Hydroxyeicosapentaenoic acids from the Pacific krill show high ligand activities for PPARs.

PPARs regulate the expression of genes for energy metabolism in a ligand-dependent manner. PPARs can influence fatty acid oxidation, the level of circulating triglycerides, glucose uptake and insulin sensitivity. Here, we demonstrate that 5-hydroxyeicosapentaenoic acid (HEPE), 8-HEPE, 9-HEPE, 12-HEPE and 18-HEPE (hydroxylation products of EPA) obtained from methanol extracts of Pacific krill (Euphausia pacifica) can act as PPAR ligands. Two of these products, 8-HEPE and 9-HEPE, enhanced the transcription levels of GAL4-PPARs to a significantly greater extent than 5-HEPE, 12-HEPE, 18-HEPE, EPA, and EPA ethyl-ester. 8-HEPE also activated significantly higher transcription of GAL4-PPARα, GAL4-PPARγ, and GAL4-PPARδ than EPA at concentrations greater than 4, 64, and 64 μM, respectively. We also demonstrated that 8-HEPE increased the expression levels of genes regulated by PPARs in FaO, 3T3-F442A, and C2C12 cells. Furthermore, 8-HEPE enhanced adipogenesis and glucose uptake. By contrast, at the same concentrations, EPA showed weak or little effect, indicating that 8-HEPE was the more potent inducer of physiological effects.

PPARs are members of a nuclear receptor superfamily that play critical roles in the regulation of storage and catabolism of lipids ( 1 ). They contribute to these regulation processes by activating gene expression in a ligand-dependent manner that involves recognition of and binding to peroxisome proliferator response elements composed of TGACCT-related direct repeats separated by one nucleotide ( 2,3 ). PPARs form heterodimers with peroxisome proliferator response elements via the retinoid-X receptor, fraction was eluted with 100% methanol. HEPEs were separated on an InertSustain ODS-3 column (20.0 mm diameter × 250 mm; GL Science Inc.) with isocratic elution of acetonitrile/water/formic acid (55/45/0.1) at a fl ow rate of 15 ml/min and detection at 235 nm ( Fig. 2A ). The fraction that contained compounds 3 and 4 was subjected to recycle-HPLC, and peaks 3 and 4 were separated on an InertSustain ODS-3 column (20.0 mm diameter × 250 mm; GL Science Inc.) with isocratic elution of acetonitrile/ water/formic acid (60/40/0.1) at a fl ow rate of 15 ml/min and detection at 235 nm ( Fig. 2B ).

Quantitative real-time PCR
FaO, C2C12, and 3T3-F442A cells were seeded in 24-well culture plates (1 × 10 5 cells/well) and cultured overnight. 8-HEPE, EPA, GW7647, GW1929, or GW501516 was then added to the culture for 6 (FaO and C2C12) or 24 h (3T3-F442A). Total RNAs were extracted with an RNeasy kit (QIAGEN, Tokyo, Japan) and used to synthesize cDNAs using a PrimeScript RT reagent kit (Takara, Shiga, Japan); all kits were used according to the manufacturers' recommendations. Quantitative real-time PCR was performed with the gene-specifi c primers listed in supplementary Table I and Fast SYBR Green master mix (Applied Biosystems, Foster City, CA).
Increasing levels of triglycerides are associated with obesity and are indicators of progressive development of insulin resistance, hypertension, and hyperlipidemia ( 24,25 ). Thus, normalization of triglyceride levels has been investigated as a possible means of prevention for these conditions. It is well-known that dietary PUFAs can ameliorate some of the deleterious effects of these disorders ( 26,27 ). This property may refl ect both activation of PPAR-dependent ␤ -and -oxidation pathways ( 28 ), as well as PUFA-dependent suppression of lipogenic and glycolytic enzymes ( 29 ). Krill are a source of PUFAs, as their oil is rich in EPA and docosahexaenoic acid. In a similar manner to fi sh oil, krill oil has been shown to have anti-infl ammatory effects and to lower the levels of triglycerides and cholesterol in the plasma ( 30,31 ). Although lipids are major bioactive compounds in krill oil, we found in a previous study that a watersoluble extract from Pacifi c krill could suppress weight gain in mice caused by a high fat diet. The levels of triglycerides in the livers of mice given a water-soluble extract from Pacifi c krill decreased ( 32 ).

Extraction and isolation from Pacifi c krill
We used dried Pacifi c krill purchased from Kawashu (Iwate, Japan). The krill were powdered and then extracted with methanol under refl ux for 4 h. The methanol extract was diluted with distilled water (1:4 by volume) and subjected to column chromatography using Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan). A fraction was eluted with 80% methanol from the HP-20. This fraction was diluted with distilled water (1:3 by volume) and applied to an InertSep C18 (GL Science Inc., Tokyo, Japan). The
Here, we demonstrate that HEPEs from Pacifi c krill can act as PPAR ␣ activators ( Fig. 2 ; supplementary Figs. II, III). Moreover, through use of Diaion HP-20 column chromatography to increase concentrations, we found that their activities varied in a concentration-dependent manner stained with Oil Red O. Stained cells were examined using a Nikon microscope (×400). An adipogenesis assay kit (Bio Vision, Mountain View, CA) was used to measure triglyceride content.

