Intracellular trafficking of vitamin E in hepatocytes: the role of tocopherol transfer protein

Abstract

The term vitamin E denotes a family of tocopherols and tocotrienols, plant lipids that are essential for vertebrate fertility and health. The principal form of vitamin E found in humans, RRR -␣ -tocopherol (TOH), is thought to protect cells by virtue of its ability to quench free radicals, and functions as the main lipid-soluble antioxidant. Regulation of vitamin E homeostasis occurs in the liver, where TOH is selectively retained while other forms of vitamin E are degraded. Through the action of tocopherol transfer protein (TTP), TOH is then secreted from the liver into circulating lipoproteins that deliver the vitamin to target tissues. Presently, very little is known regarding the intracellular transport of vitamin E. We utilized biochemical, pharmacological, and microscopic approaches to study this process in cultured hepatocytes. We observe that tocopherol-HDL complexes are efficiently internalized through scavenger receptor class B type I. Once internalized, tocopherol arrives within ‫ف‬ 30 min at intracellular vesicular organelles, where it colocalizes with TTP, and with a marker of the lysosomal compartment (LAMP1), before being transported to the plasma membrane in a TTP-dependent manner. We further show that intracellular processing of tocopherol involves a functional interaction between TTP and an ABC-type transporter.

Altered vitamin E status in Niemann-Pick type C disease

“…As shown in Fig. 3A , NBD-tocopherol was efficiently taken up by the cells and concentrated in a vesicular, perinuclear compartment, reminiscent of our previous observations in human HepG2 and rat McARH-7777 hepatocytes ( 35,54 ). We quantitated fl uorescence intensity in images from three independent experiments, and we found that cells with reduced expression of either NPC1 or NPC2 accumulated ‫ف‬ 2-fold more NBD-tocopherol compared with control cells.…”

“…Finally, modest supplementation with vitamin E has been reported to result in a mild improvement in motor performance in a mouse model of NPC disease ( 34 ). On the cellular level, it has been established that uptake of vitamin E occurs via endocytosis ( 35,36 ) and that a signifi cant portion of the vitamin is found in lysosomes ( 37 ). In light of these observations, we hypothesized that proper intracellular traffi cking of vitamin E (and in turn, adequate antioxidant protection) depends on timely egress from the lysosome and, therefore, on the functionality of NPC1/2.…”

“…Given that sphingomyelin, glycosphingolipids, and phospholipids also accumulate in NPC-affected lysosomes ( 2, 48-51 ), we hypothesized that NPC1/2 proteins participate in the endocytic processing of the lipid-soluble antioxidant ␣ -tocopherol (vitamin E). To visualize the intracellular traffi cking of ␣ -tocopherol, we utilized NBD-tocopherol, a fl uorescent analog that we previously characterized in vitro ( 42,43,52,53 ) and in vivo ( 35,54 ). Using fl uorescence microscopy, we visualized the accumulation of NBD-tocopherol in cultured fi broblasts isolated from an NPC-affected patient (harboring the c.709C>T and c.3182T>C substitutions in the NPC1 gene) and control fi broblasts.…”

“…NBD-cholesterol (Invitrogen) and NBD-tocopherol ( 42,43 ) were complexed to serum lipoprotein as described earlier ( 35,44 ) and added to the culture media to a fi nal concentration of 20 M and incubated for 17 h at 37°C. The fl uorescent lipid was "chased" by incubation in normal media for 3 h more.…”

Novel Transcriptional Activities of Vitamin E: Inhibition of Cholesterol Biosynthesis

“…Antioxidants were added as either pre-formed serum complexes (25)(26)(27) or from ethanol stocks directly to the media. Both methods resulted in comparable uptake efficiencies as determined by accumulation of radioactively labeled tocopherol.…”