The supplementation of Se in its various chemical forms has been associated with decreased plasma cholesterol concentrations in both human(Reference Kauf, Dawczynski and Jahreis1–Reference Raymand, Stranges and Griffin4) and rodent(Reference Dhingra and Bansal5–Reference Vinson, Stella and Flanagan17) studies. To date, the molecular mechanisms underlying the hypocholesterolaemic effects of high-dose supplemental selenite have not been clearly defined. Feeding trials involving rodent models of hypercholesterolaemia have shown selenite supplementation to be associated with lowered mRNA abundance of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr)(Reference Dhingra and Bansal5) and increased mRNA abundance of hepatic LDL receptor (Ldlr)(Reference Dhingra and Bansal6). To our knowledge, no studies have examined the impact of selenite supplementation on three key genes involved in the control of cholesterol absorption: the heterodimeric ATP-binding cassette transporters g5 (Abcg5) and g8 (Abcg8) and Nieman-Pick C1-like 1 protein (Npc1l1) in the Syrian hamster, which is the rodent model most similar to humans with regards to cholesterol metabolism(Reference Dietschy18, Reference Zhang, Wang and Jiao19). The heterodimeric transporters Abcg5 and Abcg8 are responsible for sterol efflux from hepatocytes(Reference Yu, Hammer and Li-Hawkins20) and enterocytes(Reference Duan, Wang and Wang21), whereas the transporter Npc1l1 is involved in cholesterol entry into the enterocyte(Reference Davis, Zhu and Hoos22). Overexpression of hepatic and intestinal Abcg5 and Abcg8 and inactivation of Npc1l1 (Reference Basso, Freeman and Ko23) are associated with plasma lipid-lowering effects in conjunction with lowered hepatic cholesterol concentrations, which are the surrogate markers for cholesterol absorption(Reference Salisbury, Davis and Burrier24).
Gene expression of Abcg5 and Abcg8 is responsive to cholesterol feeding(Reference Jia, Ebine and Demonty25), which is also associated with increased oxysterol concentrations(Reference Zhang, Li and Blanchard26). The feeding of a high-cholesterol diet to triple-knockout mice unable to synthesise 24-(S)-hydroxycholesterol (24(S)-OHC), 25-hydroxycholesterol (25-OHC) and 27-hydroxycholesterol (27-OHC) failed to induce hepatic mRNA abundance of Abcg5 and Abcg8, which implicates these oxysterols as key in vivo regulators of the mRNA expression of the heterodimers(Reference Chen, Chen and Head27). To date, however, in vivo concentrations of endogenously occurring oxysterols have generally not been examined within feeding trials investigating the effects of dietary cholesterol on the Abcg5 and Abcg8 genes.
Therefore, the aim of the present study was to examine the dose-related effect of high-dose selenite supplementation in the Syrian hamster fed 0·1 % cholesterol and 15 % saturated fat diets on hepatic and/or jejunal mRNA abundance of Abcg5, Abcg8 and Npc1l1 genes in relation to cholesterol concentrations in plasma, liver, bile and faeces. The effect of supplemental selenite on the mRNA abundance of key cholesterol metabolising genes, 7-hydroxylase (Cyp7a1), Hmgcr and Ldlr was also investigated. A secondary aim was to investigate the hypocholesterolaemic effects of selenite on in vivo tissue concentrations of 24(S)-OHC, 25-OHC and 27-OHC in relation to the hepatic and jejunal mRNA expression of the Abcg5 and Abcg8 genes.
Materials and methods
Animals and diets
A total of forty Syrian male hamsters, aged 9–10 weeks (approximate weight 110–120 g), were purchased from Charles River Laboratories (St-Constant, QC, Canada). The hamsters were housed individually in the Animal Resources Division, Food Directorate of Health Canada (Ottawa, ON, Canada) in stainless-steel wire-bottom cages and acclimatised to laboratory conditions for 10 d while being fed a standard commercial chow diet. At the end of the acclimatisation period, hamsters were weighed and randomised to four groups of ten animals each and fed their respective test diets for 4 weeks (Table 1). The dietary levels of selenite were adjusted to four different levels which included: (1) Se 0·15 parts per million (ppm); (2) Se 0·85 ppm; (3) Se 1·7 ppm; (4) Se 3·4 ppm. The minimal level of Se of 0·15 ppm in the basal diet conformed to National Research Council guidelines for Se of 0·1 ppm(28). The highest supplemental level of Se (3·4 ppm) was chosen on the basis of a previous study(Reference Poirier, Cockell and Hidiroglou9), which demonstrated that hamsters can safely tolerate this level of Se supplementation. Chronic toxicity of Se was tested in the previous work, which found levels of dietary Se up to 10 ppm to be non-toxic to Syrian hamsters(Reference Birt, Julius and Runice29–Reference Julius, Davies and Birt31). Diets were obtained from Dyets, Inc. (Bethlehem, PA, USA) in pellet form in vacuum-packed bags, which were stored at − 20°C to prevent lipid auto-oxidation. Feed was provided on a daily basis (approximately 15 g/d, accurately weighed) and hamsters had free access to tap water. Food consumption was measured daily by weighing uneaten portions. Hamsters were weighed three times per week for the initial 2 weeks of feeding and thereafter, body weight was recorded on a weekly basis. All experiments were conducted in accordance with the institutional guidelines for animal care, and all experimental procedures were approved by the Health Canada Animal Care Committee and the research was conducted according to the Canadian Council on Animal Care guidelines(32).
