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J. Biol. Chem., Vol. 277, Issue 42, 39243-39250, October 18, 2002

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Fatty Acid Regulation of Liver X Receptors (LXR) and Peroxisome Proliferator-activated Receptor  (PPAR) in HEK293 Cells^*

Anjali Pawar^§, Jinghua Xu^§, Erik Jerks, David J. Mangelsdorf^¶, and Donald B. Jump

From the  Departments of Physiology, Biochemistry, and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 and the ^¶ Department of Pharmacology and Howard Hughes Medical Institute, University of Texas, Southwestern Medical Center, Dallas, Texas 75390-9050

Received for publication, June 20, 2002, and in revised form, August 2, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Fatty acids bind to and regulate the activity of peroxisome proliferator-activated (PPAR) and liver X receptors (LXR). However, the role lipid metabolism plays in the control of intracellular fatty acid ligands is poorly understood. We have identified two strains of HEK293 cells that display differences in fatty acid regulation of nuclear receptors. Using full-length and Gal4-LBD chimeric receptors in functional assays, 20:4,n6 induced PPAR activity ~2.2-fold and suppressed LXR activity by 80% (ED₅₀ ~25-50 µM) in HEK293-E (early passage) cells but had no effect on PPAR or LXR receptor activity in HEK293-L (late passage) cells. LXR was insensitive to fatty acid regulation in both HEK293 strains. Metabolic labeling studies using [¹⁴C]20:4,n6 (at 100 µM) indicated that the uptake of 20:4,n6 and its assimilation into triacylglycerol, diacylglycerol, and polar lipids revealed no difference between the two strains. Such treatment increased total cellular 20:4,n6 (~11-fold) and its elongation product, 22:4,n6 (~3.6-fold), within 6 h. Non-esterified 20:4,n6 and 22:4,n6 represented 3% of the total cellular 20:4,n6 and 22:4,n6. In HEK293-E cells, non-esterified 20:4,n6 and 22:4,n6 increased 8- and 18-fold, respectively, by 6 h and was sustained at that level for 24 h. In HEK293-L cells, non-esterified 20:4,n6 also increased (5-fold) at 6 h but fell by 70% within 24 h. In contrast to HEK293-E cells, non-esterified 22:4,n6 did not accumulate in HEK293-L cells. Functional assays showed that 22:4,n6 was ~2-fold more effective than 20:4,n6 at inhibiting oxysterol-induced LXR activity in HEK293-E cells, but had no effect on LXR activity in HEK293-L cells. Taken together, these findings demonstrate that the rate of assimilation of exogenously added fatty acids and their metabolites into complex lipids plays an important role in regulating PPAR and LXR activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Several nuclear receptors have been identified as targets for regulation by fatty acids or their metabolites. These include the peroxisome proliferator-activated receptor family (PPAR¹ (NR1C1), PPAR (NR1C2), PPAR (NR1C3)), liver X receptors (LXR (NR1H3) and LXR (NR1H2)), RXR, and HNF-4 (1). The best characterized are members of the PPAR family. All PPARs bind 20-carbon polyunsaturated fatty acids, e.g. 20:4,n6 and 20:5,n3, with an apparent K_d of ~1-4 µM (2). As class II nuclear receptors, PPARs heterodimerize with RXR and bind direct repeats (PPREs, a DR1, rat AOX PPRE: CCGAACGTGACCTNTGTCCT) in promoters of regulated genes. PPARs play a major role in whole body lipid metabolism, inflammatory, and immune responses (3).

Liver X receptors (LXR and LXR) are also class II nuclear receptors that bind direct repeats (LXRE, a DR-4, murine Cyp7A1: TGGTCActcaAGTTCA) as a heterodimer with RXR (4). Oxysterols, like 22(R)-hydroxycholesterol or 24,25-epoxycholesterol, bind and activate LXRs (5-7). LXR/RXR heterodimers bind LXREs in promoters of enzymes involved in hepatic bile acid synthesis, e.g. 7-hydroxylase (Cyp7A), the main route for cholesterol elimination from the body. LXRs also regulate lipogenic gene expression through two mechanisms either by controlling the expression of SREBP-1c or by direct binding to promoters of certain lipogenic genes, e.g. fatty acid synthase (5, 7). In contrast to PPARs, unsaturated fatty acids act as antagonist to oxysterol activation of LXR in HEK293 and hepatoma cell lines (6). The hierarchy for the fatty acid effect on LXR is 20:4,n6 > 18:2,n6 > 18:1,n9; saturated fatty acids have no effect. The half-maximal effect for 20:4,n6 antagonism of oxysterol binding to the LXR-ligand binding domain is ~1.5 µM. This level of fatty acid is comparable to the K_d for 20-carbon PUFA binding to PPARs (3).

