Mol. Nutr. Food Res. 2015, 59, 1987–1996
1987
DOI 10.1002/mnfr.201500336
RESEARCH ARTICLE
Consumption of polyunsaturated fat improves the saturated fatty acid-mediated impairment of HDL antioxidant potential 1,4 ´ L´ıdia Cedo´ 1,2 , Jari Metso3 , David Santos1,2 , Jose Lu´ıs Sanchez-Quesada , Josep Julve1,2,4 , 1 1,4 3 ´ ´ , Josefina Mora-Brugues , Matti Jauhiainen , Annabel Garc´ıa-Leon 1,2,4∗ 1,2,4 ` and Joan Carles Escola-Gil Francisco Blanco-Vaca 1
` Institut d’Investigacions Biomediques (IIB) Sant Pau, Barcelona, Spain ´ CIBER de Diabetes y Enfermedades Metabolicas Asociadas, CIBERDEM Madrid, Spain 3 National Institute for Health and Welfare, Genomics and Biomarkers Unit, Biomedicum, Helsinki, Finland 4 ` Departament de Bioqu´ımica i Biolog´ıa Molecular, Universitat Autonoma de Barcelona, Barcelona, Spain 2
Scope: The present study aimed to compare the effects of diets containing high-fat, highcholesterol and saturated fatty acids (HFHC-SFA) and HFHC-polyunsaturated fatty acidscontaining (HFHC-PUFA) diets on two major antiatherogenic functions of HDL, the HDL antioxidant function and the macrophage-to-feces reverse cholesterol transport. Methods and results: Experiments were carried out in mice fed a low-fat, low-cholesterol (LFLC) diet, an HFHC-SFA diet or an HFHC-PUFA diet in which SFAs were partly replaced with an alternative high-linoleic and ␣-linolenic fat source. The HFHC-SFA caused a significant increase in serum HDL cholesterol and phospholipids as well as elevated levels of oxidized HDL and oxidized LDL. Replacing SFA with PUFA significantly reduced the levels of these oxidized lipoproteins and enhanced the ability of HDL to protect LDL from oxidation. The SFA-mediated impairment of HDL antioxidant potential was not associated with the cholesterol content of the diet, obesity or insulin resistance. In contrast, the effect of the HFHC diets on fecal macrophage-derived cholesterol excretion was independent of the fatty acid source. Conclusion: SFA intake impairs the antioxidant potential of HDL and increases serum levels of oxidized lipoprotein species whereas the antioxidant potential of HDL is enhanced after PUFA consumption.
Received: April 29, 2015 Revised: June 12, 2015 Accepted: June 21, 2015
Keywords: HDL / Oxidation / Reverse cholesterol transport / Saturated fatty acids / Polyunsaturated fatty acids
1
Introduction
Western dietary transition increases the incidence of noncommunicable diseases such as type 2 diabetes and coronary heart disease (CHD) [1]. The conventional western diet ` Correspondence: Joan Carles Escola-Gil E-mail: [email protected] Abbreviations: ABC, ATP-binding cassette; apo, apolipoprotein; CHD, coronary heart disease; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; HDL-C, HDL-cholesterol; HFHC diet, highfat and high-cholesterol diet; LDL-C, LDL cholesterol; LXR, liver X receptor; LFLC diet, low-fat and low-cholesterol diet; oxLDL, oxidized LDL; PAF-AH, platelet-activating factor acetyl-hydrolase; PON, paraoxonase; RCT, reverse cholesterol transport; SFA, saturated fatty acids C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
is high in saturated fatty acids (SFAs) and substantial evidence shows that dietary SFA intake is associated with an increased risk of CHD [2–4]. This effect is thought to be mediated by an increase in low-density cholesterol (LDL-C), whereas rather paradoxically with respect to CHD, SFA intake increases high-density lipoprotein cholesterol (HDL-C) (reviewed in [5]). Conversely, PUFAs usually reduces HDL-C [5], whereas they are potentially atheroprotective [6, 7]. Macrophage-specific reverse cholesterol transport (RCT) and HDL antioxidant function are considered among the most important HDL-mediated cardioprotective mechanisms [8, 9]. Dietary fatty acid composition may influence HDL antioxidant function. HDL from human subjects fed a high ∗ Additional corresponding author: Francisco Blanco-Vaca, E-mail: [email protected]
