Absence of VLDL secretion does not affect alpha-tocopherol content in peripheral tissues

alpha-Tocopherol is a lipid-soluble antioxidant that helps to prevent oxidative damage to cellular lipids. alpha-Tocopherol is absorbed by the intestine and is taken up and retained by the liver; it is widely presumed that alpha-tocopherol is then delivered to peripheral tissues by the secretion of VLDL. To determine whether VLDL secretion is truly important for the delivery of alpha-tocopherol to peripheral tissues, we examined alpha-tocopherol metabolism in mice that lack microsomal triglyceride transfer protein (Mttp) expression in the liver and therefore cannot secrete VLDL (Mttp(Delta/Delta) mice). Mttp(Delta/Delta) mice have low plasma lipid levels and increased stores of lipids in the liver. Similarly, alpha-tocopherol levels in the plasma were lower in Mttp(Delta/Delta) mice than in controls, whereas hepatic alpha-tocopherol stores were higher. However, alpha-tocopherol levels in the peripheral tissues of Mttp(Delta/Delta) mice were nearly identical to those of control mice, suggesting that VLDL secretion is not critical for the delivery of alpha-tocopherol to peripheral tissues. When fed a diet containing deuterated alpha-tocopherol, Mttp(Delta/Delta) and control mice had similar incorporation of deuterated alpha-tocopherol into plasma and various peripheral tissues. We conclude that the absence of VLDL secretion has little effect on the stores of alpha-tocopherol in peripheral tissues, at least in the mouse.     

Kinetics of transfer of alpha-tocopherol between model and native plasma lipoproteins

The rate of spontaneous transfer of alpha-tocopherol, cholesterol and beta-carotene between model and native lipoproteins was measured to determine the mechanism and kinetics of equilibration of these lipids in plasma. Cholesterol and alpha-tocopherol transfer from apolipoprotein A-I/1-palmityl-2- oleoylphosphatidylcholine ( POPC ) recombinants to bovine brain ganglioside/ POPC single bilage vesicles with half-times of approximately 20 min and 70 min, respectively. Under identical conditions, there is no significant transfer of beta-carotene even after an 18-h incubation period. alpha-Tocopherol transfers from apolipoprotein A-II/dimyristoylphosphatidylcholine recombinants with a half-time of 40 min and an activation energy of 17.2 kcal/mol. Incubation of high-density lipoproteins containing alpha-[3H]tocopherol with low-density lipoproteins or very-low-density lipoproteins results in the equilibration of the labelled lipid between the lipoprotein classes in 1 h. A comparison of the rates of transfer indicates that alpha-tocopherol equilibrates 2-3-times more slowly than cholesterol but on a time scale much shorter than the lifetime of lipoproteins in the circulation. Thus, the distribution of alpha-tocopherol is not kinetically controlled but determined thermodynamically by the partitioning between the total amount of lipid in each compartment. The spontaneous transfer of beta-carotene is too slow for this equilibration to occur.     

Antioxidant effect of simvastatin is not enhanced by its association with alpha-tocopherol in hypercholesterolemic patients

Among the pleiotropic effects of statins, their antioxidant action may be involved in their protective effects. Thus, we investigated the antioxidant effect of simvastatin, associated or not with alpha-tocopherol, on levels of electronegative low-density lipoprotein (LDL-), nitrotyrosine, thiols (homocysteine, glutathione, cysteine, methionine), and lipid-soluble antioxidants in blood plasma of hypercholesterolemic subjects. In this study, 25 hypercholesterolemic subjects were treated for 2 months with simvastatin (20 mg/day) and with simvastatin (20 mg/day) + alpha-tocopherol (400 IU/day). Concentrations of thiols were determined by high-performance capillary electrophoresis-laser-induced fluorescene. Lipid-soluble antioxidants were determined by HPLC, and LDL-, and nitrotyrosine by ELISA. Simvastatin, independent of its association with alpha-tocopherol, reduced plasma concentrations of LDL-, nitrotyrosine, total cholesterol, and LDL cholesterol and the LDL cholesterol/HDL cholesterol ratio. Neither simvastatin nor simvastatin plus alpha-tocopherol altered plasma levels of the thiols analyzed. alpha-Tocopherol did not change the antioxidant effect of simvastatin on the levels of LDL- and nitrotyrosine in hypercholesterolemic subjects. The reduction of LDL- and nitrotyrosine by simvastatin seems to be related to the pleiotropic effects of this statin, and it may have an important protective effect against endothelial dysfunction and atherosclerosis.     