Based on in vitro binding and cell culture studies both PPARs and LXR have emerged as prospective monitors of intracellular NEFA levels, and in liver these receptors would respond accordingly by altering metabolism to prevent lipid and cholesterol overload. However, there remains little information that documents changes in intracellular NEFA levels in cells or how lipid metabolism might contribute to the regulation of NEFA levels and influence nuclear receptor activity. During the course of our studies, we observed that the fatty acid regulation of PPAR and LXR in HEK293 cells was strain-dependent. This difference in fatty acid responsiveness provided us with an opportunity to identify prospective metabolic pathways that impact intracellular NEFA levels and affect nuclear receptor activity. Our studies show that while both cell types share the same metabolic pathways for 20:4,n6 assimilation onto complex lipids, there are clear quantitative differences. These differences contribute to the level of non-esterified PUFA in cells that, in turn, affects LXR and PPAR activity. Using the HEK293 cell model has provided support for the notion that both LXR and PPAR are indeed sensors of intracellular NEFA.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cells-- Two strains of HEK293 identified as HEK293-E (early passage) and HEK293-L (late passage) were obtained from K. Olson and K. Gallo at the Department of Physiology at Michigan State University. Cells were maintained in DMEM/F12 (Invitrogen) with 7.5% fetal calf serum (Intergen, Purchase, NY) in plastic tissue culture vessels (Falcon; 80- or 50-mm culture dishes or 6-well plates). Cells were grown to confluence and then incubated overnight in serum-free media before fatty acid treatment.

Plasmids-- CMX-hLXR, CMX-hLXR, CMX-Gal4-hLXR, CMX-Gal4-hLXR, TK-LXREx3-Luc, TK-MH100X4-Luc, and SG5-rPPAR were previously described (8, 9). pM-rPPAR-LBD was constructed using the Matchmaker kit (CLONTECH) fusing the PPAR-LBD to the Gal4-DBD. The primers used for this construction are: sense, ⁸⁷³GGG ATG TCA CAC AAT GCA ATC CGT and antisense, ¹⁷⁶¹TCA GTA CAT GTC TCT GTA GAT CTC. phRG-Luc was obtained from Promega and serves as an internal control for transfection efficiency. All plasmids were cesium-purified before transfection.

Transfection-- Cells were grown to confluence in 6-well culture plates. Cells in each well receive 1 µg of reporter plasmid and 1 µg of receptor expression plasmid. Cells were transfected using Lipofectin (6.6 µl/µg DNA) in serum-free media. After an overnight transfection period, cells were treated with fatty acids or drugs for 24 h in serum-free media containing bovine serum albumin (BSA). The BSA was adjusted to maintain a 5:1 molar ratio of fatty acid to BSA. LXRs were activated with 22(R)-hydroxycholesterol (Research Plus, Inc.), Manasquan, at 20 µM. The vehicle for 22(R)-hydroxycholesterol is ethanol. After treatment, cells were lysed and assayed for firefly and Renilla luciferase activity using the dual luciferase assay kit (Promega) with a dual channel Turner Luminometer. Protein was measured using the Bio-Rad Protein reagent with BSA as standard. All experiments were run in triplicate and repeated at least one time.

Fatty Acid Metabolism-- Cells were grown to confluence in 50-mm plastic dishes in DMEM/F12 plus 7.5% fetal calf serum. Prior to fatty acid treatment cells were incubated overnight in serum-free media. Cells were treated with [¹⁴C]20:4,n6 (3 ml of DMEM/F12 containing 100 µM 20:4,n6, 0.5 µCi, (1.7 Ci/mol)). ¹⁴C-labeled fatty acids were purchased from PerkinElmer Life Sciences and non-radioactive fatty acids were purchased from Nu-Chek Prep (Elysian, MN). Fatty acid-free BSA (Roche Molecular Biochemicals) is added to 20 µM. Cells were treated with fatty acids for 1.5, 6, and 24 h. At harvest, media was collected, cells were washed one time with phosphate-buffered saline + 20 µM BSA, then washed one time with PBS, and resuspended in 500 µl of 40% methanol. The methanol extract was acidified with HCl to 0.75 N, and lipids were extracted with chloroform:methanol (2:1) containing 1 mM butylated hydroxytoluene (10). Protein and aqueous phases were re-extracted with chloroform + 1 mM BHT. The organic phases were pooled, dried under nitrogen, resuspended in chloroform + 1 mM BHT, and stored at -80 °C. Lipids are separated by thin layer chromatography (LK6D Silica G 60A, Whatman) in hexane:diethyl ether:acetic acid (90:30:1). Distribution and quantifying ¹⁴C used a Molecular Dynamics PhosphorImager 820. Location of lipids was compared with authentic standards for triacylglycerol (TAG), diacylglycerol (DAG), cholesterol esters (CE), fatty acids, fatty acid (wax) esters (Sigma), and glycerol- and sphingo-phospholipids (Avanti Polar Lipids). Uptake of ¹⁴C into the organic fraction was quantified by liquid scintillation counting. Depletion of ¹⁴C-labeled fatty acids from media was quantified by scintillation counting and thin layer chromatography followed by PhosphorImager analysis as described above.

