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Cell senescence is due to the permanent cell cycle arrest that occurs as a result of the inherent limited replicative capacity toward the Hayflick limit (replicative senescence), or in response to various stressors (stress-induced premature senescence, SIPS). With the acquisition of the senescence-associated secretory phenotype (SASP), cells release several molecules (cytokines, proteases, lipids), and express the senescence-associated beta-galactosidase (SA-β-Gal). Here we tested whether vitamin E affects SA-β-Gal in an in vitro model of cell ageing. Skin fibroblasts from human subjects of different age (1, 13, 29, 59, and 88 years old) were cultured until they reached replicative senescence. At different passages (Passages 2, 9, 13, and 16), these cells were treated with vitamin E for 24 hr. Vitamin E reduced SA-β-Gal in all cells at passage 16, but at earlier passage numbers it reduced SA-β-Gal only in cells isolated from the oldest subjects. Therefore, short time treatment with vitamin E decreases SA-β-Gal in cells both from young and old subjects when reaching replicative senescence; but in cells isolated from older subjects, a decrease in SA-β-Gal by vitamin E occurs also at earlier passage numbers. The possible role of downregulation of CD36 by vitamin E, a scavenger receptor essential for initiation of senescence and SASP, is discussed.

The name vitamin E, was given by Barnett and Sure who suggested that the factor proposed by Evans and Bishop as substance "X," be termed vitamin "E" as the next vitamin after the A, B, C and D vitamins had been already described. The identification of vitamin E with a-tocopherol was made in 1936 by Evans' group. One year later β-tocopherol and 11 years later δ-tocopherol were isolated. Tocotrienol (named zetatocopherol) was first described in 1957 and later isolated in 1961. The antioxidant property of tocopherols was reported by Olcott and Emerson in 1937. Inherited vitamin E deficiency, AVED, characterized by a form of neuromyopathy was first described in 1981. The disease, was localized to chromosome 8q and found to be caused by a mutation of the a-TTP gene. The subsequent paragraphs are not a comprehensive review but only critical reflections on some important aspects of vitamin E research.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.mam.2020.100894. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Four tocopherols are available in nature and are absorbed with the diet, but only one RRR-α-tocopherol satisfies the criteria of being a vitamin. The biological activity of the different tocopherols studied in the rat by the resorption-gestation test has been inconsistently extrapolated to human beings where the tocopherols have no influence on a successful pregnancy. Diminution of RRR-α-tocopherol intake results in diseases characterized by ataxia, whose pathogenetic mechanism, despite vigorous claims, has not been clarified. The calculation of the Daily Reference Intake (DRI), necessary to prevent disease, is based on an obsolete test, the peroxide-induced erythrocyte hemolysis, called the gold standard, but of highly questioned validity. If many epidemiological studies have given positive results, showing prevention by high vitamin E containing diets of cardiovascular events, neurodegenerative disease, macular degeneration and cancer, the clinical confirmatory intervention studies were mostly negative. On the positive side, besides preventing vitamin E deficiency diseases, vitamin E has shown efficacy as anti-inflammatory and immune boosting compound. It has also shown some efficacy in protecting against nonalcoholic hepato-steatosis. At a molecular level, vitamin E and some of its metabolites have shown capacity of regulating cell signaling and modulating gene transcription.

An answer to the question posed by the title must be simple not to disturb in his tomb Albert Einstein, who wrote "Man muß die Dinge so einfach wie möglich machen. Aber nicht einfacher". A simple answer (not simpler) can be: Antioxidants are not antioxidants, they are not wonder drugs and they are not all quackery; but they are not nothing. The arguments in support of this conundrumic statement will be developed below. © 2017 BioFactors, 43(6):785-788, 2017.

The CD36 scavenger receptor binds several ligands and mediates ligand uptake and ligand-dependent signal transduction and gene expression, events that may involve CD36 internalization. Here we show that CD36 internalization in THP-1 monocytes is triggered by α-tocopherol (αT) and more strongly by α-tocopheryl phosphate (αTP) and EPC-K1, a phosphate diester of αTP and L-ascorbic acid. αTP-triggered CD36 internalization is prevented by the specific covalent inhibitor of selective lipid transport by CD36, sulfo-N-succinimidyl oleate (SSO). Moreover, SSO inhibited the CD36-mediated uptake of 14C-labelled αTP suggesting that αTP binding and internalization of CD36 is involved in cellular αTP uptake, whereas the uptake of αT was less affected. Similar to that, inhibition of selective lipid transport of the SR-BI scavenger receptor resulted mainly in reduction of αTP and not αT uptake. In contrast, uptake of αT was mainly inhibited by Dynasore, an inhibitor of clathrin-mediated endocytosis, suggesting that the differential regulatory effects of αTP and αT on signaling may be influenced by their different routes of uptake. Interestingly, αTP and EPC-K1 also reduced the neutral lipid content of THP-1 cells and the phagocytosis of fluorescent Staphylococcus aureus bioparticles. Moreover, induction of the vascular endothelial growth factor (VEGF) promoter activity by αTP occurred via CD36/PI3Kγ/Akt, as it could be inhibited by specific inhibitors of this pathway (SSO, Wortmannin, AS-605240). These results suggest that αTP activates PI3Kγ/Akt signaling leading to VEGF expression in monocytes after binding to and/or transport by CD36, a receptor known to modulate angiogenesis in response to amyloid beta, oxLDL, and thrombospondin. J. Cell. Biochem. 118: 1855-1867, 2017. © 2017 Wiley Periodicals, Inc.

