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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Tocotrienol Suppresses Adipocyte Differentiation and Akt Phosphorylation in 3T3-L1 Preadipocytes^1–3,

⁴ Internal Medicine 1, National Defense Medical College, Tokorozawa-shi, Saitama 359-8513, Japan; ⁵ Faculty of Education, Utsunomiya University, Utsunomiya-shi, Tochigi 321-8505, Japan; ⁶ Department of Applied Bioscience, Kanagawa Institute of Technology, Atsugi-shi, Kanagawa 243-0292, Japan; ⁷ Institute of Environmental Science for Human Life, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan; ⁸ Department of Education, Hirosaki University, Hirosaki-shi, Aomori 036-8560, Japan; and ⁹ Department of Life Science, Ibaraki Christian University, Hitachi, Ibaraki 319-1295, Japan

^* To whom correspondence should be addressed. E-mail: kondo.kazuo{at}ocha.ac.jp.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
In vivo studies show that -tocotrienol and -tocotrienol accumulate in adipose tissue. Furthermore, a recent study reports that the oral administration of -tocotrienol from a tocotrienol-rich fraction from palm oil (TRF) decreases body fat levels in rats. The objective of this study was to evaluate the effect of TRF and its components on adipocyte differentiation in 3T3-L1 preadipocytes, which differentiated into adipocytes in the presence of 1.8 µmol/L insulin. TRF suppressed the insulin-induced mRNA expression of adipocyte-specific genes such as PPAR, adipocyte fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein- (C/EBP) compared with the differentiation of 3T3-L1 preadipocytes into adipocytes only in the presence of insulin. To confirm the suppressive effect of TRF, the major components of TRF, such as -tocotrienol, -tocotrienol, and -tocopherol, were investigated. -Tocotrienol and -tocotrienol decreased the insulin-induced PPAR mRNA expression by 55 and 90%, respectively, compared with insulin, whereas -tocopherol increased the mRNA expression. In addition, -tocotrienol suppressed the insulin-induced aP2 and C/EBP mRNA expression, triglyceride accumulation, and PPAR protein levels compared with insulin. The current results also revealed that -tocotrienol inhibited the insulin-stimulated phosphorylation of Akt but not extracellular signal-regulated kinase (ERK)1/2 in the insulin signaling pathway of 3T3-L1 preadipocytes. Thus, the antiadipogenic effect of TRF depends on -tocotrienol and -tocotrienol, and -tocotrienol may be a more potent inhibitor of adipogenesis than -tocotrienol. Therefore, the results of this study suggest that tocotrienol suppresses insulin-induced differentiation and Akt phosphorylation in 3T3-L1 preadipocytes. Furthermore, tocotrienol could act as an antiadipogenic vitamin in the nutrient-mediated regulation of body fat through its effects on differentiation.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Several studies report that certain food components and nutrients inhibit adipogenesis in mouse adipocytes (5,6). Tea catechin suppresses adipocyte differentiation accompanied by downregulation of PPAR and C/EBP in 3T3-L1 cells (5) and the soybean isoflavone genistein suppress adipogenesis in 3T3-L1 cells (6). Provitamin A carotenoids and all-trans retinoic acid derived from dietary β-carotene potently inhibits the differentiation of 3T3-L1 cells (7–9) and all-trans retinoic acid effectively inhibits the differentiation of porcine preadipocytes in primary culture, suggesting that retinoids may regulate fat cell differentiation in growing animals (10).