Table 1 Composition of experimental diets (g/kg)*
ppm, Parts per million; BF, butterfat; USP, United States Pharmacopeia.
* All diets were formulated at McGill University and prepared in pellet form by Dyets, Inc.
† BF fatty acid composition is as follows (% by weight) as per Dyets inspection report: 4 : 0, 3·4; 6 : 0, 2·0; 8 : 0, 1·2; 10 : 0, 2·7; 12 : 0, 3·0; 14 : 0, 10·7, 14 : 1, 1·6; 16 : 0, 28·0; 16 : 1, 2·5; 18 : 0, 13·0; 18 : 1, 26·8; 18 : 2, 2·5, 18 : 3, 1·5, 20 : 0, 1·1.
‡ Safflower oil was added to prevent essential fatty acid deficiency. α-Tocopherol concentration of safflower oil is 350 ppm of α-tocopherol, 180 ppm of other tocopherols. Fatty acid profile of safflower oil included (% by weight): 14 : 0, trace; 16 : 0, 6·9; 16 : 1, trace; 18 : 0, 2·9; 18 : 1, 12·2; 18 : 2, 78·0; 18 : 3, trace.
§ Cholesterol USP standard was added to BF 4·193 g/kg.
∥ The mineral mix was free of Se and was composed of (g/kg): calcium carbonate 336·4; calcium phosphate, monobasic 285·0; magnesium oxide 2·9; potassium iodate (10 mg KIO3/g) 0·8; potassium phosphate, dibasic 40·8; NaCl 11·5; cupric carbonate 0·1; cobalt chloride 0·1; sodium fluoride 0·002; ferric citrate 25·5; manganous carbonate 0·2; ammonium paramolybdate 0·01; zinc carbonate 0·5; sucrose 296·2. Sodium selenite (10 mg Se/g sodium selenite mixture) was added separately to make the diets; for Se 0·15 ppm, 0·03; Se 0·85 ppm, 0·2; Se 1·7 ppm, 0·4; Se 3·4 ppm, 0·8.
¶ The vitamin mix was free of α-tocopherol and vitamin A and was composed of (g/kg): vitamin D3 (10 000 μg) 0·9; vitamin K1 premix (10 mg/g) 110·0; biotin 0·03; folic acid 0·3; niacin 13·5; pantothenate (Ca) 1·5; riboflavin 2·3; thiamin HCl 3·0; pyridoxine HCl 0·9; vitamin B12 (0·1 %) 1·5; sucrose 866·1; vitamin A palmitate (275 μg) was added separately to make the diets; for all diets 4·5; α-tocopheryl acetate (454 mg) was added separately to make the diets; for all diets, 0·1.
Sample collection
At the end of the feeding period, hamsters were fasted overnight and killed in a treatment-blocked randomised order within 2 d. Under isoflurane anaesthesia, blood was drawn by cardiac puncture and collected in EDTA tubes for subsequent plasma isolation. After surgical exposure of the liver, bile was aspirated from the gallbladder by tuberculin syringe, transferred to sample tubes, mixed by gentle inversion and stored at − 80°C. Immediately after removal, the liver pieces were frozen in liquid N2. The intestine was dissected out, rinsed with filtered phosphate-buffered saline solution before separating into sections and freezing in liquid N2. Both liver and jejunum were stored at − 80°C until further use. During dietary treatment, and near the end of the feeding phase, faeces was collected on three consecutive days and stored at − 20°C until further use.
Plasma lipid analysis
The measurement of plasma lipids included total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), and TAG and was carried out according to the manufacturer's instructions using commercially available kit assays. Randox enzymatic reagent kits (Randox Laboratories Limited, Antrim, UK) were used for the measurement of plasma TC, HDL-C and TAG. Plasma LDL-C was assessed using Wako L-Type LDL kit assay (Wako Pure Chemical Industries Limited, Osaka, Japan).
Liver lipid analysis
Hepatic TC, free cholesterol (FC), cholesteryl ester and TAG were determined from lipid extracts via a method developed by Carr et al. (Reference Carr, Andersen and Rudel33) using commercially available enzymatic assay kits. Hepatic TC was determined using cholesterol E reagent (Wako Pure Chemical Industries Limited). Hepatic FC was measured using free cholesterol C enzymatic colorimetric method (Wako Pure Chemical Industries Limited). The hepatic cholesteryl ester concentration was calculated by the difference between liver TC and FC. Hepatic TAG concentrations were determined using Wako L-Type TAG H Reagents 1 and 2 (Wako Pure Chemical Industries Limited). Briefly, tissue lipids were extracted from approximately 200 mg of liver tissue (wet weight) using 30 ml chloroform–methanol (2:1) according to the method of Folch et al. (Reference Folch, Lees and Sloane Stanley34). For the enzymatic determination of hepatic tissue lipids, all assays were performed using 50 μl aliquots of sample and standard using ninety-six-well microtitre plates. Separate microtitre plates were used for hepatic TC, FC and TAG determination. For the standard solution preparations, soyabean oil was used as the primary TAG standard, and cholesterol was used as the standard for both TC and FC determinations. Both soyabean oil and cholesterol were purchased from Sigma Chemical Company (St Louis, MO, USA). For all lipid assessments, assays were carried out at room temperature (25°C) and were read using a multifilter microtitre plate reader (Titertek Multiskan Plus MKII; ICN Biochemicals, Cleveland, OH, USA).
