Egg yolks inhibit activation of NF-\xce\xbaB and expression of its target genes in adipocytes after partial delipidation
How composition of egg yolk (EY) influences NF-\xce\xbaB, a key transcription pathway in inflammation, remains unclear. We performed partial delipidation of EY that removed 20\xe2\x80\x9330% of cholesterol and triglycerides. The resulting polar and non-polar fractions were termed EY-P and EY-NP. NF-\xce\xbaB activation in response to EY from different suppliers and their fractions was examined in 3T3-L1 adipocytes using a NF-\xce\xbaB response element reporter assay and by analyzing expression of 248 inflammatory genes. Although EY-P and EY contained similar level of vitamins, carotenoids, and fatty acids, only delipidated EY-P fraction suppressed NF-\xce\xbaB via down-regulation of toll like receptor-2 and up-regulation of inhibitory toll interacting protein (Tollip) and lymphocyte antigen 96 (Ly96). Our data suggest that anti-inflammatory activity of lutein and retinol were blunted by non-polar lipids in EY likely via crosstalk between SREBP and NF-\xce\xbaB pathways in adipocytes. Thus, moderate delipidation may improve their beneficial properties of regular eggs.
Introduction<!>Reagents and supplies<!>EY extraction<!>Cell culture studies<!>mRNA analyses<!>Protein assay of cell lysates<!>Lipid content analysis of EY fractions<!>Fatty acid analysis<!>HPLC- tandem mass spectrometry analysis (HPLC/MS/MS)<!>Statistical analysis<!>Hexane extraction procedure moderately reduces cholesterol and triglycerides in EY content<!>EY-P and EY-NP fractions have similar composition of major fatty acids<!>Lipophilic vitamins and carotenoids were retained exclusively in EY-P fraction<!>1. EY-P fraction inhibits the activation of NF-\xce\xbaB and its target genes in adipocytes<!>2. Inhibition of NF-\xce\xbaB activation by EY-P fraction is associated with selective regulation of Tlr receptors and their inhibitor proteins<!>3. Dissimilar regulation of expression by EY-NP and EY-P fractions influence proteins with potential binding sites for both SREBP and NF\xce\xbaB<!>Discussion<!><!>Discussion
<p>Chronic inflammation accompanies all degenerative metabolic disorders, including obesity, type 2 diabetes, cardiovascular diseases, cancer, and aging.1, 2 Moreover, inflammation is causatively linked to the development and progression of these disorders.3–5 In different diseases, inflammation is mediated by specific cytokines that are produced from different cell types. In obesity, chronic inflammation is associated with the production of inflammatory chemokines (CXC) and CC chemokine ligands (CCL) by circulating and resident immune cells, as well as by differentiated adipocytes, steatotic hepatocytes and other peripheral tissues accumulating lipid.6–8 Reduction of inflammation delays the progression of metabolic disorders and is considered to be an effective strategy for their prevention.5, 9, 10</p><p>The production of the wide spectrum of molecules mediating inflammation is under the control of several transcription factors. These include Rel, signal transducers and activators of transcription family (STAT), interferon (IRF) and other families of transcription factors.11–15 Among these transcription factors, NF-κB has been established as a key regulator of acute and chronic inflammation.12 NF-κB is a heterodimeric complex of Rel proteins (e.g. p65/p50). Inhibitory IκB proteins can bind to the p65/p50 complex and sequester this inactive complex in the cytosol. NF-κB activation is mediated by a family of pattern recognition receptors that includes nine receptors toll-like receptors 1-9 (TLR1-9).12 Activation of TLR receptors leads to the binding of adaptor molecules and activation of signaling cascades, which results in the phosphorylation of IκB and its degradation.12 The free NF-κB complex binds to the cognate response elements (Nfκb-RE) in the promoter or enhancer regions of target genes. NF-κB target genes include families of CXC and CCL cytokines, chemokines, adhesion