Linalool is a PPARα ligand that reduces plasma TG levels and rewires the hepatic transcriptome and plasma metabolome.

We investigated the hypotriglyceridemic mechanism of action of linalool, an aromatic monoterpene present in teas and fragrant herbs. Reporter gene and time-resolved fluorescence resonance energy transfer assays demonstrated that linalool is a direct ligand of PPARα. Linalool stimulation reduced cellular lipid accumulation regulating PPARα-responsive genes and significantly induced FA oxidation, and its effects were markedly attenuated by silencing PPARα expression. In mice, the oral administration of linalool for 3 weeks reduced plasma TG concentrations in Western-diet-fed C57BL/6J mice (31%, P < 0.05) and human apo E2 mice (50%, P < 0.05) and regulated hepatic PPARα target genes. However, no such effects were seen in PPARα-deficient mice. Transcriptome profiling revealed that linalool stimulation rewired global gene expression in lipid-loaded hepatocytes and that the effects of 1 mM linalool were comparable to those of 0.1 mM fenofibrate. Metabolomic analysis of the mouse plasma revealed that the global metabolite profiles were significantly distinguishable between linalool-fed mice and controls. Notably, the concentrations of saturated FAs were significantly reduced in linalool-fed mice. These findings suggest that the appropriate intake of a natural aromatic compound could exert beneficial metabolic effects by regulating a cellular nutrient sensor.

puncture under general anesthesia with 2.5% tribromoethanol (20 ml/kg, ip). Food intake was measured twice a week during the feeding period. After the feeding period, the mice were euthanized after overnight fasting, and their livers were removed, snap-frozen in liquid nitrogen, and stored at Ϫ 80°C prior to analysis. All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of Korea University (animal protocol number: KUIACUC-20090421-2).

Transcriptional reporter assay
CHO-K1 cells were seeded in a 24-well plate (1 × 10 5 cells per well) and incubated overnight before being transfected using Metafectene (Biontex, Munich, Germany) with 0.3 g each of PPAR ␣ expression vector and DR-1 (PPAR recognition site) luciferase reporter plasmids and 20 ng of a ␤ -galactosidase reporter plasmid. Transfected cells were incubated for 24 h and then treated with linalool or fenofi brate for 24 h. The cells were lysed, and luciferase activity was measured with the Firefl y Luciferase Assay Kit (Biotium, Hayward, CA) using a luminometer (Victor3, PerkinElmer, Waltham, MA). Relative promoter activity was computed by normalization to ␤ -galactosidase activity, as determined with the ␤ -Galactosidase Enzyme Assay System (Promega Corp., Madison, WI).

PPAR ␣ knockdown by a lentivirus carrying PPAR ␣selective shRNA
HEK293T cells were cotransfected for 4 h with a lentiviral pGIPZ plasmid carrying PPAR ␣ -selective shRNA (mature antisense sequence 5 ′ -TGAGTCGAATCGTTCGCCG-3 ′ ; Open Biosystems, Huntsville, AL), a psPAX2 packaging plasmid, and a pMD2G envelop plasmid using HilyMax reagent (Dojindo, Kumamoto, Japan). The medium containing the transfection reagent was removed and replaced with fresh complete DMEM supplemented with 10% FBS and penicillin/streptomycin. Twelve hours later, the culture medium containing lentiviral particles was harvested from HEK293T cells, fi ltered through a 0.45 m fi lter, and transferred to a polypropylene storage tube. This procedure was repeated three times. The virus was stored in aliquots at Ϫ 80°C until use. HepG2 cells were then infected with appropriate amounts of lentiviral particles in medium supplemented with polybrene for 8 h. Fresh complete medium was added, and the infection was repeated twice. Infected HepG2 cells were selected with puromycin, and immunoblotting was performed to examine the effi ciency of the protein knockdown. As a control, scrambled shRNA was used following the same procedure.

