The molecular function of vitamin E has been the object of intensive investigations during at least the last 50 years (Zingg and Azzi 2004). After the original molecular understanding of vitamin E as an antioxidant at the level of cells and organs, it has become clear that, beyond and above this, vitamin E could act in in vitro cell systems as well as in vivo as a regulator of gene expression and cell signal transduction. More recent work has also shown that vitamin E can undergo phosphorylation in cells and experimental animals. The phosphorylated form of vitamin E, alpha-TP, was recently also found in human plasma and tissues. It has been also described that supplementation with alpha-TP in humans is able to increase the amounts found in plasma (Zingg, Libinaki et al. 2010). Alpha-TP, naturally present in the body or after supplementation, appears to be in amounts (nanomolar concentrations) not compatible with a storage form of vitamin E. The hypothesis has been then formulated that alpha-TP is a signaling molecule, similar in nature to other lipid-phosphate compounds such as inositol phosphates or sphingosine phosphate. Although very stable, alpha-TP can be somewhat hydrolyzed both in cells and in the body. However, the amounts of free alpha-T produced after administration to cells or to experimental animals are not sufficient to explain the superior function of alpha-TP relative to alpha-T (Zingg, Meydani et al. 2010). In fact, in a number of experiments ranging from cell signaling to protection against atherosclerosis or heart infarction, alpha-TP was shown, at the same concentrations, to be more effective than alpha-T (Libinaki, Tesanovic et al. 2010). The function assigned to alpha-T, alternative to the antioxidant one, has been to modulate the expression of a number of genes both in the sense of up and down regulation. In the attempt to understand the molecular function of alpha-TP a gene array analysis of the two molecules, alpha-T and alpha-TP has been made. Also in this case it was shown that, at the same concentration, alpha-TP appeared to be, in some cases, several folds more potent than the non-phosphorylated compound. The analysis of the genes over expressed in the presence of alpha-TP by THP-1 cells indicated that a surprising number of them belonged to a group of genes involved in cell lipid storage (See figure 6.1) and in particular, REDD-1, TRB3, C8FW [anti-G-Protein-Coupled Receptor Induced Protein GIG2 (C8FW) (TRIB1)], Sestrin-2, and Insulin Induced Gene 1, INSIG (Zingg, Libinaki et al. 2010). As a proof of concept, the transcription/translation of the VEGF gene was analyzed by quantitative RT-PCR and by Western blot, and the results were fully consistent with the gene array outcome. In the course of this study, NIH3T3-L1 pre-adipocytes have been employed, cells that have the ability to differentiate from fibroblasts to adipocytes (see Figure 5.2). In parallel experiments alpha-TP appears to be more effective than alpha-T in inhibiting the proliferation of NIH3T3-L1 at the same concentrations and incubation time. This experiment confirms that alpha-TP acts as such under these conditions, and not as a precursor of free alpha-T; in fact the parent compound is much less active, excluding the possibility that the effects of alpha-TP be caused by its conversion to alpha-T. Alpha-T is not able to induce lipid accumulation both in NIH3T3-L1 pre-adipocytes and in adipocytes. On the other hand, alpha-TP was capable of inducing a significant lipid accumulation in adipocytes and it had only a small effect in NIH3T3-L1. To evaluate the effect of alpha-T and of alpha-TP on the gene expression, we performed RT-PCR using specific primers for the genes which are most relevant in the process of fat accumulation in cells, namely Sestrin 2, TRB3 and INSIG. These genes were shown to be strongly up-regulated in THP-1 cells by alpha-TP . Sestrins potentiate AMPK and inhibit activation of target of mTOR increased lipolysis and increased fatty acid oxidation may result. At the same time inhibition of fatty acid synthesis is also occurring. TRB3 has been shown to suppress adipocytes differentiation by negative regulation of PPAR alpha and to facilitate the proteasome degradation of acetyl-CoA-carboxylase. INSIG1 plays an important role in the SREBP-mediated regulation of cholesterol biosynthesis, its action resulting in a decreased expression of HMG-CoA-reductase and in increased degradation of the enzyme. These genes are more relevant in the lipid metabolism. All these genes pathways, should increase lipid degradation and inhibition of lipid synthesis, but more studies are, however, needed to substantiate this hypothesis. In all cases, it is surprising that the effects of alpha-T are opposite to those of alpha-TP. In particular a clear dose-dependent up regulation of Sestrin by alpha-TP is observed in NIH3T3-L1 pre-adipocytes as well as a clear suppression by alpha-T in the same cells. Also TRB3 and INSIG transcription is up-regulated by alpha-TP and down regulated by alpha-T. It appears therefore that in NIH3T3-L1 pre-adipocytes, the presence of alpha-T diminishes the transcription of fat catabolic enzymes while alpha-TP activates the transcription of the same genes therefore making this cell metabolically more active and less prone to accumulate lipid. When the NIH3T3-L1 differentiated to adipocytes the opposite picture becomes visible. Sestrin transcription is clearly diminished by alpha-TP. The transcription of the other two genes appears also to be down regulated although to a lesser extent. On the other side alpha-T was capable of stimulating transcription of genes. Consequently, also in adipocytes alpha-T appears to have an effect opposite to that of alpha-TP the former being efficient in up regulating the gene set intended to limit lipid accumulation. The simplest conclusions that can be drown from the experiments presented here is that the presence of alpha-TP (but not alpha-T) in NIH3T3-L1 pre-adipocytes appears to activate a transcriptional gene set potentially preventing fat accumulation in these cells. In undifferentiated adipocytes, alpha-TP appears to be responsible for activation of those potentially protective genes. Using cell lines in vitro may not give reliable indications of the complex metabolic disorder resulting in obesity. However, in vitro results are important to understand, at least in part, the complexity of the disease. The use of high concentrations (micromolar) of alpha-TP may be criticized on the basis of the amounts physiologically found in plasma (nanomolar). However, the presence of large amounts of divalent cations needed in the incubation media for cell activities and survival makes the actual concentration of the free alpha-TP several orders of magnitude lower than the added amounts due to the sequestration properties of the divalent metal ions. Consequently, the used amounts of alpha-TP are comprised within a physiological range. Altogether, describing and understanding the effects of the two physiological compounds alpha-T and alpha-TP on single genes or a set of genes may be useful to focus upon some details of the complex picture of the control of pathological fat accumulation. Clinical studies are, however, needed to substantiate this hypothesis.