molecules, integrins, differentiation factors.16 The activation of TLR by viral/bacterial metabolic products and cytokines induces acute inflammatory response.12</p><p>In metabolic disorders, NF-κB activation can be provoked by high-fat, high-cholesterol diets.12 Studies demonstrate that fatty acids and lipoproteins can bind to TLR receptors,12 whereas cholesterol can trigger the activation of NF-κB by mechanisms that remain unclear.17 In experimental models, the inhibition of NF-κB improves insulin resistance and hepatic steatosis and decreases obesity and atherosclerosis.10 Inhibition of NF-κB can also be achieved through the diet. Polyunsaturated fatty acid (PUFA), antioxidants, and lipophilic vitamins can inhibit NF-κB activation via a variety of mechanisms. PUFA and bioactive hormone-like metabolites of vitamin A, retinoic acid, and vitamin D3 serve as cognate ligands for anti-inflammatory transcription factors peroxisome proliferator-activated receptors (PPARs), retinoic acid receptor (RAR), and vitamin D receptor (VDR), respectively.18–21 Activated PPARs, RAR, and VDR interfere with NF-κB activation by sequestering p65 in the nucleus, encoding inhibitory IκB, promoting catabolism of pro-inflammatory lipids, decreasing reactive oxygen species (ROS) production and other mechanisms.21–23 Vitamin E and polyphenols have been shown to inhibit NF-κB activation by disabling signaling cascades, counteracting ROS production as antioxidants, and through activation of redox-active transcription factors, e.g. nuclear factor (erythroid-derived 2)-like 2 (NRF2).22</p><p>Many dietary products have complex bioactive macro- and micronutrient composition. Recent research highlighted the anti-inflammatory24 and pain-reducing25 properties of egg yolk (EY). These effects have been attributed to specific lipid-soluble extracts25 and water-soluble proteins 24. EY contains an unique combination of vitamins A, D, E, carotenoids, such as lutein and zeaxanthin, and phospholipids that can potentially diminish inflammation and oxidative stress.26 However, the EY also contains cholesterol and fatty acids that can contribute to pro-inflammatory pathways.27 The role of EY composition on the regulation of NF-κB has not been investigated. In this paper, we show that partial delipidation of the EY without changes to the EY's carotenoid/vitamin content, is sufficient to markedly reduce activation of NF-κB and its target cytokines.</p><!><p>We purchased reagents for cell culture from Invitrogen (Carlsbad, CA). Other reagents were acquired from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Vectors were purchased: Cignal Lenti NFκB Reporter (GFP) Kit from SABiociences/Qiagen (Valencia, CA); mCherry constitutive expression vector from Genecopoeia (Rockville, MD). We bought four types of regular eggs from Sauder's egg (EY1), Kroger medium egg (EY2), Kroger large egg (EY3), and EggLand's Best egg (EY4). Cholesterol was purchased from MP Biomedicals (Santa Ana, CA).</p><!><p>15mL whole EY was diluted in 35mL phosphate buffered saline (PBS) (1:3.33, v/v). This solution was termed 'EY'. Hexane (4mL) was added to 1mL of EY and inverted vigorously by hand for 1min. The hexane phase was removed and dried under nitrogen, and then resuspended in 1mL ethanol. The hexane extract of the EY was termed 'Non-polar, (EY-NP)' and the remaining phase was 'Polar, (EY-P)' (Fig 1A). These EY, EY-NP, and EY-P extracts were used for cholesterol/cholesteryl ester quantification, triglyceride quantification, gas chromatography, high performance liquid chromatography (HPLC), and cell stimulation.</p><!><p>Nfkb-RE/GFP cell line was derived from murine 3T3-L1 preadipocyte clone stably transfected with lentiviral Nfkb-RE GFP reporter construct and control mCherry overexpression construct as described.28 The Nfkb-RE/GFP cell line derivation procedure and its characterization in presence of pro- and anti-inflammatory mediators was described before.28