Liver selenium analysis
For liver Se analysis, hepatic tissue was digested with nitric acid and the Se content was measured using flame atomic absorption spectrophotometry (Hitachi, Polarized Zeeman AAS, Z-8200 Mississauga, Canada)(Reference Poirier, Cockell and Ratnayake10).
Liver oxysterol analysis
Oxysterol determination was performed by GC/MS as described previously(Reference Cockell, Wotherspoon and Belonje35). Briefly, 19-hydroxycholesterol was added to the samples as an internal standard before lipid extraction. Artifactual oxidation of cholesterol was minimised by the incorporation of l-ascorbic acid and sodium acetate to scavenge oxygen and acidic species, respectively. The lipid extract was saponified and unsaponified lipids were extracted with diethyl ether and NEFA were removed using KOH. Bulk cholesterol was removed by solid-phase extraction and oxysterols were eluted with 2-propanol in hexane. Samples were evaporated at room temperature under N2 and converted to trimethylsilyl ethers for GC/MS analysis (Agilent 6890 GC System with 5973 Mass Selective Detector, Agilent Technologies, Wilmington, DE, USA) using a J&W DB-1 capillary column with flow rate of He carrier gas of 1·0 ml/min. The injector was operated in splitless mode and with an initial temperature of 290°C. After injection, oven temperature began at 80°C, and was then programmed at a rate of 30°C/min to a final temperature of 215°C, held for 2 min, followed by a rate of 2°C/min to a final temperature of 280°C, held for 10 min. A volume of 1 μl per sample was injected. Oxysterol analysis was carried out using selected ion monitoring. The multiple ion detector was focused on m/z 145, 353 and 366 for 19-hydroxycholesterol; m/z 367 and 472 for 7-ketocholesterol; 145, 413 and 456 for 24(S)-OHC; 131, 327 and 456 for 25-OHC; m/z 129, 417 and 456 for 27-OHC.
RNA isolation and real-time quantitative reverse transcription-PCR
Total RNA was extracted from frozen hamster liver tissue and jejunal samples using two passes of Trizol reagent (Invitrogen Life Technologies, Burlington, ON, Canada) for each sample. The isolated RNA was purified and DNase I treated on RNeasy mini columns (Qiagen, Mississauga, ON, Canada) using the manufacturer's recommended conditions. Purified RNA was quantified using RiboGreen RNA Quantification Reagent and Kit (Molecular Probes, Eugene, OR, USA), and subsequent complementary DNA synthesis was performed with Retroscript Kit (Ambion, Streetsville, ON, Canada) in accordance with the manufacturer's instructions. Real-time quantitative PCR was performed using the Mx4000 Multiplex Quantitative PCR System and Brilliant SYBR green quantitative PCR Core Reagent Kit (Stratagene, La Jolla, CA, USA). Real-time quantitative PCR was carried out for Abcg5, Abcg8, Cyp7a1, Hmgcr, Ldlr, Npc1l1 and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) genes using primers obtained from the literature(Reference Yu, Pandit and Klett36, Reference Shimomura, Bashmakov and Shimano37) or primers newly designed with PrimerQuest software (White Head Institute for Biomedical Research, Cambridge, MA, USA). To verify that the primers (Table 2) were specific for each gene of interest (GOI), sequences were analysed using the Basic Local Alignment and Search Tool on the National Center for Biotechnology Information website(38). In addition, the specificity of the PCR was confirmed by dissociation curve analysis of the products and by size verification of the PCR products on agarose gel. A non-template reaction and a no-RT reaction were included as negative controls for each experiment. Gapdh expression was not affected by Se treatment in this study and was therefore considered a valid housekeeping gene (data not shown). Standard curves for each GOI, as well as for Gapdh, were used to calculate the relative levels of mRNA for each gene. The relative amounts of each GOI were normalised to Gapdh expression levels in the liver or jejunum as an endogenous internal standard. Normalised values (GOI/Gapdh) were then calibrated to the hamsters fed the control diet (set as 1·0).
Table 2 Oligonucleotide primers used for real-time PCR
Abcg5, ATP binding cassette transporter g5; Abcg8, ATP binding cassette transporter g8; Cyp7a1, 7-hydroxylase; Gapdh, glyceraldehyde-3-phosphate-dehydrogenase; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; Ldlr, LDL receptor; Npc1l1, Niemann-pick C1-like 1 protein.