Reverse Phase-High Pressure Liquid Chromatography (RP-HPLC) Analysis of PUFA-- Confluent HEK293 cells in 80-mm plastic dishes were treated with 100 µM fatty acid (non-radioactive) as described above. Afterward, cells were recovered in 40% methanol and [¹⁴C]16:0 was added (0.2 µCi/plate) as a recovery standard. Total lipid was extracted as described above. Total lipids were saponified (0.5 N KOH in 80% methanol, 1 h at 50 °C.), neutralized, extracted in diethyl ether, dried, and resuspended in methanol + 0.1 mM BHT for RP-HPLC analysis (reverse phase C18 column, Symmetry Shield, 2487 UV detector set to 192 nm with a 600 Controller; Waters Corp.). A linear gradient of 10% acetonitrile + 0.1% acetic acid to 100% acetonitrile + 0.1% acetic acid over 40 min was used to fractionate unsaturated fatty acids. Verification and quantification of unsaturated fatty acids by RP-HPLC used authentic fatty acid standards (Nu-Chek Prep) and Win-flow Radio HPLC software for windows from IN/US Systems, Inc., Tampa, FL. The identity of specific fatty acids was verified by gas chromatography-mass spectrometry at the mass spectroscopy facility at Michigan State University.

The NEFA fraction in total cellular lipids was fractionated on amino-propyl columns [Alltech, Inc] (11). Lipid extracts in chloroform + 1 mM BHT were applied to a 0.1-ml amino-propyl column and washed extensively with chloroform:isopropanol (2:1) to remove neutral lipids. NEFA were eluted with diethyl ether + 2% acetic acid. Phospholipids were retained on the column. The diethyl ether-2% acetic acid fraction was dried under nitrogen, resuspended in methanol + 0.1 mM BHT, and used directly for RP-HPLC fractionation and quantification of unsaturated fatty acids. The fractional recovery of NEFA from whole cells extracts was >95%.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Fatty Acid Regulation of LXR, LXR, and PPAR in HEK293 Cells-- PPAR is a well established target for fatty acid regulation (1-3). However, LXR has only recently been identified as a target for fatty acid regulation (6). In Figs. 1-4, we characterized the fatty acid regulation of oxysterol activation of LXR and LXR and fatty acid regulation of PPAR in two strains of HEK293 cells. These studies will show that these two strains of HEK293 cells give very different results for fatty acid effects on LXR and PPAR activity. The two strains of HEK293 cells differ in passage number; HEK293-E are early passage cells and HEK293-L are late passage cells. HEK293-L cells grow faster than HEK293-E with a doubling time of ~1 day versus 2 days, respectively. Moreover, HEK293-L cells grow to a higher density (3.8 × 10⁶ cells/well) than HEK293-E (2.8 × 10⁶ cells/well). However, all transfections and fatty acid metabolism studies described below were carried out at full confluence in serum-free media when cell growth was minimized.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Fatty acid effects on oxysterol induction of LXR activity in HEK293-E cells. A, confluent HEK293-E cells were transfected with expression vectors containing full-length hLXR and hLXR. The reporter plasmid was TK-LXREx3-Luc, and the internal control for transfection efficiency was phRG-Luc. Mean ± S.D., n > 9. B, confluent HEK293-E cells were transfected with expression vectors containing LXR-LBD fused to the Gal4 DBD: CMX-Gal4-hLXR, CMX-Gal4-hLXR. The reporter plasmid was TK-MH100X4-Luc, which contains four copies of the Gal4 binding motif. After an overnight transfection, cells were treated without or with 22(R)-hydroxycholesterol and without and with fatty acids (16:0, 18:1,n9, 20:4,n6) at 100 µM for 24 h. Cells were lysed and assayed for firefly and Renilla luciferase activity and protein. Mean ± S.D.; n = 3. C, cells were transfected as in B, and after an overnight transfection, cells were treated without or with 20 µM 22(R)-hydroxycholesterol and without and with fatty acids (20:4,n6, 18:3,n3, 20:5,n3, and 22:6,n3) at 100 µM for 24 h. Mean ± S.D.; n > 6. These results are representative of at least two separate studies.

Fatty acid regulation of nuclear receptors was evaluated by transfection analysis using either full-length receptors or Gal4-LBD chimeric receptors. Accordingly, cells were transfected with the LXRE-TK-Luc reporter plasmid and expression vectors containing full-length LXR or LXR (Fig. 1A). The LXR ligand, 22(R)-hydroxycholesterol induced a 6- and 2-fold increase in LUC activity in HEK293-E cells transfected with full-length LXR and LXR, respectively. 22(R)-hydroxycholesterol had no effect on LUC activity when LXR expression vectors were omitted from the transfection mixture. Treatment of cells with 100 µM 16:0 had no effect on either basal or oxysterol-induction of LUC activity. Treatment of cells with 18:1,n9 or 20:4,n6 reduced the inductive effect of 22(R)-hydroxycholesterol on LUC by 50 and 75%, respectively. LXR was induced 2-fold by 22(R)-hydroxycholesterol induction (~2-fold). The oxysterol induction of LXR was not affected by fatty acid treatment. Thus, the effect of fatty acids on LXRs is specific for LXR.

