Tocotrienols regulate cholesterol production in mammalian cells by post-transcriptional suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Parker RA, Pearce BC, Clark RW, Gordon DA, Wright JJ.

J Biol Chem. 1993 May 25;268(15):11230-8.

Tocotrienols are natural farnesylated analogues of tocopherols which decrease hepatic cholesterol production and reduce plasma cholesterol levels in animals. For several cultured cell types, incubation with gamma-tocotrienol inhibited the rate of [14C]acetate but not [3H] mevalonate incorporation into cholesterol in a concentration- and time-dependent manner, with 50% inhibition at approximately 2 microM and maximum approximately 80% inhibition observed within 6 h in HepG2 cells. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase total activity and protein levels assayed by Western blot were reduced concomitantly with the decrease in cholesterol synthesis. In HepG2 cells, gamma-tocotrienol suppressed reductase despite strong blockade by inhibitors at several steps in the pathway, suggesting that isoprenoid flux is not required for the regulatory effect. HMG-CoA reductase protein synthesis rate was moderately diminished (57% of control), while the degradation rate was increased 2.4-fold versus control (t1/2 declined from 3.73 to 1.59 h) as judged by [35S]methionine pulse-chase/immunoprecipitation analysis of HepG2 cells treated with 10 microM gamma-tocotrienol. Under these conditions, the decrease in reductase protein levels greatly exceeded the minor decrease in mRNA (23 versus 76% of control, respectively), and the low density lipoprotein receptor protein was augmented. In contrast, 25-hydroxycholesterol strongly cosuppressed HMG-CoA reductase protein and mRNA levels and the low density lipoprotein receptor protein. Thus, tocotrienols influence the mevalonate pathway in mammalian cells by post-transcriptional suppression of HMG-CoA reductase, and appear to specifically modulate the intracellular mechanism for controlled degradation of the reductase protein, an activity that mirrors the actions of the putative non-sterol isoprenoid regulators derived from mevalonate.

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Long-term administration of tocotrienols and tumor-marker enzyme activities during hepatocarcinogenesis in rats

Rahmat A, Ngah WZ, Shamaan NA, Gapor A, Abdul Kadir K.

Nutrition. 1993 May-Jun;9(3):229-32.

The effects of long-term administration of tocotrienol on hepatocarcinogenesis in rats induced by diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) were investigated by determining the activities of gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), glutathione S-transferases (GSTs), and glutathione (GSH) levels in blood and liver. Twenty-eight male 7- to 8-wk-old Rattus norwegicus rats, weighing 120-160 g, were used in this study. The rats were divided into four treatment groups: a control group on a basal diet, a group fed a basal diet supplemented with tocotrienol (30 mg/kg food), a group treated with DEN/AAF, and a group treated with DEN/AAF and fed a diet supplemented with tocotrienol (30 mg/kg food). Blood was collected monthly, and GGT, ALP, and GSH levels were determined. The rats were killed after 9 mo, and the livers were examined morphologically. Grayish white nodules (2/liver) were found in all the DEN/AAF-treated rats (n = 10), but only one of the rats treated with DEN/AAF and supplemented with tocotrienol (n = 6) had liver nodules. A significant increase in the level of blood and liver GSH, ALP, and GGT activities was observed in the DEN/AAF-treated rats. Liver GSTs were similarly increased with DEN/AAF treatment. Tocotrienol supplementation attenuated the impact of the carcinogens in the rats.

Comparative antioxidant activity of tocotrienols and other natural lipid-soluble antioxidants in a homogeneous system, and in rat and human lipoprotein

Suarna C, Hood RL, Dean RT, Stocker R.

Biochim Biophys Acta. 1993 Feb 24;1166(2-3):163-70.

The antioxidant activity of tocotrienols toward peroxyl radicals was compared with that of other natural lipid-soluble antioxidants in three different systems by measuring the temporal disappearance of antioxidants and the formation of lipid hydroperoxides. In homogeneous solution, the initial rates of consumption of the various antioxidants, assessed by competition experiments between pairs of antioxidants for radicals, decreased in the order: ubiquinol-10 approximately ubiquinol-9 > alpha-tocopherol approximately alpha-tocotrienol > beta-carotene approximately lycopene > gamma-tocopherol approximately gamma-tocotrienol. Following in vitro incubation of human plasma with alpha-tocotrienol, this form of vitamin E was present in all classes of lipoproteins isolated from the supplemented plasma. Dietary supplementation of rats and humans with a tocotrienol-rich preparation resulted in a dose-dependent appearance of alpha- and gamma-tocotrienols in plasma and all circulating lipoproteins, respectively. Exposure of such enriched rat plasma to aqueous peroxyl radicals resulted in simultaneous consumption of the alpha- and then gamma-isomers of vitamin E. The sequence of radical-induced consumption of antioxidants in freshly isolated, in vitro and in vivo tocotrienol-enriched low density lipoprotein (LDL) was again ubiquinol-10 > alpha-tocotrienol approximately alpha-tocopherol > carotenoids > gamma-tocopherol approximately gamma-tocotrienol. Under conditions where radicals were generated at constant rates, the rate of lipid hydroperoxide formation in LDL was not constant. It proceeded in at least three stages separated by the phase of ubiquinol-10 consumption and, subsequently, that of alpha-tocopherol/alpha-tocotrienol. Our results show that dietary tocotrienols become incorporated into circulating human lipoproteins where they react with peroxyl radicals as efficiently as the corresponding tocopherol isomers.