Quantification of biliary and faecal bile acids and cholesterol
The analysis of biliary total bile acids was performed using a combination of two previously published methods as described by Chijiiwa & Nakayama(Reference Chijiiwa and Nakayama39) and Batta et al. (Reference Batta, Salen and Rapole40). Briefly, 5 μl bile were added to a 5 ml volumetric flask containing 2 ml ethanol and heated until boiling point in a water bath. After cooling to room temperature, ethanol was added to make 5 ml volume and centrifuged to separate precipitated protein. To 2 ml supernatant were added internal standards (nor-cholic acid, 10 μg in 100 μl ethanol and 5α-cholestane, 10 μg in 100 μl ethanol), 1·86 % EDTA (1 ml), 0·87 % mercaptoethanol (1 ml), 0·1 mg cholyglycine hydrolase and 0·1 mg β-glucuronidase (suspended together in 2 ml acetate buffer, pH 5·6); and the resulting suspension was incubated in a dry bath at 37 °C for 18 h. After hydrolysis, the contents were acidified to pH 1·0 using HCl followed by extraction with ethyl acetate (3 × 5 ml). Pooled ethyl acetate was evaporated to dryness and the residue was subjected to n-butyl ester formation with the addition of 200 μl n-butanol and 20 μl HCl, and the contents were heated at 60 °C for 4 h and the solvents were evaporated under N2. The esterified bile acids and cholesterol were reacted with 100 μl of Sil-prep (hexamethyldisilazane–trimethylchlorosilane–pyridine (3:1:9); Alltech Associates, Deerfield, IL, USA) for 30 min at 55 °C and the solvents were evaporated under N2. The trimethylsilyl ether derivatives formed were taken in 100 μl of hexane, transferred to a sample vial and capped. Then, 1 μl was injected into the GLC column for analysis in the 20:1 split mode.
The analysis of faecal bile acids was performed as described by Batta et al. (Reference Batta, Salen and Rapole41). To 10–15 mg freeze-dried stool (weighed exactly) were added internal standards (nor-cholic acid, 20 μg in 100 μl n-butanol) and (5α-cholestane, 20 μg in 100 μl n-butanol), followed by 20 μl concentrated HCl. The contents were subjected to n-butyl ester formation by heating contents at 60 °C for 4 h and evaporation under N2. Dried residue was reacted with 100 μl of Sil-prep for 30 min at 55 °C, and the solvents were evaporated under N2. The trimethylsilyl ether derivatives formed were taken in 200 μl of hexane, and centrifuged to separate the stool debris. The clear supernatant (100 μl) was transferred to a sample vial and capped. And then, 1 μl was injected into the GLC column for analysis in the 20:1 split mode.
Internal standards – nor-cholic acid (23-NOR-5β-cholanic acid-3α,7α,12α-triol) and cholestane (5α-cholestane), and standards – chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholanoic acid), cholic acid (3α,7α,12α-trihydroxy-5β-cholanoic acid), lithocholic acid (3α-hydroxy-5β-cholanoic acid), deoxycholic acid (3α, 12α-dihydroxy-5β-cholanoic acid), ursocholanic acid (5β-cholanic), ursodeoxycholic acid (3α,7β-dihydroxy-5β-cholanoic acid), hyodeoxycholic acid (3α,6α-dihydroxy-5β-cholanoic acid) and cholesterol (5-cholesten-3β-ol) were purchased from Steraloids, Inc. (Newport, RI, USA). All standards contained the internal standards nor-cholic acid and 5α-cholestane. Standard concentrations ranged between 0·02 and 0·2 μg bile acid per 1 μl hexane injected and were subjected to n-butyl ester formation and trimethylsilylation as delineated previously.
Identification and quantification of bile acids were achieved using a Hewlett-Packard model 6890 gas chromatograph equipped with a flame ionisation detector and injector with a split/splitless device for capillary columns. The chromatographic column used was a J&W 122-1031 capillary column (30 m × 0·250 mm internal diameter). Helium was used as the carrier gas. The gas chromatograph operating conditions were as follows: injector and detector temperatures were 260 and 290°C, respectively. After injection, oven temperature was kept at 150°C for 1 min, programmed at a rate of 7°C/min to reach a final temperature of 272°C.
Hepatic protein determination
Protein concentrations were determined using the Bradford reagent (Sigma). Bovine serum albumin stock in saline was used as the standard(Reference Poirier, Cockell and Ratnayake10).
Statistical analysis
Statistical analyses were performed using the mixed model procedure (MIXED) for all analyses using SAS version 9.1, with a significance level less than or equal to 0·05 (SAS Institute, Cary, NC, USA)(42). Blocking was an integral part of the experimental design owing to kill order and assaying, which occurred in distinct blocks over time, and to the use of microtitre plates for hepatic and plasma lipid analysis and for mRNA analysis. Blocking can be viewed as another independent variable introduced only for the purpose of controlling error variation. Normality was measured based on residuals using the Shapiro–Wilk test. The statistical significance of the differences between least square means was determined using the protected least squares means test. Correlations between tissue Se and biochemical measurements were examined by using Spearman's correlation coefficient by rank.
Results
Selenite supplementation increased hepatic selenium concentrations and did not affect biliary or faecal bile acid and cholesterol concentrations, average daily intake, final body weight or liver weight of hamsters
Each increment of dietary selenite resulted in significantly higher liver Se concentrations (μmol/g wet weight) of 0·51 (sem 0·03); 0·62 (sem 0·03); 0·81 (sem 0·03); 1·03 (sem 0·03), with the Se 0·15 ppm, Se 0·85 ppm, Se 1·7 ppm and Se 3·4 ppm diets, respectively. No effect of selenite treatment was noted on biliary or faecal TC or bile acid concentrations (data not shown). Hamsters consuming the Se 0·15 ppm, Se 0·85 ppm, Se 1·7 ppm and Se 3·4 ppm diets showed biliary TC concentrations (μmol/ml bile) of 13 (sem 1·5); 11 (sem 1·3); 12 (sem 1·4); 10 (sem 1·4) and faecal TC concentrations (μmol/g faeces) of 5·8 (sem 1·1), 7·0 (sem 2·6), 8·3 (sem 2·5) and 9·2 (sem 1·9), respectively.