Further characterization of the fatty acid effects on LXR used Gal4-chimeric receptors containing the LXR LBD fused to the Gal4 DNA binding domain (Fig. 1B). Transfection of HEK293-E cells with Gal4-LXR-LBD led to a comparable level of induction of LUC activity (~4-fold) following 22(R)-hydroxycholesterol treatment as seen with the full-length receptor. Moreover, the effect of fatty acids on the oxysterol regulation of LXR-LBD was essentially the same as that seen with the full-length receptor. Again, LXR, but not LXR, was responsive to unsaturated fatty acid regulation. Substituting TO-091317 (at 1 µM), a potent LXR agonist (9), for 22(R)-hydroxycholesterol yielded essentially the same results for both the LXR and LXR (not shown) as seen in Fig. 1, A and B. Finally, n3-PUFA were tested for their effects on Gal4-LXR-LBD activation (Fig. 1C). 22:6,n3 was as effective as 20:4,n6 at suppressing oxysterol-activated LXR, while 18:3,n3 was the least effective at antagonizing oxysterol-regulation of LXR.

These results confirm previous reports on the antagonistic effect of unsaturated fatty acids on the oxysterol regulation of LXR (6). We have extended these observations to include 20- and 22-carbon n3 PUFAs as effective inhibitors of oxysterol activation of LXR. However, neither n3 nor n6 PUFA affected oxysterol activation of LXR.

LXR and PPAR Are Not Responsive to Fatty Acid Regulation in HEK293-L Cells-- A similar analysis in the HEK293-L cells (Fig. 2) displayed a very different response pattern to fatty acids. Oxysterol 22(R)-hydroxycholesterol induced LUC activity by 8- and 2-fold in cells transfected with the LXR and LXR expression vectors. This response is comparable to that seen in the HEK293-E cells (Fig. 1A). In contrast to the HEK293-E cells, oxysterol activation of LXR and LXR in HEK293-L cells was insensitive to fatty acid regulation. In addition, both the LXR and  chimeric Gal4-LBD receptors were insensitive to fatty acid regulation in HEK293-L cells. Even higher levels of fatty acids (300 µM) had no effect on LUC activity (Fig. 8).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Fatty acid effects on oxysterol induction of LXR activity in HEK293-L cells. Confluent HEK293-L cells were transfected with expression vectors containing full-length hLXR and hLXR as described in Fig. 1A. The reporter plasmid was TK-LXREx3-Luc, and the internal control for transfection efficiency was phRG-Luc. Mean ± S.D., n > 9. These results are representative of three separate studies.

Because fatty acid regulation of LXR was cell-dependent, we examined the fatty acid regulation of a second fatty acid-regulated nuclear receptor, i.e. PPAR, in the two HEK293 strains. As noted above, the fatty acid regulation of PPAR is well established (2, 3). The two HEK293 cell strains were transfected with a reporter vector containing the acyl CoA oxidase PPRE fused upstream from the TK promoter and the CAT reporter gene (AOX-PPRE-TK-CAT). Cells were co-transfected with the SG5-PPAR expression vector. Cells were treated with 100 µM 20:4,n6 or WY14,643 (a strong PPAR agonist) for 24 h. In HEK293-E transfected with SG5-PPAR, CAT activity was induced ~2.2-fold by 20:4,n6 or WY14,643 (Fig. 3). While WY14,643 induced CAT activity 2.7-fold in HEK293-L cells, 20:4,n6 had no effect on CAT activity. In the absence of SG5-PPAR transfection, CAT activity was not affected by either 20:4,n6 or WY14,643. Thus, like LXR, PPAR is not sensitive to fatty acid regulation in HEK293-L cells.

View larger version (8K): [in this window] [in a new window]   Fig. 3.   Effect of 20:4,n6 and WY14,643 on PPAR-regulated gene expression in HEK293-E and HEK293-L. Cells are transfected with SG5-PPAR and the AOX-PPRE-TK-CAT reporter. Cells are treated with 20:4,n6 or WY14,643 at 100 µM overnight. CAT activity and protein was measured (8) and expressed as fold induction. A second group of cells were transfected with pM-Gal4-rPPAR. The reporter plasmid was TK-MH100X4-Luc. Mean ± S.D., n > 6. Results are expressed as fold change in LUC or CAT activity. These results are representative of two separate studies.

PPAR and LXR Display Equal Sensitivity to Exogenous Fatty Acid-- We evaluated the fatty acid sensitivity of LXR and PPAR in HEK293-E cells by carrying out a dose-response analysis (Fig. 4, A and B). In this study, the chimeric receptor Gal4-LXR-LBD was used to assess the sensitivity of LXR to fatty acid regulation. The maximum effect of 20:4,n6 on 22(R)-hydroxycholesterol-induced LUC activity was an 80% inhibition at 200 µM 20:4,n6. Fifty percent inhibition (IC₅₀) of oxysterol-mediated induction of LXR by 20:4,n6 was at ~60 µM. Assessment of fatty acid activation of PPAR used both the full-length SG5-PPAR expression vector and the Gal4-PPAR-LBD to activate the CAT or LUC reporter genes, respectively. At 200 µM, 20:4,n6 induced reporter gene activity 2.2-fold. This level was comparable to the effect of WY14,643 (100 µM) on PPAR-regulated reporter gene activity. The ED₅₀ for the inductive effect of 20:4,n6 was ~50 µM. These results indicate that in HEK293-E cells, both LXR and PPAR display a similar sensitivity to exogenously added 20:4,n6.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Dose response of 20:4,n6 effects on hLXR and rPPAR in HEK293-E cells. A, cells were transfected with CMX-Gal4-h LXR as described in Fig. 1B. B, one set of cells were transfected with either pM-Gal4-rPPAR and TK-MH100X4-Luc (LUC) as reporter, and another set of cells were transfected with SG5-PPAR (full-length PPAR) and AOX-PPRE-TK-CAT (CAT) as reporter. After an overnight transfection, cells were treated with varying doses of 20:4,n6 or 100 µM WY14,643 (PPAR only) for 24 h. Results are expressed as fold induction of LUC or CAT activity; Mean ± S.D., n = 3. These results are representative of two separate studies.