Nuclear protein preparation and PPAR ␣ promoter binding assay
Nuclear proteins were prepared from HepG2 cells treated with linalool for 24 h using the Nuclear Extraction Kit (Cayman, Linalool is a major and common terpenoid contained in most herbal essential oils and teas, including both green and black teas, and has been implicated in aroma and fl avoring ( 14,15 ). Oral exposure to linalool from formulated food products, including beverages, was estimated at up to 140 g/kg/day, including the dietary intake of linalool from natural sources such as vegetables and spices ( 16 ). Linalool has been traditionally used for medicinal purposes because of its potent antioxidative activities ( 17,18 ). Recent studies have suggested that linalool may have a novel biological activity in TG metabolism, as the oral administration of fragrant herbal essential oils containing linalool, including Plantago asiatica and Melissa offi cinalis essential oils, was shown to improve dyslipidemia by reducing plasma TG concentrations ( 13,19 ).
The metabolic functions of the nuclear receptor and nutrient sensor peroxisome PPAR ␣ in the regulation of plasma TG concentrations have been intensively studied. It is well known that PPAR ␣ activation is a favorable drug target for liver and plasma TG level reductions ( 20,21 ). Similar to other nuclear receptors, PPAR ␣ is activated by ligand binding. It then heterodimerizes with retinoid X receptor and binds to PPAR response elements in the promoter regions of target genes responsible for FA uptake, FA oxidation, and TG-rich VLDL secretion (20)(21)(22)(23). Conformational changes during the ligand-dependent activation of PPAR ␣ facilitate the coordinated dissociation of corepressors and the recruitment of coactivator proteins to enable transcriptional activation ( 24,25 ).
Previous luciferase-based activity screening with ‫ف‬ 900 natural compounds and extracts revealed that linalool is a potential PPAR ␣ agonist. Subsequently, we performed two preliminary animal feeding experiments with linalool, and the results consistently included signifi cant reductions in TG concentrations. Therefore, in this study, we investigated the biological mechanism of action of linalool as a novel PPAR ␣ agonist that is responsible for the hypotriglyceridemic effects of teas and aromatic herbs. To this end, we examined the agonistic activity of linalool for PPAR ␣ and performed three independent animal studies to confi rm hypotriglyceridemic activities. In addition, we performed transcriptome and metabolome analyses and adopted selected conventional biomarker approaches.

Quantitative real-time PCR analysis
Total RNA was extracted from the livers and HepG2 cells using RNAiso Plus (Takara, Shiga, Japan). cDNA was synthesized from 2 g of total RNA using M-MLV Reverse Transcriptase (Mbiotech, Seoul, Korea) and oligo(dT) primers. The levels of gene expression were measured using the iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) and RealMasterMix SYBR ROX reagent (5 Prime, Hamburg, Germany). Relative levels of gene expression were calculated using iQ5 Optical System Software version 2 (Bio-Rad), with the expression of each target gene being normalized to that of ␤ -actin or GAPDH.

Oligonucleotide microarray analysis
Two-color oligonucleotide microarray experiments were performed using untreated control and lipid-accumulated cells or control and lipid-accumulated cells stimulated with either linalool (1 mM) or fenofi brate (0.1 mM). Total RNA was prepared from HepG2 cells using RNAiso Plus, RNase-free DNase I Set (Qiagen, Valencia, CA), and the RNeasy MinElute Cleanup Kit (Qiagen). During reverse transcription, cDNA was labeled with the Cy3-dUTP and Cy5-dUTP (GeneChem Inc., Seoul, Korea) and purifi ed with QIAquick PCR Purifi cation Kit (Qiagen) before being hybridized to the 32 K Human OneArray™ (Phalanx Ann Arbor, MI), according to the manufacturer's instructions. Sample protein contents were determined by the Bradford method. PPAR ␣ promoter binding activity in the nuclear proteins was measured using an ELISA-based PPAR ␣ transcription factor assay kit (Cayman) to detect PPAR ␣ bound to PPAR response element (PPRE) -containing double-stranded DNA sequences, according to the manufacturer's instructions.

PPAR ␣ coactivator recruitment assay
PPAR ␣ ligand binding activity was determined by Lan-thaScreen™ TR-FRET PPAR ␣ Coactivator Assay (catalog no. PV4684, Invitrogen, Carlsbad, CA), as described previously ( 26 ). Recombinant human PPAR ␣ ligand binding domain (LBD; amino acids 192-468; NP_005027) was synthesized and tagged to glutathione S-transferase , terbium-labeled anti-PPAR ␣ antibodies were provided (catalog no. PV4691; included in the PV4684 kit), and fl uorescein-labeled coactivator [PPAR ␥ coactivator 1, (PGC1 ␣ )] peptides (EAEEPSLLKKLLLAPANTQ; catalog no. PV4421) were provided in the PV4684 kit. Recruitment of fl uorescein-PGC1 ␣ peptides in response to linalool or fenofi brate treatment was measured by monitoring the fl uorescence resonance energy transfer (FRET) from the terbium anti-glutathione S-transferase antibody to the fl uorescein on the peptide. The FRET signal was determined by excitation at 340 nm and emission at 520 nm for terbium, as well as 490 nm for fl uorescein, using a SpectraMax spectrophotometer (Molecular Devices, Sunnyvale, CA). Data were analyzed using GraphPad Prism software (La Jolla, CA).