No effect of selenite treatment was observed on average daily intake, final body weight or liver weight. Final body weights (g) for hamsters consuming the diets were 116·1 (sem 3·03); 109·4 (sem 3·03); 111·3 (sem 3·03); 108·8 (sem 3·03) for the Se 0·15 ppm, Se 0·85 ppm, Se 1·7 ppm and Se 3·4 ppm diets, respectively. Hamsters consuming the Se 0·15 ppm, Se 0·85 ppm, Se 1·7 ppm and Se 3·4 ppm diets consumed on average on a daily basis (g) 7·2 (sem 0·18), 6·6 (sem 0·18), 6·9 (sem 0·18) and 7·2 (sem 0·18), respectively.
Selenite supplementation decreased plasma cholesterol concentrations
Hamsters consuming the Se 3·4 ppm and Se 0·85 ppm diets as compared to the Se 0·15 ppm diet showed lower plasma TC concentrations (Table 3). The effect of dietary selenite on plasma LDL-C was significant, with hamsters consuming the Se 3·4 ppm and Se 1·7 ppm diets showing lower plasma concentrations of LDL-C in comparison to hamsters consuming the Se 0·15 ppm control diet. Decreased plasma HDL-C concentrations were noted in hamsters consuming the Se 0·85 ppm diet as compared to hamsters consuming the Se 0·15 ppm diet. Hamsters receiving the Se 0·85 ppm diet showed significantly higher LDL-C:HDL-C ratios as compared to hamsters consuming the Se 0·15 ppm diet. Increased plasma TAG concentrations were noted in hamsters consuming the Se 1·7 ppm diet versus hamsters fed the Se 0·15 ppm diet.
Table 3 Effect of dietary selenite supplementation on plasma total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), TAG and LDL-C:HDL-C ratio in adult male Syrian hamsters fed high-cholesterol and high-saturated fat diets for 4 weeks
(Mean values with their standard errors, n 10)
ppm, Parts per million.
a,b,c Mean values within a row with unlike superscript letters were significantly different (P ≤ 0·05; Mixed model).
* Plate significant in statistical model.
Selenite supplementation increased liver TAG concentrations
Hamsters consuming the Se 3·4 ppm and Se 1·7 ppm diets showed significantly greater concentration of hepatic TAG as compared to hamsters consuming the Se 0·15 ppm diet (Table 4).
Table 4 Effect of dietary selenite supplementation on liver total cholesterol (TC), cholesteryl ester (CE), free cholesterol (FC) and TAG in adult male Syrian hamsters fed high-cholesterol and high-saturated fat diets for 4 weeks
(Mean values with their standard errors, n 10)
ppm, Parts per million; wt, weight.
a,b,c Mean values within a row with unlike superscript letters were significantly different (P ≤ 0·05; Mixed model).
* Plate significant in statistical model.
Selenite supplementation up-regulated hepatic ATP binding cassette transporter g8 and LDL receptor mRNA expression
Hamsters consuming the Se 3·4 ppm, Se 1·7 ppm and Se 0·85 ppm diets showed significantly greater levels of Abcg8 mRNA in their livers as compared to hamsters consuming the control Se 0·15 ppm diet (Fig. 1). Similarly, hamsters consuming the Se 3·4 ppm and Se 1·7 ppm diets both had higher hepatic levels of Ldlr mRNA in comparison to hamsters fed the control Se 0·15 ppm diet.
Fig. 1 Effect of selenite supplementation on hepatic ATP-binding cassette transporter g5 (Abcg5), ATP-binding cassette transporter g8 (Abcg8), 7-hydroxylase (Cyp7a1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) and LDL receptor (Ldlr) mRNA expression levels in adult Syrian hamsters fed high-cholesterol and high-saturated fat (HCHS) diets for 4 weeks. Data are relative amounts of genes of interest normalised to glyceraldehyde 3-phosphate dehydrogenase expression levels as an endogenous internal standard. Normalised values are calibrated to hamsters fed control (selenium (Se) 0·15 parts per million (ppm)) diet, with control expression set at 1. □, Se 0·15 ppm; 
, Se 0·85 ppm; 
, Se 1·7 ppm; 
, Se 3·4 ppm. Values are means, with their standard errors represented by vertical bars (n 10). * Mean value was significantly different from that for the control Se 0·15 diet (P ≤ 0·05; Mixed model).
Selenite supplementation up-regulated jejunal Ldl receptor and down-regulated Niemann-pick C1-like 1 protein mRNA expression
Hamsters consuming the Se 3·4 ppm and Se 1·7 ppm diets showed higher jejunal levels of Ldlr mRNA as compared to hamsters fed the control Se 0·15 ppm diet (Fig. 2). Hamsters consuming the Se 3·4 ppm and Se 1·7 ppm diets had lower jejunal levels of Npc1l1 mRNA expression as compared to hamsters fed the control Se 0·15 ppm diet. The supplementation of Se showed a tendency to increase Abcg8 mRNA expression levels in the jejunum of hamsters consuming the Se diets (P = 0·08).