Plasma phospholipid transfer protein prevents vascular endothelium dysfunction by delivering alpha-tocopherol to endothelial cells.

alpha-tocopherol, the most potent antioxidant form of vitamin E, is mainly bound to lipoproteins in plasma and its incorporation into the vascular wall can prevent the endothelium dysfunction at an early stage of atherogenesis. In the present study, the plasma phospholipid transfer protein (PLTP) was shown to promote the net mass transfer of alpha-tocopherol from high density lipoproteins (HDL) and alpha-tocopherol-albumin complexes toward alpha-tocopherol-depleted, oxidized low density lipoproteins (LDL). The facilitated transfer reaction of alpha-tocopherol could be blocked by specific anti-PLTP antibodies. These observations indicate that PLTP may restore the antioxidant potential of plasma LDL at an early stage of the oxidation cascade that subsequently leads to cellular damages. In addition, the present study demonstrated that the PLTP-mediated net mass transfer of alpha-tocopherol can constitute a new mechanism for the incorporation of alpha-tocopherol into the vascular wall in addition to the previously recognized LDL receptor and lipoprotein lipase pathways. In ex vivo studies on rabbit aortic segments, the impairment of the endothelium-dependent arterial relaxation induced by oxidized LDL was found to be counteracted by a pretreatment with purified PLTP and alpha-tocopherol-albumin complexes, and both the maximal response and the sensitivity to acetylcholine were significantly improved. We conclude that PLTP, by supplying oxidized LDL and endothelial cells with alpha-tocopherol through a net mass transfer reaction may play at least two distinct beneficial roles in preventing endothelium damage, i.e., the antioxidant protection of LDL and the preservation of a normal relaxing function of vascular endothelial cells.     

Alpha-tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats

To test the hypothesis that supra-elevated hepatic alpha-tocopherol concentrations would up-regulate mechanisms that result in increased hepatic alpha-tocopherol metabolism and excretion, rats received daily subcutaneous alpha-tocopherol injections (10 mg/100 g body wt) and then were sacrificed on Day 0 or 12 h following their previous injection on Days 3, 6, 9, 12, 15, and 18. Liver alpha-tocopherol concentrations increased from 12 +/- 1 nmol/g (mean +/- SE) to 819 +/- 74 (Day 3), decreased at Day 9 (486 +/- 67), and continued to decrease through Day 18 (338 +/- 37). alpha-Tocopherol metabolites and their intermediates increased and decreased similarly to alpha-tocopherol albeit at lower concentrations. There were no changes in known vitamin E regulatory proteins, i.e., hepatic alpha-tocopherol transfer protein or cytochrome P450 (CYP) 4F. In contrast, both CYP3A and CYP2B, key xenobiotic metabolizing enzymes, doubled by Day 6 and remained elevated, while P450 reductase increased more slowly. Consistent with the decrease in liver alpha-tocopherol concentrations, a protein involved in biliary xenobiotic excretion, p-glycoprotein, increased at Day 9, doubling by Day 15. Thus hepatic alpha-tocopherol concentrations altered hepatic proteins involved in metabolism and disposition of xenobiotic agents.     

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma

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.     

Endogenous adipocyte apolipoprotein E is colocalized with caveolin at the adipocyte plasma membrane

Apolipoprotein (apo)E is well established as a secreted protein that plays an important role in systemic lipoprotein metabolism and vascular wall homeostasis. Recently, endogenous expression of apoE in adipocytes has been shown to play an important role in adipocyte lipoprotein metabolism and gene expression consistent with a nonsecreted cellular itinerary for apoE. We designed studies to evaluate if adipocyte apoE was retained as a constituent protein in adipocytes and to identify a cellular retention compartment. Using confocal microscopy, coimmunoprecipitation, and sucrose density cellular fractionation, we establish that endogenous apoE shares a cellular itinerary with the constituent protein caveolin-1. Altering adipocyte caveolar number by modulating cellular cholesterol flux or altering caveolin expression regulates the distribution of cellular apoE between cytoplasmic and plasma membrane compartments. A mechanism for colocalization of apoE with caveolin was established by demonstrating a noncovalent interaction between an aromatic amino acid-enriched apoE N-terminal domain with the caveolin scaffolding domain. Absent apoE expression in adipocytes alters caveolar lipid composition. These observations provide evidence for an interaction between two proteins involved in cellular lipid metabolism in a cell specialized for lipid storage and flux, and rationalize a biological basis for the impact of adipocyte apoE expression on adipocyte lipoprotein metabolism.     