Fatty Acid Metabolism Studies-- The fatty acid regulation of LXR and PPAR is clearly different in the two HEK293 strains. As noted above, there are several factors that can account for this difference such as cell growth and density. However, the use of cells at confluence and fatty acid treatments in serum-free media minimized these differences. An alternative explanation focuses on metabolic differences in the cell and relates to how these two cell types metabolize exogenous fatty acids. Because both receptors bind NEFA (3, 6) we hypothesize that this difference in response can be attributed to higher levels of non-esterified 20:4,n6 in HEK293-E cells than in HEK293-L cells. To test this hypothesis, two approaches were used to examine 20:4,n6 metabolism in these cells. The first approach used a kinetic analysis of [¹⁴C]20:4,n6 uptake and metabolism. The second approach quantified mass changes of specific fatty acids in cells following treatment with fatty acids.

HEK293 cells were treated with [¹⁴C]20:4,n6 at 100 µM under the same conditions as used for the transfection studies. Fig. 5A illustrates the assimilation of [¹⁴C]20:4,n6 into the chloroform/methanol-soluble fraction of cells and the fractional distribution of [¹⁴C]20:6,n6 into various lipid fractions. No significant difference in fatty acid uptake or its assimilation into the chloroform-methanol-soluble fraction was observed. Thin-layer chromatographic separation of lipids allowed for the analysis of the fractional distribution of [¹⁴C]20:4,n6 into TAG, DAG, and polar lipids (lysophosphatidic acid, phosphatidic acid, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, sphingomyelin) (Fig. 5B). At all time points examined, there was no significant differences in the fractional distribution of [¹⁴C]20:4,n6 between TAG, DAG, or polar lipids between the two cells. However the fraction labeled "Other" is clearly different between the two cell types at all time points examined. This fraction is composed of cholesterol esters, neutral plasmalogens and NEFA (Fig. 5C). There are clear differences in the proportional distribution of ¹⁴C-labeled fatty acids in these three components. First, the non-esterified ¹⁴C-labeled fatty acid represents 3% of the total ¹⁴C-labeled fatty acid incorporated into the chloroform/methanol extract of these cells. HEK293-E cells assimilate a greater fraction of the ¹⁴C-labeled fatty acid into cholesterol ester, while HEK293-L have a greater proportion of exogenous fatty acid assimilated into a fraction having chromatographic properties characteristic of neutral plasmalogens. In fact the level of 20:4,n6 assimilation into neutral plasmalogens far exceeds the level of fatty acid assimilation into CE. Together, these studies reveal differences in the level of ¹⁴C-fatty acid retained in the NEFA pool and its assimilation into two neutral lipid fractions, i.e. cholesterol ester and neutral plasmalogen.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Thin-layer chromatography of [14C]20:4,n6-labeled lipids in HEK293-E and HEK293-L cells. Cells were incubated overnight in serum-free media and then treated with 100 µM 20:4,n6 (14C) as described under "Materials and Methods." At the designated time, cells were harvested for total lipid extraction. Twenty µl of a total 500 µl of extract was applied to silica TLC plates, dried, and developed in hexane:ether:acetic acid (90:30:1). Plates were dried, and radioactivity was detected and quantified by phosphorimaging. A, a phosphorimage of TLC plates. Samples from triplicate cell culture plates were applied for each time point. Labels are: CE, cholesterol ester; NP, neutral plasmalogen; TAG, triacylglycerol; NEFA, non-esterified fatty acid; 1,2-DAG, 1,2-diacylglycerol; and PL, polar lipids containing glycero- and sphingolipids. The bands above 1,2-DAG are not identified but may represent 1,3-DAG resulting from TAG remodeling. B and C, distribution of [14C]20:4,n6 among triacylglycerol, diacylglycerol, polar lipids, and other cholesterol esters, neutral plasmalogens, and NEFA. Nanomoles of 20:4,n6 in the various fractions were quantified using the following information: specific activity of [14C]20:4,n6 366 CPM/nmol; 300 nmol/plate; CPM/mg protein in a 20-µl sample applied to the TLC plate and the fractional distribution of radioactivity determined by phosphorimaging. Results are expressed as nmol of 20:4,n6/mg of protein. Mean ± S.D., n = 3. The study shown is representative of two independent studies.