2-Deoxyglucose uptake measurement
C2C12 cells were seeded in 24-well culture plates (2 × 10 4 cells/well) and cultured overnight. The medium was then replaced with DMEM containing 2% horse serum and antibiotic antimycotic solution and the cells were cultured for a further 5 days. Following this treatment to induce myogenesis, they were cultured in DMEM containing 8-HEPE, EPA, or GW501516 for 6 h. A 2-deoxyglucose uptake measurement kit (Cosmo Bio, Tokyo, Japan) was used to quantify glucose uptake into the C2C12 cells.

Statistical analysis
Statistically signifi cant differences between the experimental groups were identifi ed using one-way ANOVA and Tukey's post hoc tests. Data are shown as means ± SD.

8-HEPE induces adipogenesis in mouse preadipocyte cells
PPAR ␥ plays a central role in regulation of gene expression and differentiation in adipocytes ( 11 ). We therefore examined whether 8-HEPE could act as a PPAR ␥ ligand in (supplementary Fig. IIB). Measurements of HEPEs and EPA indicated that HP-20 chromatography was suffi cient to concentrate HEPEs (supplementary Table IV) and that InertSep C18 chromatography removed EPA. Methanol extract of Pacifi c krill contained 0.72 g/mg 8-HEPE, 0.24 g/mg 9-HEPE, and 7.1 g/mg EPA (supplementary Table IV). A cocktail that contained the same relative amounts of 8-HEPE and 9-HEPE, as in the methanol extract, showed about 60% of the activity of the methanol extract. However, addition of EPA to the cocktail did not increase activity (supplementary Fig. IV). These results indicate that 8-HEPE and 9-HEPE are the major PPAR ␣ activators in Pacifi c krill extracts.

8-HEPE increases expression of genes related to fatty acid oxidation in mitochondrial and peroxisomal pathways
PPAR ␣ regulates the expression of genes encoding enzymes and proteins responsible for fatty acid oxidation. To examine the activity of 8-HEPE as a PPAR ␣ ligand, we investigated whether 8-HEPE increased the expression of genes

8-HEPE increases Angptl4 expression and enhances glucose uptake in mouse myoblast cells
PPAR ␦ is the predominant isoform found in skeletal muscle; the agonist GW501516, which is PPAR ␦ -specifi c, increases the expression of angiopoietin-like protein 4 ( Angptl4 ) ( 34,35 ) and enhances glucose uptake ( 36 ) in C2C12 cells, a mouse myoblast cell line. Angptl4 is one of the major targets of PPAR ␦ , and Angptl4 is the gene that is most highly induced by long-chain fatty acids in human myotubes ( 34 ). Here, we examined the relative level of Angptl4 expression by realtime PCR. We cultured C2C12 cells with EPA 8-HEPE or GW501516 for 6 h and found that expression of Angptl4 was increased to a greater extent by 8-HEPE than EPA ( Fig. 7A ). GSK0660, a PPAR ␦ -specifi c antagonist, inhibited 8-HEPE-induced expression of Angptl4 ( Fig. 7A ). these cells. We cultured 3T3-F442A cells, a mouse preadipocyte cell line, with EPA, 8-HEPE, or GW1929, a PPAR ␥specifi c agonist, for 24 h. We found that expression of Fabp4 and Pparg was increased by 8-HEPE, but not by EPA ( Fig. 6A, B ). Both 8-HEPE and GW1929 increased the level of Fabp4 expression ( Fig. 6A ), but only 8-HEPE had an effect on Pparg expression ( Fig. 6B ). T0070907, a PPAR ␥specifi c antagonist, inhibited 8-HEPE-induced expression of Fabp4 ( Fig. 6A ).
Next, we cultured 3T3-F442A cells with 50 M EPA or 8-HEPE, or 1 M GW1929 for 7 days, and measured triglyceride accumulation to evaluate adipocyte differentiation. Compared with the control or EPA-treated cells, a greater level of triglyceride accumulation was observed in 8-HEPE-treated cells ( Fig. 6C-E ).  Values represent the mean ± SD from four independent cultures. a Signifi cant differences from the control are indicated by P < 0.01. b Signifi cant differences from EPA are indicated by P < 0.01. uptake was higher, albeit nonsignifi cantly, than that induced by EPA ( Fig. 6B ). These results indicate that 8-HEPE acts as a PPAR ␦ ligand. Overall, our analyses show that although 8-HEPE has a greater activity than EPA in upregulating We then measured glucose uptake in C2C12 cells that had been induced to undergo myotube differentiation for 5 days. 8-HEPE enhanced the uptake of glucose in these cells ( Fig. 6B ); the level of 8-HEPE-enhanced glucose  important for activation of PPAR ␣ in both HETEs and HEPEs. However, 8-HETE and 8-HEPE activities with regard to PPAR ␥ and PPAR ␦ have not previously been identifi ed ( 8,20 ). The likely reason is that the concentrations of 8-HETE and 8-HEPE tested in the experiments were too low to activate PPAR ␥ or PPAR ␦ . For example, earlier studies used 3 M 8-HETE and 1 M 8-HEPE ( 8,20 ), whereas, in the present study, activation of PPAR ␥ and PPAR ␦ was observed at concentrations greater than 5 M 8-HEPE ( Fig. 4 ). For PPAR ␣ and PPAR ␦ , 8-HEPE showed a greater effect than 8-HETE, while both 8-HETE and 8-HEPE were similarly active with regard to for PPAR ␥ ( Fig. 4 ). Previous studies did not compare the relative ligand activity of 8-HEPE and EPA for PPARs. As shown here, 8-HEPE had a greater ability than EPA to activate PPAR ␣ , PPAR ␥ , and PPAR ␦ at concentrations of more than 4 M, 64 M, and 64 M, respectively ( Table 1 ). The concentration of total fatty acids in serum is about 700 M ( 37 ), while serum concentrations of specifi c fatty acids that are abundant in Angptl4 expression, its effect on glucose uptake is comparable to EPA.

