Regulation of inflammatory and lipid metabolism genes by eicosapentaenoic acid-rich oil.

Omega-3-PUFAs, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are associated with prevention of various aspects of metabolic syndrome. In the present studies, the effects of oil rich in EPA on gene expression and activation of nuclear receptors was examined and compared with other ω3-PUFAs. The EPA-rich oil (EO) altered the expression of FA metabolism genes in THP-1 cells, including stearoyl CoA desaturase (SCD) and FA desaturase-1 and -2 (FASDS1 and -2). Other ω3-PUFAs resulted in a similar gene expression response for a subset of genes involved in lipid metabolism and inflammation. In reporter assays, EO activated human peroxisome proliferator-activated receptor α (PPARα) and PPARβ/γ with minimal effects on PPARγ, liver X receptor, retinoid X receptor, farnesoid X receptor, and retinoid acid receptor γ (RARγ); these effects were similar to that observed for purified EPA. When serum from a 6 week clinical intervention with dietary supplements containing olive oil (control), DHA, or two levels of EPA were applied to THP-1 cells, the expression of SCD and FADS2 decreased in the cells treated with serum from the ω3-PUFA-supplemented individuals. Taken together, these studies indicate regulation of gene expression by EO that is consistent with treating aspects of dyslipidemia and inflammation.

oil (EO, triglyceride form) used in this study is a proprietary product manufactured under contract and was provided by the study sponsor, DuPont Applied Biosciences, Wilmington, DE. DuPont has developed an oleaginous yeast that produces an oil rich in EPA at 35% of FA content and linoleic acid at 20% of content and which is low in all other FAs (<7%).

Preparation of BSA-conjugated FAs
Due to the aqueous insolubility, all free FAs were conjugated to FA-free BSA (BSA) for treatments (molar ratio of 4:1 FA:BSA). The EO was saponifi ed, and FAs were extracted as described previously (13) . Molecular weight of EO was determined by average of the components and estimated at 290 g/mol. FAs were weighed and dissolved in ethanol as a stock concentration of 0.5 M. A total of 32 l stock solution was transferred to a brown glass vial and dried under argon, whereas an equal volume of ethanol was dried in another vial as a vehicle control. A total of 132 l of 0.15 M KOH was added to both vials, vortexed, and incubated for 1 h at 70°C under argon. Following the incubation, 2 ml of fi ltersterilized BSA (2 mM) in PBS was added to the FAs and the vehicle control to achieve a fi nal concentration of 8 mM. The pH was adjusted to 7.2 to 7.4. The BSA-conjugated FA and its BSA control were stored at Ϫ 20°C under argon until use.

Cell culture and treatments
THP-1 ( Homo sapiens monocyte) cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and cultured in RPMI 1640 with 10% heat-inactivated FBS, 50 M 2-mercaptoethanol, 1 mM sodium pyruvate, and antibiotics. These cells were seeded in 24-well plates at a density of 3 × 10 5 /well and differentiated into macrophages with 100 nmol/l phorbol 12-myristate 13-acetate (Sigma) for 48 h. For in vitro treatment experiments, THP-1 cells were grown to 75% confl uency and treated with 0, 1, 10, and 100 µM of the EPA-enriched oil or FAs as BSA conjugates. Eighteen hours after treatment, the cells were stimulated with 10 ng/ml LPS for 6 h. For ex vivo experiments, THP-1 cells were cultured as described above and treated with 10% (v/v) human serum from individual subjects for 18 h, after which the cells were stimulated with LPS for 6 h.

RNA extraction, reverse transcription, and real-time PCR
Total RNA was isolated using a Qiagen RNeasy Mini Kit (Qiagen; Valencia, CA) according to the manufacturer's instructions. The total RNA was reverse transcribed using the ABI high-capacity cDNA archive kit (Applied Biosystems; Foster City, CA). Standard curves were made using serial dilutions from pooled cDNA samples. Real-time PCR was performed with the use of the SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol and amplifi ed on the ABI Prism 7000 sequence detection system. Detailed information on primer sequences is provided in Supplementary Materials and Methods.