www.mnf-journal.com
1988
L. Cedo´ et al.
PUFA diet, mainly containing linoleic acid (C18:2, n-6), showed a reduced susceptibility to lipid oxidation when compared with MUFA-enriched diet [10, 11]; however, the potential of HDL particles to inhibit LDL oxidation was similar [12]. A diet containing high-fat, high-cholesterol and saturated fatty acids (HFHC-SFA) accelerated HDL lipid oxidation in hypercholesterolemic mice. However, that diet contained sodium cholate which produces significant hepatotoxic effects and the reference diet was a low-fat, low-cholesterol (LFLC) diet containing large amounts of carbohydrates [13]. On the other hand, several human intervention studies showed no effect on the in vitro ability of HDL to stimulate macrophage cholesterol efflux, the first RCT step, when dietary SFA intake was replaced by PUFA [14, 15]. In contrast, n-6 PUFA promoted HDL-mediated cholesterol efflux from human fibroblast, although the effects of n-3 and n-6 PUFA, mainly ␣-linolenic (C18:3) and linoleic acids, respectively, were divergent [16]. Nonetheless, the entire RCT pathway from macrophages back to the liver via HDL and its fecal excretion comprises various susceptible regulatory steps in different body compartments; thus, the changes in cholesterol efflux might not reflect the flux of [3 H]cholesterol through different compartments [17]. We demonstrated, in mice, that a HFHC-SFA diet promoted a sustained compensatory in vivo macrophage-to-feces RCT that was dependent on dietary cholesterol-mediated liver X receptor (LXR) signaling on liver ATP-binding cassette (ABC) G5/G8 expression. However, our study did not address whether the differences in the sources of fatty acids altered the macrophage RCT pathway [18]. It has also been reported that an HFHC diet containing eicosapentaenoic acid (EPA; C20:5, n-3) and docosahexaenoic acid (DHA; C22:6, n-3) from fish oil promoted macrophage RCT in mice when compared with mice fed an HFHC diet containing mainly linoleic acid; these changes were consistent with liver ABCG5/G8 upregulation [19]. In the present study, we evaluated the effect of an HFHCSFA diet on HDL antioxidant function and on the in vivo macrophage-specific RCT pathway compared with those produced by an HFHC-PUFA diet in which SFA was partly replaced by an alternative PUFA source containing mainly the linoleic and ␣-linolenic acids. Our results demonstrate that SFA intake impairs HDL antioxidant potential but not the macrophage-to-feces RCT pathway, whereas concomitant PUFA consumption improved the antioxidant potential of HDL particles.
Mol. Nutr. Food Res. 2015, 59, 1987–1996
Table 1. Diet nutrient information and fatty acid profile for fat sources in LFLC, HFHC-SFA and HFHC-PUFA diets
LFLC
HFHC-SFA
HFHC-PUFA
Protein (% by weight) Carbohydrate (% by weight) Fat (% by weight) Cholesterol (%)
16.1
17.3
17.3
59.9
48.7
48.7
3.1
21.2 0.2
21.2 0.2
Fatty acid (% total fatty acids)
LFLC
HFHC-SFA
HFHC-PUFA
SFA MUFA PUFA C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
23.5 17.9 58.6
65.2 31.3 3.5 3.8 2.3 1.1 2.0 3.1 11.7 26.2 1.9 12.5 28.2 2.9 0.5
40.2 27.4 32.4 1.9 1.2 0.6 1.0 1.6 5.9 18.6 1.0 8.3 25.9 28.1 4.3
21.3 0.5 2.2 17.4 54.2 4.3
the following diets for eight additional weeks: (i) a LFLC diet (A04; Safe, Scientific Animal Food & Engineering, Augy, France) containing 3% fat and 0.02% cholesterol; (ii) an HFHC-SFA diet (Western-type diet TD.09821; Harlan Teklad, Madison, WI) containing 21% anhydrous milkfat and 0.2% cholesterol; (iii) an HFHC-PUFA diet (Western-type diet TD.130185; Harlan Teklad, Madison, WI) containing 10.5% anhydrous milkfat, 10.5% soybean fat and 0.2% cholesterol; or (iv) a high-fat and low-cholesterol (HFLC)SFA diet (Western-type diet TD.130184; Harlan Teklad, Madison, WI) containing 21% anhydrous milkfat and 0.05% cholesterol. The HFHC-PUFA and the HFLC-SFA diets were prepared exactly as the HFHC-SFA diet with the only differences being the fat source in the HFHC-PUFA diet and the cholesterol supplementation in the HFLC-SFA diet. Detailed descriptions of the diet composition are provided in Table 1.