The use of ¹⁴C-labeled fatty acids provides important clues as to how exogenous fatty acids are distributed to various lipid fractions. However, to determine how addition of exogenous fatty acids affect levels of endogenous fatty acids as well as the generation of fatty acid metabolites, we examine the fatty acid composition in the total chloroform/methanol extract as well as the fractionated NEFA pool. Accordingly, cells were treated with non-radioactive 20:4,n6 at 100 µM (as described above) and total lipid was extracted from cells at 0, 1.5, 6, and 24 h after initiating treatment. Total lipids in the chloroform-methanol extract were saponified and fractionated by RP-HPLC. Although this analysis is restricted to unsaturated fatty acids, all fatty acid profiles were confirmed using gas chromatography-mass spectrometry (see "Methods and Materials"). 18:1,n9 is a major unsaturated fatty acid (~1200 nmol/mg protein) in both cell types (Fig. 6A). The gas chromatography-mass spectrometry analysis indicated that both 16:0 and 18:0 are prominent lipids in HEK293 cells (not shown). Linoleic (18:2,n6), arachidonic (20:4,n6), and adrenic (22:4,n6) are minor PUFA at < 100 nmol/mg protein. Treatment of cells with 100 µM 20:4,n6 leads to a 6.3, 11.8, and 8.7-fold increase in total 20:4,n6 in HEK293-E cells and a 8.1, 12, and 11.8-fold increase in HEK293-L cells after 1.5, 6, and 24 h, respectively. Adrenic acid (22:4,n6) increased in each cell type after 20:4,n6 addition, reflecting the enzymatic conversion of 20:4,n6 to 22:4,n6 by an elongase. The fold changes in 22:4,n6 were 3.6, 4.3, and 9.2-fold for HEK293-E cells and 5.5, 8.8, 12.5-fold for HEK293-L cells. Clearly, the addition of 20:4,n6 leads to major changes in cellular 20:4,n6 and 22:4,n6 levels in both cell types. Addition of 20:4,n6 to cells did not induce major changes in cellular levels of 18:1,n9, 18:2,n6, or 22:6,n3 during the time course of this study.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Unsaturated fatty acid profile in HEK293-E and HEK293-L cells treated with 20:4,n6. A, unsaturated fatty acid analysis of saponified lipids from total cell extract. Confluent cells on 80-mm plastic dishes were incubated overnight in serum-free medium and then treated with 100 µM 20:4,n6. Cells were harvested at 0, 1.5, 6, and 24 h after initiating treatment for total lipid extraction. Extracted lipids were pooled from three plates, saponified, fractionated, and quantified by RP-HPLC (see "Materials and Methods"). Results are represented as nmol of fatty acid/mg of protein. The analysis is representative of two separate studies. In B, the NEFA fraction was prepared from a pool of extracts from three plates as described under "Materials and Methods." The recovered NEFA fraction (see "Materials and Methods") was not saponified but fractionated directly by RP-HPLC to quantify levels of fatty acids. The results are presented as nmol of fatty acid/mg of protein and is representative of two separate studies. E = HEK293-E; L = HEK293-L.

To determine the NEFA fraction of the total lipid extract, the chloroform-methanol extract was fractionated on amino-propyl columns and analyzed by RP-HPLC (Fig. 6B). The results of this analysis are represented as a percent of total fatty acid extracted from the cell (Fig. 7, A and B). The relative abundance of 18:1,n9, 18:2,n6, 20:4,n6, and 22:4,n6 in the NEFA pool parallels the distribution of these fatty acids in the total cell extract. In untreated HEK293-E cells, these fatty acids were at 17.9, 1.5, 1.1 and 0.15 nmol/mg protein and in untreated HEK293-L cells the fatty acids were 26.2, 1.8, 1.2, and 0.3 nmol/mg protein. Each of these non-esterified fatty acids represents  3% of total saponified 18:1,n9, 18:2,n6, 20:4,n6, 22:4,n6, or 22:6,n3 in the cell. After initiating treatment, 20:4,n6 remains between 1.7 and 2.8% of the total cellular 20:4,n6 over the 24-h treatment period in HEK293-E cells (Fig. 7A). In contrast, non-esterified 20:4,n6 in HEK293-L cells falls from 2.5% in untreated cells to 0.5% after 24 h of treatment. This difference reflects more rapid assimilation of 20:4,n6 into complex lipids in HEK293-L cells than HEK293-E cells.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Fraction of the total cellular 20:4,n6 and 22:4,n6 in the NEFA pool. The mass of 20:4,n6 (A) and 22:6,n6 (B) in the NEFA extract were measured as described in Fig. 6 and "Materials and Methods." The data is represented as percent of total 20:4,n6 (or 22:4,n4) as NEFA = 100 × (mass of fatty acid (20:4,n6 or 22:4,n6) in the NEFA pool/mass of total cellular fatty acid (20:4,n6 or 22:4,n6)).

A similar analysis of 22:4,n6 indicates that this fatty acid increases from 0.5% at the start of the experiment to 3.1% of the total 22:4,n6 within 6 h in HEK293-E cells, but remains between 0.3 and 0.5% in HEK293-L cells. As with 20:4,n6, 22:4,n6 is more rapidly removed from the NEFA pool and assimilated into complex lipids in HEK293-L cells than HEK293-E cells. Other unsaturated fatty acids in the NEFA pool, i.e. 18:1,n9, 18:2,n6, 22:6,n3, remain unchanged during the 24-h treatment period.

