alpha-tocopherol transfer protein stimulates the secretion of alpha-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway

Abstract

Vitamin E (alpha-tocopherol) is a fat-soluble antioxidant that is transported by plasma lipoproteins in the body. alpha-Tocopherol taken up by the liver with lipoprotein is thought to be resecreted into the plasma in very low density lipoprotein (VLDL). alpha-Tocopherol transfer protein (alphaTTP), which was recently identified as a product of the causative gene for familial isolated vitamin E deficiency, is a cytosolic liver protein and plays an important role in the efficient recycling of plasma vitamin E. To throw light on the mechanism of alphaTTP-mediated alpha-tocopherol transfer in the liver cell, we devised an assay system using the hepatoma cell line McARH7777. Using this system, we found that the secretion of alpha-tocopherol was more efficient in cells expressing alphaTTP than in matched cells lacking alphaTTP. Brefeldin A, which effectively inhibits VLDL secretion by disrupting the Golgi apparatus, had no effect on alpha-tocopherol secretion, indicating that alphaTTP-mediated alpha-tocopherol secretion is not coupled to VLDL secretion. Among other agents tested, only 25-hydroxycholesterol, a modulator of cholesterol metabolism, inhibited alpha-tocopherol secretion. This inhibition is most likely mediated by oxysterol-binding protein. These results suggest that alphaTTP present in the liver cytosol functions to stimulate secretion of cellular alpha-tocopherol into the extracellular medium and that the reaction utilizes a novel non-Golgi-mediated pathway that may be linked to cellular cholesterol metabolism and/or transport.

Figure 1

(A) Structure of α-[¹⁴C]tocopheryl acetate. (B) Assay of α-tocopherol secretion from McARH7777 cells.

Figure 2

Effect of expression of αTTP on the secretion of α-tocopherol from McARH7777 cells. (A) Expression of αTTP by transformed McARH7777 cells. McARH7777 cells were stably transfected with an αTTP expression plasmid. After selection in medium supplemented with G418, surviving colonies were subcultured and the level of αTTP was assessed by Western blotting using an anti-αTTP polyclonal antibody. The transfected cells were named McA-TTP, and one of these transformants, named McA-TTP21, was tested. Lane 1, McARH7777; lane 2, McA-TTP21. (B) McARH7777 and McA-TTP21 cell monolayers were incubated with α-[¹⁴C]tocopheryl acetate in liposomes. After incubation for the indicated times, the amount of α-[¹⁴C]tocopherol in the cell (black) and medium (shaded) fractions were measured. (C) The amounts of α-[¹⁴C]tocopheryl acetate in the cell fraction were also measured.

Figure 3

Effect of BFA on the secretion of α-tocopherol from McA-TTP21 cells. McA-TTP21 cell monolayers were incubated with α-[¹⁴C]tocopheryl acetate and [¹⁴C]oleate in the presence (+) or absence (−) of BFA (1.0 μg/ml). After a 24-h incubation, the radioactivities corresponding to α-tocopherol and triglyceride of the cell and medium fractions were measured. The results are expressed as percentages of the secretion level obtained in the absence of BFA. Each point represents the mean ± SE of triplicate determinations.

Figure 4

Effect of 25-hydroxycholesterol on the secretion of α-tocopherol from McA-TTP21 cells. McA-TTP21 cell monolayers were incubated with α-[¹⁴C]tocopheryl acetate and [¹⁴C]oleate in the presence (+) or absence (−) of 25-hydroxycholesterol (2.5 μg/ml). After a 24-h incubation, the radioactivities corresponding to α-tocopherol and triglyceride of the cell and medium fractions were measured. The results are expressed as percentages of the secretion level obtained in the absence of 25-hydroxycholesterol. Each point represents the mean ± SE of triplicate determinations.

Figure 5

Effect of 25-hydroxycholesterol is probably mediated by OSBP. (A) Effects of various oxysterols on the secretion of α-tocopherol from McA-TTP21 cells. McA-TTP21 cells were incubated in the presence of 25-hydroxycholesterol (2.5 μg/ml, lane 1), 20-hydroxycholesterol (lane 2), or 7-ketocholesterol (lane 3). After a 24-h incubation, the radioactivities of the cell and medium fractions were measured. The results are expressed relative to values for cells incubated without additives. Each point represents the mean ± SE of triplicate determinations. (B) 25-Hydroxycholesterol-mediated suppression of the secretion of α-tocopherol from McA-TTP21 cells in the presence of BFA. McA-TTP21 cells were incubated in the presence of 25-hydroxycholesterol (2.5 μg/ml) with various concentrations of BFA. After a 24-h incubation, the radioactivities of the cell and medium fractions were measured. The results are expressed relative to values for cells incubated without additives. Each point represents the mean ± SE of triplicate determinations.