Vitamin E is abundant in cereal grains, soybeans, barley, oats, rice bran, and palm oil. In nature, compounds with vitamin E activity include -, β-, -, and - tocopherols and -, β-, -, and - tocotrienols, which differ in the number and position of methyl groups on the chroman ring (11). Tocopherols have saturated tails, whereas tocotrienols have 3 double bonds in their phytyl tails. The biological activity of these vitamin E isoforms depends on their structures, and their chemical properties include antioxidative activities. -Tocopherol has the highest biological activity of the tocopherols. The findings of various studies suggest that tocotrienols exert a hypocholesterolemic or antiatherotic effect on humans, rats, and mice (12–17). In vitro studies show that tocotrienols act as 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors and consequently reduce cholesterol synthesis (12). The tocotrienol-rich fraction from palm oil (TRF), which includes -tocopherol and - and -tocotrienol, decreases serum total cholesterol, LDL-cholesterol, apolipoprotein B, and triglyceride levels compared with the baseline values (13). From our previous study, we know that -tocotrienol stimulates sodium excretion in vivo, suggesting that -tocotrienol possesses a hormone-like natriuretic function (14) and a recent study by others found that the oral administration of -tocotrienol derived from TRF decreases the body fat of rats (15). Although the results of in vivo studies have shown that -tocotrienol and -tocotrienol are present in the adipose tissue of rats, nude mice, and hairless mice fed a diet containing TRF (16,17), not much is known about the effects of TRF or vitamin E homologs on adipose cells. The objective of this study was to evaluate the effect of TRF and its components on adipocyte differentiation in 3T3-L1 cells. We also investigated the mechanisms of the effect of TRF on the insulin signaling pathway promoting adipogenesis in 3T3-L1 cells. Our findings suggest that the major components of TRF, such as -tocotrienol and -tocotrienol, suppress adipocyte differentiation and Akt phosphorylation in 3T3-L1 preadipocytes.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Materials. TRF consisting of 227 mg/g -tocopherol, 353 mg/g -tocotrienol, and 497 mg/g -tocotrienol was kindly provided by Lion. It was dissolved in ethanol. -Tocotrienol, -tocotrienol, and -tocopherol were donated by Eisai Food and Chemical and they were also dissolved in ethanol. Polyclonal antibodies against PPAR (H-100) were purchased from Santa Cruz Biotechnology and polyclonal antibodies against Akt, phospho-Akt (Ser473), extracellular signal-regulated kinase (ERK)1/2, and phosphor-ERK1/2 (Thr202/Tyr204) were purchased from Cell Signaling Technology. Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were obtained from GE Healthcare UK for use as secondary antibodies. Recombinant mouse insulin was purchased from US Biological. All reagents used in this study were of reagent grade.

    Adipocyte differentiation. 3T3-L1 preadipocytes, which had been grown and maintained in DMEM supplemented with 10% bovine serum, penicillin (200,000 U/L), and streptomycin (200 mg/L), were seeded in 12-well plates at a density of 2 x 10⁵ cells per well. 3T3-L1 preadipocytes were cultured for 3 d postconfluency and maintained in DMEM supplemented with 10% fatal bovine serum, penicillin (200,000 U/L), and streptomycin (200 mg/L) in the presence of 1.8 µmol/L insulin.

    Real-time RT-PCR. Total cellular RNA was prepared using TRIZOL reagent (Invitrogen). One microgram of total RNA was reverse transcribed into cDNA using an Omniscript Reverse Transcriptase kit (Qiagen). The concentration and quality of the purified total RNA were determined spectrophotometrically at 260 nm and by the OD260:280 ratio, and 28s:18s using agarose gel electrophoresis, respectively. mRNA expression was quantified using an ABI 7300 instrument and the SYBR green reagent (Applied Biosystems). Results are expressed as copy number ratio of the target mRNA:β-actin mRNA. The primers for PPAR, C/EBP, aP2, and β-actin were as follows: PPAR, 5'-GGCGATCTTGACAGGAAAGAC-3', 5'-CCCTTGAAAAATTCGGATGG-3'; C/EBP, 5'-GGTTTTGCTCTGATTCTTGCC-3', 5'-CGAAAAAACCCAAACATCCC-3'; aP2, 5'-AGCATCATAACCCTAGATGGCG-3', 5'-CATAACACATTCCACCACCAGC-3'; and β-actin, 5'-TCACCCACACTGTGCCCATCTA-3', 5'-TTGCTGATCCACATCTGCTGG-3'.

    Adipocyte triglyceride. The content of cellular protein was determined using a bicinchoninic acid protein assay (Pierce Laboratories) (18). Adipocyte triglyceride was extracted with hexane:isopropyl alcohol (2:3, v:v) for 10 min at 4°C, emulsified with 2% Triton X-100 in isopropyl alcohol for 20 min at 70°C, and measured using a triglyceride test kit (Kyowa Medex).

    Oil Red O staining. 3T3-L1 adipocytes were washed twice with PBS and fixed with 4% buffered formalin for at least 30 min at 4°C. The cells were then stained for 30 min at room temperature with a filtered oil red O solution (0.3% oil red O in 60% isopropyl alcohol), washed twice with distilled water, and visualized.