Fig. 2 Effect of selenite supplementation on jejunal ATP-binding cassette transporter g5 (Abcg5), ATP-binding cassette transporter g8 (Abcg8), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr), LDL receptor (Ldlr) and Niemann-pick C1-like 1 protein (Npc1l1) mRNA expression levels in adult Syrian hamsters fed high-cholesterol and high-saturated fat diets for 4 weeks. Data are relative amounts of genes of interest normalised to glyceraldehyde 3-phosphate dehydrogenase expression levels as an endogenous internal standard. Normalised values are calibrated to hamsters fed control selenium (Se) 0·15 parts per million (ppm) diet, with control expression set at 1. □, Se 0·15 ppm; , Se 0·85 ppm; , Se 1·7 ppm; , Se 3·4 ppm. Values are means, with their standard errors represented by vertical bars (n 10). * Mean value was significantly different from that for the control Se 0·15 ppm diet (P ≤ 0·05; Mixed model).
Selenite supplementation increased hepatic 27-hydroxycholesterol concentrations
Hamsters fed the Se 3·4 ppm and Se 1·7 ppm diets showed increased hepatic concentrations of 27-OHC as compared to hamsters fed the control Se 0·15 ppm diet (Fig. 3). Hepatic 27-OHC concentrations showed a tendency to correlate with hepatic mRNA abundance of Abcg8 (r 2 0·3, P = 0·10).
Fig. 3 Effect of selenite supplementation on hepatic oxysterol concentrations in adult male Syrian hamsters fed high-cholesterol and high-saturated fat diets for 4 weeks. □, Selenium (Se) 0·15 parts per million (ppm); , Se 0·85 ppm; , Se 1·7 ppm; , Se 3·4 ppm. Values are means, with their standard errors represented by vertical bars (n 10). * Mean values were significantly different from control (P ≤ 0·05; Mixed model). 7-Keto, 7-ketocholesterol; 24(S)-OHC, 24-hydroxycholesterol; 25-OHC, 25-hydroxycholesterol; 27-OHC, 27-hydroxycholesterol.
Discussion
The major aim of the present study was to elucidate the mechanisms of cholesterol-lowering action of Se supplementation by examining the dose–response effect of supplemental selenite on the mRNA expression of Abcg5, Abcg8 and Npc1l1 transporters in relation to tissue cholesterol concentrations(Reference Kauf, Dawczynski and Jahreis1–Reference Vinson, Stella and Flanagan17). A notable finding was the association between the three doses of Se (0·85, 1·7 and 3·4 ppm) and the increased expression of hepatic Abcg8 despite no change in hepatic Abcg5 mRNA abundance (Fig. 1). Likewise, selenite supplementation was associated with a tendency to increased Abcg8 with no effect on Abcg5 mRNA expression levels in the jejunum (Fig. 2). The combination of unchanged hepatic Abcg5 mRNA expression and up-regulation of hepatic Abcg8 mRNA abundance is in concordance with the lack of effect of selenite supplementation on biliary cholesterol content, as both Abcg5 and Abcg8 are required for biliary cholesterol secretion(Reference Duan, Wang and Wang21). The present study thus indicates that biliary cholesterol secretion through up-regulation of the heterodimer of Abcg5 and Abcg8 transporters is not a primary cholesterol-lowering mechanism associated with selenite supplementation. As selenite supplementation was also not associated with altered faecal TC concentrations, it appears that increased faecal excretion of cholesterol mediated via the heterodimer of Abcg5 and Abcg8 transporters is not involved as a primary cholesterol-lowering mechanism of selenite supplementation. Selenite supplementation was not associated with altered biliary or faecal bile acid concentrations (data not shown) or hepatic Cyp7a1 mRNA expression levels (Fig. 1), which is compatible with the unchanged liver cholesterol content(Reference Horton, Cuthbert and Spady43). The present findings are in contrast to the observations of Iizuka et al. (Reference Iizuka, Sakurai and Tanaka7), who showed significant lowering of hepatic cholesterol concentrations in Se-supplemented rats fed a 1 % cholesterol diet and 0·5 % cholic acid for 10 weeks. The addition of dietary cholic acid may account for the aforementioned study differences since cholic acid can stimulate the Abcg5/Abcg8 pathway to increase biliary cholesterol secretion(Reference Yu, Gupta and Xu44).