2.2 Lipid and apolipoprotein analyses
2
Materials and methods
2.1 Mice and diets All animal procedures were approved by the Institutional Animal Care Committee of the Institut de Recerca of the Hospital de la Santa Creu i Sant Pau. Eight-week-old male C57BL/6 wild-type mice (#000664; Jackson Laboratories, Bar Harbor, ME) were randomized into four groups and fed C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
At the end of the study, mice were fasted overnight (o/n), euthanized and exsanguinated via cardiac puncture and livers and epididymal fat pads were removed. Serum lipid and lipoprotein analyses were determined enzymatically using commercial kits adapted to a COBAS 6000 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland) [18]. Mouse apoA-I assay was quantified with an ELISA coated with a polyclonal rabbit antibody against mouse apoA-I [20]. HDL was isolated www.mnf-journal.com
1989
Mol. Nutr. Food Res. 2015, 59, 1987–1996
by sequential ultracentrifugation at 100 000 × g for 24 h at a density of 1.063–1.21 g/mL [21]. The composition of isolated HDL was determined using the commercial methods adapted to the COBAS 6000 autoanalyzer. HDL protein concentrations were determined with the bicinchoninic acid (BCA) method (TermoScientific, Rockford, IL). In order to analyze HDL fatty acid composition, a lipid extraction from HDL isolated by ultracentrifugation was performed with isopropyl alcohol-hexane (2:3, v/v). After hexane evaporation of the lipid phase, samples were saponified with KOH/MeOH 6% o/n, extracted with hexane and KOH/MeOH phase was decanted and acidified with HCl. Samples were then extracted with hexane; after evaporation, BF3 /MeOH was added and left to react o/n. Samples were extracted again with hexane; after evaporation, fatty acids were determined by gas chromatography/MS (Shimadzu QP2010, Kyoto, Japan) using a BPX70 column measuring 30 m x 0.25 mm x 0.25 m. Liver lipids were extracted with isopropyl alcohol–hexane. The lipid layer was collected, evaporated and resuspended in cholate 0.5% (w/v) for lipid determinations [18].
2.3 Enzyme activities Total plasma arylesterase activity was measured using phenylacetate as substrate [22]. EDTA-sensitive serum arylesterase activity was calculated by subtracting the EDTA-resistant arylesterase, which was used to determine paraoxonase (PON) 1 activity. PON1 activity was also measured using paraoxon (Sigma, St. Louis, MO) as substrate [23]. Plateletactivating factor acetyl-hydrolase (PAF-AH) activity was determined using a commercial kit (Cayman Chemical, Ann Arbor, MI) [22]. ␣-tocopherol was determined by HPLC as previously described [24].