Nfkb-RE/GFP cells were cultured in standard culture medium (high glucose DMEM containing 10% CS and 1% penicillin-streptomycin). Similar to the parent 3T3-L1 cell line, Nfkb-RE/GFP cells preadipocytes were capable of differentiation.28 Differentiation was induced (day 0) in confluent preadipocytes using differentiation media I (DMI) containing 3-isobutyl-1-methylxanthine (0.5mM), dexamethasone (1μM), insulin (1.7μM), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin in DMEM. Differentiation media II (DMII) contained 10% FBS, insulin (1.7μM), and 1% penicillin-streptomycin in DMEM. Every 48h post adipogenesis induction, cell media was replaced with DMII using standard protocol.29 Prior to measuring fluorescence, cells were washed twice with PBS and lysed with an appropriate volume (i.e. 120μL/well for 24-well plate, 150μL/P60 dish) of RIPA buffer (Boston BioProducts, Ashland, MA) containing complete protease inhibitor (Roche Diagnostics Corporation, Indianapolis, IN). Cells were incubated on ice for 15min, and then the plate was scratched using a tip to lyse residual cells. Cell lysate (80μL/well) was transferred to a black 96-well plate (Fisher Scientific Company, Hanover, IL) to measure fluorescence at wavelengths Ex/Em 485/528 for GFP, and then, at Ex/Em 587/640 for mCherry using Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT). Nfkb-RE activation was calculated as ratio of GFP to mCherry control fluorescence (GFP/mCherry), or GFP/protein if mCherry fluorescence was below detection limit. 3T3-L1 preadipocytes were cultured and differentiated under the same conditions as Nfkb-RE/GFP preadipocytes.</p><!><p>The quantitative analysis of mRNA was performed using the nCounter mouse inflammation expression V2 Panel kit (NanoString Technologies, Seattle, WA). NanoString's nCounter technology is based on direct detection of target molecules using color-coded molecular barcodes, providing a digital simultaneous quantification of the number of target molecules.29 All data were normalized to 6 housekeeping genes quantified in the same samples. Total mRNA (100ng in 5ul) was hybridized overnight with nCounter Reporter (20μl) probes in hybridization buffer and in excess of nCounter Capture probes (5μL) at 65°C for 16–20h. The hybridization mixture containing target/probe complexes was allowed to bind to magnetic beads containing complementary sequences on the Capture Probe. After each target found a probe pair, excess probes were washed followed by a sequential binding to sequences on the Reporter Probe. Biotinylated capture probe-bound samples were immobilized and recovered on a streptavidin-coated cartridge. The abundance of specific target molecules was then quantified using the nCounter Digital Analyzer. Individual fluorescent barcodes and target molecules present in each sample were recorded with a CCD camera by performing a high-density scan (600 fields of view). Images were processed internally into a digital format and were normalized using the NanoString nSolver software analysis tool. Counts were normalized for all target RNAs in all samples based on the positive control RNA to account for differences in hybridization efficiency and post-hybridization processing, including purification and immobilization of complexes. The average was normalized by background counts for each sample obtained from the average of the eight negative control counts. Subsequently, a normalization of mRNA content was performed based on six internal reference housekeeping genes Cltc, Gapdh, Gusb, Hprt1, Pgk1, and Tubb using nSolver Software (NanoString Technologies, Seattle, WA).</p><!><p>The protein content was measured using a BCA kit (Thermo Fisher Scientific; Rockford, IL). The absorbance (562nm) was measured using the Synergy H1 Hybrid Multi-Mode Microplate Reader.</p><!><p>The EY, EY-P, and EY-NP fractions were prepared by hexane extract procedure as described above. To enable enzymatic reaction, EY-NP fraction was resuspended in 200μL chloroform and 300μL Cholesterol Assay Buffer (from the Cholesterol Quantitation Kit), instead of 1mL ethanol. The suspension was mixed well by tapping, and then sonicated to evaporate chloroform. 