Metabolomics reveal 1-palmitoyl lysophosphatidylcholine production by peroxisome proliferator-activated receptor α.

PPARα is well known as a master regulator of lipid metabolism. PPARα activation enhances fatty acid oxidation and decreases the levels of circulating and cellular lipids in obese diabetic patients. Although PPARα target genes are widely known, little is known about the alteration of plasma and liver metabolites during PPARα activation. Here, we report that metabolome analysis-implicated upregulation of many plasma lysoGP species during bezafibrate (PPARα agonist) treatment. In particular, 1-palmitoyl lysophosphatidylcholine [LPC(16:0)] is increased by bezafibrate treatment in both plasma and liver. In mouse primary hepatocytes, the secretion of LPC(16:0) increased on PPARα activation, and this effect was attenuated by PPARα antagonist treatment. We demonstrated that Pla2g7 gene expression levels in the murine hepatocytes were increased by PPARα activation, and the secretion of LPC(16:0) was suppressed by Pla2g7 siRNA treatment. Interestingly, LPC(16:0) activates PPARα and induces the expression of PPARα target genes in hepatocytes. Furthermore, we showed that LPC(16:0) has the ability to recover glucose uptake in adipocytes induced insulin resistance. These results reveal that LPC(16:0) is induced by PPARα activation in hepatocytes; LPC(16:0) contributes to the upregulation of PPARα target genes in hepatocytes and the recovery of glucose uptake in insulin-resistant adipocytes.

randomly divided into four groups (n = 4/group) and fed SD (16%kcal fat) or HFD for 8 weeks. The mice were then fed SD, HFD, SD plus fenofi brate, and HFD plus fenofi brate (200 mg/kg body weight/day, oral administration) for 2 weeks. At the end of the treatment period, anesthetized mice were euthanized by cervical dislocation after overnight fasting, and blood and organs samples were collected. Plasma TG and glucose levels were determined by the TG E-test and glucose CII-test (Wako), respectively.

Extraction of mouse plasma, tissues, and cell culture sample
Mouse heparin-blood samples were centrifuged at 10,000 rpm for 10 min at 4°C. After centrifugation, plasma samples (5 µl) were dissolved in 95 µl of extraction solvent (80% methanol containing HMF as an internal standard). Mouse liver samples (10 mg) were homogenized in 1 ml of extraction solvent. The partial cell culture medium (300 µl/well) was evaporated to dryness and redissolved in 100 µl of extraction solvent. The hepatocyte extraction was washed with PBS and dissolved in extraction solvent (200 µl/well). The hepatocyte extraction was ultrasonically fragmented.

Metabolomic analysis by HPLC-Orbitrap MS
LC/MS for metabolomics was performed using an HPLC system (Agilent) coupled to an LTQ Orbitrap XL-MS system (Thermo Fisher Scientifi c Inc., San Jose, CA), equipped with an electrospray source operating in the positive-and negative-ion modes. The spray voltage and capillary temperature were 4 kV and 250°C, respectively. This analysis consists of two scan events. Scan Event 1 is full mass type (Analyzer; FTMS, Resolution; 60,000). Scan Event 2 is MS/MS type (Analyzer; Ion Trap MS, Act Type; collision-induced dissociation, Normalized Collision Energy; 35.0). An aliquot of the extracted sample (10 µl) was injected into an Inertsil ODS-4 reversed-phase column (column size, 3.0 × 250 mm; particle size, 3.0 µm; GL Sciences Inc., Tokyo, Japan). The column temperature was set at 30°C. Mobile phases A (0.1% formic acid) and B (acetonitrile including 0.1% formic acid) were used. The buffer gradient consisted of 30.0% to 90.0% B for 0 to 30 min, 95.0% B for 30.0 to 60.0 min, 95.0% to 30.0% B for 60.0 to 60.1 min, and 30.0% B for 24.9 min before the next injection, at a fl ow rate of 250 µl/min. These data were acquired with X-Calibur software (Thermo Fisher Scientifi c Inc.) and Power-Get software (Kazusa DNA Research Institute, Japan) using a previously described methods ( 22 ; http://www.kazusa.or.jp/komics/ software/PowerGet). Briefl y, PowerGet is a Java software package for detection, alignment, and annotation of metabolite features from data obtained using LC/high-resolution MS. The peak area was divided by the area of the internal standard. This value was used to calculate the rate of change for the control group. Differences between groups were compared with the Student's t -test.