Absorption of linalool in Caco-2 cells
The assay was performed as previously described ( 27 ), and its reproducibility was confi rmed with a reference compound, propranolol. Caco-2 cells were seeded at a density of 1.6 × 10 5 cells/ cm 2 on collagen-coated Transwell fi lter inserts (Corning Costar Corp., Cambridge, MA). Cells were grown and differentiated into confl uent monolayers for 21 days. The integrity of the monolayers was evaluated by measuring transport of Lucifer yellow, as described previously ( 28 ). HBSS containing linalool (0.25 M in 0.5 ml) was added to the apical side, while the basolateral side received 1.5 ml of HBSS. After incubation, apical and basolateral HBSS buffers containing linalool were collected and analyzed using a GC-mass selective detector (GC-MSD; Agilent 7890A GC/5975C MSD, Agilent Technologies, Avondale, PA) equipped with a nonpolar fused silica capillary DB-5MS column (30 m × 25 mm, 0.25 m fi lm thickness; J and W Scientifi c, Folsom, CA). Data were acquired and processed using ChemStation software (Agilent Technologies). The apparent permeability coeffi cient (P app ) was used to predict the absorption potential of linalool ( 29 ).

Cellular and plasma lipids, lipoprotein profi ling, and DiI and Oil Red O staining
Lipid-loaded HepG2 cells were treated with linalool for 24 h. Cellular TG and cholesterol concentrations were quantifi ed as described previously, and cellular lipids were extracted with 2 ml of a 2:1 (v/v) mixture of hexane and isopropanol at room temperature ( 30 ). Plasma TG concentrations were determined enzymatically using the Cobas C111 analyzer. Lipoproteins were analyzed using a fast protein LC (FPLC) system (AKTA Purifi er 10; GE Healthcare, Piscataway, NJ) equipped with two Superose 6 10/300 GL columns (GE Healthcare) connected in series. Pooled mouse plasma (150 and 200 l for C57BL/6J and apoE2 transgenic mice, respectively) was injected onto the column and separated with elution buffer containing 154 mM NaCl, 1 mM EDTA, and 0.02% NaN 3 (pH 8.2) at a fl ow rate of 0.35 ml/min. The effl uent was collected, and TG levels were determined enzymatically. DiI (1,1 ′ -dioctadecyl-3,3,3 ′ 3 ′ -tetramethylindocarbocyanine than the L -isoform. In this study, we investigated the effects of a mixture of L -and D -linalool (linalool) on the PPAR ␣dependent lipid metabolism because linalool occurs naturally as two isomeric forms that were both signifi cantly effective. The binding of activated PPAR ␣ to the PPRE sequence was confi rmed by the binding assay ( Fig. 1B ). PPAR ␣ binding to PPRE was induced signifi cantly in HepG2 cells stimulated with linalool. Ligand binding to PPAR ␣ caused conformational changes and induced coactivator recruitment, which can be assessed by the timeresolved (TR)-FRET assay. In the TR-FRET assay, linalool displayed a dose-dependent induction of the recruitment of a PGC1 ␣ coactivator peptide to PPAR ␣ LBD (EC 50 = 5.45 M, Fig. 1C ), suggesting that linalool could act as a PPAR ␣ ligand to induce target gene transcription. In the Caco-2 cell-based in vitro bioavailability assay, 0.5 mM linalool showed a time-dependent transport from the apical to the basolateral side of Caco-2 monolayers ( Fig. 1D ). After 3 h of incubation, the amounts of linalool in the apical and basolateral compartments were 0.1 and 0.07 M, respectively. The apparent permeability coeffi cient (P app ) of linalool was calculated as 11.3 ± 2.1 × 10 Ϫ 6 cm/s at the end of 3 h, which is considered indicative of a moderate permeability ( 34 ). FA oxidation, which can be induced by PPAR ␣ activation, was signifi cantly increased in cells stimulated with linalool ( Fig. 1E ). In silico molecular docking analysis suggested that D -linalool interacts directly in the active site pocket of PPAR ␣ LBD via hydrogen bonding with His440 and Tyr464, thus acting as a PPAR ␣ ligand to induce target gene transcription ( Fig. 1F ). These results demonstrate that linalool is bioavailable, interacts with PPAR ␣ protein directly, and thus induces PPAR ␣ transactivation.