10μL EY-NP from this stock (in 300μL cholesterol assay buffer) was diluted with DI water (to 33μL) to achieve an original concentration in 1ml of EY. All EY fractions were then diluted in Cholesterol Assay Buffer according to manufacturer's instruction and L-Type Triglyceride M Kit (Wako Diagnostics; Richmond, VA) was performed. The absorbance (650 nm) was measured using the Synergy H1 Hybrid Multi-Mode Microplate Reader.</p><p>Cholesterol Enzymatic Kit (Wako Diagnostics) was performed according to manufacturer's description. The absorbance (600nm) was measured using the Synergy H1 Hybrid Multi-Mode For cholesterol ester analysis, the EY and EY-NP fractions were prepared by hexane extract procedure as described above; however, EY-NP fraction was resuspended in 200μL Cholesterol Assay Buffer (NP). 0.3μL EY, 0.5μL EY-P, and 10μL EY-NP were diluted with Cholesterol Assay Buffer (total volume 50μL). Cholesterol Quantitation Kit was performed according to manufacturer's description. The absorbance (570nm) was measured using the Synergy H1 Hybrid Multi-Mode Microplate Reader.</p><!><p>Total lipids were extracted from all EY fractions with 2:1 (v/v) chloroform:methanol and washed with 0.88% KCl.30 Fatty acid methyl esters were prepared using 5% hydrochloric acid in methanol at 76°C.31 Analysis of fatty acid methyl esters was completed by gas chromatography with column and conditions as previously described.32 Retention times were compared to standards (Matreya, LLC, Pleasant Gap, PA, Supelco, Bellefonte, PA, and Nu-Check Prep Inc, Elysian, MN) and fatty acids are reported as percent of total identified fatty acids.</p><!><p>Prior to analysis, EY-P fractions were further extracted using hexane/ethanol/acetone/toluene (10:6:7:7, v/v/v/v, termed HEAT).33 The EY-NP extracts and HEAT-extracted EY-P fractions were injected on a YMC C30 column (4.6×150mm, 5um, Waters Corp.) and separated with a gradient solution 1 (80/18/2 MeOH/water/2% ammonium acetate) and solution 2 (78/20/2 MtBE/MeOH/2% ammonium acetate at 1.8mL/min and 40°C. Diode array spectra were collected with an Agilent 1260 diode-array detector with 60mm path length flow cell (Agilent Technologies). Eluent was introduced to a QTrap 5500 (ABSciex, Concord, Canada) mass spectrometer via an atmospheric pressure chemical ionization (APCI) probe operated in positive ion mode. Multiple reaction monitoring (MRM) MS/MS transitions were used to detect vitamins and carotenoids. Lutein/zeaxanthin and retinol were quantified based on external standards.</p><!><p>Data was shown as mean ± SD based on 3 to 4 independent experiments. Statistical comparison of groups was examined by both Student's t test and Mann-Whitney U test. Correlation analysis was performed using Pearson test. P<0.05 was considered statistically significant.</p><!><p>EY contains many lipophilic compounds that can potentially promote and suppress inflammation.26, 27 To assess how lipophilic compounds influence properties of whole EY, we performed hexane extraction procedure on the EY (Fig. 1A) and characterized the composition of the non-polar (EY-NP), and polar (EY-P) fractions. To account for variability related to egg production, we performed experiments using EY obtained from 4 different suppliers. Due to a high level of amphipathic phospholipids in EY, hexane extraction removed approximately 20% of total cholesterol from EY (100%) (Fig. 1B). EY contained 65% of free cholesterol (43% in EY-P and 22% in EY-NP fractions) and 35% of cholesteryl ester (27% in EY-P and 8% in EY-NP fractions). Hexane extraction also removed 30% of triglycerides that were found in the EY (100%) (Fig. 1B, insert). Thus, the majority of cholesterol was retained in the EY-P fraction, while the EY-NP fraction contained only 20–30% of cholesterol and triglycerides content from the EY.</p><!