Quantifi cation of lysoGPs by ultraperformance liquid chromatography quadrupole time-of-fl ight mass spectrometry (UPLC-QTOF-MS)
LC/MS for target metabolites analysis was performed using a Acquity UPLC system coupled to a Xevo QTOF-MS system (Waters, Milford, MA), equipped with an electrospray source in the positive-ion mode operating with a lock-spray interface for accurate mass measurement. Leucine enkephalin was used as the lockmass compound. It was infused straight into the MS system at a fl ow rate of 20 µl/min at 200 pg/µl (in 50% acetonitrile, 0.1% slows the progression of focal coronary atherosclerosis, and reduces the number of coronary events in young survivors of myocardial infarction ( 17 ). It is interesting to note that fi brates, including bezafi brate, contribute to the improvement of both dyslipidemia and glucose metabolism disorder (18)(19)(20). Although PPAR ␣ target genes are widely known, little is known about the variation of metabolites in plasma and liver during the activation of these genes.
Over the past decade, the use of metabolomic techniques, including liquid chromatography coupled with ultraprecise mass spectrometry, to investigate animal or plant metabolism has increased dramatically ( 21,22 ). These techniques provide molecular weight data with the precision of a few ppm, permitting the determination of chemical formulae. Metabolite investigation based on precise molecular weight data enables the large-scale analysis of animal metabolite dynamics during treatment with the PPAR ␣ agonist. The application of metabolomics is useful for monitoring the metabolism and helpful for identifying novel PPAR ␣ agonist-responsive pathways.
The aim of the present study is to identify the metabolite that is regulated by PPAR ␣ and to clarify its function. In the present study, metabolomics approach revealed that 1-palmitoyl lysophosphatidylcholine [LPC(16:0)] is induced by PPAR ␣ activation in the liver. Furthermore, LPC(16:0) activates PPAR ␣ in the hepatocytes and stimulates glucose uptake in insulin-resistant 3T3-L1 adipocytes. To the best of our knowledge, this is the fi rst report stating that LPC(16:0) induces PPAR ␣ activation and is capable of improving dyslipidemia and hyperglycemia.

Animal experiments
All the animal experiments were approved by the Kyoto University Animal Care Committee. The mice were kept in individual cages in a temperature-controlled room at 23 ± 1°C and maintained under a constant 12 h light/dark cycle. Male KK-Ay mice were purchased from CLEA Japan (Tokyo, Japan). The 4-weekold mice were maintained for 7 days on a standard diet (SD) and then divided into two groups of similar average body weights. Each group was maintained on a 60%kcal high-fat diet (HFD) and HFD containing 0.2% (w/w) bezafi brate for 4 weeks. The energy intake of all the mice was adjusted by pair feeding. Male C57BL/6J mice were purchased from CLEA Japan. Mice were The medium was replaced with serum-free DMEM for 18 h before glucose-uptake experiments. RAW264.7 macrophage (RAW) cell lines were cultured in a growth medium, DMEM with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO 2 . RAW cells were seeded on 12-well plates at a cell density of 3.0 × 10 5 cells/well for 24 h in growth medium and then serumfree DMEM containing 0.5% BSA for 24 h. The medium was used as RAW-conditioned medium (RAW-CM).