Adrenic Acid Is More Potent than 20:4,n6 at Inhibiting LXR-- Finding that adrenic acid (22:4,n6) is formed in cells following 20:4,n6 treatment indicates active elongase activity in both cell types. To determine the effect of adrenic acid on oxysterol-regulated LXR activity, a dose-response analysis was carried out. Adrenic acid (22:4,n6; ED₅₀ ~20 µM) was ~2-fold more potent than 20:4,n6 (ED₅₀ ~40 µM) at inhibiting LXR activity in HEK293-E cells. Neither fatty acid inhibited LXR activity in HEK293-L cells, even up to 300 µM. Based on these metabolism studies, we conclude that non-esterified 20:4,n6 and its metabolite, 22:4,n6, are more rapidly assimilated into complex lipids in HEK293-L cells than in HEK293-E cells. This difference in clearance of exogenous fatty acid and its metabolite from the NEFA pool may account for the differences in fatty acid regulation of LXR and PPAR in these two cell lines.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study was initially undertaken to evaluate the fatty acid regulation of LXRs. After beginning these studies we quickly discovered that cell specific-factors significantly affected the outcome of these studies. Because PPAR and LXR bind NEFA with an apparent K_d of 1-4 µM (3, 6), both receptors should respond to a rise in intracellular NEFA following administration of fatty acids to cells. However, our studies with two strains of HEK293 cells reveal very different responses to exogenous fatty acid challenge. The notion that cell-specific lipid metabolism contributes to nuclear receptor regulation has been described in the context of eicosanoid regulation of PPARs (12, 13) or the differential effects on mono- and polyunsaturated fatty acids on lipogenic gene expression (1, 14, 15). However, we are unaware of any report that has examined the rate of change in intracellular NEFA or their metabolites following administration of fatty acids to cells and related these changes to nuclear receptor control.

Our studies confirm an earlier report (6) by showing that oxysterol regulation of LXR is suppressed by unsaturated fatty acids. We have extended this observation to include the 20- and 22-carbon n3-PUFA as regulators of LXR; at 100 µM 20:4,n6 = 22:6,n3 > 20:5,n3 > 18:1,n9 = 18:3,n3 > 16:0. While 20:4,n6 binds the LXR LBD with an apparent K_d of 1-5 µM (6), the IC₅₀ for the inhibitory effect on LXR activity is ~40-60 µM (6) (Figs. 4 and 8). Both the K_d for fatty acid binding and the ED₅₀ for the activation of PPAR is in this same range (3) (Fig. 4). Thus, at a given intracellular level of non-esterified 20- or 22-carbon n6 or n3 PUFA, both PPAR and LXR regulatory networks will be affected. Our studies with primary hepatocytes indicate that this concept applies to some PPAR- and LXR-regulated genes⁵.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   A comparison of the effects of 20:4,n6 and 22:4,n6 on oxysterol activation of hLXR in HEK293-E A, and HEK293-L cells. B, cells were transfected with CMX-Gal4-hLXR and TK-MH100X4-Luc as described in Fig. 1B. After an overnight transfection, cells were treated with varying doses of 20:4,n6 or 22:4,n6 for 24 h. Results are expressed as fold induction of LUC activity. Mean ± S.D., n = 6. These results are pooled data from two separate studies.

Although LXR is known to be less sensitive than LXR to oxysterol activation (9), we were surprised to find that, in contrast to LXR, LXR was insensitive to fatty acid regulation (Fig. 1). The amino acid sequence of the LBD of the two receptors differs at several locations. Presumably these differences account for the levels of oxysterol, fatty acid, and co-activator binding. This observation also points out that in liver, where both receptors are expressed, only LXR will be sensitive to fatty acid control. Our studies with primary hepatocytes have revealed some LXR-regulated genes are sensitive to PUFA regulation, while others are not.² Whether these two receptors regulate different sets of genes may be resolved using LXR and LXR knockout mice (16).

The differences we described in nuclear receptor regulation by fatty acids in the two HEK293 strains prompted the analysis of how these cells handle exogenous 20:4,n6. The use of both metabolic labeling and mass analysis of 20:4,n6 and its elongation product, 22:4,n6, by RP-HPLC provides insight into fatty acid metabolism and rates of clearance of exogenously added fatty acids from the intracellular NEFA pool. Both methods provide clear evidence for a more rapid assimilation of exogenous fatty acid as well as its metabolite, 22:4,n6, in the HEK293-L cells than in HEK293-E cells. This evidence is based on the rate of reduction of [¹⁴C]20:4,n6 (Fig. 5C) as well as the mass of 20:4,n6 and 22:4,n6 (Fig. 7, A and B) in the NEFA pool. We found no evidence of enhanced oxidation of [¹⁴C]20:4,n6 in HEK293-L cells. This was based on the finding that nearly equal levels of fatty acid were assimilated into cells at the various time points (Fig. 5B) and that the mass of 20:4,n6/mg of protein was actually higher in HEK293-L cells (Fig. 6A). In addition, analysis of culture media for rates of depletion of [¹⁴C]-20:4,n6 or the generation of lipid oxidization products failed to reveal major differences between these two cell lines. Instead, the difference between these two cell lines is in the assimilation of exogenously added fatty acid into complex lipids. This difference may reflect levels of expression of acyl CoA synthetase. At least five acyl CoA isoforms have been identified, each capable of forming fatty acyl CoA thioesters using saturated and unsaturated fatty acids of chain lengths ranging from 12-20 carbons (17). However, ACS4 is most active on 20- and 22-carbon PUFA (17). Whether the difference in fatty acid sensitivity between the HEK293-E and HEK293-L cells is due to levels of expression of a specific acyl CoA synthetase remains to be determined.