><p>Next we compared the composition of fatty acids in the EY, EY-P, and EY-NP fractions after their hydrolysis from complex lipids. The major fatty acids in all fractions were saturated palmitic (16:0), monounsaturated oleic (18:1), and polyunsaturated omega 6 linoleic (18:2) acids that comprised approximately 27%, 37%, and 19% of all fatty acids, respectively, in the EY, EY-P, or EY-NP fractions (Fig. 2 A–C). We found significant, but minor differences between different fractions (less than 2% compared to total fatty acid content) in the less abundant saturated (18:0, 14:0, 20:0), monounsatured (16:1n7), and polyunsaturated (22:5n3) fatty acids. In the EY-P fraction, 22:5n3 was less compared to EY (Fig. 2D), while the content of saturated (18:0, 14:0) and monounsatured (16:1n7) fatty acids was higher in EY-P compared to EY-NP fractions. Overall, the relative composition of fatty acids was similar among the EY, EY-P, and EY-NP fractions.</p><!><p>Simultaneous determination of vitamins E (α-tocopherol), D (D3), A (retinol and retinyl esters), and carotenoids (free and esterified lutein and zeaxanthin esters) was performed using HPLC/MS/MS. Carotenoids and retinol were quantified based on external standards. The EY-NP fraction contained no detectable levels of lipophilic vitamins and carotenoids (Fig. 3). All lipid soluble vitamins and carotenoids were found in the EY-P fraction, probably due to their interaction with phospholipids. The contents of lutein and zeaxanthin were the most variable (~25%) among the EY-P fractions from different EY suppliers (Fig. 3A). The variability of retinol content in EY was less than 6% (Fig. 3B). Vitamins E and D3 were not quantified but were detected in the EY-P fractions from all suppliers (Fig. 3C). Thus, the EY-P fraction contained all lipophilic vitamins and carotenoids from the EY.</p><p>In summary: The EY-NP fraction contained 20–30% of cholesterol and triglycerides compared to EY (100%). The EY-P fraction contained 70–80% of cholesterol and triglycerides and all lipophilic vitamins and carotenoids seen in EY (100%).</p><!><p>To assess whether similar composition of the EY and EY-P fractions exerted comparable activation of NF-κB, we performed reporter assays in the 3T3-L1-Nfκb-RE preadipocyte cell line. This reporter cell line was previously validated using both pro-inflammatory agents and anti-inflammatory antioxidants.28 We compared Nfκb-RE activation in adipocytes stimulated with the EY, EY-P, and EY-NP fractions. Although both the EY and EY-P fractions contained carotenoids and vitamins, Nfκb-RE activation was suppressed only in adipocytes stimulated with the EY-P fractions. Compared to Nfκb-RE activation in non-treated adipocytes, stimulation with EY increased Nfκb-RE activation to 126%. In contrast, the EY-P fraction from the same EYs reduced Nfκb-RE activation 40% compared to control adipocytes (Fig. 4A). The suppression of Nfκb-RE activation by the EY-P fraction was seen in eggs from different suppliers. The variability of response to the EY, EY-P, and EY-NP fractions from EY obtained from different suppliers reached 33%, 20%, and 30%, respectively (data not shown). The suppression of Nfκb-RE activation by EY-P fraction occurs in a concentration-dependent manner (Fig. 4B).</p><p>The inhibitory effect EY-P fraction on NF-κB target genes was further examined in the classical preadipocyte 3T3-L1 cell line. We stimulated differentiated 3T3-L1 preadipocytes with the EY, EY-P, and EY-NP. Then we simultaneously quantified expression of multiple genes in these cells. First, we analyzed the expression of classical NF-κB target genes, such as MCP1 (CCL2). MCP1 was the most abundantly expressed cytokine in 3T3-L1 adipocytes followed by MCP3 (CCL7) and MCP2 (CCL8) (Fig. 4C). In spite of differences in basal expression, all Mcp genes were markedly suppressed by the EY-P fraction. EY-P inhibited expression of Mcp1 by 59% (Fig. 4D), Mcp2 by 61% (Fig. 4E), and Mcp3 by 49% (Fig. 4F) compared to non-stimulated adipocytes. The EY and EY-NP fractions did not significantly influence expression of these genes.