results are expressed as mean ± SEM of 5 individual animals per group. *P b 0.05 vs. vehicle mice.

Fractional catabolic rate (pools/h) Secretion rate (μmol HDLc/h/100 g bw) Liver [3H]cholesterol (% injected dose) Fecal [3H]cholesterol (% injected dose) Fecal [3H]bile acids (% injected dose)

Vehicle

Fenofibrate

Gemfibrozil

0.081 ± 0.015

0.076 ± 0.015

0.082 ± 0.011

1.2 ± 0.2

2.5 ± 0.5⁎

1.7 ± 0.2

2.4 ± 0.2

3.2 ± 0.4

2.5 ± 0.6

0.21 ± 0.03

0.26 ± 0.05

0.18 ± 0.02

0.16 ± 0.07

0.14 ± 0.04

0.19 ± 0.06

Values are mean ± SEM of 5 animals per group. ⁎ P b 0.05 vs. vehicle mice.

N. Rotllan et al. / Biochimica et Biophysica Acta 1811 (2011) 104–110

Relative liver mRNA levels

A

5

Fenofibrate 4 Gemfibrozil 3

2

**

*

* *

*

1

0

B

*

Vehicle

PPARα

HapoA-I ABCA1 ABCG1

LCAT

PLTP

5

Relative liver mRNA levels

4

*

3

2

*

* *

*

1

0

Relative small intestine mRNA levels

C

Α ΑBCG5

ABCG8

SR-BI

CYP7A1

5

4

109

gemfibrozil to raise hepatic lipase activity [14] which could increase hApoA-I catabolism [35]. Consistent with plasma PLTP activity, our data also indicated that fenofibrate has a significant and differential effect on the expression of liver PLTP. It is interesting to note that fenofibrate is chemically different from gemfibrozil [36] and, thus, the nature of fibrates may influence coactivator recruitment and expression of this gene [11,12]. An intriguing observation was the moderate effects of fibrates on liver ABCA1, considerably less than that seen in HepG2 cells and mouse hepatocytes [37]. This could be related to the activation of certain ABCA1 repressors such as SREBP1 by PPARα in vivo [38,39]. In contrast to female data, our results show that the overall macrophage-specific RCT rate in male hApoA-ITg mice (determined as net [3H]cholesterol + bile acid excretion) was not significantly increased by fenofibrate (Online Figure 2). This was concomitant with a major increase in the liver content of cholesterol in fenofibratetreated hApoA-ITg male mice (see Online Table 1 and Online Figure 2). The different accumulation of cholesterol in liver between mouse sexes has been attributed to sex-specific hormones [40,41] and fenofibrate treatment results in pronounced hepatomegaly in rodents, particularly in males [42]. Thus, an accumulation and enhanced re-esterification of liver [3H]cholesterol may constitute an explanation for the comparatively lower excretion of [3H]cholesterol + bile acids in feces of male hApoA-ITg mice given fenofibrate. In conclusion, our data indicate that fenofibrate promoted increased excretion of macrophage-derived cholesterol into feces in female hApoA-ITg mice. Our data would be consistent with a protective clinical effect of fenofibrate that remains to be clearly demonstrated in interventional studies directed at reducing the residual risk in high-risk patients after statin treatment. Supplementary materials related to this article can be found online at doi:10.1016/j.bbcan.2010.11.003.

3

Disclosures 2

The authors declare that there is no duality of interest associated with this article.

1

Acknowledgments 0

ΑBCG5

ABCG8

NPC1L1

Fig. 5. Real-time RT–PCR quantification of relative mRNA expression in livers and small intestines. (A and B) Relative mRNA expression of liver genes involved in HDL remodeling and hepatobiliary cholesterol excretion in female hApoA-ITg mice treated with a vehicle, fenofibrate or gemfibrozil solution for 17 days. (C) Small intestine relative mRNA expression of genes involved in intestinal cholesterol absorption. The signal of vehicle-treated hApoA-ITg mice was set at a normalized value of 1 arbitrary unit. GAPDH and 18S was used as internal control. Results are expressed as mean ± SEM of 5 individual animals per group. *Significant differences between mice treated with fenofibrate or gemfibrozil vs. those treated with a vehicle solution are indicated (P b 0.05).

intestinal cholesterol absorption [33]. However, one key point should be taken into account. That report did not determine the effect of fenofibrate in hApoA-ITg mice [33] and HDL metabolism is regulated in an opposite manner by fibrates in wild-type rodents due to sequence divergences in the mouse apoA-I promoter [34]. It should also be noted that cholesterol originating in macrophages is minimal compared to that of remaining tissues and, therefore, the total fecal cholesterol or bile acid excretion does not reflect macrophagespecific RCT [2,3]. As expected, both fibrates increased PPARα and two key liver regulated genes involved in cholesterol transport to bile and feces, ABCG5 and G8 [21]. HApoA-I mRNA levels were associated with plasma protein levels in fenofibrate-treated hApoA-ITg mice. However, gemfibrozil did not affect hApoA-I levels despite increasing mRNA levels. These findings may be related to the ability of

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