2.4 Susceptibility to lipoprotein oxidation Mouse serum oxidized (ox) LDL and oxHDL levels were assayed with two specific ELISA kits (Cloud-Clone, Houston, TX and MyBioSource, San Diego, CA, respectively). The ability of HDL to protect against LDL oxidation in vitro was based on an assay in which human LDL is oxidized alone by CuSO4 or in the presence of HDL and the oxidation kinetics is followed by continuous monitoring of the formation of conjugated diene [21, 22]. Briefly, oxidation was started by adding 2.5 M CuSO4 to LDL, mouse HDL or LDL+HDL (0.1 mM of choline-containing phospholipids). Continuous monitoring of 234 nm absorbance was performed in a microplate reader (BioTek Synergy, Winooski, VT) at 37⬚C for 4 h, using a 96-microwell plate for UV detection (Greiner, Hannover, Germany). The lag phase was calculated from the intersection point between the maximal slope of the curve and initial absorbance [21]. The kinetics of the LDL in the LDL+HDL mixture was calculated by subtracting the kinetics of HDL incubated without LDL. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.5 Macrophage-specific RCT pathway Mouse peritoneal macrophages were isolated from C57BL/6 wild-type mice three days after intraperitoneal (i.p.) injection of 2.5 mL of 3% thioglycollate medium [21]. The mouse macrophages were incubated in 75-cm tissue culture plates for 48 h in the presence of 5 Ci/mL of [1␣,2␣(n)3 H]cholesterol (GE Healthcare, Little Chalfont, UK), 100 g/mL of acetylated LDL and 10% lipoprotein-depleted serum [18, 21]. The cells were equilibrated with medium containing 0.2% BSA, detached by gently pipetting, resuspended in PBS and pooled before being injected i.p. into mice (7.5 × 105 macrophages containing 3 × 105 cpm; cell viability was 91%). The mice were then housed individually in metabolic cages and their stools collected over the next two days. Plasma radioactivity was determined at 48 h by liquid scintillation counting. HDL-associated [3 H]cholesterol was measured after precipitation of apoB-containing lipoproteins. Liver and fecal lipids were extracted with isopropyl alcoholhexane (2:3, v/v). The lipid layer was collected and evaporated, and [3 H]cholesterol radioactivity measured [18,21]. [3 H]tracer in the fecal bile acids was determined in the remaining aqueous portion of the fecal material extracts.
2.6 Glucose tolerance test and glucose and insulin determination Glucose tolerance tests were performed by i.p. administration of glucose (1.3 mg/g of body mass) after a 12-h fast and subsequent measurement of plasma glucose at t = 0, 15, 30, 60, 120 and 180 min. The area under the curve (AUC) was calculated [18]. Serum glucose was determined enzymatically using commercial kits adapted to a COBAS 6000 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland) and serum insulin was assayed by ELISA (Mercodia, Uppsala, Sweden). HOMA-IR index was calculated by multiplying the fasting values of glucose (mM) and insulin (U/mL) and dividing by 22.5. 2.7 Quantitative real time-PCR analyses Total RNA was extracted from livers and small intestine using TRIzol LS Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions and purified using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). cDNA was generated using Oligo(dT)23 and dNTPs mix (Sigma Diagnostics) and M-MLV reverse transcriptase RNase H minus point mutant (Promega, Madison, WI) and it was subjected to quantitative real-time PCR amplification using TaqMan Master Mix (Applied Biosystems, Foster City, CA). Specific TaqMan probes (Applied Biosystems) were used for each gene and Gapdh was used as the internal control gene. Reactions were run on a CFX96TM Real-Time System (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Relative mRNA expression levels were calculated using the ⌬⌬Ct method [18]. www.mnf-journal.com
1990
L. Cedo´ et al.
Mol. Nutr. Food Res. 2015, 59, 1987–1996
Table 2. Serum and liver lipids in LFLC-, HFHC-SFA- and HFHC-PUFA-fed C57BL/6 mice
LFLC Total serum cholesterol (mmol/L) Total serum free cholesterol (%) Total serum phospholipids (mmol/L) Serum triglycerides (mmol/L) HDL cholesterol (mmol/L) HDL phospholipids (mmol/L) Liver weight (g) Liver cholesterol (mol/g of liver) Liver triglycerides (mol/g of liver)
2.51 21.38 2.60 0.88 2.35 2.20 0.96 4.40 20.38