Linalool signifi cantly reduces cellular TG concentrations and activates PPAR ␣ target genes in HepG2 cells
The effects of linalool on TG metabolism and PPAR ␣ target gene expression were examined in cultured hepatocytes. Linalool reduced cellular TG concentrations in a dose-dependent manner compared with controls (e.g., 37% at 1 mM, P < 0.05; Fig. 2A ). Oil Red O and DiI staining also showed a signifi cant reduction in intracellular TG accumulation by linalool ( Fig. 2B ). The quantitative realtime PCR (qPCR) and immunoblotting analysis revealed that the gene ( Fig. 2C ) and protein ( Fig. 2D ; supplementary Fig. I) expressions of PPAR ␣ and the target genes responsible for the hypotriglyceridemic effects were significantly altered by linalool stimulation (e.g., FATP4 and ACS1 for FA uptake; ACOX 1 and UCP2 for FA oxidation; and LPL and APOC3 for TG hydrolysis). Linalool dose-dependently increased the mRNA and protein expression of PPAR ␣ (1.9-and 1.8-fold for mRNA and protein expression, respectively, at 1 mM, P < 0.05). The increase in PPAR ␣ expression induced by linalool stimulation contributed to the regulation of the expression of its target genes, including upregulation of FATP4, ACS1, ACOX 1, UPC2, and LPL expression and downregulation of APOC3 expression ( P < 0.05). In addition, the PPAR ␣ -dependent mechanism of action of linalool was further confi rmed by specifi c PPAR ␣ knockdown in HepG2 cells using a lentivirus carrying Biotech Group, Hsinchu, Taiwan), which contains 30,968 human genome probes. Hybridized arrays were scanned with a GenePix 4000B scanner (Axon Instruments, Palo Alto, CA) and analyzed with GenePix 5.1 software (Axon Instruments). Probe-level gene expression values were computed, background corrected, and normalized by the Loess method using GenePix 5.1 and Acuity 4.0 software (Axon Instruments). Genes showing signifi cant changes in expression in response to lipid accumulation, linalool, or fenofi brate were determined by Student's t -test ( P < 0.05), and commonly detected genes were used to compare the effects of the three treatments on transcriptional profi les, assessed with the HeatMap Viewer tool in GenePattern software (http://genepattern.broadinstitute.org/pages/index.jsf).

Metabolite analysis
The extraction, derivatization, and analysis of metabolites from plasma were conducted as previously described ( 32,33 ). A total of 30 µL of each plasma sample was extracted through incubation at 20°C for 5 min with 1 ml of an extraction solvent (3:3:2, v/v/v, isopropanol-acetonitrile-water). After centrifugation (16,000 rpm, 4°C for 5 min), 0.5 ml of supernatant was removed into a 1.5 ml tube and then dried in a centrifugal vacuum concentrator at 25°C for 6 h. The completely dried pellet was subsequently derivatized prior to GC/MS analysis. The carbonyl functional groups were protected by methoximation using 10 l of a 40 mg/ml methoxyamine hydrochloride in pyridine (Sigma-Aldrich) at 30°C for Restek, Bellefonte, PA). For metabolite analysis, 1 l of the derivatized sample was injected in splitless mode, and the sample was subjected to the following conditions: 50°C for 1 min, followed by an increase to 330°C at 20°C per min, and incubation at 330°C for 5 min. Mass spectra were acquired in a scan range of m/z 85-500, with the electron impact and temperature of the ion source set to 70 eV and 250°C, respectively. The mass spectra were preprocessed by automated peak detection and mass spectral deconvolution using LECO ChromaTOF software (version 2.32) and then matched against the retention index and mass spectrum information of customized reference mass spectrum libraries from the BinBase database, which were acquired using authentic standard compounds with identical data acquisition parameters. The identifi ed metabolites were used for the unsupervised principal components analysis (PCA) of multivariate statistics using Statica software (version 7.1; StatSoft, Tulsa, OK, USA).

Statistical analysis
Data are presented as the means ± SE. The values of the treatment groups were compared with those of controls by t-test. Differences with P values < 0.05 were considered statistically signifi cant.

