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

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 PPARα activity ∼2.2-fold and suppressed LXRα activity by 80% (ED50 ∼25–50 μm) in HEK293-E (early passage) cells but had no effect on PPARα or LXRα receptor activity in HEK293-L (late passage) cells. LXRβ 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 LXRα activity in HEK293-E cells, but had no effect on LXRα 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 PPARα and LXRα activity.

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 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 chromatographymass spectrometry at the mass spectroscopy facility at Michigan State University.
The NEFA fraction in total cellular lipids was fractionated on aminopropyl columns [Alltech, Inc] (11). Lipid extracts in chloroform ϩ 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 ϩ 2% acetic acid. Phospholipids were retained on the column. The diethyl ether-2% acetic acid fraction was dried under nitrogen, resuspended in methanol ϩ 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 Ͼ95%.

RESULTS
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 ϳ1 day versus 2 days, respectively. Moreover, HEK293-L cells grow to a higher density (3.8 ϫ 10 6 cells/well) than HEK293-E (2.8 ϫ 10 6 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.
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 M 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 (ϳ2-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 (ϳ4-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 M), 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 oxysterolregulation 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 20and 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 M) had no effect on LUC activity (Fig. 8).
Because fatty acid regulation of LXR␣ was cell-dependent, we examined the fatty acid regulation of a second fatty acidregulated 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 cotransfected with the SG5-PPAR␣ expression vector. Cells were treated with 100 M 20:4,n6 or WY14,643 (a strong PPAR␣ agonist) for 24 h. In HEK293-E transfected with SG5-PPAR␣, CAT activity was induced ϳ2.2-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.
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 doseresponse 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 M 20:4,n6. Fifty percent inhibition (IC 50 ) of oxysterol-mediated induction of LXR␣ by 20: 4,n6 was at ϳ60 M. 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 M, 20:4,n6 induced reporter gene activity 2.2-fold. This level was comparable to the effect of WY14,643 (100 M) on PPAR␣-regulated reporter gene activity. The ED 50 for the inductive effect of 20:4,n6 was ϳ50 M. These results indicate that in HEK293-E cells, both LXR␣ and PPAR␣ display a similar sensitivity to exogenously added 20:4,n6.
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 [ 14 C]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 [ 14 C]20:4,n6 at 100 M under the same conditions as used for the transfection studies. Fig. 5A illustrates the assimilation of [ 14 C]20:4,n6 into the chloroform/methanol-soluble fraction of cells and the fractional distribution of [ 14 C]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. Thinlayer chromatographic separation of lipids allowed for the analysis of the fractional distribution of [ 14 C]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 [ 14 C]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 14 C-labeled fatty acids in these three components. First, the non-esterified 14 C-labeled fatty acid represents Յ3% of the total 14 C-labeled fatty acid incorporated into the chloroform/methanol extract of these cells. HEK293-E cells assimilate a greater fraction of the 14 C-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 14 Cfatty acid retained in the NEFA pool and its assimilation into two neutral lipid fractions, i.e. cholesterol ester and neutral plasmalogen.
The use of 14 C-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 M (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 (ϳ1200 nmol/mg protein) in both cell types (Fig. 6A). The gas chromatography-mass spectrometry analysis indicated that  To determine the NEFA fraction of the total lipid extract, the chloroform-methanol extract was fractionated on aminopropyl 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 nonesterified fatty acids represents Յ 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.
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 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 ϭ HEK293-E; L ϭ HEK293-L. 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; ED 50 ϳ20 M) was ϳ2-fold more potent than 20:4,n6 (ED 50 ϳ40 M) at inhibiting LXR␣ activity in HEK293-E cells. Neither fatty acid inhibited LXR␣ activity in HEK293-L cells, even up to 300 M. 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
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 M (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.
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 [ 14 C]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 [ 14 C]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 [ 14 C]-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 14 C-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).