Microarray experiments and statistical analysis
THP-1 cells were treated with EO at three concentrations (1, 10, and 100 M) or control (BSA) for 16 h, followed by stimulation with LPS for 6 h. RNA was extracted using Qiagen RNeasy, and quality was assessed by RNA Nano Chips on the Agilent Bioanalyzer. Each sample was labeled using the Affymetrix IVT Express Kit according to the manufacturer's protocol. The GeneChip Human Genome U133A 2.0 (Affymterix), representing 14,500 well-characterized genes, was hybridized with the labeled RNA using GeneChip Hybridization Wash and Stain Kit (#702232) in the Affymetrix GeneChip Hybridization Oven 640, according to the manufacturer's instructions. Following hybridization the arrays were washed and stained using the Affymetrix thrombosis, and improving endothelial function. As noted in Hu and Willett ( 3 ), several studies have shown association of fi sh intake and/or fl axseed oil (high in ALA) with decreased fatalities from CVD. Importantly, blood levels of EPA and DHA are strongly associated with decreased risk of death, myocardial infarction, and stroke ( 3 ).
In addition to playing a major role in CVD, chronic infl ammation is a contributor to many human diseases. Omega-3 PUFAs play an important role in the regulation of infl ammation by decreasing the production of infl ammatory eicosanoids, cytokines, and reactive oxygen species and the expression of adhesion molecules ( 4,5 ). EPA and DHA supplementation has proven effective in decreasing intestinal damage and improving gut histology in infl ammatory bowel disease ( 6,7 ). Fish oil supplementation decreases joint pain, number of tender and swollen joints, duration of morning stiffness and, as a result, use of nonsteroidal anti-infl ammatory drugs ( 8 ).
Proteins of the nuclear receptor (NR) superfamily act as intracellular transcription factors that directly regulate gene expression in response to lipophilic molecules (9)(10)(11)(12). They affect a wide variety of functions, including FA metabolism, infl ammatory responses, cancer, reproductive development, and detoxifi cation of foreign substances. Several NRs respond to dietary lipids and include the FA receptors peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), retinoid X receptors (RXRs), and farnesoid X receptors (FXRs). This particular subset of proteins may be considered constituents of a large group of NRs, the "metabolic nuclear receptors" that act as overall sensors of metabolic intermediates, xenobiotics, and compounds in the diet and allow cells to respond to environmental changes by inducing the appropriate metabolic genes and pathways ( 9,12 ). The goals of the present study were to use multiple approaches to examine the effects of -3 PUFAs on gene expression in macrophages and to extrapolate these fi ndings to a clinical study examining a dietary supplement enriched in EPA. Altered gene expression by several 3-PUFAs was examined in a commonly employed human macrophage cell line (differentiated THP-1 cells), followed by an infl ammatory challenge [lipopolysaccharide (LPS) stimulation]. In addition, nuclear receptor activity profi ling was employed to compare potential molecular targets of n3-PUFAs. Together, these studies show differences in gene expression patterns and NR specifi city among the 3-PUFAs. Also, genes particularly sensitive to 3-PUFA were identifi ed that could aid in examining the clinical effectiveness of dietary supplements.

Chemicals
ALA, steariodonic acid (SDA), EPA, docosapentaenoic acid (DPA), and DHA were purchased from Sigma-Aldrich (St. Louis, MO). FBS was purchased from HyClone (Logan, UT). Geneticin was purchased from Invitrogen (Grand Island, NY). The EPA-rich with serum obtained from the clinical study diluted in media (10% v/v) ( 13 ) for 16 h, followed by treatment with LPS for 6 h. RNA was extracted, and quantitative PCR was performed as described above.

Specialized laboratory measures
Using plasma aliquots, frozen at Ϫ 80°C and never thawed, obtained at the baseline and at 6 week visits, we carried out a wide variety of biochemical assays. Total cholesterol, triglyceride, and HDL cholesterol were measured as previously described ( 15 ). Direct LDL cholesterol and small, dense LDL cholesterol levels were measured using kits obtained from Denka-Seiken Corporation, Tokyo, Japan ( 16 ). Triglyceride-rich lipoprotein cholesterol was calculated by subtraction of direct LDL and HDL cholesterol values from total cholesterol. Analysis of the FA profi le of total serum phospholipids was determined at Nutrasource Diagnostics, Guelph, Canada in a blinded fashion as described ( 17,18 ) Serum was subjected to Folch extraction, and phospholipids were separated by thin-layer chromatography. FA methyl esters were prepared from the phospholipid fraction and measured on a Varian 3400 gas-liquid chromatograph using a 60 meter DB-23 capillary column (0.32 mm internal diameter).