</p><p>We also explored the specificity of cytokine production in response to the EY fractions. All INFs (Infa1: 7±3, Infb1: 51±6, Infg: 7±3 AU) were expressed at low levels in 3T3-L1 adipocytes. The EY fractions did not significantly influence expression of all Infs (data not shown). Cxcl5 and Cxcl1 cytokines co-regulated by NF-κB and STAT1 transcription factors,3, 14 whereas mitogene-inducible gene (Mig), interferon gamma-induced protein 10 (Ip10), and Rantes are co-regulated by NF-κB, STAT1, and IRF transcription factors11, 34–37 (Fig. 5A). Stimulation of 3T3-L1 adipocytes with the EY-P fraction markedly decreased expression of Cxcl5 (Fig. 5B) and Cxcl1 (Fig. 5C) to 9% and 22%, respectively, compared to non-treated control adipocytes. In contrast, all EY, EY-P and EY-NP fractions lead to a modest (less than 40%) reduction in expression of Ip10 (Fig. 5D), Mig (data not shown), or Rantes (Fig. 5E) compared to the control. The reduction of Ip10 and Mig expression was only significant in adipocytes stimulated with the EY-P fraction, and Rantes only with the EY fraction compared to control adipocytes. None of the EY fractions significantly influenced the expression of other Ccl cytokines, Tnfa, and Il6 (data not shown). Given that the EY-P fraction led to the most potent (10-fold) reduction in the expression of Cxcl5, which was known to be regulated by both STAT1 and NF-κB,3, 14, 38 we also analyzed the expression of Stat1. We found 10-fold reduction of Stat1 expression in presence of EY-P in adipocytes (Fig. 5F). Thus, in spite of similar content of lipophilic vitamins in the EY-P and EY fractions, EY-P inhibited the activation of Nfκb-RE and expression of NF-κB target genes and Stat1, whereas EY and EY-NP exerted similar level of NF-κB activation as non-treated adipocytes. The expression of genes regulating apoptosis, such as Bcl2l1 and Fasl, was not different in control and treated cells (Data are not shown).</p><!><p>Among nine Tlr receptors, Tlr2 was the most abundantly expressed receptor in 3T3-L1 adipocytes followed by Tlr3 and Tlr4 (Fig. 6A). Tlr2 expression was similar in non-treated adipocytes and adipocytes treated with EY-NP fraction (Fig. 6B). EY moderately reduced expression of Tlr2 (to 75%), whereas the EY-P fraction decrease Tlr expression to 31% compared to control adipocytes. The expression of two other major, Tlr3 and Tlr4 receptors was not significantly affected by the treatment with any EY fractions (Fig. 6C,D). However, the EY-P fraction significantly increased the expression of Ly96 (to 224%) compared to control adipocytes (Fig. 6E). The interaction of Ly96 with TLR4 prevents ligand binding to this receptor and the activation of NF-κB. Similar up-regulation (to 145%) of expression by the EY-P fraction was also observed in an inhibitory adaptor protein, Tollip, compared to control adipocytes (Fig. 6F). Therefore, decreased activation of NF-κB was associated with inhibited expression of major Tlr either via direct inhibition of expression of Tlr2 or indirectly via up-regulation of the expression of inhibitory Ly96 and Tollip by the EY-P but not the EY-NP and EY fractions.</p><!><p>To obtain a clue for specific regulation of Tlr and inhibitory Ly96 and Tollip by the EY-P, but not the EY and EY-NP fractions, we analyzed the putative binding sites of Srebp. This transcription factor has been established as a sensor of cholesterol/phospholipid ratio on the membranes or by signaling cascades (e.g. insulin).39 We found 3 potential high score SREBP binding sites in Tlr2 (score 92–93), and 2 SREBP binding sites in Tollip (score 86) and one in Ly96 (score 88). Tlr2 and Ly96 could be potentially regulated by NF-κB, because they also contained 5 and 2 potential NF-κB binding sites, respectively. Stat1 contains 2 SREBP and 9 NF-κB binding sites. To examine the putative contribution of SREBP to dissimilar responses among the EY, EY-P and EY-NP fractions we examined the effect of SREBP inhibitor, fatostatin,40 on NF-κB activation in Nfkb-RE/GFP preadipocyte cell line. Fatostatin