Effect of alpha-tocopherol on adrenal cortex functions under stress]

alpha-Tocopherol has been studied for its effect on lipid peroxidation and steroidogenesis in the adrenal cortices of rat and rabbit under stress. The vitamin is shown to exert an inhibitory effect on the lipid peroxidation developing under chronic stress. A biphasic pattern of the alpha-tocopherol effect on the steroidogenesis in the adrenal cortex is established: a decrease in the release of the steroids under the acute stress and maintaining of their levels under the chronic stress. A conclusion is drawn about a potential alpha-tocopherol application to correct the adrenal cortex function under stress.     

Cell calcium, vitamin E, and the thiol redox system in cytotoxicity

The controversial role of extracellular Ca2+ in toxicity to in vitro hepatocyte systems is reviewed. Recent reports demonstrate that extracellular Ca2+-related cytotoxicity is dependent on Ca2+-influenced vitamin E (alpha-tocopherol) content of isolated hepatocytes. Based on a Ca2+-omission model of in vitro oxidative stress, the role of vitamin E in cytotoxicity is further explored. This model demonstrates the interdependence of the GSH redox system and vitamin E as protective agents during oxidative stress. Following chemical oxidant-induced depletion of intracellular GSH, cell morphology and viability are maintained by the continuous presence of cellular alpha-tocopherol above a threshold level of 0.6-1.0 nmol/10(6) cells. alpha-Tocopherol threshold-dependent cell viability is directly correlated with the prevention of the loss of cellular protein thiols in the absence of intracellular GSH. Potential mechanisms for this phenomenon are explored and include a direct reductive action of alpha-tocopherol on protein thiyl radicals, and the prevention of oxidation of protein thiols by scavenging of lipid peroxyl radicals by alpha-tocopherol. It is suggested that in light of the threshold phenomenon of vitamin E prevention of potentially severe oxidative stress-induced cytotoxicity, its use as a protective agent against an oxidative challenge in vivo should be reassessed.     

Autophagy in obesity and atherosclerosis: Interrelationships between cholesterol homeostasis,lipoprotein metabolism and autophagy in macrophages and other systems

The incidence of diseases characterized by a dysregulation of lipid metabolism such as obesity, diabetes and atherosclerosis is rising at alarming rates, driving research to uncover new therapies to manage dyslipidemias and resolve the metabolic syndrome conundrum. Autophagy and lipid homeostasis – both ancient cellular pathways – have seemingly co-evolved to share common regulatory elements, and autophagy has emerged as a prominent mechanism involved in the regulation of lipid metabolism. This review highlights recent findings on the role of autophagy in the regulation of cellular cholesterol homeostasis and lipoprotein metabolism, with special emphasis on macrophages. From modulation of inflammation to regulation of cellular cholesterol levels, a protective role for autophagy in atherosclerosis is emerging. The manipulation of autophagic activity represents a new possible therapeutic approach for the treatment complex metabolic disorders such as obesity and the metabolic syndrome.     

The effect of concentration on the antioxidant effectiveness of alpha-tocopherol in lipid peroxidation induced by superoxide free radicals

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.     

The effects of alpha-tocopherol on site-specific lipid peroxidation induced by iron in charged micelles

alpha-Tocopherol inhibited H2O2-Fe2+-induced lipid peroxidation of linoleic acid (LA) by scavenging OH radicals in tetradecyltrimethylammonium bromide (TTAB) micelles. The inhibiting ability of alpha-tocopherol was much greater than that of OH-radical scavengers mannitol and t-butanol. In contrast, alpha-tocopherol enhanced linoleic acid hydroperoxide (LOOH)-Fe2+-induced lipid peroxidation through regeneration of Fe2+ in sodium dodecyl sulfate (SDS) micelles containing LA. alpha-Tocopherol was oxidized by Fenton's reagent (FeSO4 + H2O2) at a higher rate in SDS micelles than in TTAB micelles. The likely oxidants were OH radicals in the former and Fe3+ in the latter. Both reagents formed in the Fenton reaction. Ferrous ion catalyzed in a dose-dependent manner the decomposition of LOOH and conjugated diene compounds in SDS but not in TTAB micelles. alpha-Tocopherol and Fe3+ individually had no effect on the decomposition of LOOH, but together were quite effective. The rate of the decomposition was a function of the concentration of alpha-tocopherol. The mechanism of "site-specific" antioxidant action of alpha-tocopherol in charged micelles is discussed.     