    Western blot analysis. 3T3-L1 adipocytes were washed with ice-cold PBS containing 1 mmol/L Na₃VO₄, lysed with M-PER Mammalian Protein Extraction reagent (Pierce Biotechnology) including proteinase inhibitor cocktail (Pierce Biotechnology). After centrifugation at 20,400 x g for 20 min at 4°C, the protein content in the supernatant was determined using a bicinchoninic acid protein assay (Pierce Laboratories) and aliquots of the proteins were separated by 7.5% or 12.5% SDS-PAGE gels (e-PAGEL; ATTO). The proteins were electroblotted onto a poly-vinyligene difluoride membrane (ATTO) and detected using an ECL Plus detection kit (GE Healthcare UK) and a LAS 3000 image analyzer (Fuji Photo Film).

    Statistical analysis. Statistical analyses were performed using the Stat View version 5.0 software (SAS Institute). All results were expressed as the means ± SD. Significance among individual treatment groups was evaluated using 2-way ANOVA with the Scheffé post hoc test in the 2 x 2 designs (Fig. 1, 3–6) and the 2 x 4 designs (Fig. 2) with insulin and TRF, insulin and tocotrienol, or insulin and tocopherol as the independent variables. Differences were considered significant at P < 0.05.

View larger version (21K): [in this window] [in a new window]   FIGURE 1  TRF suppressed the differentiation of 3T3-L1 preadipocytes into adipocytes. Cells were precultured for 0, 7, or 14 d with 1.8 µmol/L insulin and then cultured with 1 mg/L TRF (TRF) with or without 1.8 µmol/L insulin (Ins) for 14 d. Results are means ± SD, n = 3 separate experiments. Within each treatment group, means without a common letter differ, P < 0.05. Asterisks indicate different from 0 d at that group, *P < 0.05.

View larger version (15K): [in this window] [in a new window]   FIGURE 3  TRF and -tocotrienol suppressed the aP2 and C/EBP mRNA expression in the differentiation of 3T3-L1 preadipocytes into adipocytes. Cells were cultured for 14 d with 1 mg/L TRF (TRF) (A,D), 2.4 µmol/L of - tocotrienol (-T3) (B,E), or - tocotrienol (-T3) (C,F) with or without 1.8 µmol/L insulin (Ins). Results are means ± SD, n = 3 separate experiments. Within each treatment group, means without a common letter differ, P < 0.05.

View larger version (11K): [in this window] [in a new window]   FIGURE 4  Tocotrienol suppressed the triglyceride accumulation in the differentiation of 3T3-L1 preadipocytes into adipocytes. Cells were cultured for 21 d in the presence of control medium (Cont), 2.3 µmol/L -tocopherol (-Toc), 2.4 µmol/L - tocotrienol (-T3), or - tocotrienol (-T3) with (+ Ins) or without 1.8 µmol/L insulin (–Ins). Results are means ± SD, n = 3 separate experiments. Within each treatment group, means without a common letter differ, P < 0.05.

View larger version (29K): [in this window] [in a new window]   FIGURE 5  Tocotrienol suppressed PPAR protein levels in the differentiation of 3T3-L1 preadipocytes into adipocytes. Cells were cultured for 14 d in the presence of control medium (Cont), 2.4 µmol/L -tocotrienol (-T3), or -tocotrienol (-T3) with or without 1.8 µmol/L insulin (Ins). Results are expressed as fold of control and are means ± SD, n = 3 separate experiments. Within each treatment group, means without a common letter differ, P < 0.05. A representative Western blot is shown in the insert.

View larger version (33K): [in this window] [in a new window]   FIGURE 6  Tocotrienol inhibited the tyrosine phosphorylation of Akt, but not ERK1/2, in the differentiation of 3T3-L1 preadipocytes. Cells were precultured for 24 h in the presence of control medium (Cont), 2.4 µmol/L -tocotrienol (-T3), or -tocotrienol (-T3) and subsequently stimulated with or without 100 nmol/L insulin (Ins) for 15 min. The abundances of phosphorylated Akt relative to total Akt (A) and phosphorylated ERK1/2 relative to total ERK1/2 (B) were evaluated. Results are expressed as fold of control and are means ± SD, n = 3 separate experiments. Within each treatment group, means without a common letter differ, P < 0.05. Representative Western blots are shown in the inserts.