The discovery of vitamin E will have its 100th anniversary in 2022, but we still have more questions than answers regarding the biological functions and the essentiality of vitamin E for human health. Discovered as a factor essential for rat fertility and soon after characterized for its properties of fat-soluble antioxidant, vitamin E was identified to have signaling and gene regulation effects in the 1980s. In the same years the cytochrome P-450 dependent metabolism of vitamin E was characterized and a first series of studies on short-chain carboxyethyl metabolites in the 1990s paved the way to the hypothesis of a biological role for this metabolism alternative to vitamin E catabolism. In the last decade other physiological metabolites of vitamin E have been identified, such as α-tocopheryl phosphate and the long-chain metabolites formed by the ω-hydroxylase activity of cytochrome P-450. Recent findings are consistent with gene regulation and homeostatic roles of these metabolites in different experimental models, such as inflammatory, neuronal and hepatic cells, and in vivo in animal models of acute inflammation. Molecular mechanisms underlying these responses are under investigation in several laboratories and side-glances to research on other fat soluble vitamins may help to move faster in this direction. Other emerging aspects presented in this review paper include novel insights on the mechanisms of reduction of the cardiovascular risk, immunomodulation and antiallergic effects, neuroprotection properties in models of glutamate excitotoxicity and spino-cerebellar damage, hepatoprotection and prevention of liver toxicity by different causes and even therapeutic applications in non-alcoholic steatohepatitis. We here discuss these topics with the aim of stimulating the interest of the scientific community and further research activities that may help to celebrate this anniversary of vitamin E with an in-depth knowledge of its action as vitamin.

Older studies of the phosphoric acid ester of α-tocopherol (TP) in enzymes and animal models have given no conclusive results. More recently, the molecule has been the object of new scientific attention as an extension to the renewed popularity of vitamin E (α-tocopherol). α-Tocopherol is a micronutrient that is needed to prevent a form of cerebellar ataxia, but several alleged functions have been attributed to it, including protection against neurodegeneration, atherosclerosis, cancer and aging. Initially, the biological function of TP was seen as a pro-vitamin E capable of releasing α-tocopherol in the body. Subsequent studies have indicated that the nanomolar amount of TP in the body is not compatible with functioning as a reserve of vitamin E, whose concentration in plasma is in the micromolar order. On the other hand, its existence in humans, animals and plants has prompted studies on TP's molecular functions, and these have revealed that it can be synthesized and hydrolyzed in cells and in animals. The enzymes responsible for α-tocopherol kinase and tocopheryl phosphate phosphatase cellular activities have not been purified. TP inhibits cell proliferation and regulates gene expression more potently than α-tocopherol; furthermore, some genes are exclusively regulated by TP. These signaling effects of TP are in connection with phosphatidyl inositol kinase. In animal models, TP has shown more potency than α-tocopherol against atherosclerosis and inflammation. TP has been proposed to be an activated form of α-tocopherol.

Curcumin, a polyphenol from turmeric (Curcuma longa), reduces inflammation, atherosclerosis, and obesity in several animal studies. In Ldlr-/- mice fed a high-fat diet (HFD), curcumin reduces plasma lipid levels, therefore contributing to a lower accumulation of lipids and to reduced expression of fatty acid transport proteins (CD36/FAT, FABP4/aP2) in peritoneal macrophages. In this study, we analyzed the molecular mechanisms by which curcumin (500, 1000, 1500 mg/kg diet, for 4 months) may influence plasma and tissue lipid levels in Ldlr-/- mice fed an HFD. In liver, HFD significantly suppressed cAMP levels, and curcumin restored almost normal levels. Similar trends were observed in adipose tissues, but not in brain, skeletal muscle, spleen, and kidney. Treatment with curcumin increased phosphorylation of CREB in liver, what may play a role in regulatory effects of curcumin in lipid homeostasis. In cell lines, curcumin increased the level of cAMP, activated the transcription factor CREB and the human CD36 promoter via a sequence containing a consensus CREB response element. Regulatory effects of HFD and Cur on gene expression were observed in liver, less in skeletal muscle and not in brain. Since the cAMP/protein kinase A (PKA)/CREB pathway plays an important role in lipid homeostasis, energy expenditure, and thermogenesis by increasing lipolysis and fatty acid β-oxidation, an increase in cAMP levels induced by curcumin may contribute to its hypolipidemic and anti-atherosclerotic effects. © 2016 BioFactors, 43(1):42-53, 2017.