Quantifi cation of mRNA expression levels
Total RNA was prepared from primary hepatocytes, as well as liver and skeletal muscle, using Sepasol (Nacalai Tesque Inc., Kyoto, Japan), according to the manufacturer's protocols. Using M-MLV reverse transcriptase (Life Technologies Japan Ltd.), total RNA was reverse-transcribed using a thermal cycler (Takara PCR Thermal Cycler SP; Takara Bio Inc., Shiga, Japan). To determine mRNA expression levels, real-time quantitative RT-PCR analysis was performed with a Light Cycler System (Roche Diagnostics) using SYBR green fl uorescence signals, as described previously ( 25,26 ). The oligonucleotide primer sets of mouse 36B4 and PPARa target genes were designed using a PCR primer selection program at the website of the Virtual Genomic Center from the GenBank database as follows: mouse Cpt1a (Fwd: mouse Pla 2 g12b (Fwd: 5 ′ -ATCAAGGTACCAGGAAGTATGGAC-3 ′ ; Rev: 5 ′ -TCATAGCTCTTCTTTCTCCTCCTC-3 ′ ), and mouse 36B4 as an internal control (Fwd: 5 ′ -TCCTTCTTCCAGGCTTTGGG-3 ′ ; Rev: 5 ′ -GACACCCTCCAGAAAGCGAG-3 ′ ). All data indicating mRNA expression levels are presented as a ratio relative to a control in each experiment.

Induction of insulin resistance
Two different insulin-resistant adipocyte models were established as previously described with modifi cations ( 27,28 ). One of these used TNF-␣ . On the sixth day after induction, the fully differentiated 3T3-L1 adipocytes were treated with serum-free DMEM containing 10 ng/ml recombinant mouse TNF-␣ (Peprotech, Rocky Hill, NJ) for 18 h. In another method used, RAW-CM 3T3-L1 adipocytes were incubated with control medium (basal medium of serum-free DMEM containing 0.5% BSA) or basal medium conditioned by RAW-CM for 18 h. The 3T3-L1 adipocytes exposed to TNF-␣ or RAW-CM became insulin resistant, as assessed by the ability of insulin to stimulate glucose uptake. The level of uptake of 2-deoxy-D - [1,[2][3] H]glucose ([1,2-3H]-2DG) was measured, as previously described ( 29 ).

Luciferase assay
Luciferase assays were performed as previously described, using a GAL4/PPAR chimera system ( 25,30 ). We transfected p4x-UASg-tk-luc (a reporter plasmid), pM-hPPARa (an expression plasmid for a chimera protein for the GAL4 DNA-binding domain and each human PPAR-ligand-binding domain), and pRL-CMV (an internal control for normalizing transfection effi ciency) into monkey CV1 kidney cells by using Lipofectamine (Life Technologies Japan Ltd.), according to the manufacturer's protocol. Luciferase activity was assayed using the dual luciferase system (Promega, MO) according to the manufacturer's protocol. formic acid). During lysoGP analysis, the capillary, sampling cone, and extraction cone voltages were set at 3,500, 35, and 3.0 V, respectively. The source and desolvation temperatures were 120°C and 450°C, respectively. The cone and desolvation gas fl ow rates were set at 50 l/h and 800 l/h, respectively. Scan event is MS mode (scan range: 100-1,000 Da; scan time: 0.2 s). An aliquot of the extracted sample (3 µl) was injected into an Acquity UPLC BEH-C18 reversed-phase column (column size, 2.1 × 100 mm; particle size, 1.7 µm). Mobile phases A and B were used. The column temperature was set at 40°C. The buffer gradient of lysoGP analysis consisted of 5.0% to 99.0% B for 0 to 10 min, 99.0% B for 10 to 15 min, 99.0% to 5.0% B for 15 to 15.5 min, and 5.0% B for 4.5 min before the next injection, at a fl ow rate of 300 µl/min. These data were acquired with the MassLynx software (Waters). The amount of LPC(16:0) was estimated from calibration curves obtained using analytical-grade standard compound. The peak area of m/z [M-H] ± 0.05 Da was divided by the area of the internal standard. This value was used to generate the calibration curves.