Linalool directly binds to the PPAR ␣ LBD, induces transactivation, and is bioavailable
We fi rst investigated PPAR ␣ activity regulated with linalool. Linalool ( L / D -linalool) induced PPAR ␣ transactivation in a dose-dependent manner ( Fig. 1A ). The results confi rmed that each isomeric form, L -and D -, could contribute to the effects; however, D -linalool was more potent C57BL/6J and apoE2 mice was lower than that of controls after linalool or fenofi brate feeding for 3 weeks with more marked changes in apoE2 mice than C57BL/6J mice (supplementary Fig. III). Linalool administration reduced plasma TG levels by 31% and 50% in C57BL/6J ( Fig. 3A ) and apoE2 mice ( Fig. 4A ), respectively, compared with those in control groups ( P < 0.05). When the same amount of fenofi brate (100 mg/kg body weight/day) was orally administered to mice, 76% and 89% of the reductions in plasma TG levels were observed in C57BL/6J and apoE2 mice, respectively, which were more profound than those of the linalool group. Lipoprotein profi ling analysis by FPLC showed that the TG concentrations in VLDL were dramatically reduced in the linalool group relative to controls ( Figs. 3B, 4B ; supplementary Fig. IV). Prominent effects of linalool in plasma cholesterol concentrations were also found in our previous study, with Ϫ 12% and Ϫ 45% in the total and LDL cholesterol levels of linaloolsupplemented group (0.57 mg linalool/mouse/day for a PPAR ␣ -selective shRNA. PPAR ␣ shRNA reduced endogenous PPAR ␣ protein expression by 80%, and under these conditions, linalool (0.05 and 0.1 mM) did not alter the protein expression of PPAR ␣ or the expression of its responsive genes (supplementary Fig. II). Combined, these results demonstrate that linalool is a PPAR ␣ ligand and regulates cellular lipid metabolism by altering target gene transcription in vitro.
Linalool reduces plasma TG concentrations by activating PPAR ␣ in Western-diet-fed C57BL/6J and human apoE2 transgenic mice but has no effect in PPAR ␣ -defi cient mice In vivo experiments were performed with C57BL/6J-, apoE2-, and PPAR ␣ -defi cient mice. Western-diet-fed animals were orally administered with linalool (100 mg/kg body weight/day) for 3 weeks. Cumulated food intake in both C57BL/6J and apoE2 mice was decreased by linalool or fenofi brate supplementation compared with controls as previously reported (data not shown) ( 35 ). Body weight of

Linalool stimulation of lipid-loaded HepG2 cells rewired the hepatic transcriptome profi le, with the effects of linalool being comparable to those of fenofi brate
Next, transcriptome profi ling was assessed with microarray analysis in lipid-loaded HepG2 cells stimulated with linalool. A total of 8,988 genes were commonly significantly expressed in all experimental groups, including control (CON), lipid-loaded (LL), and lipid-loaded hepatocytes stimulated with either linalool (1 mM; LL+LN) or the hypotriglyceridemic drug fenofi brate (0.1 mM; LL+FF). These genes were analyzed to compare the transcriptome profi les of cells treated with linalool and those treated with fenofi brate. The 13 genes associated with lipid metabolism from the selected genes were measured by qPCR analysis to verify the results of the microarray. The expression patterns of the selected genes in qPCR analyses were similar to those in the microarray analysis ( Fig. 6A ). Nonbiased 3 weeks), respectively, versus the control group ( P < 0.05) ( 12 ). In the qPCR and immunoblotting assays of selected biomarkers, hepatic PPAR ␣ expression was signifi cantly increased in C57BL/6J mice (+2.1-fold, Fig. 3C ) and apoE2 mice (+1.4-fold, Fig. 4C ). Liver mRNA expression of PPAR ␣ target genes, including FATP4, ACS1, ACOX 1, UCP2, and LPL, was upregulated, whereas the APOC3 mRNA expression was suppressed. Protein expression, which was assessed by immunoblotting, showed trends similar to those for mRNA expression ( Figs. 3D, 4D ; supplementary Fig. V). However, hyportriglyceridemic activities and PPAR ␣ regulation were not observed in PPAR ␣ -defi cient mice ( Fig. 5 ). Plasma TG concentration and the expression of PPAR ␣ and of its responsive genes and proteins were not altered in PPAR ␣ -defi cient mice fed linalool ( Fig. 5A-C ). These data demonstrate that linalool ameliorates hypertriglyceridemia by PPAR ␣ -dependent mechanisms. lipid-loading conditions after fenofi brate stimulation ( Fig. 6B ). These results indicate that linalool and fenofibrate shifted aberrant gene expression patterns under lipid-loading conditions toward those of controls without lipid loading. In addition, the gene expression patterns of a high concentration of linalool-treated cells were comparable to those of fenofi brate-stimulated cells.
An excessive accumulation of TG in hepatocytes is mainly linked to defects in the energy metabolism ( 36,37 ). Pathway analysis indicated that linalool caused a significant alteration in energy metabolism, including FA oxidation and synthesis, glycolysis, and TCA cycle. Key genes involved in the metabolic pathways were detectable (supplementary Table I). Decreases in the transcription factor sterol regulatory element binding protein (SREBP)-1C and lipogenic target gene Acc1 were found in the linalool-treated group. As we showed in the PPAR ␣ -targeted mechanism of genome profi ling analysis revealed that lipid loading in hepatocytes notably changed the hepatic transcriptome profi le relative to controls. The total of 6,922 genes (77% of the selected genes) showed >20% changes in expression [fold change >1.2 (blue color) or fold change <0.8 (red color)], suggesting that hepatic lipid accumulation could induce dramatic alternations in the global gene expressions compared with normal conditions. However, linalool-stimulated lipid-loaded (LL+LN) cells showed expression changes of >20% in 48% (4,314 genes) of the selected genes relative to controls. Thus, 29% of the selected genes (2,608 genes) resulted in <20% changes in expression after linalool stimulation, compared with the lipidloading conditions. Fenofi brate-stimulated cells (LL+FF) showed expression changes of >20% in 46% of the selected genes (4,134 genes). Thus, 31% of the selected genes (2,788 genes) fell into the <20% change category compared with Fig. 3. Linalool ameliorates hypertriglyceridemia in Western-diet-fed C57BL6 mice by hepatic PPAR ␣ activation. Western-diet-fed hypertriglyceridemic C57BL6J mice (n = 3-5 per group) were orally administered with vehicle, linalool (100 mg/kg body weight/day), or fenofi brate (100 mg/kg body weight/day) for 3 weeks, as described in Materials and Methods. A: Fasting plasma TG levels. B: Plasma TG profi ling with FPLC analysis. FPLC fractions obtained from overnight fasting plasma were determined as described in Materials and Methods. C: PPAR ␣ and its responsive gene expressions. D: PPAR ␣ and its target protein expressions. Total RNA and protein were extracted from liver tissues, and the expression levels of PPAR ␣ and targets were analyzed by real-time PCR and immunoblotting, respectively (n = 3 per group). mRNA and protein expression levels were normalized to ␤ -actin mRNA and ␣ -tubulin protein, respectively. Data represent means ± SE. * P < 0.05 versus the control group. In C and D, means without common letters differed, P < 0.05.