This review deals with the expectations of vitamin E ability of preventing or curing, as a potent antioxidant, alleged oxidative stress based ailments including cardiovascular disease, cancer, neurodegenerative diseases, cataracts, macular degeneration and more. The results obtained with clinical intervention studies have highly restricted the range of effectiveness of this vitamin. At the same time, new non-antioxidant mechanisms have been proposed. The new functions of vitamin E have been shown to affect cell signal transduction and gene expression, both in vitro and in vivo. Phosphorylation of vitamin E, which takes place in vivo, results in a molecule provided with functions that are in part stronger and in part different from those of the non-phosphorylate compound. The in vivo documented functions of vitamin E preventing the vitamin E deficiency ataxia (AVED), slowing down the progression of non-alcoholic steato-hepatitis (NASH), decreasing inflammation and potentiating the immune response are apparently based on these new molecular mechanisms. It should be stressed however that vitamin E, when present at higher concentrations in the body, should exert antioxidant properties to the extent that its chromanol ring is unprotected or un-esterified.

Background Curcuminoids, mainly present in the plant rhizomes of turmeric (Curcuma longa), consist of mainly three forms (curcumin (CUR), bisdemethoxycurcumin (BDMC) and demethoxycurcumin (DMC)). It has been reported that different forms of curcuminoids possess different biological activities. However, the mechanisms associated with these differences are not well-understood. Recently, our laboratory found differences in the cellular uptake of these curcuminoids. Therefore, it has been inferred that these differences contribute to the different biological activities. Purpose In this study, we investigated the mechanisms of differential cellular uptake of these curcuminoids. Method Based on our previous study, we hypothesized the differential cellular uptake is caused by (I) polarity, (II) transporters, (III) metabolism rate of curcuminoids and (IV) medium components. These four hypotheses were each investigated by (I) neutralizing the polarities of curcuminoids by encapsulation into poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs), (II) inhibition of polyphenol-related absorption transporters, (III) analysis of the cellular curcuminoids and their metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (IV) use of different mediums in cell study. Results The differential cellular uptake was not affected by (I-III). However, when investigating (IV), not only CUR but also BDMC and DMC were incorporated into cells when serum free media was used. Furthermore, when we used the serum free medium containing bovine serum albumin (BSA), only CUR was taken up but BDMC and DMC were not. Therefore, we identified that the differential cellular uptake of curcuminoids is caused by the medium components, especially BSA. Also, the fluorescence quenching study suggested that differential cellular uptake is due to the different interaction between BSA and each curcuminoid. Conclusion The differential cellular uptake of curcuminoids was caused by the different interaction between curcuminoids and BSA. The results from this study might give clues on the mechanisms by which curcuminoids exhibit different physiological activities.

α-Tocopherol is the only tocopherol that has been shown to prevent the human deficiency disease Ataxia with Isolated Vitamin E Deficiency (AVED), and thus it is the only one that, for humans, can be called vitamin E. Vitamin E in addition to preventing AVED has documented immune boosting properties and an activity against nonalcoholic hepatosteatosis and low-grade inflammation. Epidemiological studies indicating that vitamin E could prevent cardiovascular events, neurodegenerative disease, macular degeneration, and cancer were in general not confirmed by clinical intervention studies. Vitamin E and some of its metabolites modulate cell signaling and gene transcription. Future research is needed to achieve a better understanding of the molecular events leading to gene regulation by vitamin E, especially in its phosphorylated form. Isolation and characterization of the vitamin E kinase and vitamin E phosphate phosphatase will help in the understanding of cell regulation processes modulated by vitamin E. A clarification of the pathogenesis of AVED remains an important goal to be achieved. © 2018 IUBMB Life, 71(4):411-415, 2019.

Background Dietary bioactive compounds capable of improving metabolic profiles would be of great value, especially for overweight individuals undergoing a caloric restriction (CR) regimen. Curcumin (Cur), a possible anti-obesity compound, and piperine (Pip), a plausible enhancer of Cur’s bioavailability and efficacy, may be candidate agents for controlling body fat, metabolism and low grade inflammation. Methods 47 eight-week-old male C57BL/6 mice were fed a high fat diet (HFD) for 23 weeks to induce obesity. Then, mice were divided into 5 groups. Group 1 continued on HFD ad libitum. The other 4 groups underwent CR (reduced 10% HFD intake for 10 weeks, 20% for 20 weeks) with Cur, Pip, Cur + Pip or none of these. Percent body fat, plasma inflammatory markers associated with obesity (interferon (IFN)-γ, interleukin (IL)-10, IL-12 p70, IL-1β, IL-6 and KC/GRO), plasma Cur metabolites and liver telomere length were measured. Results Compared to the other groups, obese mice who underwent CR and received Cur + Pip in their diet lost more fat and had significantly lower IL-1β and KC/GRO. Tandem mass spectrometry analysis of plasma from obese mice under CR showed no difference in Cur metabolite levels between groups supplemented with Cur alone or combined with Pip. However, plasma IL-1β levels were inversely correlated with curcumin glucuronide. Minor modulation of telomere length were observed. Conclusions It is plausible that supplementing the high fat diet of CR mice with Cur + Pip may increase loss of body fat and suppresses HFD induced inflammation. Combination of Cur and Pip has potential to enhance CR effects for the prevention of metabolic syndrome.