The role of the kidney in lipid metabolism

PURPOSE OF REVIEW: Cellular uptake of plasma lipids is to a large extent mediated by specific membrane-associated proteins that recognize lipid-protein complexes. In the kidney, the apical surface of proximal tubules has a high capacity for receptor-mediated uptake of filtered lipid-binding plasma proteins. We describe the renal receptor system and its role in lipid metabolism in health and disease, and discuss the general effect of the diseased kidney on lipid metabolism. RECENT FINDINGS: Megalin and cubilin are receptors in the proximal tubules. An accumulating number of lipid-binding and regulating proteins (e.g. albumin, apolipoprotein A-I and leptin) have been identified as ligands, suggesting that their receptors may directly take up lipids in the proximal tubules and indirectly affect plasma and tissue lipid metabolism. Recently, the amnionless protein was shown to be essential for the membrane association and trafficking of cubilin. SUMMARY: The kidney has a high capacity for uptake of lipid-binding proteins and lipid-regulating hormones via the megalin and cubilin/amnionless protein receptors. Although the glomerular filtration barrier prevents access of the large lipoprotein particles to the proximal tubules, the receptors may be exposed to lipids bound to filtered lipid-binding proteins not associated to lipoprotein particles. Renal filtration and receptor-mediated uptake of lipid-binding and lipid-regulating proteins may therefore influence overall lipid metabolism. The pathological mechanisms causing the pronounced atherosclerosis-promoting effect of uremia may involve impairment of this clearance pathway.     

Cardiac-specific knock-out of lipoprotein lipase alters plasma lipoprotein triglyceride metabolism and cardiac gene expression

Fatty acids are the primary energy source for the heart. The heart acquires fatty acids associated with albumin or derived from lipoprotein lipase (LpL)-mediated hydrolysis of lipoprotein triglyceride (TG). We generated heart-specific LpL knock-out mice (hLpL0) to determine whether cardiac LpL modulates the actions of peroxisome proliferator-activated receptors and affects whole body lipid metabolism. Male hLpL0 mice had significantly elevated plasma TG levels and decreased clearance of postprandial lipids despite normal postheparin plasma LpL activity. Very large density lipoprotein-TG uptake was decreased by 72% in hLpL0 hearts. However, heart uptake of albumin-bound free fatty acids was not altered. Northern blot analysis revealed a decrease in the expression of peroxisome proliferator-activated receptor alpha-response genes involved in fatty acid beta-oxidation. Surprisingly, the expression of glucose transporters 1 and 4 and insulin receptor substrate 2 was increased and that of pyruvate dehydrogenase kinase 4 and insulin receptor substrate 1 was reduced. Basal glucose uptake was increased markedly in hLpL0 hearts. Thus, the loss of LpL in the heart leads to defective plasma metabolism of TG. Moreover, fatty acids derived from lipoprotein TG and not just albumin-associated fatty acids are important for cardiac lipid metabolism and gene regulation.     

Vitamin E: action, metabolism and perspectives

Natural vitamin E includes four tocopherols and four tocotrienols. RRR-alpha-tocopherol is the most abundant form in nature and has the highest biological activity. Although vitamin E is the main lipid-soluble antioxidant in the body, not all its properties can be assigned to this action. As antioxidant, vitamin E acts in cell membranes where prevents the propagation of free radical reactions, although it has been also shown to have pro-oxidant activity. Non-radical oxidation products are formed by the reaction between alpha-tocopheryl radical and other free radicals, which are conjugated to glucuronic acid and excreted through the bile or urine. Vitamin E is transported in plasma lipoproteins. After its intestinal absorption vitamin E is packaged into chylomicrons, which along the lymphatic pathway are secreted into the systemic circulation. By the action of lipoprotein lipase (LPL), part of the tocopherols transported in chylomicrons are taken up by extrahepatic tissues, and the remnant chylomicrons transport the remaining tocopherols to the liver. Here, by the action of the "alpha-tocopherol transfer protein", a major proportion of alpha-tocopherol is incorporated into nascent very low density lipoproteins (VLDL), whereas the excess of alpha-tocopherol plus the other forms of vitamin E are excreted in bile. Once secreted into the circulation, VLDL are converted into IDL and LDL by the action of LPL, and the excess of surface components, including alpha-tocopherol, are transferred to HDL. Besides the LPL action, the delivery of alpha-tocopherol to tissues takes place by the uptake of lipoproteins by different tissues throughout their corresponding receptors. Although we have already a substantial information on the action, effects and metabolism of vitamin E, there are still several questions open. The most intriguing is its interaction with other antioxidants that may explain how foods containing small amounts of vitamin E provide greater benefits than larger doses of vitamin E alone.     