HFHC-SFA ± ± ± ± ± ± ± ± ±
0.11 0.76 0.11 0.10 0.07 0.04 0.04 0.23 3.36
4.36 22.41 3.69 1.19 3.80 3.18 1.15 6.90 24.78
± ± ± ± ± ± ± ± ±
0.22* 0.28 0.14* 0.13 0.22* 0.13* 0.04* 0.90* 4.26
HFHC-PUFA 3.70 22.24 3.21 1.29 2.81 2.56 1.09 5.73 18.00
± ± ± ± ± ± ± ± ±
0.09* 0.86 0.10* 0.13 0.09† 0.06† 0.06* 0.85 4.16
† †
Values are mean ± SEM (n = 8). *p
1987
DOI 10.1002/mnfr.201500336
RESEARCH ARTICLE
Consumption of polyunsaturated fat improves the saturated fatty acid-mediated impairment of HDL antioxidant potential 1,4 ´ L´ıdia Cedo´ 1,2 , Jari Metso3 , David Santos1,2 , Jose Lu´ıs Sanchez-Quesada , Josep Julve1,2,4 , 1 1,4 3 ´ ´ , Josefina Mora-Brugues , Matti Jauhiainen , Annabel Garc´ıa-Leon 1,2,4∗ 1,2,4 ` and Joan Carles Escola-Gil Francisco Blanco-Vaca 1
` Institut d’Investigacions Biomediques (IIB) Sant Pau, Barcelona, Spain ´ CIBER de Diabetes y Enfermedades Metabolicas Asociadas, CIBERDEM Madrid, Spain 3 National Institute for Health and Welfare, Genomics and Biomarkers Unit, Biomedicum, Helsinki, Finland 4 ` Departament de Bioqu´ımica i Biolog´ıa Molecular, Universitat Autonoma de Barcelona, Barcelona, Spain 2
Scope: The present study aimed to compare the effects of diets containing high-fat, highcholesterol and saturated fatty acids (HFHC-SFA) and HFHC-polyunsaturated fatty acidscontaining (HFHC-PUFA) diets on two major antiatherogenic functions of HDL, the HDL antioxidant function and the macrophage-to-feces reverse cholesterol transport. Methods and results: Experiments were carried out in mice fed a low-fat, low-cholesterol (LFLC) diet, an HFHC-SFA diet or an HFHC-PUFA diet in which SFAs were partly replaced with an alternative high-linoleic and ␣-linolenic fat source. The HFHC-SFA caused a significant increase in serum HDL cholesterol and phospholipids as well as elevated levels of oxidized HDL and oxidized LDL. Replacing SFA with PUFA significantly reduced the levels of these oxidized lipoproteins and enhanced the ability of HDL to protect LDL from oxidation. The SFA-mediated impairment of HDL antioxidant potential was not associated with the cholesterol content of the diet, obesity or insulin resistance. In contrast, the effect of the HFHC diets on fecal macrophage-derived cholesterol excretion was independent of the fatty acid source. Conclusion: SFA intake impairs the antioxidant potential of HDL and increases serum levels of oxidized lipoprotein species whereas the antioxidant potential of HDL is enhanced after PUFA consumption.
Received: April 29, 2015 Revised: June 12, 2015 Accepted: June 21, 2015
Keywords: HDL / Oxidation / Reverse cholesterol transport / Saturated fatty acids / Polyunsaturated fatty acids
1
Introduction
Western dietary transition increases the incidence of noncommunicable diseases such as type 2 diabetes and coronary heart disease (CHD) [1]. The conventional western diet ` Correspondence: Joan Carles Escola-Gil E-mail: [email protected] Abbreviations: ABC, ATP-binding cassette; apo, apolipoprotein; CHD, coronary heart disease; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; HDL-C, HDL-cholesterol; HFHC diet, highfat and high-cholesterol diet; LDL-C, LDL cholesterol; LXR, liver X receptor; LFLC diet, low-fat and low-cholesterol diet; oxLDL, oxidized LDL; PAF-AH, platelet-activating factor acetyl-hydrolase; PON, paraoxonase; RCT, reverse cholesterol transport; SFA, saturated fatty acids C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
is high in saturated fatty acids (SFAs) and substantial evidence shows that dietary SFA intake is associated with an increased risk of CHD [2–4]. This effect is thought to be mediated by an increase in low-density cholesterol (LDL-C), whereas rather paradoxically with respect to CHD, SFA intake increases high-density lipoprotein cholesterol (HDL-C) (reviewed in [5]). Conversely, PUFAs usually reduces HDL-C [5], whereas they are potentially atheroprotective [6, 7]. Macrophage-specific reverse cholesterol transport (RCT) and HDL antioxidant function are considered among the most important HDL-mediated cardioprotective mechanisms [8, 9]. Dietary fatty acid composition may influence HDL antioxidant function. HDL from human subjects fed a high ∗ Additional corresponding author: Francisco Blanco-Vaca, E-mail: [email protected]
