α-Tocopherol modulates the low density lipoprotein receptor of human HepG2 cells

Pal Sebely, Thomson, Bottema, Roach

Description

The aim of this study was to determine the effects of vitamin E (α-tocopherol) on the low density lipoprotein (LDL) receptor, a cell surface protein which plays an important role in controlling blood cholesterol. Human HepG2 hepatoma cells were incubated for 24 hours with increasing amounts of α, δ, or γ-tocopherol. The LDL receptor binding activity, protein and mRNA, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA, cell cholesterol and cell lathosterol were measured. The effect of α-tocopherol was biphasic. Up to a concentration of 50 μM, α-tocopherol progressively increased LDL receptor binding activity, protein and mRNA to maximum levels 2, 4 and 6-fold higher than control, respectively. The HMG-CoA reductase mRNA and the cell lathosterol concentration, indices of cholesterol synthesis, were also increased by 40% over control by treatment with 50 μM α-tocopherol. The cell cholesterol concentration was decreased by 20% compared to control at 50 μM α-tocopherol. However, at α-tocopherol concentrations higher than 50 μM, the LDL receptor binding activity, protein and mRNA, the HMG-CoA reductase mRNA and the cell lathosterol and cholesterol concentrations all returned to control levels. The biphasic effect on the LDL receptor was specific for α-tocopherol in that δ and γ-tocopherol suppressed LDL receptor binding activity, protein and mRNA at all concentrations tested despite the cells incorporating similar amounts of the three homologues. In conclusion, α-tocopherol, exhibits a specific, concentration-dependent and biphasic "up then down" effect on the LDL receptor of HepG2 cells which appears to be at the level of gene transcription. Cholesterol synthesis appears to be similarly affected and the cell cholesterol concentration may mediate these effects.

Sujets

• Vitamin E
• LDL receptor
• HMG-CoA reductase
• Cholestérol

Informations

• Publié par: biomed
• Publié le: 01 janvier 2003
• Nombre de lectures: 10
• Langue: English