Probucol inactivates ABCA1 in the plasma membrane with respect to its mediation of apolipoprotein binding and high density lipoprotein assembly and to its proteolytic degradation

Probucol has been shown to inhibit the release of cellular lipid by helical apolipoprotein and thereby to reduce plasma high density lipoprotein. We attempted to explore the underlying mechanism for this effect in human fibroblast WI-38. Probucol inhibited the apoA-I-mediated cellular lipid release and binding of apoA-I to the cells in a dose-dependent manner. It did not influence cellular uptake of low density lipoprotein, transport of cholesterol to the cell surface whether de novo synthesized or delivered as low density lipoprotein, and overall cellular content of cholesterol, although biosynthesis of lipids from acetate was somewhat increased. Probucol did not affect the mRNA level of ABCA1, and ABCA1 was recovered along with marker proteins for plasma membrane regardless of the presence of probucol. However, the protein level of ABCA1 increased, and the rate of its decay in the presence of cycloheximide was slower in the probucol-treated cells. ABCA1 in the probucol-treated cells was resistant to digestion by calpain but not by trypsin. We concluded that probucol inactivates ABCA1 in the plasma membrane with respect to its function in mediating binding of and lipid release by apolipoprotein and with respect to proteolytic degradation by calpain.     

Mechanism of the stabilization of synaptosomes with alpha-tocopherol during activation of lipid peroxidation

Using the fluorescent probe technique, it was shown that activation of lipid peroxidation decreases the value of transmembrane potential of rat brain synaptosomes. Depolarization of synaptosomes may be due to the impairment of the "barrier" properties of synaptosomal membranes and the decrease in Na,K-ATPase activity. alpha-Tocopherol and its model derivative devoid of the phytol chain--2,2,5,7,8-pentamethyl-6-oxychromanol--stabilize the transmembrane potential value during inhibition of lipid peroxidation. alpha-Tocopherol acetate causes no stabilizing or inhibiting effects. Unlike 2,2,5,7,8-pentamethyl-6-oxychromanol, alpha-tocopherol exerts a structuralizing action which manifests itself in the stabilization of the synaptosomal membrane potential during incomplete inhibition of lipid peroxidation. The previously established ability of alpha-tocopherol to protect synaptosomes from the damaging action of phospholipases and the experimental results of this work permit to regard vitamin E as a universal stabilizer of brain synaptosomal membranes.     

Cytoprotective properties of alpha-tocopherol are related to gene regulation in cultured D-galactosamine-treated human hepatocytes

Vitamin E (alpha-tocopherol) has demonstrated antioxidant activity and gene-regulatory properties. d-Galactosamine (D-GalN)-induced cell death is mediated by nitric oxide in hepatocytes, and it is associated with hepatic steatosis. The beneficial properties of alpha-tocopherol and their relation to oxidative stress and gene regulation were assessed in D-GalN-induced cell death. Hepatocytes were isolated from human liver resections by a collagenase perfusion technique. alpha-Tocopherol (50 microM) was administered at the advanced stages (10 h) of D-GalN-induced cell death in cultured hepatocytes. Cell death, oxidative stress, alpha-tocopherol metabolism, and NF-kappaB-, pregnane X receptor (PXR)-, and peroxisome proliferator-activated receptor (PPAR-alpha)-associated gene regulation were estimated in the hepatocytes. D-GalN increased cell death and alpha-tocopherol metabolism. alpha-Tocopherol exerted a moderate beneficial effect against apoptosis and necrosis induced by D-GalN. Induction (rifampicin) or inhibition (ketoconazole) of alpha-tocopherol metabolism and overexpression of PXR showed that the increase in PXR-related CYP3A4 expression caused by alpha-tocopherol enhanced cell death in hepatocytes. Nevertheless, the reduction in NF-kappaB activation and inducible nitric oxide synthase expression and the enhancement of PPAR-alpha and carnitine palmitoyl transferase gene expression by alpha-tocopherol may be relevant for cell survival. In conclusion, the cytoprotective properties of alpha-tocopherol are mostly related to gene regulation rather than to antioxidant activity in toxin-induced cell death in hepatocytes.