www.mnf-journal.com
1988
L. Cedo´ et al.
PUFA diet, mainly containing linoleic acid (C18:2, n-6), showed a reduced susceptibility to lipid oxidation when compared with MUFA-enriched diet [10, 11]; however, the potential of HDL particles to inhibit LDL oxidation was similar [12]. A diet containing high-fat, high-cholesterol and saturated fatty acids (HFHC-SFA) accelerated HDL lipid oxidation in hypercholesterolemic mice. However, that diet contained sodium cholate which produces significant hepatotoxic effects and the reference diet was a low-fat, low-cholesterol (LFLC) diet containing large amounts of carbohydrates [13]. On the other hand, several human intervention studies showed no effect on the in vitro ability of HDL to stimulate macrophage cholesterol efflux, the first RCT step, when dietary SFA intake was replaced by PUFA [14, 15]. In contrast, n-6 PUFA promoted HDL-mediated cholesterol efflux from human fibroblast, although the effects of n-3 and n-6 PUFA, mainly ␣-linolenic (C18:3) and linoleic acids, respectively, were divergent [16]. Nonetheless, the entire RCT pathway from macrophages back to the liver via HDL and its fecal excretion comprises various susceptible regulatory steps in different body compartments; thus, the changes in cholesterol efflux might not reflect the flux of [3 H]cholesterol through different compartments [17]. We demonstrated, in mice, that a HFHC-SFA diet promoted a sustained compensatory in vivo macrophage-to-feces RCT that was dependent on dietary cholesterol-mediated liver X receptor (LXR) signaling on liver ATP-binding cassette (ABC) G5/G8 expression. However, our study did not address whether the differences in the sources of fatty acids altered the macrophage RCT pathway [18]. It has also been reported that an HFHC diet containing eicosapentaenoic acid (EPA; C20:5, n-3) and docosahexaenoic acid (DHA; C22:6, n-3) from fish oil promoted macrophage RCT in mice when compared with mice fed an HFHC diet containing mainly linoleic acid; these changes were consistent with liver ABCG5/G8 upregulation [19]. In the present study, we evaluated the effect of an HFHCSFA diet on HDL antioxidant function and on the in vivo macrophage-specific RCT pathway compared with those produced by an HFHC-PUFA diet in which SFA was partly replaced by an alternative PUFA source containing mainly the linoleic and ␣-linolenic acids. Our results demonstrate that SFA intake impairs HDL antioxidant potential but not the macrophage-to-feces RCT pathway, whereas concomitant PUFA consumption improved the antioxidant potential of HDL particles.
Mol. Nutr. Food Res. 2015, 59, 1987–1996
Table 1. Diet nutrient information and fatty acid profile for fat sources in LFLC, HFHC-SFA and HFHC-PUFA diets
LFLC
HFHC-SFA
HFHC-PUFA
Protein (% by weight) Carbohydrate (% by weight) Fat (% by weight) Cholesterol (%)
16.1
17.3
17.3
59.9
48.7
48.7
3.1
21.2 0.2
21.2 0.2
Fatty acid (% total fatty acids)
LFLC
HFHC-SFA
HFHC-PUFA
SFA MUFA PUFA C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
23.5 17.9 58.6
65.2 31.3 3.5 3.8 2.3 1.1 2.0 3.1 11.7 26.2 1.9 12.5 28.2 2.9 0.5
40.2 27.4 32.4 1.9 1.2 0.6 1.0 1.6 5.9 18.6 1.0 8.3 25.9 28.1 4.3
21.3 0.5 2.2 17.4 54.2 4.3
the following diets for eight additional weeks: (i) a LFLC diet (A04; Safe, Scientific Animal Food & Engineering, Augy, France) containing 3% fat and 0.02% cholesterol; (ii) an HFHC-SFA diet (Western-type diet TD.09821; Harlan Teklad, Madison, WI) containing 21% anhydrous milkfat and 0.2% cholesterol; (iii) an HFHC-PUFA diet (Western-type diet TD.130185; Harlan Teklad, Madison, WI) containing 10.5% anhydrous milkfat, 10.5% soybean fat and 0.2% cholesterol; or (iv) a high-fat and low-cholesterol (HFLC)SFA diet (Western-type diet TD.130184; Harlan Teklad, Madison, WI) containing 21% anhydrous milkfat and 0.05% cholesterol. The HFHC-PUFA and the HFLC-SFA diets were prepared exactly as the HFHC-SFA diet with the only differences being the fat source in the HFHC-PUFA diet and the cholesterol supplementation in the HFLC-SFA diet. Detailed descriptions of the diet composition are provided in Table 1.