In several studies, vitamin E has been observed to influence angiogenesis and vasculogenesis. We recently showed that the phosphorylated form of α-tocopherol (αT), α-tocopheryl phosphate (αTP), increases the expression of the vascular endothelial growth factor (VEGF). Thus, αTP may act as an active lipid mediator increasing VEGF expression, angiogenesis, and vasculogenesis. Here, we investigated the molecular signaling mechanisms by which αTP induces VEGF expression using cultured HEK293 cells as model system. αT and more so αTP increased VEGF-promoter activity in a phosphatidylinositol-3-kinase gamma (PI3Kγ)-dependent manner. In contrast, after overexpression of PI3Kγ and/or protein kinase B (PKB), VEGF promoter activity was inhibited by αT and more so by αTP. Inhibition by αT and αTP was dependent on the lipid kinase activity of PI3Kγ, whereas an induction was seen with the protein kinase activity, consistent with a model in which PKB inhibition by αT or αTP occurs only when activated at the plasma membrane and possibly involves a phosphatase such as PHLPP1. PI3Kγ-induced VEGF expression was reduced when the human tocopherol-associated protein 1 (hTAP1/SEC14L2) was overexpressed suggesting formation of an inactive PI3Kγ/hTAP1 heterodimer, that could be reactivated by αT and more so by αTP. We suggest a novel signaling mechanism by which αTP stimulates PI3Kγ activity by stimulating hTAP-mediated phosphatidylinositol exchange and presentation to the enzyme and/or dissociation of an inactive heterodimer. At cellular level, hTAP may act as sensor for intracellular lipid information (location, type, and amount of lipid) and translate it into responses of PI3K-mediated signaling and gene expression.

Curcumin, a polyphenol from turmeric (Curcuma longa), reduces inflammation, atherosclerosis, and obesity in several animal studies. In Ldlr

We have previously shown that curcumin (CUR) may increase lipid accumulation in cultured human acute monocytic leukaemia cell line THP-1 monocytes/macrophages, but that tetrahydrocurcumin (THC), an in vivo metabolite of CUR, has no such effect. In the present study, we hypothesised that the different cellular uptake and/or metabolism of CUR and THC might be a possible explanation for the previously observed differences in their effects on lipid accumulation in THP-1 monocytes/macrophages. Chromatography with tandem MS revealed that CUR was readily taken up by THP-1 monocytes/macrophages and slowly metabolised to hexahydrocurcumin sulphate. By contrast, the uptake of THC was low. In parallel with CUR uptake, increased lipid uptake was observed in THP-1 macrophages but not with the uptake of THC or another CUR metabolite and structurally related compounds. From these results, it is possible to deduce that CUR and THC are taken up and metabolised differently in THP-1 cells, which determine their biological activity. The remarkable differential cellular uptake of CUR, relative to THC and other similar molecules, may imply that the CUR uptake into cells may occur via a transporter.

Of the 8 different analogues (α-, β-, γ-, δ-tocopherols and tocotrienols) designated as vitamin E, alpha-tocopherol (α-T) has been mostly studied, together with gamma-tocopherol (γ-T) which is abundant in the US diet. We compared the effect of dietary supplementation with adequate or high doses of α-T or γ-T on the number and type of genes expressed following T cell activation. C57BL/6 mice were fed diets containing adequate (30 ppm) or high (500 ppm) amounts of α-T or γ-T for 4 weeks. Spleen T cells were stimulated ex vivo with plate-bound anti-CD3 and soluble anti-CD28, and gene expression changes were assessed by gene array analysis. The data obtained indicated significant qualitative and quantitative differences between the two analogs in regulating gene expression induced by T cell stimulation. Genes were found uniquely responding to either high α-T (e.g. induced: CD40 ligand, lymphotoxin A) or γ-T (e.g. repressed: poliovirus receptor-related-2). Interestingly, in stimulated T-cells from mice supplemented with high amounts of α-T a bigger number of genes were activated than in mice supplemented with the same amounts of γ-T; under the same conditions γ-T repressed the expression of a number of genes larger than α-T. It is possible that the observed diminution in gene expression in T cells after high γ-T in vivo supplementation modulates inflammation or other T cell mediated functions.