Statistical analysis
One-way ANOVA, followed by Dunnett's posthoc test, was used to test the difference between treatments ( P < .05). The values were expressed as mean ± SEM. All data analyses were performed by JMP 7.0 (SAS Institute; Cary, NC) and data were plotted by Prism 5.01 (GraphPad Software, Inc.; San Diego, CA).

Comprehensive analysis of gene expression
The design of the present experiments was aimed to understand the anti-infl ammatory effects of 3-PUFA oil and to identify sensitive biomarkers for subsequent studies. The THP-1 monocytes were treated with EO for 16 h, followed by stimulation with LPS, and the RNA was used to examine gene expression via high-density microarray. A total of 798 genes were signifi cantly regulated (corrected P value 0.01) by EO in a dose-dependent manner, as assessed by one-way ANOVA, of which 58 were 2-fold different than control at a minimum of one dose of EO ( Table 1 , Fig. 1 ) . The dose-dependent regulation of gene expression followed three predominant trends: genes that are increased at the highest dose of EO (i.e., PLAU, TGF ␤ 1, COL10A); genes that are decreased at the highest dose of EO [i.e., stearoyl CoA desaturase 1 (SCD-1), CYP51A, IDI1, ACAT2]; and genes that are decreased at low doses but increased at high doses of EO (i.e., ILF3, SLC16A3) ( Fig. 1 ). The predominant function of EO-regulated genes was steroid, sterol, and lipid isoprenoid biosynthesis and metabolism (see supplementary Table II ). This subset of genes was examined for common transcriptional regulators using Ingenuity Pathway Analysis (IPA 9.0, Ingenuity Systems, Inc.; Redwood City, CA), as shown in supplementary Fig. I . Several nuclear receptors, including the PPARs and the LXRs, as well as the nuclear factor B complex, were implicated in the gene expression patterns observed.
GeneChip Fluidics Station 450 according to the manufacturer's protocol and scanned using the GeneChip Scanner 3000 7G. The scanned image fi le (DAT) and the intensity data (CEL) were imported into GeneSpring 10.0 (Agilent Technologies). The Robust Multi-array Average was used to normalize the data (22,277 entities) and was fi ltered on expression (>20% percentile in at least 1 of 12 samples, 18,497 entities). The 12 slides were grouped based on dose (four doses; 0, 1, 10, 100 M, n = 3 per dose) and one-way ANOVA with asymptotic P value, and Benjamini-Hochberg multiple corrections was performed. At a P value of 0.01, a total of 798 entities were signifi cantly regulated, of which 58 exhibited expression differences of 2-fold when compared with the control group. The group of genes was examined by hierarchical clustering using complete linkage analysis of the normalized data (JMP 7.0, SAS Institute; Cary, NC).

Effects of omega-3 FA supplementation, ex vivo
Serum samples for ex vivo studies were collected from subjects enrolled in a double-blinded, placebo-controlled, parallel-design study of 121 subjects randomized into four treatment groups. There were 26, 27, 29, and 28 completers in the placebo olive oil group, low-dose EPA group (600 mg/day), high-dose EPA group (1,800 mg/day), and DHA group (600 mg/day), respectively. Of the completing subjects, the mean age was 52 years, 67% were male, 33% were female (all postmenopausal), 70% were white, 26% were black, 3% were Asian, and 1% were Hispanic. The mean body mass index of the group was 27.4 kg/m 2 .The human protocol was approved by Schulman Associates Institutional Review Board, Cincinnati, OH. This study was registered at Clini-calTrials.gov as NCT00988585, and was given the identifi cation number DuPont-0609. Study capsules were manufactured under contract and were provided by the study sponsor, DuPont Applied Biosciences, Wilmington, DE. DuPont has developed an oleaginous yeast that produces an oil rich in EPA at 38% of FA content and linoleic acid at 20% of content and which is low in all other FAs (<7%). At the time of the enrollment visit, all qualifying and consenting subjects were randomly allocated into a protocol, where they were required to take two 1 g capsules three times daily, which contained a total of either: 1 ) 6 g/day of olive oil placebo, 2 ) 600 mg/day of EPA/day and 4.42 g/day of olive oil, 3 ) 1,800 mg of EPA/day and 1.26 g/day of olive oil, and 4 ) 600 mg of DHA/day. The olive oil placebo, the low-dose EPA oil, the high-dose EPA oil, and the DHA oil supplements, in total milligrams, contained:  supplementary Table II for additional details). Over the entire 42 days, the study subjects were expected to have consumed a total of 252 capsules. Compliance was calculated as a percentage of consumed capsule count/expected capsule count based on the number of days the subject was in the study.
At the time of the enrollment visit, all qualifying and consenting subjects were randomly allocated into the four treatment groups described above. Subjects were asked to fast for 12 h prior to blood draws that occurred prior to intervention (baseline) and after 6 weeks of consuming the capsules. Serum was prepared and frozen until examined. THP-1 cells were cultured and treated the lowest dose (1 µM). Transcripts for ABCG1, HMCGR, CYP51, FADS1, PLAU, and TGF ␤ 2 showed intermediate sensitivity, with signifi cant alterations in quantity starting at 10 µM EO.