View larger version (18K): [in this window] [in a new window]   FIGURE 2  Tocotrienol suppressed PPAR mRNA expression in the differentiation of 3T3-L1 preadipocytes into adipocytes. Cells were cultured for 14 d with various concentrations of -tocotrienol (A), -tocotrienol (B), or -tocopherol (C) with (+ Ins) or without 1.8 µmol/L insulin (– Ins). Results are means ± SD, n = 3 separate experiments. Within each treatment group, means without a common letter differ, P < 0.05. *Different from – Ins or #+ Ins at various concentrations of each vitamin E homologs, P < 0.05.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

    Tocotrienol suppressed adipocyte differentiation of 3T3-L1 preadipocytes into adipocytes. Because the major components of TRF are -tocotrienol, -tocotrienol, and -tocopherol, we studied the effects of these vitamin E homologs on adipocyte-specific gene expression to further clarify the inhibitory effect of TRF on the differentiation of 3T3-L1 preadipocytes into adipocytes. There was no change in the mRNA expression of PPAR, aP2, and C/EBP in 3T3-L1 preadipocytes treated with 2.4 µmol/L -tocotrienol or 2.4 µmol/L -tocotrienol compared with the control differentiation of 3T3-L1 preadipocytes into adipocytes in all experiments (Figs. 2 and 3). The mRNA expression of PPAR was lower in cells treated with 1.8 µmol/L insulin and 2.4 µmol/L -tocotrienol (55 ± 4%; P < 0.001) or 0.24 µmol/L -tocotrienol (50 ± 8%; P < 0.001) than in cells treated only with insulin (Fig. 2A). However, treatment with 2.4 µmol/L -tocotrienol and 1.8 µmol/L insulin produced no changes in the mRNA expression of aP2 (41 ± 19%; P = 0.082) and C/EBP (28 ± 14%; P = 0.098) compared with insulin (Fig. 3E,B). In addition, the PPAR mRNA expression in cells treated with 1.8 µmol/L insulin and 2.4 µmol/L -tocotrienol (90 ± 2%; P < 0.001), 0.24 µmol/L -tocotrienol (73 ± 3%; P < 0.001), or 0.024 µmol/L -tocotrienol (70 ± 4%; P < 0.001) was less than in cells treated only with insulin (Fig. 2B). The mRNA expression of other adipocyte-specific genes, including aP2 (42 ± 10%; P < 0.01) and C/EBP (64 ± 10%; P < 0.01), was also decreased by -tocotrienol (Fig. 3C,F). In addition, treatment of 3T3-L1 preadipocytes with 2.4 µmol/L -tocotrienol or 2.4 µmol/L -tocotrienol produced no changes in the mRNA expression of PPAR, aP2, and C/EBP compared with the control differentiation of 3T3-L1 preadipocytes into adipocytes. Furthermore, compared with insulin, the level of adipocyte triglycerides was lower in 3T3-L1 cells treated with 2.4 µmol/L -tocotrienol and 1.8 µmol/L insulin (40 ± 12%; P < 0.01), although there was no change with 2.4 µmol/L -tocotrienol and 1.8 µmol/L insulin (30 ± 19%) (Fig. 4). The suppression of insulin-induced adipocyte differentiation by -tocotrienol or -tocotrienol compared with insulin alone was also confirmed by oil red O staining (Supplemental Fig. 1). However, 2.3 µmol/L -tocopherol and insulin enhanced the mRNA expression of PPAR (162 ± 35%; P < 0.001) compared with the mRNA expression for insulin (Fig. 2C). Interestingly, the PPAR mRNA expression in cells treated with just 2.3 µmol/L -tocopherol increased by 205 ± 32% (P < 0.01) compared with the control. In addition, oil red O staining revealed that -tocopherol also slightly increased the insulin-induced accumulation of intracellular triglycerides compared with insulin (Supplemental Fig. 1).