Linalool altered the plasma FA metabolite
Plasma metabolomic analysis was performed to examine the metabolic effects of linalool in C57BL/6J mice. A total of 103 metabolites were detected in both control and linalool-fed mice. PCA of the 103 metabolites showed a differential distribution of control and linalool-treated mice along the principal component (PC) 1 and PC2 dimensions, describing 38% and 23% of the total variance, respectively ( Fig. 7A ). PC1 primarily contributed to the separation of control and linalool-treated groups. Interestingly, the results showed that the top loading metabolites for PC1 included long-chain FAs, such as oleic and lauric acids, which were major contributors to the differentiation of PCA values between control and linalool-fed groups and were thus associated with the hypotriglyceridemic effects of linalool (supplementary Table II).
linalool, the expression of the genes involved in FA oxidation, including Echs1, Acaa, Acox1, and Acadm, was induced by linalool. Microarray data also showed that the impaired glycolysis under hepatic lipid accumulation conditions could be improved by linalool by signifi cant reductions in glucokinase (Gck), pyruvate kinase (Pk), and phosphofructokinase (Pkfl). The expression of TCA-cycle-related genes, which link glycolysis and fat oxidation to respiration, was increased under linalool-treated conditions (i.e., Sdhb, Mdh, and Suclg1). Linalool also positively affected hepatic lipid metabolism through the upregulation of Hsd17b4, Cyp27a1, Hsd3b7, and Akr1c3 expression for the conversion of excessive cholesterols to bile acids. Therefore, the linalool-mediated regulation of these key genes might play a prominent role in the improvement of the aberrant hepatic transcriptome by lipid accumulation and eventual protection against hypertriglyceridemia . Fig. 4. Linalool activates hepatic PPAR ␣ and attenuates hypertriglyceridemia in Western-diet-fed human apoE2 knock-in transgenic mice. Hypertriglyceridemic apoE2 mice (n = 3-7 per group) were fed a Western diet supplemented or not (control group) with 100 mg/kg body weight/day linalool or fenofi brate by oral gavage for 3 weeks. A: Analysis of fasting plasma TG levels. B: TG levels in lipoprotein fractions isolated by FPLC analysis from overnight fasting plasma, as described in Materials and Methods. C: PPAR ␣ and its responsive gene expressions. D: PPAR ␣ and its target protein expressions. Total RNA and protein were isolated from the livers, and the expression levels of PPAR ␣ and its targets were quantifi ed by real-time PCR and immunoblotting, respectively (n = 3 per group). ␤ -actin and ␣ -tubulin were used for the normalization of mRNA and protein expression, respectively. Data represent means ± SE. * P < 0.05 versus the control group. In C and D, means without common letters differed, P < 0.05.

