**3.1. Effects of vitamin E**

### *3.1.1. Intracellular handling of vitamin E*

Key factors in the hepatic handling of vitamin E have been outlined in the previous sections. This section will focus on the action of vitamin E on its own intracellular handling. As indicated above, the key enzyme for the intracellular trafficking of vitamin E is α-TTP, and the rate-limiting enzymes in vitamin E metabolism are CYP4F2 and CYP3A4. Hence, we will here focus on the known actions of vitamin E on these key players.

The key protein of the hepatic handling of vitamin E is α-TTP, with its implications in cellular trafficking, metabolism, and release of vitamin E. Hence, several studies have been conducted to elucidate a possible feedback regulation of α-TTP in response to vitamin E intake, resulting in alterations of the metabolism or the distribution of the vitamin. In principle, research is focused on three levels of regulation: mRNA expression, protein expression, and stabilization of α-TTP protein. However, contradictory results from rodent models have been reported. Fechner et al. found that hepatic α-TTP mRNA expression was strongly induced in rats depleted from vitamin E for 5 weeks after the intake of a TOH-supplemented diet for 24 h [72]. However, rats fed a vitamin E-depleted diet, control diet, or vitamin E-enriched diet for 20 weeks showed upregulation of α-TTP mRNA when vitamin E is deprived, but a downregulation when vitamin E was repleted. Hepatic α-TTP protein levels were comparable for depletion and control, but lowest in rats fed the repleted diet [73]. A similar study reported no differences in hepatic α-TTP mRNA levels of rats fed either a control diet or a diet rich in or low in vitamin E. However, in contrast to the aforementioned study, downregulation of α-TTP protein was reported in the vitamin E-depleted group, while high vitamin E intake did not alter the levels compared to control [74]. The lack of an effect of a vitamin E deficient diet for 290 days on hepatic α-TTP mRNA levels was also reported in another rat model [75]. In line with this, subcutaneous injection of vitamin E for up to 18 days did not alter α-TTP protein levels in rats [76]. However, mice fed a diet rich in vitamin E showed 20% higher hepatic α-TTP protein levels than mice fed a low vitamin E diet [77]. Taken together, some studies report elevated α-TTP levels due to a higher intake of vitamin E [72, 77], but some revealed no effect [74–76] or even lower levels [73]. Hence, further studies are needed to clarify the role of vitamin E in the regulation of α-TTP. In addition, an *in vitro* study suggested that vitamin E does not regulate α-TTP at the level of gene expression, but stabilizes α-TTP at the protein level upon binding and thus protects the protein from degradation, leading to higher α-TTP protein levels [78]. Reports on the hepatic mRNA levels might thus be of minor importance for the interpretation of the contribution of vitamin E to α-TTP action; however, the findings on α-TTP protein expression are also inconsistent.

the loading of lipoproteins with α-TOH from the plasma membrane has not been elucidated yet, although the involvement of ABC transporters has been suggested [56, 67, 70]. However, ABC transporters are fueling HDL particles, which is in contrast to the assumption that the hepatic release of α-TOH is mediated via VLDL. In turn, two explanations have evolved: first, α-TOH translocates spontaneously from the membrane to VLDL like free cholesterol [65], and second, α-TOH is transported to HDL via ABCA1 and is then spontaneously transferred to VLDL [71]. However, both hypotheses need evaluation. A recent report on the self-assembly of α-TTP to form nanoparticles and transport vitamin E to tissues protected by endothelial barriers like the brain [34] opens another possible way for the distribution of vitamin E

Key factors in the hepatic handling of vitamin E have been outlined in the previous sections. This section will focus on the action of vitamin E on its own intracellular handling. As indicated above, the key enzyme for the intracellular trafficking of vitamin E is α-TTP, and the rate-limiting enzymes in vitamin E metabolism are CYP4F2 and CYP3A4. Hence, we will here