A correct function of adipocytes in connection with cellular fatty acid loading and release is a vital aspect of energy homeostasis; dysregulation of these reactions can result in obesity and type 2 diabetes mellitus. In addition, adipocytes have been proposed to play a major role in preventing lipotoxicity by removing excess fatty acids from the circulation and converting them into triglycerides and thus decreasing the exposure of other cells to their potentially harmful effects. We report here that the addition of α-tocopheryl phosphate (but not α-tocopherol) to NIH3T3-L1 preadipocytes transcriptionally activates a set of genes TRB3 (Tribbles Homolog 3), Sestrin-2 (SESN2), and Insulin-Induced Gene 1 (INSIG1)] potentially preventing fat accumulation in these cells. In contrast, in differentiated adipocytes, α-tocopheryl phosphate is responsible for the transcriptional inhibition of the same genes, possibly facilitating fat uptake and storage. In conclusion, it appears that in proliferating preadipocytes α-tocopheryl phosphate foils fat accumulation, whereas in adipocytes it enhances it. These processes may be relevant in the regulation of excess fat accumulation and in prevention of lipotoxicity.

Recent evidence suggests potential benefits from phytochemicals and micronutrients in protecting against atherosclerosis and inflammation, but the molecular mechanisms of these actions are still unclear. Here, we investigated whether the dietary polyphenol curcumin can modulate the accumulation of lipids in monocytes/macrophages. Curcumin increased the expression of two lipid transport genes, the fatty acids transporter CD36/FAT and the fatty acids binding protein 4 (FABP4/aP2; P < 0.05), leading to increased lipid levels in THP-1 and RAW264.7 monocytes and macrophages (P < 0.05). To investigate the molecular mechanisms involved, we assessed the activity of Forkhead box O3a (FOXO3a), a transcription factor centrally involved in regulating several stress resistance and lipid transport genes. Curcumin increased FOXO3a-mediated gene expression by twofold (P < 0.05), possibly as a result of influencing FOXO3a phosphorylation and nuclear translocation. The curcumin derivative, tetrahydrocurcumin (THC), with similar chemical antioxidant activity as curcumin, did not show any measurable effects. In contrast to the in vitro results, curcumin showed a trend for reduction of lipid levels in peritoneal macrophages in LDL receptor knockout mice fed a high fat diet for 4 months, suggesting additional regulatory mechanisms in vivo. Thus, the up-regulation of FOXO3a activity by curcumin could be a mechanism to protect against oxidant- and lipid-induced damage in the inflammatory cells of the vascular system.

Vitamin E was originally discovered as a dietary factor essential for reproduction in rats. Since then, vitamin E has revealed many important molecular properties such as the scavenging of reactive oxygen and nitrogen species or the modulation of signal transduction and gene expression in antioxidant and nonantioxidant manners. A congenital disease, ataxia with vitamin E deficiency, which is characterized by impaired enrichment of α-tocopherol (αT) in plasma due to mutations in the α-tocopherol transfer protein gene, has been discovered. An effect of vitamin E on angiogenesis and vasculogenesis has been observed in several studies, and recently, it has been demonstrated in the placenta of pregnant ewes, possibly involving the stimulation of vascular endothelial growth factor (VEGF) expression. We recently observed that the phosphorylated form of αT, α-tocopheryl phosphate (αTP), increases the expression of VEGF. We propose that the stimulatory effect of αT on angiogenesis and vasculogenesis is potentiated by phosphorylation to αTP, which may act as a cofactor or active lipid mediator increasing VEGF expression. Increased VEGF expression and consequent enhanced angiogenesis and vasculogenesis induced by αTP may explain not only the essential roles of vitamin E on reproduction, but also its beneficial effects against pre-eclampsia, ischemia/reperfusion injury, and during wound healing. It may also serve as a survival factor for brain and muscle cells. The finding that αTP may regulate vasculogenesis may indicate potential, important pathophysiological implications.

Mice were supplemented with low and high doses of natural and synthetic vitamin E, T cells from the spleen isolated and stimulated with plate-bound anti-CD3 and soluble anti-CD28, and gene expression changes assessed by gene array experiments. The data obtained indicate significant qualitative and quantitative differences between the two vitamin forms in regulating gene expression in response to T-cell stimulation. Marker genes have been found whose expression can be considered significant in establishing the level of, and response to vitamin E for both natural and synthetic vitamin E supplementation; unique markers for synthetic vitamin E supplementation and unique markers for natural vitamin E supplementation have been identified.

Protection by vitamin E against free radical-induced DNA mutations appears not to be an effective occurrence. On the other hand, in vitro evidence that different tocopherols slow down cell proliferation is an accepted observation. However, such an event may not be sufficient to result in beneficial clinical outcomes. Tocopheryl phosphate, a more active, natural derivative of tocopherol, endowed with prevention and therapeutic potential, represents a possible key to the understanding of the present conflict between laboratory and clinical results.