Regulation of gene expression in pre-adipocytes and adipocytes: role of alpha tocopheryl-phosphate / Melania Lirangi - : . , 2012 Apr 11. ((24. ciclo

Regulation of gene expression in pre-adipocytes and adipocytes: role of alpha tocopheryl-phosphate

2012-04-11

Abstract

The molecular function of vitamin E has been the object of intensive investigations during at least the last 50 years (Zingg and Azzi 2004). After the original molecular understanding of vitamin E as an antioxidant at the level of cells and organs, it has become clear that, beyond and above this, vitamin E could act in in vitro cell systems as well as in vivo as a regulator of gene expression and cell signal transduction. More recent work has also shown that vitamin E can undergo phosphorylation in cells and experimental animals. The phosphorylated form of vitamin E, alpha-TP, was recently also found in human plasma and tissues. It has been also described that supplementation with alpha-TP in humans is able to increase the amounts found in plasma (Zingg, Libinaki et al. 2010). Alpha-TP, naturally present in the body or after supplementation, appears to be in amounts (nanomolar concentrations) not compatible with a storage form of vitamin E. The hypothesis has been then formulated that alpha-TP is a signaling molecule, similar in nature to other lipid-phosphate compounds such as inositol phosphates or sphingosine phosphate. Although very stable, alpha-TP can be somewhat hydrolyzed both in cells and in the body. However, the amounts of free alpha-T produced after administration to cells or to experimental animals are not sufficient to explain the superior function of alpha-TP relative to alpha-T (Zingg, Meydani et al. 2010). In fact, in a number of experiments ranging from cell signaling to protection against atherosclerosis or heart infarction, alpha-TP was shown, at the same concentrations, to be more effective than alpha-T (Libinaki, Tesanovic et al. 2010). The function assigned to alpha-T, alternative to the antioxidant one, has been to modulate the expression of a number of genes both in the sense of up and down regulation. In the attempt to understand the molecular function of alpha-TP a gene array analysis of the two molecules, alpha-T and alpha-TP has been made. Also in this case it was shown that, at the same concentration, alpha-TP appeared to be, in some cases, several folds more potent than the non-phosphorylated compound. The analysis of the genes over expressed in the presence of alpha-TP by THP-1 cells indicated that a surprising number of them belonged to a group of genes involved in cell lipid storage (See figure 6.1) and in particular, REDD-1, TRB3, C8FW [anti-G-Protein-Coupled Receptor Induced Protein GIG2 (C8FW) (TRIB1)], Sestrin-2, and Insulin Induced Gene 1, INSIG (Zingg, Libinaki et al. 2010). As a proof of concept, the transcription/translation of the VEGF gene was analyzed by quantitative RT-PCR and by Western blot, and the results were fully consistent with the gene array outcome. In the course of this study, NIH3T3-L1 pre-adipocytes have been employed, cells that have the ability to differentiate from fibroblasts to adipocytes (see Figure 5.2). In parallel experiments alpha-TP appears to be more effective than alpha-T in inhibiting the proliferation of NIH3T3-L1 at the same concentrations and incubation time. This experiment confirms that alpha-TP acts as such under these conditions, and not as a precursor of free alpha-T; in fact the parent compound is much less active, excluding the possibility that the effects of alpha-TP be caused by its conversion to alpha-T. Alpha-T is not able to induce lipid accumulation both in NIH3T3-L1 pre-adipocytes and in adipocytes. On the other hand, alpha-TP was capable of inducing a significant lipid accumulation in adipocytes and it had only a small effect in NIH3T3-L1. To evaluate the effect of alpha-T and of alpha-TP on the gene expression, we performed