Improved glucose metabolism in mice lacking alpha-tocopherol transfer protein.

BACKGROUND: Conflicting evidence suggests a possible role for vitamin E in mammalian glucose metabolism and the protection from type 2 diabetes. The alpha-tocopherol transfer protein (alpha-TTP) mediates the transfer of alpha-tocopherol (alpha-TOH) from hepatocytes to very-low-density lipoproteins, thereby controlling plasma levels of alpha-TOH. AIM OF THE STUDY: The aim of this study was to investigate the putative impact of alpha-TTP knock-out on glucose metabolism in mice. METHODS: Mice deficient for alpha-TTP and wild-type control littermates were fed a diet containing 200 mg alpha-tocopheryl acetate per kg to ameliorate alpha-TOH deficiency in knock-out mice. We investigated fasting and postprandial plasma glucose, insulin and triglyceride levels of both groups of mice at different ages. All genotypes and age groups were further subjected to glucose and insulin tolerance tests, and number of insulin-producing islets of Langerhans were determined. RESULTS: Plasma alpha-TOH levels of knock-out mice were 34% the levels of wild-type controls: Any signs of alpha-TOH deficiency were absent at any age. Unexpectedly, serum glucose levels both in the fasted and in the fed state were lower in alpha-TTP-deficient mice at any age. Removal rates for intraperitoneally injected glucose were found to be significantly increased in young alpha-TTP-deficient mice. This improved glucose tolerance was caused by increased insulin secretion in response to an intraperitoneal glucose challenge due to an increased number of pancreatic islets, as well as by increased sensitivity to intraperitoneally injected insulin, both significantly promoting glucose metabolism in alpha-TTP-deficient mice. CONCLUSIONS: Our findings suggest that alpha-TTP-deficiency in states of alpha-TOH supplementation unexpectedly promotes glucose tolerance in mice due to both increased insulin secretion and insulin action, suggesting differential roles of alpha-TTP and alpha-TOH in the pathogenesis of type 2 diabetes mellitus.

PCR-verified microarray analysis and functional in vitro studies indicate a role of alpha-tocopherol in vesicular transport.

Global gene expression profiles of livers from mice, fed diets differing in alpha-tocopherol content, were compared using DNA microarray technology. Three hundred and eighty nine genes were found to significantly differ in their expression level by a factor of 2 or higher between the high and the low alpha-tocopherol group. Functional clustering using the EASE software identified 121 genes involved in transport processes. Twenty-one thereof were involved in (synaptic) vesicular trafficking. Up-regulation of syntaxin 1C (Stx1c), vesicle-associated membrane protein 1 (Vamp1), N-ethylmaleimide-sensitive factor (Nsf) and syntaxin binding protein 1 (Stxbp1, Munc18-1) was verified by real time PCR. At a functional level, alpha-tocopherol increased the secretory response in RBL and PC12 cells. Although here detected in liver, the alpha-tocopherol-responsive pathways are also relevant to neurotransmission. A role of alpha-tocopherol in the vesicular transport might not only affect its own absorption and transport but also explain the neural dysfunctions observed in severe alpha-tocopherol deficiency.

Modulation of Cyp3a11 mRNA expression by alpha-tocopherol but not gamma-tocotrienol in mice.

Metabolism of vitamin E is initiated by cytochrome P450 (CYP) enzymes usually involved in the metabolism of xenobiotics. Like other CYP substrates, vitamin E induced a reporter gene under the control of the pregnane X receptor (PXR) which regulates the expression of CYPs including CYP3A4. gamma-Tocotrienol, the most effective PXR activator, also induced endogenous CYP3A4 mRNA in HepG2 cells. Since these findings imply an interference of vitamin E with drug metabolism it was deemed necessary to investigate their in vivo relevance. Therefore, mice were grown for 3 months with alpha-tocopherol-deficient, -adequate, and -supranutritional diet, i.e. 2, 20 and 200 mg RRR-alpha-tocopheryl acetate/kg diet, respectively. Half of them received 250 microg gamma-tocotrienol/day for the last 7 days. After 3 months, hepatic levels of Cyp3a11 mRNA, the murine homolog to human CYP3A4, were about 2.5-fold higher in the 20 and 200 mg alpha-tocopherol groups than in the 2 mg group. After feeding 200 mg alpha-tocopherol for 9 months, Cyp3a11 mRNA was 1.7-fold higher than after 3 months. In contrast, gamma-tocotrienol did not induce Cyp3a11 mRNA. This could be explained by its high metabolism as demonstrated by the 20- to 25-fold increase in the urinary excretion of gamma-CEHC, the final metabolite of gamma-tocotrienol degradation. In conclusion, alpha-tocopherol maintains an adequate level of xenobiotic-metabolizing enzymes. If fed in supranutritional dosages, especially for longer times, alpha-tocopherol induces Cyp3a11 to levels which might interfere with drug metabolism.

Vitamin E: underestimated as an antioxidant.

Some 80 years after its discovery, vitamin E has experienced a renaissance which is as surprising as it is trivial. Although vitamin E is essential for reproduction, in rats at least, and deficiency causes neurological disorders in humans, the main interest in the last decades has concentrated on its antioxidant functions. This focus has highly underestimated the biological importance of vitamin E, which by far exceeds the need for acting as a radical scavenger. Only recently has it become clear that vitamin E can regulate cellular signaling and gene expression. Out of the eight different tocols included in the term vitamin E, alpha-tocopherol often exerts specific functions, which is also reflected in its selective recognition by proteins such as the alpha-tocopherol transfer protein and alpha-tocopherol-associated proteins. Vitamin E forms other than alpha-tocopherol are very actively metabolised, which explains their low biopotency. In vivo, metabolism may also attenuate the novel functions of gamma-tocopherol and tocotrienols observed in vitro. On the other hand, metabolites derived from individual forms of vitamin E have been shown to exert effects by themselves. This article focuses on the metabolism and novel functions of vitamin E with special emphasis on differential biological activities of individual vitamin E forms.