The emerging potential of alpha-tocopheryl phosphate, a phosphoric acid ester of alpha-tocopherol, in health benefits was tested gavaging this compound (5 mg/kg body wt) to a group of rats for a period of thirty days while the control rats were given water only. After thirty days, the rats were sacrificed, the hearts excised, and the isolated hearts were perfused by working mode. Both control and experimental hearts were subjected to 30-min global ischemia followed by 2 h of reperfusion. The tocopheryl phosphate fed rats exhibited significant cardioprotection as evidenced by improved ventricular performance and reduced myocardial infarct size and cardiomyocyte apoptosis. Supplementation with alpha-tocopheryl phosphate converted MAP kinase-induced death signal into a survival signal by enhancing anti-apoptotic p42/44 ERK kinase and p38 MAPKbeta and reducing pro-apoptotic proteins p38 MAPKalpha and JNK. In concert, the phosphorylation of pro-apoptotic c-Src was also reduced. Tocopheryl phosphate increased the DNA binding of the redox-sensitive transcription factor NFkappaB and potentiated the activation of anti-death protein Bcl-2 and survival signaling protein Akt. The results of this study demonstrated for the first time that tocopheryl phosphate could ameliorate myocardial ischemic reperfusion injury by converting ischemia/reperfusion-mediated death signal into a survival signal by modulating MAP kinase signaling.

Polymorphisms in genes involved in vitamin E uptake, distribution, metabolism, and molecular action may be important determinants for the protective effects of vitamin E supplementation. The haptoglobin 2-2 polymorphism is associated with increased production of oxygen free radicals and reduces levels of vitamin E and C; the consequent elevated risk for cardiovascular disease can be prevented by vitamin E supplementation.

The inability of other antioxidants to substitute for α-tocopherol in a number of cellular reactions, the lack of a compensatory antioxidant response in the gene expression under conditions of α-tocopherol deficiency, the unique uptake of α-tocopherol relative to the other tocopherols and its slower catabolism, and the striking differences in the molecular function of the different tocopherols and tocotrienols, observed in vitro, unrelated to their antioxidant properties, are all data in support of a nonantioxidant molecular function of α-tocopherol. Furthermore, in vivo studies have also shown that α-tocopherol is not able, at physiological concentrations, to protect against oxidant-induced damage or prevent disease allegedly caused by oxidative damage. α-Tocopherol appears to act as a ligand of not yet identified specific proteins (receptors, transcription factors) capable of regulating signal transduction and gene expression.

The tocopherols (alpha, beta-, gamma-, and delta-tocopherol) and resveratrol are phytochemicals with alleged beneficial effects against atherosclerosis, vascular diseases and different cancers. They both can act as antioxidants, but they also modulate signal transduction and gene expression by non-antioxidant mechanisms. Here we wanted to determine whether the combined treatment of mast cells with the two compounds inhibits cell proliferation more efficiently when compared to individual treatments. Both compounds inhibit HMC-1 mastocytoma cell proliferation and reduce the activity of Protein Kinase B (PKB/Akt) by inhibiting its Ser473-phosphorylation. The combination of 50 microM delta-tocopherol and 50 microM resveratrol inhibits proliferation of HMC-1 cells more efficiently when compared to single treatments. In line with this, PKB Ser473-phosphorylation is inhibited best by delta-tocopherol and resveratrol combinatory treatment. Resveratrol acts more efficiently as an inhibitor of PKB phosphorylation than alpha-, beta-, gamma-tocopherols, whereas delta-tocopherol shows a stronger inhibition possibly as a result of its apoptotic secondary effects. Our data suggest that delta-tocopherol and resveratrol can act additively in reducing cell proliferation and PKB phosphorylation. The combination of phytochemicals with relatively broad specificity on enzymes involved in signal transduction and gene expression may increase their activity in disease prevention by modulating several different molecular targets.

The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. gamma-tocopherol at 50 microM concentration exerted more inhibitory effect than alpha-tocopherol at the same concentration on glioma cell proliferation. Integrin alpha5 and beta1 protein levels were increased upon both alpha- and gamma-tocopherol treatments. In parallel, an increase in the alpha5beta1 heterodimer cell surface expression was observed. The tocopherols inhibited glioma cell-binding to fibronectin where gamma-tocopherol treatment induced glioma cell migration. Taken together, the data reported here are consistent with the notion that the inhibition of glioma cell proliferation induced by tocopherols may be mediated, at least in part, by an increase in integrin alpha5 and beta1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the alpha5beta1 heterodimer. Cell migration is stimulated by gamma-tocopherol. It is concluded that alpha5 and beta1 integrin expression and surface appearance are not sufficient to explain all the observations and that other integrins or in general other factors may be associated with these events.