    Tocotrienol suppressed PPAR protein level in differentiation of 3T3-L1 preadipocytes into adipocytes. The PPAR protein levels in 3T3-L1 preadipocytes were analyzed in the presence of -tocotrienol and -tocotrienol. The PPAR protein levels in cells treated with 1.8 µmol/L insulin increased compared with the control differentiation of 3T3-L1 preadipocytes into adipocytes (P < 0.001). PPAR protein levels were lower in cells treated with 2.4 µmol/L -tocotrienol and 1.8 µmol/L insulin (54 ± 14%; P < 0.05), although there was no change with 2.4 µmol/L -tocotrienol and insulin (40 ± 15%; P = 0.066) compared with cells treated only with insulin (Fig. 5). These results were consistent with the results from the analysis of mRNA expression.

    Examination of the inhibitory mechanism using a protein phosphorylation assay of -tocotrienol and -tocotrienol of 3T3-L1 preadipocytes. Because Akt and ERK1/2 in the insulin signaling pathway are upstream of PPAR and adipocyte differentiation, we examined the effects of tocotrienols on the levels of phosphorylated Akt and phosphorylated ERK1/2. The tyrosine phosphorylation of Akt (P < 0.001) and ERK 1/2 (P < 0.001) in 3T3-L1 preadipocytes treated with 100 nmol/L insulin increased compared with the control. The phosphorylation of Akt stimulated by 100 nmol/L insulin was inhibited by the presence of 2.4 µmol/L -tocotrienol (43 ± 16%; P < 0.05), although -tocotrienol did not change (27 ± 10%) compared with stimulation by insulin only (Fig. 6A). On the other hand, the insulin-stimulated phosphorylation of ERK1/2 was not affected by the presence of -tocotrienol (113 ± 20%) or -tocotrienol (100 ± 14%) compared with insulin alone (100%) (Fig. 6B).

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
The findings of this study showed that through its effects on differentiation, tocotrienol could be an antiadipogenic vitamin similar to vitamin A in the nutrient-mediated regulation of body fat. PPAR and C/EBP play roles in the early stage of adipocyte differentiation, because they are transcription factors for numerous genes (20). Further, PPAR forms a heterodimer with retinoic acid X-receptor (21) and regulates the transcription of adipocyte-specific genes (22–24).Retinoids and carotenoids, which are ligands of retinoic acid X-receptor, inhibit the early stage of differentiation in 3T3-L1 preadipocytes (9). These observations are consistent with the findings of the current study that tocotrienols suppressed PPAR mRNA expression when 3T3-L1 preadipocytes were postcultured in media supplemented with1.8 µmol/L insulin for 0 d but not for 7 or 14 d. Therefore, tocotrienols might suppress adipogenesis in preadipocytes but not adipocytes.

In the current study, treatment with 2.4 µmol/L -tocotrienol and 1.8 µmol/L insulin produced no significant change in the mRNA expression of aP2 and C/EBP compared with insulin alone, and -tocotrienol seemed to be a more potent inhibitor of adipogenesis than -tocotrienol. However, both aP2 and C/EBP mRNA expression were suppressed in vitro with -tocotrienol treatment at much higher doses.

Previous in vivo studies showed that -tocotrienol and -tocotrienol accumulate in adipose tissue and the concentration of these tocotrienols in rats fed a diet containing TRF is 0.024 µmol/g adipose tissue (16,17). Our study revealed that having 0.024 µmol/L -tocotrienol and 1.8 µmol/L insulin in the culture media suppressed PPAR mRNA expression compared with insulin alone in the differentiation of 3T3-L1 preadipocytes into adipocytes. Considering that the oral administration of -tocotrienol decreases the body fat of rats in another study (15), -tocotrienol could decrease adipose tissue mass, although the effects of TRF on body fat mass was not determined in these studies (15–17).

Insulin is known to induce the differentiation of adipocytes from preadipocytes in adipose tissues (25) and these changes are associated with the sequential activation of pro-adipogenic transcription factors, including the mRNA expression of PPAR and C/EBP (20,26). To test the antiadipogenic mechanism of tocotrienol, we investigated Akt and ERK1/2 phosphorylation in the insulin signaling pathway. The adipogenic actions of insulin are mediated by the insulin signaling pathway and the binding of insulin to insulin receptor (IR) at the cell surface. This event activates the intrinsic tyrosine kinase activity residing in the β-subunit of the IR and leads to autophosphorylation of the cytoplasmic portion of the β-subunit and further activation of its tyrosine kinase toward several intermediate IR substrate-1 (IRS-1) with several downstream signaling molecules. Two major pathways are located downstream of IRS-1: the Akt and ERK1/2 pathways (27,28). Other studies, however, imply that this is still controversial (29–32). ERK1/2 induces cell growth and potentially affects cell differentiation. Interestingly, retinoic acid activation of the ERK pathway is required for embryonic stem cell commitment to the adipocyte lineage despite its inhibition of the differentiation of clonal preadipocyte cell lines (33,34). In addition, tea polyphenol, which reduces body fat in vivo, and (-)-epigallocatechin gallate inhibit insulin-stimulated phosphorylation of IR and IRS-1 in rat hepatoma cells (35–37). (-)-Epigallocatechin gallate also downregulates resistin mRNA expression via a pathway that is dependent on the ERK pathway (38).