Structure-activity relationships
The genes regulated by EO were compared with that of an equal dose (100 µM) of the -3 PUFAs ALA, DHA, DPA, EPA, and SDA. THP-1 cells were treated with the FAs and subsequently stimulated with LPS or control. Figure 3 depicts the effects of -3 PUFA treatment on the LPS-stimulated cells. The repression of gene expression for SCD-1, ABCG1, HMGCR, CYP51, and FADS1 and -2 was similar Quantitative real-time PCR was used to confi rm a subset of transcripts identifi ed by the microarray experiments. Care was taken to choose genes that were both increased and decreased by EO treatment, as well as those with known biological functions. In addition, because the desire was to fi nd sensitive biomarkers of EO response genes that were signifi cantly affected at the 1 M EO in the microarray experiment were of particular interest. As shown in Fig. 2 , all genes studied were signifi cantly regulated by EPA oil, albeit to a different extent and with varying potency. Some mRNAs, such as ADRP and SLC16A, were induced only at the highest concentration of EO (100 µM), whereas SCD-1 and FADS2 were signifi cantly repressed at other -3 PUFAs, whereas TGF ␤ 1 mRNA was increased by EO, DPA, and EPA treatment. The effects of EO (1, 10, and 100 µM) and -3 PUFAs at 100 µM, with or without LPS stimulation of gene expression, are shown in supplementary Fig. I . In general, the alterations in gene expression were more evident in the LPS-treated THP-1 cells, due in part to higher variability in the unstimulated cells.

Transcription factor profi ling
The subset of genes regulated by EO in the microarray experiments was examined for common transcriptional regulators using Ingenuity Pathway Analysis (see supplementary Fig. II ). Several NRs were identifi ed as being transcriptional regulators of EO-responsive genes, including LXR ␣ and -␤ (NR1H3 and 2), glucocorticoid receptor, NR3C1, thyroid hormone receptor ␤ (TR ␤ ), PPAR ␣ and -␥ (NR1C1 and -3), and estrogen receptor (ER) ␣ (NR3B1). These ligand-activated transcription factors and several others identifi ed or speculated to be FA receptors were examined in whole-cell receptor assays for regulation by EO. Signifi cant dose-response regulation by EO of the PPARs ( ␣ , ␤ / ␦ , and ␥ ), RXR ␤ , farnesoid X receptor (FXR), and retinoic acid receptor ␥ (RAR ␥ ) was seen ( Fig. 4 ). In contrast, ER ␣ , ER ␤ , LXR ␣ , LXR ␤ , TR ␣ , vitamin D receptor (VDR), and constitutive androstane receptor, variant 3 (CAR3) activity was not affected by EPA oil treatment (data not shown). PPAR ␣ (10-fold) and PPAR ␤ / ␦ (13-fold) were the most activated by EO, followed by RXR ␣ (6-fold), RAR ␥ (3-fold), and FXR (2-fold). The dose-response activation of these six nuclear receptors was examined for -3 PUFAs (see supplementary Fig. III ), and the activation in comparison to EO at the 100 µM concentration is shown in Fig. 5 . PPAR ␣ was responsive to all FAs examined with activation of approximately 10-fold. The largest response observed was for EPA activation of PPAR ␤ / ␦ (35-fold). PPAR ␥ showed a distinct preference for DHA and DPA relative to the other FAs. EO was the most effi cacious activator of FXR, albeit only a 2-fold activation.