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

We have detected alpha-tocopheryl phosphate in biological tissues including liver and adipose tissue, as well as in a variety of foods, suggesting a ubiquitous presence in animal and plant tissue. Alpha-tocopheryl phosphate is a water-soluble molecule that is resistant to both acid and alkaline hydrolysis, making it undetectable using standard assays for vitamin E. A new method was therefore developed to allow the extraction of both alpha-tocopheryl phosphate and alpha-tocopherol from a single specimen. We used ESMS to detect endogenous alpha-tocopheryl phosphate in biological samples that also contained alpha-tocopherol. Due to the significance of these findings, further proof was required to unequivocally demonstrate the presence of endogenous alpha-tocopheryl phosphate in biological samples. Four independent methods of analysis were examined: HPLC, LCMS, LCMS/MS, and GCMS. Alpha-tocopherol phosphate was identified in all instances by comparison between standard alpha-tocopheryl phosphate and extracts of biological tissues. The results show that alpha-tocopheryl phosphate is a natural form of vitamin E. The discovery of endogenous alpha-tocopheryl phosphate has implications for the expanding knowledge of the roles of alpha-tocopherol in biological systems.

Vitamin E was discovered in 1922, but in more than 60 years many new findings have added to the early one. However, its description in many textbooks has not been proportionally updated. The result is an inaccurate, incomplete, and often incorrect description of the function of this vitamin. In some other textbooks, vitamin E is absolutely neglected. In almost all books the existence of vitamin E deficiency diseases is ignored. Recent findings on vitamin E molecular functions and its related diseases are described in this article.

Therapies with antiretroviral protease inhibitors (ARPI) are correlated with a higher risk for dyslipidemia, hypercholesterolemia, and atherosclerosis. The original aim of this study was to establish whether alpha-tocopherol can reduce CD36 scavenger receptor overexpression occurring after treatment of monocytes with the ARPI ritonavir. We show here that treatment of THP-1 monocytes with ritonavir increases total protein and surface expression of CD36; however, only weak changes are observed at the mRNA level, suggesting that CD36 overexpression occurs mainly at the posttranscriptional level. Concentrations of ritonavir that upregulate CD36 expression inhibit proteasome activity in THP-1 cells, indicating a possible regulatory role of the proteasome in CD36 overexpression. Similar to ritonavir, the proteasome inhibitor ALLN increases the CD36 surface expression on THP-1 cells. alpha-Tocopherol efficiently normalizes CD36 protein overexpression after ritonavir treatment and reduces oxLDL uptake. Furthermore, in THP-1 monocytes, alpha-tocopherol reverses the proteasome activity inhibited by ritonavir. This study indicates that an increased CD36 protein expression in THP-1 monocytes induced by ritonavir can be normalized by alpha-tocopherol. CD36 overexpression is caused by inhibition of proteasome activity by ritonavir, which is efficiently restored by alpha-tocopherol.

A robust database shows that dietary supplements of vitamins E and C are safe for the general population. Because these nutrients supply antioxidant and other functions for homeostasis and protection against free radical damage, supplementation has been intensively studied. Because of perceived benefits, many persons consume quantities of vitamins E and C well above the recommended dietary allowances. As safety guidance, tolerable upper intake levels have been established by the Food and Nutrition Board, Institute of Medicine, at 1000 mg for vitamin E and 2000 mg for vitamin C in adults. Many clinical trials with these vitamins have involved subjects with various diseases, and no consistent pattern of adverse effects has occurred at any intake. Numerous studies of vitamin C supplementation have provided no pattern of evidence to support concerns about safety other than occasional gastrointestinal upset or mild diarrhea resulting from the osmotic effects of unabsorbed quantities of vitamin C. Evidence of bleeding effects and other potential adverse effects of high vitamin E intakes in humans is not convincing. Evidence of adverse effects of vitamin C that result from its effects on iron absorption and metabolism has not been confirmed in clinical trials. Thus, we conclude from clinical trial evidence that vitamin E supplements appear safe for most adults in amounts

alpha-Tocopherol modulates two major signal transduction pathways centered on protein kinase C and phosphatidylinositol 3-kinase. Changes in the activity of these key kinases are associated with changes in cell proliferation, platelet aggregation, and NADPH-oxidase activation. Several genes are also regulated by tocopherols partly because of the effects of tocopherol on these two kinases, but also independently of them. These genes can be divided in five groups: Group 1. Genes that are involved in the uptake and degradation of tocopherols: alpha-tocopherol transfer protein, cytochrome P450 (CYP3A), gamma-glutamyl-cysteine synthetase heavy subunit, and glutathione-S-transferase. Group 2. Genes that are implicated with lipid uptake and atherosclerosis: CD36, SR-BI, and SR-AI/II. Group 3. Genes that are involved in the modulation of extracellular proteins: tropomyosin, collagen-alpha-1, MMP-1, MMP-19, and connective tissue growth factor. Group 4. Genes that are connected to adhesion and inflammation: E-selectin, ICAM-1 integrins, glycoprotein IIb, IL-2, IL-4, IL-1b, and transforming growth factor-beta (TGF-beta). Group 5. Genes implicated in cell signaling and cell cycle regulation: PPAR-gamma, cyclin D1, cyclin E, Bcl2-L1, p27, CD95 (APO-1/Fas ligand), and 5a-steroid reductase type 1. The transcription of p27, Bcl2, alpha-tocopherol transfer protein, cytochrome P450 (CYP3A), gamma-glutamyl-cysteine sythetase heavy subunit, tropomyosin, IL-2, and CTGF appears to be upregulated by one or more tocopherols. All the other listed genes are downregulated. Gene regulation by tocopherols has been associated with protein kinase C because of its deactivation by alpha-tocopherol and its contribution in the regulation of a number of transcription factors (NF-kappaB, AP1). A direct participation of the pregnane X receptor (PXR) / retinoid X receptor (RXR) has been also shown. The antioxidant-responsive element (ARE) and the TGF-beta-responsive element (TGF-beta-RE) appear in some cases to be implicated as well.