Few studies have considered the inhibitory effects of the phosphorylation of Akt in adipose cells, although the tyrosine phosphorylation of Akt (but not ERK1/2) is inhibited by -tocotrienol after insulin stimulation in 3T3-L1 preadipocytes. It should also be noted that tocotrienol inhibited Akt signaling in neoplastic mammary epithelial cells and human umbilical vein endothelial cells (39,40). These previous studies and the current one suggest that -tocotrienol might be a potential Akt inhibitor. Because we did not examine insulin signaling in 3T3-L1 adipocytes, further study is needed to evaluate this.

In contrast to the inhibitory effect of the tocotrienols, -tocopherol enhanced insulin-induced differentiation of 3T3-L1 preadipocytes into adipocytes. These findings are consistent with those of a previous study that found that -tocotrienol reduces body fat mass and -tocopherol is ineffective in this respect in rats fed -tocotrienol and -tocotrienol for 8 wk (18). It seems that the opposing effects of -tocopherol and -tocotrienol on adipocyte differentiation may depend on differences between them, which are a saturated tail in -tocopherol and 3 double bonds in the phytyl tail of -tocotrienol. Also, -tocopherol (600 mg/kg) increased glucose uptake and ameliorated the inhibitory effect of diabetes on skeletal muscle in vivo (41). Skeletal muscles are a major site of blood glucose utilization and, together with adipose tissue, are a target tissue for insulin action. Based on these findings, -tocopherol may effectively prevent diabetes but not obesity.

-Tocotrienol is metabolized into 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (-CEHC), which possesses a hormone-like natriuretic function (14,42). Although it is unclear whether -CEHC is incorporated into adipose tissues, the addition of 264.3 µmol/L -CEHC and 1.8 µmol/L insulin to the culture media suppressed the PPAR mRNA expression compared with the insulin of 3T3-L1 preadipocytes into adipocytes. However, the inhibitory effect of -CEHC was less than that of -tocotrienol (data not shown).

In conclusion, tocotrienols may prevent obesity through suppression of the differentiation of preadipocytes into adipocytes, and the inhibitory effect of TRF depends on both -tocotrienol and -tocotrienol but not -tocopherol. These data show the possibility that tocotrienol could be an antiadipogenic vitamin similar to vitamin A in regard to nutrient-mediated regulation of body fat through its effects on differentiation. Further study is required on determine whether tocotrienol promotes the loss of body fat in humans.

	ACKNOWLEDGMENTS

 
We thank Dr. K. Shimokado of Tokyo Medical and Dental University for his helpful discussions. We also thank T. Kido for secretarial assistance and H. Yoshimura and M. Ichikawa of Eisai Food and Chemical Co., Ltd. for providing the tocotrienols.

	FOOTNOTES

 
¹ Supported by special funds from the Ministry of Education of Japan.

² Author disclosures: H. Uto-Kondo, R. Ohmori, C. Kiyose, Y. Kishimoto, H. Saito, O. Igarashi, and K. Kondo, no conflicts of interest.

³ Supplemental Figure 1 is available with the online posting of this paper at jn.nutrition.org.

¹⁰ Abbreviations used: aP2, adipocyte fatty acid-binding protein; C/EBP, CCAAT/enhancer-binding protein ; ERK, extracellular signal-regulated kinase; TRF, tocotrienol-rich fraction from palm oil; IR, insulin receptor; IRS-1, insulin receptor substrate-1; -CEHC, 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman.

Manuscript received 15 July 2008. Initial review completed 12 August 2008. Revision accepted 22 October 2008.

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