Preparation of mouse primary hepatocytes
Mouse hepatocytes were prepared as previously described ( 23 ). Briefl y, C57BL/6J male mice (wild type and PPAR ␣ Ϫ / Ϫ ) were anesthetized with intraperitoneal administration of pentobarbital, and the liver was perfused with liver perfusion medium (Life Technologies Japan Ltd., Tokyo, Japan), followed by liver digestion medium (Life Technologies Japan Ltd.). After fi ltration through a 100 mm nylon mesh fi lter, hepatocytes were isolated by repeated centrifugation at 50 g for 3 min (three times). The isolated hepatocytes were cultured in type-1 collagen-coated 12-well plates at a cell density of 2.0 × 10 5 cells/well. After 5 h incubation at 37°C in 5% CO 2 atmosphere, hepatocytes were cultured in serum-free DMEM with or without bezafi brate, fenofi brate, GW7647, or GW6471 for 24 h. The hepatocytes were used for mRNA quantifi cation and LC/MS assay.

siRNA experiments
Mouse siRNA was chemically synthesized by Qiagen (Tokyo, Japan). Negative control siRNA (Block-it NC siRNA) and lipofectamine 2000 were purchased from Life Technologies Japan Ltd. The primary hepatocytes were seeded in 12-well plates and transfected with 40 nmol/well synthesized siRNA targeting mouse Pla 2 g7 . The primary hepatocytes were transfected with siRNAlipofectamine complexes and incubated for 12 h at 37°C in 5% CO 2 atmosphere and then used in the experiments.

Cell culture
FAO cell lines from rat liver were cultured in growth medium, DMEM with 5% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO 2 . FAO cells were seeded on 12-well plates at a cell density of 2.0 × 10 5 cells/well for 24 h in growth medium and then serum-free DMEM with or without bezafi brate for 24 h.
The 3T3-L1 cells were cultured, maintained, and differentiated using a previously described method ( 24 ). Briefl y, 3T3-L1 murine preadipocytes were cultured in a growth medium, DMEM supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO 2 . At 2 days after reaching confl uence, the cells were incubated in a differentiation medium containing 0.25 M dexamethasone, 1 µM troglitazone, 10 g/ml insulin, and 0.5 mM IBMX for 48 h, and then in a medium containing 1 µM troglitazone and 5 g/ml insulin for another 2 days. On the fourth day, the medium was replaced with the growth medium containing insulin (5 g/ml).
suggest that LPC(16:0) in plasma was increased by bezafibrate treatment.

Secretion of LPC(16:0) was increased by PPAR ␣ activation in liver
It is well known that PPAR ␣ is highly expressed in liver and skeletal muscle ( 10,11 ). To elucidate the source of plasma LPC(16:0) in mice treated with bezafi brate, we quantifi ed LPC(16:0) in these tissues.
Quantitative analysis of LPC(16:0) in control mice tissue revealed that the high concentration of LPC(16:0) was

Statistical analyses
Data are presented as the mean ± SEM. Differences between groups were compared with the Student's t -test (for two groups), Pearson correlation coeffi cient, and one-way ANOVA, followed by least-signifi cant multiple comparison methods. Values of P < 0.05 were considered statistically signifi cant.

LPC in plasma was increased by PPAR ␣ activation
We confi rmed that plasma TG was decreased (to ‫ف‬ 70% vs. control; Table 1 ), and the expression of PPAR ␣ target genes in the liver was increased by bezafi brate treatment for 4 weeks ( Table 1 ). These data suggest that bezafi brate treatment for 4 weeks was suffi cient to activate hepatic PPAR ␣ in KK-Ay mice.
To identify the metabolites that were infl uenced by PPAR ␣ activation, we investigated the plasma metabolites profi le in mice treated with bezafi brate for 4 weeks by metabolome analysis based on LC/MS and by using metabolite databases, including the Kyoto Encyclopedia of Genes and Genomes and Lipid Maps. We detected 887 peaks of metabolites in mice plasma (data not shown). Metabolome analysis was used to elucidate the patterns of plasma metabolic change in the control versus bezafi brate-fed mice. Differences between these groups were compared with the statistical analysis. This analysis revealed that 27 metabolites including lysoGP species infl uenced by PPAR ␣ activation ( Fig. 1A ; supplementary Table 1). Metabolome analysis showed that LPC(16:0) was one of the metabolites that was predicted to be upregulated by PPAR ␣ activation (C 24 H 50 N 1 O 7 P 1 ; Fig. 1A ; supplementary Table 1). A previous study reported that LPC(16:0) increased adipocyte glucose uptake ( 31 ). We confi rmed that plasma glucose was decreased (to ‫ف‬ 60% vs. control) by bezafi brate treatment ( Table 1 ).
To further elucidate the mechanism of regulation of LPC(16:0) production, we analyzed the content of LPC(16:0) in primary hepatocytes treated with bezafi brate for 24 h. The content of LPC(16:0) in primary hepatocytes and culture medium increased in a dose-dependent manner ( Fig. 3A , B ). The concentration of LPC(16:0) in primary hepatocyte medium increased with GW7647 and fenofi brate (PPAR ␣ -specifi c agonist, respectively) in a dose-dependent manner ( Fig. 3C, D ). We also confi rmed this effect in FAO rat hepatocytes treated with bezafi brate ( Fig. 3E ). Furthermore, the increase in LPC(16:0) secretion in primary hepatocytes treated with bezafi brate was diminished by GW6471 (PPAR ␣ antagonist) treatment ( Fig. 3F ). These fi ndings show that production and secretion of LPC(16:0) were induced by PPAR ␣ activation in hepatocytes.