2.2 Lipid and apolipoprotein analyses
2
Materials and methods
2.1 Mice and diets All animal procedures were approved by the Institutional Animal Care Committee of the Institut de Recerca of the Hospital de la Santa Creu i Sant Pau. Eight-week-old male C57BL/6 wild-type mice (#000664; Jackson Laboratories, Bar Harbor, ME) were randomized into four groups and fed C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
At the end of the study, mice were fasted overnight (o/n), euthanized and exsanguinated via cardiac puncture and livers and epididymal fat pads were removed. Serum lipid and lipoprotein analyses were determined enzymatically using commercial kits adapted to a COBAS 6000 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland) [18]. Mouse apoA-I assay was quantified with an ELISA coated with a polyclonal rabbit antibody against mouse apoA-I [20]. HDL was isolated www.mnf-journal.com
1989
Mol. Nutr. Food Res. 2015, 59, 1987–1996
by sequential ultracentrifugation at 100 000 × g for 24 h at a density of 1.063–1.21 g/mL [21]. The composition of isolated HDL was determined using the commercial methods adapted to the COBAS 6000 autoanalyzer. HDL protein concentrations were determined with the bicinchoninic acid (BCA) method (TermoScientific, Rockford, IL). In order to analyze HDL fatty acid composition, a lipid extraction from HDL isolated by ultracentrifugation was performed with isopropyl alcohol-hexane (2:3, v/v). After hexane evaporation of the lipid phase, samples were saponified with KOH/MeOH 6% o/n, extracted with hexane and KOH/MeOH phase was decanted and acidified with HCl. Samples were then extracted with hexane; after evaporation, BF3 /MeOH was added and left to react o/n. Samples were extracted again with hexane; after evaporation, fatty acids were determined by gas chromatography/MS (Shimadzu QP2010, Kyoto, Japan) using a BPX70 column measuring 30 m x 0.25 mm x 0.25 m. Liver lipids were extracted with isopropyl alcohol–hexane. The lipid layer was collected, evaporated and resuspended in cholate 0.5% (w/v) for lipid determinations [18].
2.3 Enzyme activities Total plasma arylesterase activity was measured using phenylacetate as substrate [22]. EDTA-sensitive serum arylesterase activity was calculated by subtracting the EDTA-resistant arylesterase, which was used to determine paraoxonase (PON) 1 activity. PON1 activity was also measured using paraoxon (Sigma, St. Louis, MO) as substrate [23]. Plateletactivating factor acetyl-hydrolase (PAF-AH) activity was determined using a commercial kit (Cayman Chemical, Ann Arbor, MI) [22]. ␣-tocopherol was determined by HPLC as previously described [24].