The effect of a mixture of a-tocopheryl phosphate and di-a-tocopheryl phosphate (TPm) was studied in vitro on two cell lines, RASMC (from rat aortic smooth muscle) and human THP-1 monocytic leukaemia cells. Inhibition of cell proliferation by TPm was shown in both lines and occurred with TPm at concentrations lower than those at which a-tocopherol was equally inhibitory. TPm led in non-stimulated THP-1 cells to inhibition of CD36 mRNA and protein expression, to inhibition of oxidized low density lipoprotein surface binding and oxLDL uptake. In non-stimulated THP-1 cells, a-tocopherol had only very weak effects on these events. Contrary to a-tocopherol, TPm was cytotoxic to THP-1 cells at high concentrations. Thus, TPm is able to inhibit the major aggravating elements involved in the progression of atherosclerosis. The higher potency of TPm may be due to a better uptake of the molecule and to its intracellular hydrolysis, providing more a-tocopherol to sensitive sites. Alternatively, a direct effect of the phosphate ester on specific cell targets may be considered. 2004 Elsevier Inc. All rights reserved.

Molecules in biological systems often can perform more than one function. In particular, many molecules have the ability to chemically scavenge free radicals and thus act in the test tube as antioxidant, but their main biological function is by acting as hormones, ligands for transcription factors, modulators of enzymatic activities or as structural components. In fact, oxidation of these molecules may impair their biological function, and cellular defense systems exist which protect these molecules from oxidation. Vitamin E is present in plants in 8 different forms with more or less equal antioxidant potential (alpha-, beta-, gamma-, delta-tocopherol/tocotrienols); nevertheless, in higher organisms only alpha-tocopherol is preferentially retained suggesting a specific mechanism for the uptake for this analogue. In the last 20 years, the route of tocopherol from the diet into the body has been clarified and the proteins involved in the uptake and selective retention of alpha-tocopherol discovered. Precise cellular functions of alpha-tocopherol that are independent of its antioxidant/radical scavenging ability have been characterized in recent years. At the posttranslational level, alpha-tocopherol inhibits protein kinase C, 5-lipoxygenase and phospholipase A2 and activates protein phosphatase 2A and diacylglycerol kinase. Some genes (e. g. scavenger receptors, alpha-TTP, alpha-tropomyosin, matrix metalloproteinase-19 and collagenase) are modulated by alpha-tocopherol at the transcriptional level. alpha-Tocopherol also inhibits cell proliferation, platelet aggregation and monocyte adhesion. These effects are unrelated to the antioxidant activity of vitamin E, and possibly reflect specific interactions of alpha-tocopherol with enzymes, structural proteins, lipids and transcription factors. Recently, several novel tocopherol binding proteins have been cloned, that may mediate the non-antioxidant signaling and cellular functions of vitamin E and its correct intracellular distribution. In the present review, it is suggested that the non-antioxidant activities of tocopherols represent the main biological reason for the selective retention of alpha-tocopherol in the body, or vice versa, for the metabolic conversion and consequent elimination of the other tocopherols.

Scavenger receptors recently have been related to Alzheimer's disease, although it is still unclear whether they contribute to the pathogenesis of the disease or reflect an inflammatory response to the deposition of amyloid beta-protein (Abeta). In this study we demonstrate that CD36, a class B scavenger receptor, is highly expressed in the cerebral cortex of Alzheimer's disease patients and cognitively normal aged subjects with diffuse amyloid plaques compared with age-matched amyloid-free control brains. Moreover, in vitro experiments indicated that Abeta is able to induce CD36 expression in neuronal cells after 24 h treatment. The interaction between CD36 and Abeta has been reported to trigger oxidant production by macrophages and microglia. In line with this observation, we found an increased presence of nitrated proteins in brains showing Abeta loads and CD36 overexpression, independent of the occurrence of Alzheimer's disease pathologic features.

What is an antioxidant? Can one, at a cellular level, speak of direct and indirect antioxidants? Can oxidative stress be quantified and characterized? What are the oxidant species that may have regulatory functions in a cell? Since the above concepts have become of frequent use in all Journals it may be appropriate if some critical thinking outlined in this review could become available to a broad public.