Other differences in fatty acid metabolism are seen in the assimilation of ¹⁴C-labeled fatty acid into cholesterol esters and a fraction consistent with the chromatographic properties of neutral plasmalogens (Fig. 5). These differences likely reflect levels of expression of key enzymes involved in the biosynthesis of these complex neutral lipids; i.e. microsomal acyl-CoA:cholesterol acyltransferase 1 or 2 for cholesterol ester synthesis or peroxisomal alkyldihydroxyacetone phosphate synthase and microsomal phosphatidate phosphatase for neutral plasmalogens synthesis (18, 19). The higher capacity of the HEK293-L cells to assimilate exogenously added fatty acid into neutral lipids provides a convenient explanation for the disappearance of non-esterified 20:4,n6 and 22:4,n6 from the NEFA pool of cells. The more rapid depletion of 20- and 22-carbon n6 PUFA from the NEFA pool apparently reduces these NEFA to a level insufficient to affect LXR or PPAR activity as measured by reporter assays.

A third mechanism that might account for the difference in nuclear receptor responsiveness between the two cell types is the assimilation of 22:4,n6 into complex lipids (Figs. 6 and 7). Both cell types equally elongate exogenously added 20:4,n6 to 22:4,n6. However, when compared with HEK293-L cells, HEK293-E cells are slow to assimilate 22:4,n6 into complex lipids. A dose response shows that 22:4,n6 is 2-fold more potent than 20:4,n6. Thus, the generation of a more potent ligand coupled with its slow assimilation in to complex lipids can account for the increased sensitivity of HEK293-E cells to fatty acid regulation of nuclear receptor.

Finally, the question remains as to whether the mechanisms reported here have any bearing on PPAR or LXR regulation in vivo, e.g. in liver or primary hepatocytes. Many of the same studies on lipid metabolism reported here have been carried out in primary hepatocytes.² An added advantage of primary hepatocytes over HEK cells is that we can carry detailed kinetic analyses of key genes targeted by PPAR and LXR rather than rely on receptor activation assays. It is clear from these kinetic analyses that intracellular NEFA levels are elevated in response to extracellular fatty acid load and fall as a result of metabolism. Kinetic analysis of PUFA metabolism in primary hepatocytes is very dynamic.² Analysis of key target genes for PPAR and LXR reveals dynamic changes in expression.

In summary, we have identified two strains of HEK293 cells that display differences in nuclear receptor signaling. This difference can be ascribed to different rates of assimilation of exogenous fatty acid (20:4,n6) and its elongation product (22:4,n6) into complex lipids. Both PPAR and LXR, but not LXR, display comparable sensitivity to fatty acid regulation. This finding suggests that as PPAR is activated by elevated intracellular NEFA, LXR will be inhibited. If this same mechanism prevails in tissues like the liver that encounter major changes in plasma lipid levels (as triacylglycerols and NEFA), then elevated intracellular NEFA will likely affect both pathways. In the liver, it is well established that both n6 and n3 PUFA are feed forward activators PPAR and stimulate pathways leading to enhanced fatty acyl CoA formation and the flow of fatty acids into various oxidation pathways (1, 2). Elevated NEFA serve as feed back regulators of LXR and may lead to the suppression of transcription of SREBP-1c, a key transcription factor controlling de novo lipogenesis. PUFA also regulate SREBP-1c mRNA levels by enhanced mRNA turnover (20-22). Thus, PPAR and LXR should be viewed as monitors of intracellular NEFA levels and respond accordingly to prevent lipid overload (23).

	FOOTNOTES

^* This research was supported by National Institutes of Health Grant DK43220, United States Department of Agriculture Grant 98-35200-6064, and the Michigan Agriculture Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Both authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Physiology, 3165 Biomedical and Physical Science Bldg., Michigan State University, East Lansing, MI 48824. Tel.: 517-355-6475, Ext. 1133; Fax: 517-355-5125; E-mail: Jump@msu.edu.

Published, JBC Papers in Press, August 2, 2002, DOI 10.1074/jbc.M206170200

² A. Pawar, J. Xu, E. Jerks, D. J. Mangelsdorf, and D. B. Jump, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator activated receptor; LXR, liver X receptor; RXR, retinoid X receptor; PUFA, polyunsaturated fatty acids; NEFA, non-esterified fatty acids; BHT, butylated hydroxytoluene; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; LBD, ligand binding domain; TAG, triacylglycerol, DAG, Diacylglycerol; CE, cholesterol esters; RP-HPLC, reverse phase-high pressure liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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