Tocotrienol and fatty acid composition of barley oil and their effects on lipid metabolism

Wang L, Newman RK, Newman CW, Jackson LL, Hofer PJ.

Plant Foods Hum Nutr. 1993 Jan;43(1):9-17.

Barley oil was extracted with hexane from the grain of a high oil waxy hull-less barley. Twelve male broiler chicks were fed corn-based diets with either 10% barley oil, 10% corn oil or 10% margarine ad libitum for ten days. Total plasma cholesterol concentration of the chicks fed barley oil was 34% lower (p < 0.05) than that of the chicks fed margarine. Plasma low density lipoprotein cholesterol concentration of chicks fed barley oil was 53% and 59% lower (p < 0.05) than those of chicks fed corn oil and margarine, respectively. Plasma high density lipoprotein cholesterol and triglyceride concentration of the barley oil group were similar to those of the margarine but higher (p < 0.05) than those of the corn oil group. Chicks fed the barley oil gained more (p < 0.05) body weight than those fed the corn oil and margarine. Barley oil had an effect in suppression of TC and LDLC in chicks compared to margarine. Barley oil suppressed LDLC but not HDLC in chicks compared to corn oil. A greater weight gain of the chicks fed barley oil suggested that these chicks had normally functioning digestion and absorption. alpha-Tocotrienol and gamma-tocotrienol content of the barley oil were 24 and 17 times greater, respectively, than those observed in the corn oil, while the same fractions were not detectable in the margarine. Polyunsaturated fatty acid content of the barley oil was more than threefold that of margarine. These data suggest that alpha-tocotrienol and polyunsaturated fatty acids are hypocholesterolemic components in barley oil.

Vitamin E inhibits protein oxidation in skeletal muscle of resting and exercised rats

Reznick AZ, Witt E, Matsumoto M, Packer L.

Biochem Biophys Res Commun. 1992 Dec 15;189(2):801-6.

It is well known that exercise induces lipid peroxidation in skeletal muscle and that vitamin E prevents exercise-induced lipid damage. In this study we show for the first time, an increase in protein oxidation in skeletal muscle after a single bout of exercise, related to an exercise-induced decrease in lipophilic antioxidants, and substantial protection against both resting and exercise-induced protein oxidation by supplementation with various isomers (alpha-tocopherol, alpha-tocotrienol) of vitamin E.

Hypocholesterolemic activity of synthetic and natural tocotrienols

Pearce BC, Parker RA, Deason ME, Qureshi AA, Wright JJ.

J Med Chem. 1992 Oct 2;35(20):3595-606.

Tocotrienols are farnesylated benzopyran natural products that exhibit hypocholesterolemic activity in vitro and in vivo. The mechanism of their hypolipidemic action involves posttranscriptional suppression of HMG-CoA reductase by a process distinct from other known inhibitors of cholesterol biosynthesis. An efficient synthetic route to tocotrienols and their isolation from palm oil distillate using an improved procedure is presented. gamma-Tocotrienol exhibits a 30-fold greater activity toward cholesterol biosynthesis inhibition compared to alpha-tocotrienol in HepG2 cells in vitro. The synthetic (racemic) and natural (chiral) tocotrienols exhibit nearly identical cholesterol biosynthesis inhibition and HMG-CoA reductase suppression properties as demonstrated in vitro and in vivo.

Antioxidant functions of vitamins. Vitamins E and C, beta-carotene, and other carotenoids

Sies H, Stahl W, Sundquist AR.

Ann N Y Acad Sci. 1992 Sep 30;669:7-20

Tocopherols and tocotrienols (vitamin E) and ascorbic acid (vitamin C) as well as the carotenoids react with free radicals, notably peroxyl radicals, and with singlet molecular oxygen (1O2), this being the basis of their function as antioxidants. RRR-alpha-tocopherol is the major peroxyl radical scavenger in biological lipid phases such as membranes or low-density lipoproteins (LDL). L-Ascorbate is present in aqueous compartments (e.g. cytosol, plasma, and other body fluids) and can reduce the tocopheroxyl radical; it also has a number of metabolically important cofactor functions in enzyme reactions, notably hydroxylations. Upon oxidation, these micronutrients need to be regenerated in the biological setting, hence the need for further coupling to nonradical reducing systems such as glutathione/glutathione disulfide, dihydrolipoate/lipoate, or NADPH/NADP+ and NADH/NAD+. Carotenoids, notably beta-carotene and lycopene as well as oxycarotenoids (e.g. zeaxanthin and lutein), exert antioxidant functions in lipid phases by free-radical or 1O2 quenching. There are pronounced differences in tissue carotenoid patterns, extending also to the distribution between the all-trans and various cis isomers of the respective carotenoids. Antioxidant functions are associated with lowering DNA damage, malignant transformation, and other parameters of cell damage in vitro as well as epidemiologically with lowered incidence of certain types of cancer and degenerative diseases, such as ischemic heart disease and cataract. They are of importance in the process of aging. Reactive oxygen species occur in tissues and cells and can damage DNA, proteins, carbohydrates, and lipids. These potentially deleterious reactions are controlled in part by antioxidants that eliminate prooxidants and scavenge free radicals. Their ability as antioxidants to quench radicals and 1O2 may explain some anticancer properties of the carotenoids independent of their provitamin A activity, but other functions may play a role as well. Tocopherols are the most abundant and efficient scavengers of peroxyl radicals in biological membranes. The water-soluble antioxidant vitamin C can reduce tocopheroxyl radicals directly or indirectly and thus support the antioxidant activity of vitamin E; such functions can be performed also by other appropriate reducing compounds such as glutathione (GSH) or dihydrolipoate. The biological efficacy of the antioxidants is also determined by their biokinetics.