DISCUSSION
Our analyses here demonstrate that 8-HEPE and 9-HEPE are PPAR ligands and that their activities are greater than those of 5-HEPE, 12-HEPE, 18-HEPE, EPA, and EPA-Et ( Fig. 3 ). These results indicate that the position of hydroxylation on EPA is correlated with the strength of ligand activity for PPARs and that hydroxylation at the C-8 or C-9 positions produces more effective ligands. A similar phenomenon has been found in HETEs in which the position of hydroxylation infl uences ligand activity for PPAR ␣ ( 20 ). It was also reported that 8-HETE has a signifi cantly greater ability to activate PPAR ␣ compared with 5-HETE, 11-HETE, 12-HETE, and 15-HETE ( 20 ). This report and the present results show that hydroxylation at the C-8 position is properties of HEPEs, including 8-HEPE. It was shown that Escherichia coli hemolysin induced production of 8-HEPE in rabbit macrophages ( 48 ). However, no enzyme with the capability of transforming EPA into 8-HEPE has been identifi ed to date. In 8-HETE, 8(S)-lipoxygenase can transform AA into 8(S)-HETE ( 52 ), a compound that can stereoselectively activate PPAR ␣ ( 8,20 ). Future studies will be need to address the questions of 8-HEPE stereoselectivity and the identity of the enzyme that can transform EPA into 8-HEPE. Our study indicates that 8-HEPE and 9-HEPE induce fatty acid oxidation, adipogenesis, and glucose uptake via activation of PPARs in vivo. These metabolic responses are very interesting areas of research for the regulation of obesity and diabetes. The content of 8-HEPE in krill is more than 10 times higher than in the Japanese pilchard ( Sardinops melanostictus ) or the Pacific saury ( Cololabis saira ), species that are generally regarded as possessing high EPA contents (supplementary Table V). Our study shows that Pacifi c krill will be a valuable source of HEPEs for addressing the physiological functions of HEPEs.
the diet, such as linoleic acid and AA, generally fall into the range 25-30 M ( 8 ). Physiologically, most free fatty acids in serum are bound and buffered with albumin. The concentration of unbound free fatty acids is in the nanomolar range ( 38 ).
These results indicate that 8-HEPE acts as a ligand for intrinsic PPAR ␣ , PPAR ␥ , and PPAR ␦ .
Eicosanoids are a family of biologically active oxygenated derivatives of C20 PUFAs. Two pathways are principally involved in the production of eicosanoids. The lipoxygenase pathway transforms PUFAs into lipoxins, leukotrienes, and monohydroxy fatty acids, whereas the cyclooxygenase pathway produces prostaglandins and thromboxanes. Production of HEPEs has been observed in platelets ( 47 ), macrophages ( 48,49 ), and lung cells ( 50 ); and Tomio et al. ( 51 ) showed that 12-HEPE and 15-HEPE are decreased in 12/15-lipoxygenase knockout mice. In general, the physiological effects of HEPEs are unclear; however, it has been suggested that 12-HEPE and 15-HEPE have a suppressive effect on the development of endometriotic lesions ( 51 ). There have been comparatively few investigations of the biochemical or physiological