significantly inhibited Nfκb-RE activation by 52% compared to the activation induced by the EY-NP fraction (Fig. 7A). Thus, SREBP partially contributed to the activation of Nfκb-RE associated with the EY-NP fraction. Next we stimulated of Nfkb-RE/GFP preadipocytes with water soluble cholesterol during differentiation. Cholesterol induced concentration-dependent NF-κB activation that reached 170% plateau compared to non-stimulated adipocytes (Fig. 7B). Stimulations of Nfkb-RE/GFP preadipocytes with α-tocopherol (Fig. 7E), all trans retinol (Fig. 7C) and lutein had the opposite effect. However the significant concentration-dependent inhibition of NF-κB occurred only in presence of all trans retinol and lutein. Each of these compounds suppressed 30% of NF-κB activation compared to non-stimulated adipocytes.</p><!><p>Our data demonstrate that EY contained a mixture of compounds that can effectively suppress the key inflammatory transcription pathway NF-κB in vitro; however, these effects were blunted in the presence of non-polar cholesterol and TG. EY-P compounds efficiently inhibited:</p><!><p>Initiation of receptor-mediated signaling activating NF-κB pathway;</p><p>Binding of transcriptional complex to regulatory DNA sequences; and</p><p>Expression of target cytokines.</p><!><p>The receptors regulating NF-κB in adipocytes exhibit specific pattern. 3T3-L1 adipocytes expressed predominantly three Tlrs: Tlr2>Tlr3=Tlr4. EY-P fraction suppressed expression of Tlr2, whereas the expression of seven other Tlrs remained unchanged. The inhibition of other TLRs, was mediated via inhibitory adaptor protein Tollip and TLR4 inhibitor Ly96. Polar lipids in the EY-P fraction induced expression of these inhibitory proteins. These changes in TLRs were associated with inhibition of NF-κB activation and the expression of NF-κB-dependent genes by EY-P compounds. Unexpectedly, EY supported NF-κB activation, even though the EY and EY-P fractions shared similar content of vitamins, carotenoids, and fatty acids. Partial removal of non-polar cholesterol and TG from the EY was required to reveal its inhibitory effects on the NF-κB pathway. Although these studies were performed in adipocytes, EY fractions expect to exert similar effects in other cells that are under NF-κB and SREBP transcriptional control. Example includes mucosal epithelial cells exposed to EY-containing products such as mayonnaise. In other pathophysiological situations in vivo, EY can potentially have beneficial effects without delipidation.41, 42</p><p>The inhibition of Nfκb-RE activation together with the suppressed expression of NF-κB-regulated cytokines, and TLR2 receptor suggest that EY-P may exert anti-inflammatory properties. The association of anti-inflammatory properties with EY-P, but not EY fractions, appears to be independent of the variation in EY composition, since we showed these effects using regular quality eggs from four different suppliers. The highest variability in composition was associated with cholesterol content in the EY-NP fraction and lutein and zeaxanthin content in the EY-P fraction; however, all EY-P fractions inhibited expression of genes in the NF-κB cascade after partial delipidation. Although numerous studies showed that NF-κB pathways are regulated by fatty acids,12 in our study similar fatty acid composition among the EY fractions suggest their unlikely role in dissimilar NF-κB activation. The mechanism by which cholesterol regulates NF-κB remains unclear. In macrophages, cholesterol mobilization to lipid rafts contributes to TLR activation.43 Activation of NF-κB via TLR4 is blocked by mevinolin, suggesting the activation of TLR4 may require binding of cholesterol or its metabolic precursors.44 Decades of studies with statins, inhibitors of cholesterol synthesis, showed that anti-inflammatory effects of these pharmaceuticals depend on inhibition of NF-κB in a variety of tissues.45–47 The regulation of cholesterol metabolism and fatty acid synthesis is controlled by a SREBP family of transcription factors. In adipocytes, SREBP is activated by insulin/JNK pathways39 and glucose/JAK/STAT pathways48 that regulate differentiation, glycolytic pathways, fatty acid synthesis, and lipid droplet formation in adipocytes.49 In our studies, all proteins participating in NF-κB activation cascade, TLR2, Tollip, and Ly96, had putative binding sites for SREBP. Given that detailed analysis of promoters was beyond the scope of our first study, we accessed the global impact of SREBP in the regulation of NF-κB by EY-NP fraction using its specific inhibitor fatostatin.40