Interactions among antioxidants in health and disease: Vitamin E and its redox cycle

Packer L.

Proc Soc Exp Biol Med. 1992 Jun;200(2):271-6.

Probably most diseases at some point during their course involve free radical reactions in tissue injury. In some cases, free radical reactions may be involved in multiple sites and at different stages of a chronic disease. So, both acute and degenerative diseases are thought to involve free radical reactions in tissue injury. An overview will be given of the evidence for the occurrence of free radicals and the importance of antioxidant interventions, with particular reference to the lipophilic antioxidant vitamin E (tocopherols and tocotrienols).

Recycling of vitamin E in human low density lipoproteins

Kagan VE, Serbinova EA, Forte T, Scita G, Packer L.

J Lipid Res. 1992 Mar;33(3):385-97.

Oxidative modification of low density lipoproteins (LDL) and their unrestricted scavenger receptor-dependent uptake is believed to account for cholesterol deposition in macrophage-derived foam cells. It has been suggested that vitamin E that is transported by LDL plays a critical role in protecting against LDL oxidation. We hypothesize that the maintenance of sufficiently high vitamin E concentrations in LDL can be achieved by reducing its chromanoxyl radicals, i.e., by vitamin E recycling. In this study we demonstrate that: i) chromanoxyl radicals of endogenous vitamin E and of exogenously added alpha-tocotrienol, alpha-tocopherol or its synthetic homologue with a 6-carbon side-chain, chromanol-alpha-C6, can be directly generated in human LDL by ultraviolet (UV) light, or by interaction with peroxyl radicals produced either by an enzymic oxidation system (lipoxygenase + linolenic acid) or by an azo-initiator, 2,2′-azo-bis(2,4-dimethylvaleronitrile) (AMVN; ii) ascorbate can recycle endogenous vitamin E and exogenously added chromanols by direct reduction of chromanoxyl radicals in LDL; iii) dihydrolipoic acid is not efficient in direct reduction of chromanoxyl radicals but recycles vitamin E by synergistically interacting with ascorbate (reduces dehydroascorbate thus maintaining the steady-state concentration of ascorbate); and iv) beta-carotene is not active in vitamin E recycling but may itself be protected against oxidative destruction by the reductants of chromanoxyl radicals. We suggest that the recycling of vitamin E and other phenolic antioxidants by plasma reductants may be an important mechanism for the enhanced antioxidant protection of LDL.

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Nutritional and biochemical aspects of the hypolipidemic action of rice bran oil: A review

Rukmini C, Raghuram TC.

J Am Coll Nutr. 1991 Dec;10(6):593-601.

In this paper, we review the effects of rice bran oil (RBO), an unconventional oil recently introduced onto the Indian market for human use. RBO contains oleic acid (38.4%), linoleic acid (34.4%), and linolenic acid (2.2%) as unsaturated fatty acids, and palmitic (21.5%) and stearic (2.9%) acids as saturated fatty acids. The unsaponifiable fraction (4.2%) has total tocopherols (81.3 mg%), gamma-oryzanol (1.6%), and squalene (320 mg%). Oryzanol is a mixture of ferulic acid esters of triterpene alcohols such as cycloartenol (CA) (106 mg%) and 24-methylene cycloartanol (494 mg%). Studies on experimental rats demonstrated a hypolipidemic effect of RBO. The unsaponifiable fraction of RBO lowers cholesterol levels. Feeding phytosterols, CA, and 24-methylene cycloartanol in amounts present in RBO to hypercholesterolemic rats for 8 weeks indicates that CA alone reduces cholesterol and triglyceride levels significantly. Endogenous sterol excretion increases in animals given CA. The accumulation of CA in the liver inhibits cholesterol esterase activity, which in turn leads to reduction in circulating cholesterol levels. CA is structurally similar to cholesterol and may compete with the binding sites of cholesterol and sequestrate cholesterol, which is metabolized to its derivatives. RBO, which is rich in tocopherols and tocotrienols, may improve oxidative stability. Tocotrienols inhibit HMG CoA reductase, resulting in hypocholesterolemia. The hypolipidemic effect of RBO has also been established in human subjects. Thus, RBO could be a suitable edible oil for patients with hyperlipidemia.

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