Extrait

BioMed CentralNutrition Journal
Open AccessResearch
α-Tocopherol modulates the low density lipoprotein receptor of
human HepG2 cells
1 2 3Sebely Pal* , Andrew M Thomson , Cynthia DK Bottema and
4Paul D Roach
1 2Address: Department of Nutrition, Dietetics and Food Sciences, Curtin University of Technology, Perth, Western Australia, Laboratory for Cancer
3Medicine and University Department of Medicine, University of Western Australia, Royal Perth Hospital, Perth, Western Australia, Department
4of Animal Science Waite Campus, University of Adelaide Glen Osmond, SA 5064, Australia and CSIRO Human Nutrition, PO Box 1004, SA 5000,
Australia
Email: Sebely Pal* - [e-mail]; Andrew M Thomson - [e-mail]; Cynthia DK Bottema - [e-mail];
Paul D Roach - [e-mail]
* Corresponding author
Published: 12 May 2003 Received: 19 December 2002
Accepted: 12 May 2003
Nutrition Journal 2003, 2:3
This article is available from: http://www.nutritionj.com/content/2/1/3
© 2003 Pal et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media
for any purpose, provided this notice is preserved along with the article's original URL.
vitamin Eα-tocopherolLDL receptorHepG2 cellsHMG-CoA reductasecholesterol
Abstract
The aim of this study was to determine the effects of vitamin E (α-tocopherol) on the low density
lipoprotein (LDL) receptor, a cell surface protein which plays an important role in controlling blood
cholesterol. Human HepG2 hepatoma cells were incubated for 24 hours with increasing amounts
of α, δ, or γ-tocopherol. The LDL receptor binding activity, protein and mRNA, 3-hydroxy-3-
methylglutaryl coenzyme A (HMG-CoA) reductase mRNA, cell cholesterol and cell lathosterol
were measured. The effect of α-tocopherol was biphasic. Up to a concentration of 50 µM, α-
tocopherol progressively increased LDL receptor binding activity, protein and mRNA to maximum
levels 2, 4 and 6-fold higher than control, respectively. The HMG-CoA reductase mRNA and the
cell lathosterol concentration, indices of cholesterol synthesis, were also increased by 40% over
control by treatment with 50 µM α-tocopherol. The cell cholesterol concentration was decreased
by 20% compared to control at 50 µM α-tocopherol. However, at α-tocopherol concentrations
higher than 50 µM, the LDL receptor binding activity, protein and mRNA, the HMG-CoA reductase
mRNA and the cell lathosterol and cholesterol concentrations all returned to control levels. The
biphasic effect on the LDL receptor was specific for α-tocopherol in that δ and γ-tocopherol
suppressed LDL receptor binding activity, protein and mRNA at all concentrations tested despite
the cells incorporating similar amounts of the three homologues. In conclusion, α-tocopherol,
exhibits a specific, concentration-dependent and biphasic "up then down" effect on the LDL
receptor of HepG2 cells which appears to be at the level of gene transcription. Cholesterol
synthesis appears to be similarly affected and the cell cholesterol concentration may mediate these
effects.
tocopherol) status of rabbits can affect their plasma cho-Introduction
It has been known for over 60 years that the vitamin E (α- lesterol concentration. In 1936, Morgulis and Spencer [1]
Page 1 of 10
(page number not for citation purposes)Nutrition Journal 2003, 2 http://www.nutritionj.com/content/2/1/3
reported that the plasma cholesterol was twofold higher Methods and materials
than normal in rabbits made deficient in vitamin E and Cell culture
The HepG2 cells were grown under 5% CO at 37°C inthat dietary replenishment of the vitamin normalised the 2
cholesterol concentration. This effect was later confirmed Dulbecco's Modified Eagles Medium (DMEM) supple-
by others in the rat [2–4] as well as in the rabbit [5–7]. In mented with 12 µg/ml penicillin, 16 µg/ml gentamicin,
animal models of diet-induced hypercholesterolaemia, 20 mM HEPES buffer, 10 mM NaOH, 2 mM L-glutamine
where the animals are not deficient in vitamin E, α-toco- and 10% (v/v) fetal calf serum (FCS) (Commonwealth Se-
pherol supplementation also often decreases plasma cho- rum Laboratories, Melbourne, Australia) as previously de-
lesterol [8–12]. This is not always the case however; in scribed [21–23]. For enrichment experiments, cells were
some studies either no change [13–15] or even an increase grown to 80–90% confluency, and varying amounts of α,
[16] in plasma cholesterol was observed. In the rat how- δ or γ-tocopherol (Purity 95%; Sigma-Aldrich, Castle Hill,
ever, a concomitant deficiency in selenium may be more Australia) in ethanol were added to supplemented DMEM
relevant to increases in plasma cholesterol than the in- and the cells were incubated in the media for 24 h. The
duced deficiency in vitamin E [17] cells were then extensively washed in phosphate buffered
saline (PBS: 10 mM phosphate, 154 mM NaCl, pH 7) be-
Changes in the plasma cholesterol concentration may re- fore being scraped from the flasks and resuspended in
sult from effects the vitamin has on liver cholesterol me- PBS. Cell viability was assessed using the trypan blue dye
tabolism. Hepatic cholesterol synthesis has been found to exclusion test. Cellular protein was determined using the
be increased in vitamin E-deficient rabbits [5] and the method of Lowry et al [24].
conversion of cholesterol into bile acids was observed to
be decreased [5,6]. Such an increase in cholesterolgenesis Cellular Tocopherol Content
and a decrease in cholesterol catabolism is consistent with The tocopherol content of the cells was measured using
the increase in liver cholesterol concentration found in the method of Yang and Lee [25]. Briefly, 1.0 ml of 1%
the vitamin E-deficient rat [3,4]. ascorbic acid in 100% ethanol added to 1.0 ml of cell sus-
pension (Alpha-tocopherol acetate was used as an inter-
There is however no data on the effects of α-tocopherol, nal standard) and heated at 70°C for 2 min; then 0.3 ml
the biologically active homologue of vitamin E, [18] on of saturated KOH was added and incubated for 30 min in
the hepatic low density lipoprotein (LDL) receptor which a 70°C water bath. After cooling on ice, 1.0 ml distilled
is well known to play a major role in the control of plasma water and 4.0 ml hexane were added and shaken vigor-
cholesterol [19,20]. The importance of the LDL receptor is ously for 2 min; then the phases were separated by centrif-
most clearly seen in the human genetic disorder called fa- ugation at room temperature, 3000 × g for 10 min. An
milial hypercholesterolaemia where a deficiency in the re- aliquot of hexane phase (3.0 ml) was pipetted and dried
ceptor causes high levels of plasma cholesterol which lead under a stream of N [2] and redisolved in 0.2 ml metha-
to the premature development of atherosclerosis [20]. The nol. The aliquots (20 ml each) were injected to high per-
LDL receptor is also highly regulated in that various die- formance liquid chromatography (Waters, Milford, MA,
tary and pharmaceutical agents can affect its expression USA) for analysis on a C 18 column (5 mm 3 4.6 mm 3
[19,20] 25 cm) with the mobile phase of methanol-water (95:5)
and detected by a fluorometer set at excitation 205 and
The aim of the present study was therefore to determine emission 340 nm. The coefficient of variation over two as-
whether vitamin E could regulate the LDL receptor. Cul- sessments was less than 5%.
tured human HepG2 hepatoma cells, highly differentiat-
ed hepatocytes known to express lipoprotein receptors, LDL receptor binding assay
[21–23] were grown in the absence of added vitamin E. Human LDL, 1.025 >d > 1.050 g/ml, was isolated from 2–
Three naturally occurring vitamin E homologues, α, δ and 4 days-old blood (Red Cross, Adelaide, Australia) by se-
γ-tocopherol [18] were tested for their effects on the quential ultracentrifugation [26] and conjugated to colloi-
HepG2 cell LDL receptor mRNA, protein and LDL-bind- dal gold (LDL-gold) as described. [27,28] Freshly
ing activity. The effect of α-tocopherol on the mRNA of 3- collected and intact HepG2 cells (100 ug of protein) were
hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) re- incubated for 1 h at room temperature with LDL-gold (20
ductase, the rate-limiting enzyme in cholesterol biosyn- ug protein/ml) and buffer (60 mM Tris-HCL, pH 8.0, and
thesis, and on the cellular concentration of lathosterol, an 20 mg/ml BSA) in a total of 300 ul either in the presence
index of cholesterol synthesis, was also determined. The of 2 mM Ca(NO ) to measure total binding or 20 mM3 2
cell's cholesterol concentration was also measured. EDTA to measure calcium-independent binding. Cells
were then centrifuged at 400 × g for 10 min, resuspended
and washed in 300 ul of 2 mM Ca(NO ) for total binding3 2
or 300 ul of 20 mM EDTA (pH 8.0) for nonspecific
Page 2 of 10
(page number not for citation purposes)Nutrition Journal 2003, 2 http://www.nutritionj.com/content/2/1/3
binding. After centrifugation at 400 × g for 10 min, the gy, North Ryde, Australia) and the measurements in
cells were resuspended in 120 ul of 4% (w/v) gum arabic arbitrary absorbance units were taken as the mass of LDL
and the cell-bound LDL-gold was quantified using a silver receptor protein in the HepG2 cells. The assay was opti-
enhancement solution (IntenS