DISCUSSION
Aromatic compounds generally have potent radical scavenging activities ( 17,18 ) and possess the ability to directly interact with proteins (e.g., binding and interaction with olfactory receptors), thereby playing important biological roles ( 39,40 ). Studies have shown that the oral administration of aromatic compounds has notable biological effects in animals and humans, suggesting their potential as active therapeutic and preventive agents ( 12,19,41 ). Linalool, a major aromatic terpenoid of teas and herbal essential oils, has been traditionally used for medicinal purposes because of its potent antioxidative activities ( 17,18 ). Recent reports have shown that the oral administration of fragrant herbal essential oils containing linalool, including Plantago asiatica and Melissa offi cinalis essential oils, improved dyslipidemia by reducing plasma TG concentrations ( 13,19 ). Here, we demonstrate that the Because an association between plasma TG and FA concentrations in metabolic diseases has been reported in human studies, the effects of linalool on plasma FA composition were assessed with metabolomic analysis. We selected 13 long-chain FA metabolites that had been altered by linalool treatment (supplementary Table III), out of which four FA metabolites, lauric acid, palmitoleic acid, myristic acid, and oleic acid, showed greatly reduced levels, as described in the previous studies that reported alterations in the plasma FA metabolite composition in the plasma under TG-lowered conditions in vivo ( 38 ). However, the stearic acid concentrations were not changed. Box-whisker plots show that the intensities of the metabolic signals for FAs (n = 3) were additive in these regions and were lower in the linalool-treated group than in controls ( Fig. 7B ). These data demonstrate that linalool signifi cantly altered the plasma metabolite profi le and reduced the concentrations of most of the longchain FA concentrations as a result of PPAR ␣ activation.  5. Hypotriglyceridemic effects of linalool were not observed in PPAR ␣ -defi cient mice. Western-diet-fed PPAR ␣ -defi cient mice (n = 6-7 per group) were orally administered with vehicle, linalool (100 mg/kg body weight/day), or fenofi brate (100 mg/kg body weight/day) for 3 weeks. A: Analysis of fasting plasma TG levels. B: PPAR ␣ and its target gene expressions assessed with qPCR. ␤ -actin was used as a reference. C: Immunoblotting analysis. ␣ -tubulin was a reference for normalization. Data represent means ± SE. * P < 0.05 versus the control group .
PPAR ␣ and its responsive gene and protein expressions, thus reducing intracellular TG accumulations, although not in cells with PPAR ␣ gene knockdowns.
The role of linalool in TG and FA metabolism was investigated in vivo in three independent Western-diet-fed animal studies in C57BL/6J, human apoE2 transgenic, and PPAR ␣defi cient mice. The oral administration of linalool for 3 weeks regulated hepatic PPAR ␣ and its responsive genes, fi nally reducing plasma TG levels in both human apoE2 and wild-type (C57BL/6J) mice. Consistently, FPLC analysis showed a signifi cant suppression of TG concentrations in VLDL in the linalool-supplemented group, compared with controls. Notably, the effects of linalool in PPAR ␣ -dependent mechanisms and TG reduction were not shown in PPAR ␣defi cient mice. The results from both cultured cell and animal experiments indicate that the hypotriglyceridemic properties of linalool are achieved via PPAR ␣ activation. hypotriglyceridemic effects of linalool can be achieved by PPAR ␣ agonistic properties.
PPAR ␣ is a ligand-activated nuclear receptor regulating the plasma TG concentration. Ligand binding alters the conformation of PPAR ␣ , enabling it to recruit the coactivators required for the transcriptional activation ( 42,43 ). We confi rmed that linalool induced the transactivation of PPAR ␣ in a reporter gene assay and that it stimulated the recruitment of PGC1 ␣ coactivator peptide due to conformational changes in PPAR ␣ via ligand binding in a TR-FRET assay. These fi ndings demonstrate that linalool directly interacts with the PPAR ␣ LBD and functions as an agonistic ligand. In this case, D -linalool bound more tightly to PPAR ␣ than L -linalool. The activities of the PPAR ␤ / ␦ and ␥ subtypes were not altered by linalool (data not shown). In cultured cell studies, a radiolabeled assay confi rmed that linalool promoted FA oxidation signifi cantly and regulated subsequent hepatic TG accumulation were ameliorated by linalool. The induction of Suclg2, a key regulatory enzyme of the TCA cycle, also suggested an enhanced oxidation of acetyl-CoA. Taken together, the data from the hepatic transcriptome profi le supported the hypotriglyceridemic effects of linalool .