Nfκb-RE activation was significantly reduced by fatostatin compared to non-treated adipocytes, whereas cholesterol induced Nfκb-RE activation in a concentration-dependent manner. However, effects of fatostatin was less pronounced than that achieved by EY-P compounds. Vitamin A and lutein inhibited Nfκb-RE activation at low concentrations and could be potentially accounted for the inhibition mediated by EY-P fraction. Thus, partial delipidation of EY may lead first to the disruption of SREBP-dependent genes involved in NF-κB activation, and then enable lutein and retinol to exert their potential anti-inflammatory effects (Fig. 8).</p><p>A remaining question is: why only partial delipidation was needed to improve the inhibition of NF-κB by the EY-P fraction? Although specific studies are needed to answer this question, a possible explanation can be found in the relationship between membrane composition and activation of SREBP. Recent studies showed that SREBP activation could be triggered by reduced phospholipid concentrations in the membranes.50 After partial delipidation, the EY-P fraction has an increased ratio of phospholipid to cholesterol, which, possibly, prevents SREBP activation. The decrease in ratio of cholesterol to phospholipid could also improve the delivery of lipophilic vitamins and carotenoids to adipocytes.</p><p>Our study also revealed that partial delipidation of EY can influence inflammation via STAT1. This transcription factor is a critical mediator of interferon-mediated inflammation51 and promotes a variety of diseases, including hepatitis3 and type 1 diabetes in experimental animal models.4
Stat1 controls apoptosis in pancreatic β cells52 via transcription of Cxcl1 and Cxcl2. These cytokines which promote neutrophil migration into pancreatic islets.14 Insulin resistance in obese patients5 and patients with hepatitis3 were attributed to another STAT1-regulated cytokine, CXCL5. CXCL1 and CXCL5 are also NF-κB target genes.14, 38 The deficient NF-κB activation and Stat1 expression in our studies markedly diminished expression of Cxcl1 and Cxcl5 expression (by 80% and 90% decrease in the presence of EY-P compared to control). It is unlikely that INF was the major regulator of Stat1 in adipocytes, because the expression of all Inf was very low in adipocytes and was not regulated by EY fractions (data not shown). Accordingly, the expression of INFγ-regulated Ip10 and Rantes was only moderately regulated by EY fractions (Fig. 5). Recent studies showed that CXCL5 could be another mechanism for the activation of STAT1 in adipocytes.53 Suppression of NF-κB activation and CXCL5 expression by EY-P fraction can also result in deficient Stat1 expression. Regardless of these still speculative mechanisms, the results of our study suggest that regular EY contains compounds that can selectively regulate expression of inflammatory cytokines through two major transcriptional pathways, NF-κB and STAT1. Identification of these compounds and/or their mixtures can help to develop novel therapeutics and functional foods, which can potentially improve the treatment of metabolic diseases. Our data help to recognize the pivotal role of lipophilic compounds in EY that can sensitize inflammatory pathways and disable anti-inflammatory effects of all trans retinol and lutein associated with the EY-P fraction. We propose partial delipidation of EY as an efficient solution to improve beneficial effects of EY.</p>