The observation of unchanged tissue Abcg5 and increased Abcg8 mRNA expression with selenite supplementation contrasts with the coordinated regulation of the two transporters shown with activation of the liver X receptor(Reference Berge, Tian and Graf45), which has been suggested to be the primary regulator of Abcg5 and Abcg8 (Reference Brown and Yu46) and that acts as a cellular cholesterol sensor. To date, however, a liver X responsive element has not yet been identified in the 5′-flanking region of the Abcg5 and Abcg8 genes, and the mechanism of regulation of the genes is currently unknown. The extent to which Se-induced changes in tissue Abcg8 contributed to the observed decreased plasma lipid concentrations (Table 3) is not clear. A very recent study, investigating the effect of low levels of supplemental selenate in growing rats, observed a tendency for increased plasma TC concentrations and decreased Abcg8 mRNA expression in the livers of supplemented rats as compared to Se-sufficient rats(Reference Wolf, Mueller and Hirche47). Interestingly, polymorphisms in Abcg8 but not Abcg5 genes have been related to higher serum TC and LDL-C concentrations in humans(Reference Chen, Shin and Kuo48). In the present study, increases in hepatic Abcg8 were associated with either lowered plasma TC of hamsters receiving 0·85 ppm Se or lowered LDL-C concentrations in hamsters fed 1·7 ppm Se (Table 3). In hamsters receiving the highest Se dose of 3·4 ppm, increased tissue Abcg8 mRNA expression was associated with decreased plasma concentrations of both TC and LDL-C (Table 3). Although hepatic concentrations of 27-OHC were increased by selenite supplementation at the two highest doses (1·7 and 3·4 ppm; Fig. 3), only a weak tendency to correlate was noted between Abcg8 mRNA expression and hepatic levels of 27-OHC (r 2 0·3; P = 0·10) and no correlation was noted between hepatic 27-OHC content and hepatic expression of Abcg5. These latter results contrast with the findings of Chen et al. (Reference Chen, Chen and Head27) that suggest a regulatory role for hepatic 24(S)-OHC, 25-OHC and 27-OHC on hepatic Abcg5 and Abcg8 mRNA expression in the murine model. The aforementioned differences may be related to the species-specific responses of Abcg5 and Abcg8 expression to cholesterol treatment(Reference Dieter, Maher and Cheng49). The increased hepatic 27-OHC concentrations with selenite supplementation were probably the result of enhanced Cyp27a1 mRNA expression that we have shown previously(Reference Poirier, Cockell and Ratnayake10).
The possible mechanisms involved with the decrease in Npc1l1 mRNA abundance noted in the jejunum of hamsters consuming the higher Se doses (1·7 and 3·4 ppm) are unclear. Regulation of Npc1l1 gene expression is largely unknown and inconsistent(Reference Brown and Yu46) and similar to Abcg5/g8, a liver X responsive element in the promoter of NPC1L1 has not yet been identified(Reference Brown and Yu46). Decreased jejunal expression of Npc1l1 could be related to the decreased plasma LDL-C concentrations seen with these diets (Table 3). Ezetimibe monotherapy, which targets NPC1l1, lowers plasma LDL-C concentrations in humans(Reference Dujovne, Ettinger and McNeer50). As the levels of hepatic TC, a surrogate marker of cholesterol absorption(Reference Salisbury, Davis and Burrier24), remained unchanged with selenite supplementation (Table 4) along with no significant increases in faecal cholesterol, the possible relationship between decreased Npc1l1 expression and lowered plasma LDL-C remains speculative. As selenite supplementation has been shown to significantly enhance fermentation in the rat colon(Reference Kim and Combs51), it is conceivable that decreased Npc1l1 mRNA expression resulted in increased faecal TC concentrations producing unmeasured cholesterol metabolites via intestinal microbial conversion.
The increases in Ldlr mRNA abundance (Figs. 1 and 2) shown in the tissues of hamsters consuming the two highest doses of Se (1·7 and 3·4 ppm) might be related to the lowered plasma LDL-C concentrations. LDL-C is cleared from the circulation mainly through uptake by the hepatic LDL receptor as most plasma LDL-C turnover is accounted for by hepatic and small-intestine uptake(Reference Spady and Dietschy52). The findings of the present study are in agreement with Dhingra & Bansal(Reference Dhingra and Bansal6), who reported similar increases in hepatic Ldlr mRNA expression levels and lowered plasma LDL-C concentrations in rats fed a high cholesterol (2 %) diet receiving 1·0 ppm selenite supplementation(Reference Dhingra and Bansal6). The increased hepatic Ldlr mRNA together with unchanged hepatic cholesterol content suggests that non-sterol mechanisms might be involved in selenite-induced hepatic LDL-C clearance in the Syrian hamster. Although Ldlr mRNA expression is regulated by the cellular concentrations of cholesterol(Reference Horton, Goldstein and Brown53) and oxysterols(Reference Lund and Björkhem54) through the sterol regulatory element-binding protein 2 transcription factor pathway, other transcription factors can intervene in this regulatory process(Reference Kong, Liu and Jiang55). In support of this latter contention, a recent study showed an absence of effect of selenite supplementation on hepatic sterol regulatory element-binding protein 2 mRNA expression relative to Se-sufficient rats(Reference Wolf, Mueller and Hirche47). The unchanged liver TC concentrations by selenite supplementation might be due to the inability of selenite to modulate hepatic Hmgcr mRNA abundance (Fig. 1). The absence of effect of selenite on Hmgcr mRNA expression agrees with similar recent findings shown in growing rats fed with a low-fat, low-cholesterol diet following sodium selenate supplementation(Reference Wolf, Mueller and Hirche47), which suggests that intake of fat and cholesterol does not modulate the lack of impact of Se supplementation on hepatic Hmgcr mRNA expression. On the other hand, our findings differ from previous rat studies showing decreased hepatic Hmgcr mRNA expression in conjunction with high-cholesterol diets supplemented with 1·0 ppm selenite(Reference Dhingra and Bansal5). The above contrasting results in hepatic Hmgcr mRNA expression may be due to species differences in regulation of the Hmgcr gene when faced with a cholesterol challenge(Reference Horton, Cuthbert and Spady43).