Pla 2 g7 gene expression levels in the hepatocytes were increased by PPAR ␣ activation, and the secretion of LPC(16:0) was suppressed by Pla 2 g7 siRNA treatment
LPC(16:0) is derived from phosphatidylcholine because of the action of phospholipase A 2 (PLA 2 ), which has many subtypes. To determine the main PLA 2 subtype contributing to LPC(16:0) production in hepatocytes treated with bezafi brate, we analyzed the mRNA expression of Pla 2 subtype in the liver. We determined that Pla 2 g6 , Pla 2 g7 , and Pla 2 g12b were mainly expressed in the liver and that the mRNA expression of Pla 2 g7 was markedly elevated (to ‫ف‬ 1,500% of the control) by bezafi brate treatment ( Fig.  4A ). The mRNA expression of Pla 2 g7 levels in mice liver under SD and HFD eating conditions were also increased (to ‫ف‬ 7,500% and 5,000% of the control group, respectively) by fenofi brate treatment ( Fig. 4B ). The mRNA expression of Pla 2 g7 was also increased by PPAR ␣ activation in murine primary hepatocytes, and the increase in mRNA expression of Pla 2 g7 was diminished by GW6471 treatment  ( Fig. 4C ). These fi ndings show that PPAR ␣ is involved in the expression level of Pla 2 g7 .

LPC(16:0) activates PPAR ␣ and induces mRNA expressions of PPAR ␣ target genes in hepatocytes
To investigate the effect of Pla 2 g7 on PPAR ␣ target genes expression, we analyzed the expression of PPAR ␣ target genes in murine primary hepatocytes treated with control siRNA or Pla 2 g7 siRNA. We confi rmed that control siRNA transfection has no effect on the expression of Pla 2 g7 (supplementary Fig. 2). The mRNA expressions of Aco and Cpt1a were attenuated by Pla 2 g7 siRNA treatment (to ‫ف‬ 60% and 70% vs. control siRNA group, respectively, P < 0.05; Fig. 5B, C ). In murine primary hepatocytes treated with bezafi brate and Pla 2 g7 siRNA, we also observed that upregulated expression of PPAR ␣ target gene mRNA induced by bezafi brate treatment was suppressed on Pla 2 g7 siRNA treatment ( Fig. 5E, F ).

LPC(16:0) stimulates glucose uptake in insulin-resistant 3T3-L1 adipocytes
It is well recognized that LPC(16:0) increases adipocyte glucose uptake ( 31 ). We also confi rmed the LPC(16:0) capacity to promote glucose uptake in 3T3-L1 adipocytes in a dose-dependent manner and LPC(16:0) had an additive effect increasing glucose uptake in 3T3-L1 adipocytes cotreated with insulin and LPC(16:0) (data not shown). We hypothesized that LPC(16:0) has the ability to promote glucose uptake under conditions of insulin resistance. To elucidate the function of LPC(16:0), we investigated whether it stimulates glucose uptake in insulin-resistant adipocytes. We revealed that LPC(16:0) has the ability to recover glucose uptake in insulin-resistant adipocytes, which were induced by TNF-␣ or RAW-CM ( Fig. 8A , B ). Furthermore, we analyzed a correlation between the concentration of plasma glucose and LPC(16:0) in mice treated with or without bezafi brate for 4 weeks. The results showed strong correlation between plasma glucose and LPC(16:0) ( r = Ϫ 0.8335, P = 0.0027; Fig. 8C ). These fi ndings suggest that LPC(16:0) is capable of recovering glucose uptake in insulin-resistant adipocytes, which raises the possibility that it contributes to the decrease in the plasma glucose level.