The key protein of the hepatic handling of vitamin E is α-TTP, with its implications in cellular trafficking, metabolism, and release of vitamin E. Hence, several studies have been conducted to elucidate a possible feedback regulation of α-TTP in response to vitamin E intake, resulting in alterations of the metabolism or the distribution of the vitamin. In principle, research is focused on three levels of regulation: mRNA expression, protein expression, and stabilization of α-TTP protein. However, contradictory results from rodent models have been reported. Fechner et al. found that hepatic α-TTP mRNA expression was strongly induced in rats depleted from vitamin E for 5 weeks after the intake of a TOH-supplemented diet for 24 h [72]. However, rats fed a vitamin E-depleted diet, control diet, or vitamin E-enriched diet for 20 weeks showed upregulation of α-TTP mRNA when vitamin E is deprived, but a downregulation when vitamin E was repleted. Hepatic α-TTP protein levels were comparable for depletion and control, but lowest in rats fed the repleted diet [73]. A similar study reported no differences in hepatic α-TTP mRNA levels of rats fed either a control diet or a diet rich in or low in vitamin E. However, in contrast to the aforementioned study, downregulation of α-TTP protein was reported in the vitamin E-depleted group, while high vitamin E intake did not alter the levels compared to control [74]. The lack of an effect of a vitamin E deficient diet for 290 days on hepatic α-TTP mRNA levels was also reported in another rat model [75]. In line with this, subcutaneous injection of vitamin E for up to 18 days did not alter α-TTP protein levels in rats [76]. However, mice fed a diet rich in vitamin E showed 20% higher hepatic α-TTP protein levels than mice fed a low vitamin E diet [77]. Taken together, some studies

throughout the body starting from the liver.

*3.1.1. Intracellular handling of vitamin E*

**3.1. Effects of vitamin E**

6 Vitamin E in Health and Disease

**3. Factors influencing hepatic handling of vitamin E**

focus on the known actions of vitamin E on these key players.

The rate-limiting enzymes of vitamin E metabolism are CYP4F2 and CYP3A4. The latter was reported to be under transcriptional control of pregnane-X-receptor (PXR) [79, 80]. Hence, vitamin E might regulate its metabolism by binding to PXR and subsequent alteration of the expression of the enzymes involved in the first catabolic step. Indeed, studies using cells transfected with reporter genes provided evidence for an activation of PXR by different vitamin E structures (i.e., TOHs, T3s, and metabolites) [81, 82]. Interestingly, α-, δ-, and γ-TOH as well as α- and γ-T3 activated PXR in HepG2 liver cells transfected with human PXR and chloramphenicol acetyl transferase linked to two PXR responsive elements [81], while α- and γ-TOH as well as their metabolites α- and γ-CEHC did not in transfected colon carcinoma cells [82]. However, the LCM α-13′-COOH activated PXR in the latter cellular system and so did γ-T3 [82]. This finding implicates that the LCM of TOH are the responsible mediators of reported TOH actions via PXR. Hence, the findings in hepatic HepG2 cells [81] might be due to a higher catabolic rate of TOH and in turn the more efficient formation of the LCM than in colon cells. However, these findings were made in artificial cellular reporter systems and might not resemble the actual (hepatic) situation *in vivo*. Further, the specificity of PXR might depend on the species, as γ-T3 (the vitamin E form that activated PXR in both of the aforementioned studies) fails to bind murine PXR [83]. However, results obtained *in vivo* support the regulation of Cyp3a11 (the murine orthologue of CYP3A4) by vitamin E via PXR. Mice supplemented with α-TOH show elevated hepatic expression of Cyp3a11, while their PXRdeficient counterparts as well as mice with humanized PXR showed no upregulation of Cyp3a11 in response to α-TOH [84]. The same finding was made for Cyp4f13, the murine orthologue of CYP4F2, in this model [84]. These findings suggest that both enzymes are under the control of PXR and murine, but not human PXR is susceptible to α-TOH (or its metabolites as outlined above). Further studies reporting upregulation of hepatic Cyp3a in rodent models with α-TOH supplementation support this finding [76, 83, 85]. Interestingly, in these studies, γ-TOH and γ-T3 had no effect on Cyp3a expression [83, 85], supporting the suggested specificity of murine PXR for α-TOH. In line with this, γ-TOH did not alter the expression of Cyp4f13 in mice [85]. However, subcutaneous application of α-TOH in rats did not induce Cyp4F2 levels [76], which is in contrast to the above mentioned induction of Cyp4f13 in mice via PXR [84]. The reported induction of CYP4F2 activity in HepG2 cells by α-TOH further complicates the interpretation of the data on the effect of vitamin E on CYP4F2 [45]. Taken together, there is evidence for the regulation of CYP4F2 and CYP3A4 via PXR by vitamin E in the human liver. However, several aspects need further clarification, for instance, species and vitamin E isoform specificity of PXR, the regulation of CYP4F2 by vitamin E or the relevance of the α-LCM as true mediators of α-TOH effects via PXR.