RT-PCR using specific primers for the genes which are most relevant in the process of fat accumulation in cells, namely Sestrin 2, TRB3 and INSIG. These genes were shown to be strongly up-regulated in THP-1 cells by alpha-TP . Sestrins potentiate AMPK and inhibit activation of target of mTOR increased lipolysis and increased fatty acid oxidation may result. At the same time inhibition of fatty acid synthesis is also occurring. TRB3 has been shown to suppress adipocytes differentiation by negative regulation of PPAR alpha and to facilitate the proteasome degradation of acetyl-CoA-carboxylase. INSIG1 plays an important role in the SREBP-mediated regulation of cholesterol biosynthesis, its action resulting in a decreased expression of HMG-CoA-reductase and in increased degradation of the enzyme. These genes are more relevant in the lipid metabolism. All these genes pathways, should increase lipid degradation and inhibition of lipid synthesis, but more studies are, however, needed to substantiate this hypothesis. In all cases, it is surprising that the effects of alpha-T are opposite to those of alpha-TP. In particular a clear dose-dependent up regulation of Sestrin by alpha-TP is observed in NIH3T3-L1 pre-adipocytes as well as a clear suppression by alpha-T in the same cells. Also TRB3 and INSIG transcription is up-regulated by alpha-TP and down regulated by alpha-T. It appears therefore that in NIH3T3-L1 pre-adipocytes, the presence of alpha-T diminishes the transcription of fat catabolic enzymes while alpha-TP activates the transcription of the same genes therefore making this cell metabolically more active and less prone to accumulate lipid. When the NIH3T3-L1 differentiated to adipocytes the opposite picture becomes visible. Sestrin transcription is clearly diminished by alpha-TP. The transcription of the other two genes appears also to be down regulated although to a lesser extent. On the other side alpha-T was capable of stimulating transcription of genes. Consequently, also in adipocytes alpha-T appears to have an effect opposite to that of alpha-TP the former being efficient in up regulating the gene set intended to limit lipid accumulation. The simplest conclusions that can be drown from the experiments presented here is that the presence of alpha-TP (but not alpha-T) in NIH3T3-L1 pre-adipocytes appears to activate a transcriptional gene set potentially preventing fat accumulation in these cells. In undifferentiated adipocytes, alpha-TP appears to be responsible for activation of those potentially protective genes. Using cell lines in vitro may not give reliable indications of the complex metabolic disorder resulting in obesity. However, in vitro results are important to understand, at least in part, the complexity of the disease. The use of high concentrations (micromolar) of alpha-TP may be criticized on the basis of the amounts physiologically found in plasma (nanomolar). However, the presence of large amounts of divalent cations needed in the incubation media for cell activities and survival makes the actual concentration of the free alpha-TP several orders of magnitude lower than the added amounts due to the sequestration properties of the divalent metal ions. Consequently, the used amounts of alpha-TP are comprised within a physiological range. Altogether, describing and understanding the effects of the two physiological compounds alpha-T and alpha-TP on single genes or a set of genes may be useful to focus upon some details of the complex picture of the control of pathological fat accumulation. Clinical studies are, however, needed to substantiate this hypothesis.
alpha tocopheryl-phosphate; vitamin e
Regulation of gene expression in pre-adipocytes and adipocytes: role of alpha tocopheryl-phosphate / Melania Lirangi - : . , 2012 Apr 11. ((24. ciclo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12610/68372
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