2.4 Susceptibility to lipoprotein oxidation Mouse serum oxidized (ox) LDL and oxHDL levels were assayed with two specific ELISA kits (Cloud-Clone, Houston, TX and MyBioSource, San Diego, CA, respectively). The ability of HDL to protect against LDL oxidation in vitro was based on an assay in which human LDL is oxidized alone by CuSO4 or in the presence of HDL and the oxidation kinetics is followed by continuous monitoring of the formation of conjugated diene [21, 22]. Briefly, oxidation was started by adding 2.5 M CuSO4 to LDL, mouse HDL or LDL+HDL (0.1 mM of choline-containing phospholipids). Continuous monitoring of 234 nm absorbance was performed in a microplate reader (BioTek Synergy, Winooski, VT) at 37⬚C for 4 h, using a 96-microwell plate for UV detection (Greiner, Hannover, Germany). The lag phase was calculated from the intersection point between the maximal slope of the curve and initial absorbance [21]. The kinetics of the LDL in the LDL+HDL mixture was calculated by subtracting the kinetics of HDL incubated without LDL. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.5 Macrophage-specific RCT pathway Mouse peritoneal macrophages were isolated from C57BL/6 wild-type mice three days after intraperitoneal (i.p.) injection of 2.5 mL of 3% thioglycollate medium [21]. The mouse macrophages were incubated in 75-cm tissue culture plates for 48 h in the presence of 5 Ci/mL of [1␣,2␣(n)3 H]cholesterol (GE Healthcare, Little Chalfont, UK), 100 g/mL of acetylated LDL and 10% lipoprotein-depleted serum [18, 21]. The cells were equilibrated with medium containing 0.2% BSA, detached by gently pipetting, resuspended in PBS and pooled before being injected i.p. into mice (7.5 × 105 macrophages containing 3 × 105 cpm; cell viability was 91%). The mice were then housed individually in metabolic cages and their stools collected over the next two days. Plasma radioactivity was determined at 48 h by liquid scintillation counting. HDL-associated [3 H]cholesterol was measured after precipitation of apoB-containing lipoproteins. Liver and fecal lipids were extracted with isopropyl alcoholhexane (2:3, v/v). The lipid layer was collected and evaporated, and [3 H]cholesterol radioactivity measured [18,21]. [3 H]tracer in the fecal bile acids was determined in the remaining aqueous portion of the fecal material extracts.
2.6 Glucose tolerance test and glucose and insulin determination Glucose tolerance tests were performed by i.p. administration of glucose (1.3 mg/g of body mass) after a 12-h fast and subsequent measurement of plasma glucose at t = 0, 15, 30, 60, 120 and 180 min. The area under the curve (AUC) was calculated [18]. Serum glucose was determined enzymatically using commercial kits adapted to a COBAS 6000 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland) and serum insulin was assayed by ELISA (Mercodia, Uppsala, Sweden). HOMA-IR index was calculated by multiplying the fasting values of glucose (mM) and insulin (U/mL) and dividing by 22.5. 2.7 Quantitative real time-PCR analyses Total RNA was extracted from livers and small intestine using TRIzol LS Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions and purified using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). cDNA was generated using Oligo(dT)23 and dNTPs mix (Sigma Diagnostics) and M-MLV reverse transcriptase RNase H minus point mutant (Promega, Madison, WI) and it was subjected to quantitative real-time PCR amplification using TaqMan Master Mix (Applied Biosystems, Foster City, CA). Specific TaqMan probes (Applied Biosystems) were used for each gene and Gapdh was used as the internal control gene. Reactions were run on a CFX96TM Real-Time System (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Relative mRNA expression levels were calculated using the ⌬⌬Ct method [18]. www.mnf-journal.com
1990
L. Cedo´ et al.
Mol. Nutr. Food Res. 2015, 59, 1987–1996
Table 2. Serum and liver lipids in LFLC-, HFHC-SFA- and HFHC-PUFA-fed C57BL/6 mice
LFLC Total serum cholesterol (mmol/L) Total serum free cholesterol (%) Total serum phospholipids (mmol/L) Serum triglycerides (mmol/L) HDL cholesterol (mmol/L) HDL phospholipids (mmol/L) Liver weight (g) Liver cholesterol (mol/g of liver) Liver triglycerides (mol/g of liver)
2.51 21.38 2.60 0.88 2.35 2.20 0.96 4.40 20.38
HFHC-SFA ± ± ± ± ± ± ± ± ±
0.11 0.76 0.11 0.10 0.07 0.04 0.04 0.23 3.36
4.36 22.41 3.69 1.19 3.80 3.18 1.15 6.90 24.78
± ± ± ± ± ± ± ± ±
0.22* 0.28 0.14* 0.13 0.22* 0.13* 0.04* 0.90* 4.26
HFHC-PUFA 3.70 22.24 3.21 1.29 2.81 2.56 1.09 5.73 18.00
± ± ± ± ± ± ± ± ±
0.09* 0.86 0.10* 0.13 0.09† 0.06† 0.06* 0.85 4.16
† †
Values are mean ± SEM (n = 8). *p