Carotenoids and their metabolites are naturally occurring activators of gene expression via the pregnane X receptor.

Carotenoids are important micronutrients in the human diet and are present in human serum at micromolar concentrations. In addition to their antioxidant potential, carotenoids obtain physiologically relevant properties such as influencing cellular signal pathways, gene expression or induction of detoxifying enzymes. In this study, we determined the transactivation of PXR by cotransfection with the full-length receptor and a PXR-responsive reporter gene. Carotenoids and retinol revealed a 5-6 fold reporter gene activity in HepG2 cells in comparison to a 7-fold induction by the well-known PXR agonist rifampicin, whereas apo-carotenals and lycopene exerted less or no activation potential. The inductive efficacy was hereby concentration-dependent. In addition, carotenoid- or retinol-mediated gene expression of PXR-responsive genes like CYP3A4/CYP3A7, CYP3A5, MDR-1 and MRP-2 has been determined in HepG2 cells by RT-PCR with up-regulative properties of beta-carotene or retinol being comparable to or even higher than that of rifampicin. In conclusion, PXR-mediated up-regulation of CYP3A4/CYP3A7 and CYP3A5 as well as MDR1 and MRP2 by carotenoids points to a potential interference on the metabolism of xenobiotic and endogenous relevant compounds.

Homologous metabolic and gene activating routes for vitamins E and K.

Vitamins E and K share structurally related side chains and are degraded to similar final products. For vitamin E the mechanism has been elucidated as initial omega-hydroxylation and subsequent beta-oxidation. For vitamin K the same mechanism can be suggested analogously. omega-Hydroxylation of vitamin E is catalyzed by cytochrome p450 enzymes, which often are induced by their substrates themselves via the activation of the nuclear receptor PXR. Vitamin E is able to induce CYP3A-forms and to activate a PXR-driven reporter gene. It is shown here that K-type vitamins are also able to activate PXR. A ranking showed that compounds with an unsaturated side chain were most effective, as are tocotrienols and menaquinone-4 (vitamin K(2)), which activated the reporter gene 8-10-fold. Vitamers with a saturated side chain, like tocopherols and phylloquinone were less active (2-5-fold activation). From the fact that CYPs commonly responsible for the elimination of xenobiotics are involved in the metabolism of fat-soluble vitamins and the ability of the vitamins to activate PXR it can be concluded that supranutritional amounts of these vitamins might be considered as foreign.

Vitamin E activates gene expression via the pregnane X receptor.

Tocopherols and tocotrienols are metabolized by side chain degradation via initial omega-oxidation and subsequent beta-oxidation. omega-Oxidation is performed by cytochrome P450 (CYP) enzymes which are often regulated by their substrates themselves. Results presented here show that all forms of Vitamin E are able to activate gene expression via the pregnane X receptor (PXR), a nuclear receptor regulating a variety of drug metabolizing enzymes. In HepG2 cells transfected with the human PXR and the chloramphenicol acetyl transferase (CAT) gene linked to two PXR responsive elements, CAT activity was most strongly induced by alpha- and gamma-tocotrienol followed by rifampicin, delta-, alpha- and gamma-tocopherol. The inductive efficacy was concentration-dependent; its specificity was underscored by a lower response when cotransfection with PXR was omitted. Up-regulation of endogenous CYP3A4 and CYP3A5 mRNA was obtained by gamma-tocotrienol, the most potent activator of PXR, with the same efficacy as with rifampicin. This points to a potential interference of individual forms of Vitamin E with the metabolism and efficacy of drugs.

Identities and differences in the metabolism of tocotrienols and tocopherols in HepG2 cells.

The metabolism of alpha- and gamma-tocotrienol was investigated in HepG2 cells. Metabolites were identified by HPLC and gas chromatography/mass spectrometry. gamma-Tocotrienol was degraded to gamma-CEHC (carboxyethyl hydroxychroman), gamma-CMBHC (carboxymethylbutyl hydroxychroman), gamma-CMHenHC (carboxymethylhexenyl hydroxychroman), gamma-CDMOenHC (carboxydimethyloctenyl hydroxychroman) and gamma-CDMD(en)(2)HC (carboxydimethyldecadienyl hydroxychroman). alpha-Tocotrienol yielded alpha-CEHC, alpha-CMBHC, alpha-CMHenHC and alpha-CDMOenHC, whereas alpha-CDMD(en)(2)HC could not be detected. These findings demonstrate that the trienols are metabolized essentially like tocopherols, i.e., by omega-oxidation followed by beta-oxidation of the side chain. The failure to detect CMBHC with the original double bond in the side chain reveals that auxiliary enzymes are involved, as in the metabolism of unsaturated fatty acids. CMBHC were the most abundant metabolites obtained from the tocotrienols as well as from alpha-tocopherol. Quantitatively, the tocotrienols were degraded to a larger extent than their counterparts with saturated side chains. The pronounced quantitative differences in the metabolism between individual tocopherols as well as between tocotrienols and tocopherols in vitro suggest a corresponding lack of equivalence in vivo.