Effects of -3 PUFAs on gene expression ex vivo
The hypothesis tested herein was that bioactive molecules are present in human serum following supplementation among the FAs and EO in that all treatments signifi cantly decreased mRNA amounts. Similarly, PLAU mRNA was induced by EO, and the FAs were examined. ADRP mRNA was increased by DHA, and DPA and was not affected by  Table 1 and Fig. 1 for validation by real-time PCR. Cells were treated, and RNA was extracted as described in Methods. Gene expression following treatment with EO (0, 1, 10, 100 M) is expressed relative to a housekeeping gene (GAPDH) and normalized to vehicle control (DMSO). Asterisks denote signifi cantly different from control ( P < 0.05, n = 3). Bars represent mean and SEM.  supplementary Table IV  and supplementary Fig. IV for more details). As shown in Fig. 7 , SCD-1 and FADS2 mRNA alterations as well as IL6 and IL1 mRNA changes were signifi cantly correlated. In addition, the ratio of AA to EPA correlated with IL6 mRNA, and serum DHA affected SCD-1 mRNA expression ex vivo.
with EPA that affects gene expression. Studies performed in vitro indicated that FADS2 and SCD-1 are sensitive to EO and individual 3-PUFAs, and those were chosen for these studies along with two infl ammatory genes [interleukin 1 ␣ (IL1 ␣ ) and IL6] that are affected in vitro and in vivo by FAs. Serum from baseline and following 6 weeks of dietary supplementation was used as a treatment medium (10% v/v) for LPS-stimulated THP-1 cells. As shown in Fig. 6 , the general trend is for a decreased expression of IL1 ␣ , IL6, FADS2, and SCD-1 mRNA in THP-1 cells treated with serum obtained from the 3-PUFA-supplemented individuals compared with the olive oil-administered group; the differences were signifi cant for serum from the DHAtreated individuals.

Alteration in serum FA profi le and correlation to ex vivo gene expression
The effects of supplementation with EPA and DHA on serum FA profi le is shown in Table 2 . The placebo control had no effect on absolute or relative 3-PUFA levels. The lower dose of EPA resulted in signifi cant increases in total -3 and 3/ 6 and EPA and decreased AA/EPA ratio. When the dose of EPA was increased, in addition to the previously observed effects, the total AA and the SCD-18 index [ratio of 18:1(n-9)/18:0] decreased. DHA administration increased total 3, 3/ 6, and DHA while  genes and were decreased upon treatment. This pattern is more reminiscent of the response of adipocytes ( 30 ) than of hepatocytes to 3-PUFA treatment.
FA elongation and desaturation are two key metabolic routes for the synthesis of saturated, monounsaturated, and polyunsaturated FAs. Of these, FA desaturases have received considerable attention for their regulation by hormones and nutrients and their capacity to generate specifi c unsaturated FAs. One of these enzymes, SCD-1, or delta 9 desaturase , have emerged as key enzymes in the control of whole-body lipid composition ( 31 ). Although oleate is found ubiquitously throughout the body, endogenously derived oleate from SCD is special in terms of its preferential traffi cking through acyl-CoA:diacylglycerol acyltransferase 2 and driving triglyceride (TG) synthesis. Omega-3 FAs decrease SCD-1 mRNA expression in liver, and this effect is correlated with decreased circulating TG