DISCUSSION
In this study, metabolome analysis revealed that many types of plasma lysoGPs were increased by PPAR ␣ activation. LysoGPs have been found in a wide range of tissues and cell types ( 33 ). It has also been reported that lysoGPs play an important role in many physiological and pathophysiological processes (33)(34)(35)(36). LysoGPs are well known to be lipid mediators (autacoid) that have topical effects on cells. However, our data showed that lysoGPs have not only topical but also whole-body effects throughout the bloodstream. LPC(16:0) was one of the lysoGPs that was increased by PPAR ␣ activation in the plasma. Previous studies have demonstrated that LPC(16:0) is a major component of lysoGPs in plasma and activates glucose uptake in adipose tissue ( 31,37 ). Therefore, we focused here on LPC(16:0) and investigated the relationship between LPC(16:0) and PPAR ␣ activation.
Liver tissue is important in lipid metabolism. Although the lipidomics approach revealed in detail the hepatic lipid profi ling including that of TGs, phospholipids, and cholesteryl esters ( 21,38 ), little is known about the relationship between PPAR ␣ activation and LPC(16:0) production. In this study, we demonstrated that the activation of PPAR ␣ induced the upregulation of many types of lysoGP including LPC(16:0) production. Quantitative analysis of LPC(16:0) showed that LPC(16:0) was markedly increased in the liver, which expresses PPAR ␣ to a great extent. In the murine primary hepatocytes, secretion of LPC(16:0) was induced by PPAR ␣ agonist (bezafi brate, fenofi brate, and GW7647) treatment and suppressed by PPAR ␣ antagonist. These fi ndings suggest that synthesis of LPC(16:0) in hepatocytes is induced by PPAR ␣ activation and that secretion of LPC(16:0) from hepatocytes contributes to elevated plasma LPC(16:0) concentration. Our metabolomic data also showed that not only LPC(16:0) but also many other lysoGPs were changed by bezafi brate treatment. In particular, lysophosphatidylethanolamine (LPE) (20:4), LPC(18:2), LPC(20:4), LPE(16:0), LPE(18:1), and LPC(18:1) were changed both plasma and liver. These fi ndings suggest that the source of these lysoGPs in plasma is liver.
LPCs are mainly generated from PLA 2 -catalyzed hydrolysis of phosphatidylcholine ( 37,39 ). The various subtypes of PLA 2 , which have different localizations, individually regulate synthesis of LPC ( 37 ). In the present study, we showed that the mRNA expression of Pla 2 g7 in murine liver or primary hepatocytes was elevated by PPAR ␣ activation. Furthermore, we also showed that LPC(16:0) secretion in hepatocytes was attenuated by Pla 2 g7 siRNA treatment. We demonstrated for the fi rst time that LPC(16:0) is induced by PPAR ␣ activation via Pla 2 g7 -dependent pathway in the liver. Interestingly, the present study revealed that the expression of PPAR ␣ target genes was suppressed by Pla 2 g7 siRNA treatment and that LPC(16:0) is not only induced by PPAR ␣ activation but also has the ability to activate PPAR ␣ and upregulates the mRNA expression of PPAR ␣ target genes. These fi ndings raise the possibility is metabolized in cells. These findings raise the possibility that LPC(16:0) activates PPAR ␣ both directly and indirectly.
Adipose tissue plays a key role in glucose homeostasis ( 40 ). A preceding study has indicated that LPC(16:0) stimulates glucose uptake in 3T3-L1 adipocytes ( 31 ). It is well recognized that obesity is characterized by chronic infl ammation of adipose tissues and this infl ammation contributes to the development of insulin resistance and type 2 diabetes ( 41,42 ). Notably, macrophages contribute signifi cantly to infl ammation in adipose tissue ( 43 ). TNF-␣ is the major proinfl ammatory cytokine ( 44 ). Previous studies have reported that TNF-␣ , which is secreted by macrophages, induces additional infi ltration of macrophages into adipose tissues (44)(45)(46)(47). LPC(16:0) can increase glucose uptake in an insulin-independent manner ( 31 ). We hypothesize that LPC(16:0) has an ability to promote glucose uptake under conditions of insulin resistance. We revealed that LPC(16:0) has the ability to improve glucose uptake in insulin-resistant adipocytes induced by RAW-CM or TNF-␣ . We demonstrated for the fi rst time that the ability of LPC(16:0) to induce glucose uptake is unaffected by of positive feedback regulation of PPAR ␣ and Pla 2 g7 . The analysis of LPC(16:0) concentration in the medium and cell-associated LPC(16:0) at 24 h after addition of LPC(16:0) suggested that part of LPC(16:0) entry into cells. However, there is also the possibility that LPC(16:0)  the previous studies showed that plasma glucose level is reduced by fenofi brate ( 55 ) or Wy-14,643 [PPAR ␣ specifi c activator, ( 56 )] treatment. The results of the previous studies support our fi ndings. These data suggested that LPC(16:0) produced in liver by PPAR ␣ activation participates in the regulation of plasma glucose level.
In conclusion, metabolomics has revealed the upregulated LPC(16:0) in mice plasma and liver following bezafi brate treatment. PPAR ␣ activation induces the expression of Pla2g7 and secretion of LPC(16:0) from the hepatocytes. Furthermore, LPC(16:0) contributes to activate PPAR ␣ and induces the expression of PPAR ␣ target genes in hepatocytes. We also showed that LPC(16:0) has the ability to recover glucose uptake in insulin-resistant adipocytes. The data presented herein suggested that LPC(16:0) induced by PPAR ␣ activation improved dyslipidemia and hyperglycemia.