Supplementation of selenite at the 0·85 ppm Se dose was associated with significantly decreased plasma concentrations of HDL-C, which was not observed with the two highest doses of Se (1·7 and 3·4 ppm; Table 3). The latter result is in agreement with previous hamster work showing no effect on plasma HDL-C levels from high-dose Se supplementation(Reference Vinson, Stella and Flanagan17). Plasma concentrations of TAG were increased following Se intake at 1·7 ppm, which might be partly due to lower plasma lipoprotein lipase activity, which has previously been shown to be lowered by high dietary selenite (5 mg/kg diet) in tumour-bearing Wistar rats(Reference Chidambaram and Baradarajan11). Conversely, the highest dose of Se (3·4 ppm) showed no effect on plasma TAG levels as compared to control, which agrees with previous work with hamsters fed comparable levels of fat and cholesterol(Reference Poirier, Cockell and Hidiroglou9) and in hamsters fed comparable levels of Se in conjunction with a standard rodent diet(Reference Vinson, Stella and Flanagan17). In contrast, previous studies have shown significant decreases in plasma TAG concentrations in human subjects(Reference Djujic, Jozanov-Stankov and Milovac3), rats(Reference Crespo, Lanca and Vasconcelos12) and mice(Reference Mueller and Pallauf8). Discrepancy in results may be related to differences in species, dosage, duration of feeding trial or form of Se used. Higher hepatic TAG concentrations were observed with the higher doses (1·7 and 3·4 ppm; Table 4). The mechanisms underlying the effects of selenite on hepatic TAG concentrations are not clear but are consistent with the associations shown in rodent models between Se and increases in fatty acid synthase mRNA expression(Reference Iizuka, Sakurai and Tanaka7), PPAR-γ expression(Reference Mueller and Pallauf8) and protein tyrosine phosphatase 1B activity(Reference Mueller, Klomann and Wolf56), which have also been associated with fatty liver and insulin resistance in rats fed high-fructose diets(Reference Shimizu, Ugi and Maegawa57). Thus, it is possible that reduced insulin sensitivity could be a long-term consequence of increased liver TAG concentrations noted in the hamsters receiving the 1·7 and 3·4 ppm Se doses. These latter findings demonstrate the importance of examining liver lipid metabolism when assessing suitability of the plasma lipid-lowering effects of high-dose Se supplementation that is receiving increased research attention(Reference Stranges, Laclaustra and Ji58). The aforementioned results also call into question the long-term intake of selenite above nutrient recommendations due to potentially harmful increases in hepatic TAG that could predispose to decreased insulin sensitivity, despite the possible benefits of lowered plasma cholesterol concentrations. Future studies would need to be performed in higher primate experimental models for closer extrapolation of the aforementioned findings to humans.
The use of a rodent species to model the human response to dietary Se intake is a limitation of the present study. Regardless, the Syrian hamster is the rodent model most similar to humans with regard to cholesterol metabolism(Reference Dietschy18, Reference Zhang, Wang and Jiao19) and the present study is the first to explore the potential mechanisms of Se on cholesterol metabolism in the Syrian hamster as opposed to other models such as the rat. In contrast to the hamster, the rat is resistant to diet-induced hypercholesterolaemia due to its ability to down-regulate hepatic activity of Hmgcr and to stimulate Cyp7a1 gene abundance, and thus is highly efficient in the conversion of cholesterol into bile acids(Reference Kong, Liu and Jiang55). Another limitation concerns the dietary levels of Se ingested by the hamsters that correspond to 8-, 16- and 32-fold higher intakes than the human Se requirement, when adjusted for the average daily food intake and the final body weights of the hamsters. Although these Se doses appeared to be well-tolerated by the hamsters, such levels of Se intake are not normally seen in the human context, and so direct extrapolation to humans without additional experimentation is inappropriate. To identify the safe and efficacious levels of Se intakes, prospective epidemiological studies and randomised clinical trials are needed in different populations that take into account the ranges of intakes for the different speciated forms of Se.
In summary, this study examined the effect of supplemental selenite on hepatic and jejunal Abcg5, Abcg8 and Npc1l1 mRNA expression in the Syrian hamster in the context of its impact on cholesterol absorption and excretion, particularly in relation to plasma lipid levels. Supplemental selenite was shown to influence important genes involved in cholesterol homeostasis, Abcg8, Ldlr and Npc1l1, which provides insight into the mechanisms underlying the cholesterol-modulating effects of dietary Se in humans. The present findings indicate that the hypocholesterolaemic effects of supplemental selenite are not associated with biliary and faecal cholesterol secretion and do not appear to involve liver X receptor activation. Future studies are needed to explore the possibility that plasma cholesterol-lowering action of selenite supplementation is mediated via sterol-independent mechanisms.
Acknowledgements
The present study was supported by the National Sciences and Engineering Research Council of Canada through a grant to S. K. Additional funding for this study was awarded to K. A. C., S. K. and K. A. S., through the Innovative Science Competition, Office of the Chief Scientist, Health Canada. J. P., K. A. C., K. A. S. and S. K were responsible for the planning of the experiments and along with W. M. N. R. for evaluation of the results and writing of the manuscript. J. P. conducted the statistical evaluation of all data and all experimental analyses except for jejunal Npc1l1 and tissue Abcg5 mRNA expression analyses, which were conducted by E. S. and H. G., respectively. P. G. conducted the animal experiment and assisted with laboratory analyses along with H. R. and C. G. All authors read and approved the final manuscript. The authors declare that there are no conflicts of interest.