DISCUSSION
Animal experiments and human studies have shown that 3-PUFAs regulate genes in various tissues, including adipose tissue and peripheral blood mononuclear cells (PBMCs). There have been several comprehensive analyses of transcription responses to 3-PUFAs, including in PBMC following fi sh oil supplementation ( 19 ), in adipose tissue following a high-PUFA diet in humans ( 20 ) and mice ( 21 ), in breast cancer cell lines treated with EPA and DHA ( 22 ), and in colon cancer cells treated with DHA ( 23 ), to name a few. In the present studies, the model system was biased to examine the anti-infl ammatory responses of 3-PUFAs by using a human monocytic cell line challenged with LPS. Despite this choice of model, many of the genes regulated by EO were involved in cholesterol, sterol, and lipid metabolism. This is consistent with clinical observations whereby the predominant effect of 3-PUFA dietary supplementation is the lowering of circulating triglyceride levels (24)(25)(26)(27). In fact, the lipid-lowering effect is often seen in the absence of an anti-infl ammatory response ( 28 ). In general, the favorable effects of 3-PUFAs mainly result from the combined effects of decreased expression of lipogenesis-related genes and stimulation of FA oxidation transcripts. Fish oil decreases expression of sterol response element binding protein-1c ( 29 ), a key enzyme in controlling lipogenesis. Other lipogenic genes are decreased by 3-PUFAs, including FA synthase, malic enzyme, and glucose-6-phosphate dehydrogenase ( 11 ). In the liver, several genes involved in FA metabolism are increased by PUFAs, including apolipoproteins A-I and A-II A, acyl-CoA synthetase, acyl-CoA oxidase, liver FA-binding protein, carnitine palmitoyl transferase, and cytochrome P450 4A1 ( 11 ). Although the genomic response to EO in THP-1 cells showed a preponderance of lipid metabolism genes, the pathways affected were generally lipogenic    ( 32 ). In addition, decreased SCD-1 expression in macrophages by ALA is associated with increased cholesterol effl ux ( 33 ), a benefi cial response in terms of reverse cholesterol transport. Delta-5 desaturase [D5D, FA desaturase 2 (FADS1)] and delta-6 desaturase (D5D, FADS2) are the key enzymes for the synthesis of PUFAs such as AA and DHA. Elovl-1 (Ssc1) and Elovl-6 (LCE, FACE, rElo2) elongate saturated and monounsaturated FAs. Omega-3 PUFAs decease the expression of D6D and D5D (decreased levels of PUFAs increases their expression) as well as Elovl-6; however, the conversion of ALA to longer chain PUFAs is regulated by substrate levels to a greater extent than to expression of the synthetic enzymes ( 34 ). Nonetheless, the repression of FADS1 and FADS2 as well as SCD-1 in macrophages is among the most-sensitive biomarkers for EO response. The sensitivity of FADS2 and SCD-1 to EO treatment in vitro made them likely choices for examination in the ex vivo experiments. In addition, the infl ammatory markers IL1 and IL6 are typically decreased by 3-PUFA treatment in vitro and in vivo ( 28,34 ). In these studies, serum from individuals given various supplements were used as a treatment to THP-1 cells; this approach has been used previously by our laboratory and others as a means to assess alterations in circulating bioactive molecules following a treatment or diet ( 14, 35 -39 ). The expression of these genes would be expected to be lower following administration of serum to THP-1 cells from the 6-week treatment with EO compared with serum taken at baseline. In fact, there is a dose-dependent trend for decreased ex vivo expression of FADS2 and SCD-1, although it only reached statistical signifi cance in the DHA group. Individuals' ex vivo SCD-1 and FADS2 responses are signifi cantly correlated, as are the IL1 and IL6 responses. In contrast, the FADS2 and SCD-1 responses were not indicative of changes in interleukin expression (see supplementary Table IV). This may be refl ective of similar mechanisms of gene expression for SCD-1 and FADS2 that differ from those of IL1 and 6. Of note is the fact the one's serum DHA change (post treatment/pre-treatment) is inversely associated with the ex vivo SCD-1 mRNA (post/pre). Similarly, the change in ratio of AA to EPA in the serum is correlated with the ex vivo expression of IL1 and IL6. Principal component analysis of the individual post/pretreatment values for the FA concentration as well as the ex vivo gene expression showed a similar observation with AA/EPA being more associated with IL1 and IL6 mRNA, whereas AA/DHA clustered with SCD-1 and FADS2, as well as the SCD-16 and -18 ratios (see supplementary Fig. IV ). Together, these data show that altering DHA and EPA concentrations may impact different endpoints preferentially.