Note added in proof
The authors Rieko Nakata and Hiroyasu Inoue were inadvertently left out of the author list of the accepted version of this article. All other authors and the Journal's Editors-in-Chief approved the addition after the article was in proof stage. Drs. Nakata and Inoue will appear as authors in all forms of the article except in the originally accepted Paper in Press.
insulin resistance. This effect raises the possibility that LPC(16:0) contributes to ameliorating hyperglycemia. Many previous studies indicate that the levels of LPCs and infl ammation have crucial relevance. LPCs have been demonstrated to have both proinfl ammatory ( 48,49 ) and anti-infl ammatory ( 35,50 ) effects. Therefore, additional work is required to completely determine the relationship between LPCs and infl ammation.
It is interesting to note that fi brates, including bezafibrate, contribute to the improvement of not only dyslipidemia but also glucose metabolism disorder (18)(19)(20). However, the molecular basis of this effect remains unexplained. Excessive free fatty acids, particularly stearic acid (SA) and palmitic acid (PA), are also proinfl ammatory factors and induce insulin resistance ( 51,52 ). Bezafi brate greatly facilitates lipid metabolism, thereby anticipating the reduction of SA or PA. However, our previous study showed that bezafi brate has no apparent effect on plasma SA and PA ( 53 ). The evidence suggested that SA or PA were not involved in the improvement of hyperglycemia in KK-Ay mice treated with bezafi brate. We hypothesize that the ability of LPC(16:0) to increase glucose uptake in adipocytes improves hyperglycemia during treatment with bezafi brate. To validate this hypothesis, we analyzed a correlation between plasma LPC(16:0) and plasma glucose in mice treated with or without bezafi brate for 4 weeks. The result showed strong correlation between plasma glucose and LPC(16:0). The previous study has provided evidence that plasma LPC levels are reduced in obesity and type 2 diabetes ( 54 ). Recently, administration of LPC(16:0) has been reported to reduce plasma glucose ( 31 ). We also confi rmed that intraperitoneal injection of LPC(16:0) in mice induced reduction of plasma glucose (data not shown). Furthermore,