Plasma metabolome analysis also suggested the hypotriglyceridemic effects of linalool, showing changes in the plasma FA metabolite levels of linalool-treated mice compared with controls. Notably, among the 13 FFAs analyzed, the concentrations of 11 were lower in the linalool group than in controls, corresponding to a signifi cant TG reduction in the plasma of linalool-supplemented mice. The association between serum TG levels and FA composition under metabolic disease conditions was previously reported in several human studies. Schwab et al. (2008) reported that a plasma TG reduction affected plasma FA metabolite levels in obese subjects following a dietary intervention. These authors showed that short-chain and saturated FAs were signifi cantly downregulated, which was associated with an improvement in insulin sensitivity ( 38 ). We also found that the plasma levels of saturated FA metabolites, such as lauric acid, palmitic acid, and myristic acid, but not stearic acid, were signifi cantly reduced in linalool-treated mice. Furthermore, interestingly, our data showed very similar changes in FA metabolite compositions (especially, C16 and C18) as those in previously reported studies investigating the hypolipidemic effects of phytochemical supplementation in animals ( 44,45 ). It has been reported that the decreased circulation of saturated FAs may be considered a benefi cial effect because an increased Nonbiased genomic analysis suggested that both linalool and fenofi brate partially normalized the hyperlipidemic hepatic transcriptome. The data verifi ed again that PPAR ␣ is a target molecule of linalool. Furthermore, global gene expression patterns showed that linalool rewired the aberrant hyperlipidemic hepatic transcriptome, shifting it toward that of control conditions. Pathway analysis indicated that linalool caused a signifi cant alteration in energy metabolism, inducing FA oxidation, while suppressing the FA synthesis pathways in line with the documented biological functions of PPAR ␣ activators and the results from our experiments. These results demonstrated that linalool ameliorated the hepatic TG metabolism by rewiring the transcriptome profi le from hyperlipidemic patterns toward those found in control hepatocytes. The results of the hepatic transcriptome profi ling revealed that linalool administration was associated with the regulation of FA and carbohydrate (glucose) metabolism gene expression. Linalool activated the expression of Acadvl, Echs1, Acox1, Acaa1, and Acadm, Hadh, and Fabp1, which are key genes in FA utilization. In addition, linalool suppressed the expression of several key lipogenic genes, including Srebp-1c, Acc1, and Elovl3. Collectively, these activities decreased the number of acyl-CoA substrates for TG synthesis. In glucose metabolism, the inhibition of Pk , Gck , and Pfkl expression in the liver is known to reduce the rates of glycolysis, acetyl-CoA formation, and lipogenesis under conditions of a high energy charge. Transcriptome profi ling indicated that reductions in the expressions of Pk as well as in other Gck and Pkfl were reduced in the linalool-stimulated group, suggesting the excessive glycolysis-dependent lipogenesis and infl ux of saturated FAs into peripheral tissues can lead to the accumulation of lipids in the tissues ( 46 ). In combination with transcriptional changes, it is thought that the PPAR ␣ -targeted mechanism and regulation of gene expression involved in FA production and oxidation, as suggested in hepatic transcriptome profi ling, could very strongly support the reduction of long-chain TGs in metabolite analysis. Therefore, metabolite profi ling demonstrated a signifi cant association between the enhancement of FA metabolite composition and the attenuation of hypotriglyceridemia by linalool treatment.
The results of the in vitro permeability of human intestinal Caco-2 cells implied that linalool is effi ciently absorbed in the human intestine (P app = 11.3 ± 2.1 × 10 Ϫ 6 cm/s). For example, the hypertension drug propranolol, which shows a similar permeability to that of linalool, has been reported to display a 90% absorption in humans ( 34 ). Consistently, linalool also showed a high availability in vivo, based on the Screening Information Dataset published by United Nations Environment Programme. That study reported that linalool is rapidly and completely absorbed from intestinal tract following oral uptake in rats using 14 C-labeled substances ( 16 ). Previously, it has been demonstrated that inhalation is able to deliver aromatic compounds to the blood via the absorption of the compound through the lungs. For example, inhalation experiments with several herbal essential oils showed a signifi cant delivery of aromatic compounds in the plasma, acting in a dose-dependent manner ( 47,48 ). Thus, the aromatic compounds in teas could possibly be delivered to the blood by both inhalation and intestinal uptake to circulate in the blood and to exert metabolic effects on the target tissues, including the liver. The antioxidant activity of linalool has been wellestablished in previous studies ( 17,18 ). We reported that linalool could protect against hypercholesterolemia induced by high-fat diet through the downregulation of hepatic SREBP2-mediated cholesterol synthesis. Together with these known bioactivities of linalool, the hypotriglyceridemic effects, which we proved in this study, are able to provide strength in the availability of linalool for the prevention and treatment of CVD. Linalool might contribute to the improvement of CVD through a signifi cant reduction of TG and cholesterol production and accumulation as a PPAR ␣ ligand and through the inhibition of lipid peroxidation and infl ammation via antioxidative activities, thereby leading to the attenuation of atherosclerosis.
In conclusion, we showed that linalool, a major and common aromatic compound contained in most herbal essential oils and teas, reduced plasma TG concentrations, had PPAR ␣ agonistic effects, and rewired the hepatic transcriptome (mimicking the effects of fenofi brate) and plasma metabolome. Our results suggest that the intake of herbs and teas containing linalool may contribute to the prevention and improvement of hypertriglyceridemia.