Often the biological responses of the predominant 3-PUFAs, ALA, EPA, and DHA, are considered to be equivalent or interchangeable, although it is generally held that the marine-based FAs are more benefi cial than their plantbased counterparts ( 5,38 ). In the present studies, the altered gene expression of a small subset of genes ( Fig. 3 ) suggests that they do, in fact, lead to similar genomics responses ids, hydroxyepoxyeicosatrienoic acids (HETEs) ( 45 ), leukotriene B 4 ( 46 ), and prostaglandin D 2 (PGD 2 ) ( 47 ). The least-studied member of the PPAR family, PPAR ␤ / ␦ , was preferentially activated by EPA and showed the highest level of activation of any NR by EO. ALA was the weakest activator of PPAR ␤ / ␦ among the 3-PUFAs, with induction similar to the 6-PUFA linoleic acid (not shown). Prostaglandin A1 (PGA1), PGD2, and PGD1 can activate PPAR ␤ / ␦ in reporter assays ( 47 ). 15-HETE and the toxic lipid 4-hydroxynonenol 4-HNE are also PPAR ␤ / ␦ activators ( 48 ). PPAR ␥ has received considerable attention as a target of anti-diabetic and -infl ammatory drugs. This NR was only marginally affected by EO and 3-PUFAs, with the noted exception of DPA and DHA. Interestingly, the 5-lipoxygenase metabolite of DHA (4-hydroxy DHA) ( 49 ) and the COX-2 metabolites electrophilic oxo-derivatives ( 50 ), are more-potent PPAR ␥ activators than the parent FA and may be responsible for effects of this 3-PUFA on angiogenesis and infl ammation. Less-distinctive structureactivity relationships were noted for RXR ␣ , a previously noted DHA receptor ( 51 ), and RAR ␥ , a heretofore-unidentifi ed FA receptor. The differences in gene expression and transcription factor activation among the members of the 3-PUFA family can explain some of the differences in potency of biological and therapeutic responses and can point to specifi c recommendations for the individual FAs. For example, EPA may be of more benefi t for diseases with a PPAR ␤ / ␦ etiology, whereas DHA is more-amenable for PPAR ␥ -related therapies. This is especially important because genetic modifi cation has been used to develop a new generation of plants (e.g, corn and soy-beans) that produce seeds with a modifi ed FA profi le for use as dietary supplements with specifi c health benefi ts in mind.
Taken together, these studies have shown that EO alters gene expression in a human monocytic cell line, consistent with altered lipid metabolism and infl ammation. This particular dietary supplement contrasts with in vitro. However, more-comprehensive analysis of gene expression following treatment with 3-PUFAs shows that there are subsets of genes that are affected by particular FA structures, in particular at higher doses (Vanden Heuvel, unpublished observations). This may be the result of differential metabolism of ALA, EPA, and DHA to various bioactive molecules and/or interaction with specifi c transcription factors, enzymes, and receptors. In addition, as mentioned above, the ex vivo studies suggest that serum from individuals on the EO supplements differed from that from DHA-supplemented cohorts, further illustrating that these two 3-PUFAs are similar but are not fully interchangeable.
Of the several identifi ed FA receptors, perhaps the family that can best explain the effects of 3-PUFAs are the PPARs. The PPARs exist as three subtypes ( ␣ , ␤ , and ␥ ) that vary in expression, ligand recognition, and biological function. PPAR ␣ was the fi rst transcription factor identifi ed as a prospective FA receptor (as reviewed in Refs. [39][40][41] and is involved in FA transport, FA binding proteins, fatty acyl CoA synthesis, microsomal, peroxisomal and mitochondrial FA oxidation, ketogenesis, and FA desaturation. PPAR ␤ / ␦ is ubiquitously expressed and is often found in higher abundance than PPAR ␣ or ␥ . Examination of PPAR ␤ / ␦ -null mice has shown a role for this NR in development, myelination of the corpus callosum, lipid metabolism, and epidermal cell proliferation ( 42 ). PPAR ␥ is expressed in many tissues, including adipose, muscle, vascular cells, macrophages, and epithelial cells of the mammary gland, prostate, and colon (as reviewed in Ref. 43 ). Activated PPAR ␥ induces LPL and FA transporters (CD36), and enhances adipocyte differentiation, as well as inhibits cytokine and cylcooxygenase 2 (COX-2) expression. The EPA-enriched oil and individual 3-PUFAs activated PPAR ␣ to a similar extent, with little distinction. In fact, this NR is activated by 6-PUFAs ( 44 ), and metabolites of FAs, including epoxyeicosatrienoic ac- others on the market that are rich in other 3-PUFAs, such as DHA, SDA, and ALA. Most of the responses observed for each 3-PUFA supplement are similar and have benefi cial properties against many aspects of metabolic syndrome. However, due in part to specifi city of nuclear receptor activation as well as differential metabolism, each 3-PUFA and corresponding supplement or diet must be considered a unique entity. Evidence is provided that EOs may preferentially affect PPAR ␤ / ␦ -associated therapies, whereas DHA and other 3-PUFA supplementation have their own subset of benefi ts. Hence, additional research is warranted to provide recommendations of 3-PUFA supplements for specifi c outcomes.