IL-6 is elevated in obese individuals and participates in the metabolic dysfunction associated with that condition. However, the mechanisms that promote IL-6 expression in obesity are incompletely understood. Because elevated levels of palmitate and LPS have been reported in obesity, we investigated whether these agents interact to potentiate IL-6 production. In this study, we report that LPS induces higher levels of IL-6 in human monocytes in the presence of palmitate. Notably, the priming effect of palmitate is associated with enhanced p300 binding and transcription factor recruitment to Il6 promoter regions. Gene silencing of p300 blocks this action of palmitate. RNA polymerase II recruitment was also enhanced at the Il6 promoter in palmitate/LPS-exposed cells. Acetylation levels of H3K9 and H3K18 were increased in monocytes treated with palmitate. Moreover, LPS stimulation of palmitate-treated cells led to increased levels of the transcriptionally permissive acetylation marks H3K9/H3K18 in the Il6 promoter compared with LPS alone. The effect of palmitate on LPS-induced IL-6 production was suppressed by the inhibition of histone acetyltransferases. Conversely, histone deacetylase inhibitors trichostatin A or sodium butyrate can substitute for palmitate in IL-6 production. Esterification of palmitate with CoA was involved, whereas β-oxidation and ceramide biosynthesis were not required, for the induction of IL-6 and H3K9/H3K18 acetylation. Monocytes of obese individuals showed significantly higher H3K9/H3K18 acetylation and Il6 expression. Overall, our findings support a model in which increased levels of palmitate in obesity create a setting for LPS to potentiate IL-6 production via chromatin remodeling, enabling palmitate to contribute to metabolic inflammation.

Obesity is associated with chronic low-grade inflammation and is characterized by increased infiltration of immune cells in expanding adipose tissue, together with increased circulatory levels of proinflammatory cytokines and chemokines, including IL-6, TNF-α, and MCP-1 (1, 2). Obesity is also characterized by increased adipose lipolysis, resulting in a large increase in circulating free fatty acids, including palmitate, the most abundant saturated fatty acid in the circulation in obese individuals (3, 4). Other lipids and lipid-conjugated species are also elevated in the serum of obese humans and mice. In mice, high fat diet increased plasma LPS concentrations are 2- to 3-fold higher than those found in chow diet fed animals (5). Likewise, obese type 2 diabetic individuals have higher circulating LPS levels than do healthy controls, and weight loss reduces LPS levels (6, 7). High-fat diet intake leads to metabolic endotoxemia via increased LPS absorption involving changes in gut microbiota, increased availability of chylomicrons, and greater permeability of the gut epithelium (79). LPS induces a strong immune response via its interaction with the cell surface receptor TLR4 (10). Activation of TLR4 signaling leads to increased production of cytokines/chemokines through transcriptional mediators, including NF-κB, AP-1, and IFN regulatory factors, which promote the development of the chronic low-grade inflammation seen in obesity (1012).

IL-6 is a pleiotropic cytokine with significant effects on metabolic and inflammatory pathways under both normal and disease conditions (1315). IL-6, encoded by the Il6 gene, is produced in large amounts in response to muscle contraction following prolonged exercise, and is released into circulation (16). IL-6 levels are also chronically elevated in obesity (17). Obesity enhances gene and protein expression of IL-6 and its receptor (IL-6R) in human s.c. adipose tissue, which correlates positively with the local expression of several inflammatory markers (18). IL-6 levels are increased in the peripheral circulation with growing adiposity (1922); however, the mechanisms that regulate IL-6 overexpression during overnutrition are not completely understood.

In this study, we report that palmitate potentiates LPS-mediated IL-6 production by increasing acetylation of H3K9 and H3K18 at the Il6 locus. We also found that high levels of p300 histone acetyltransferase (HAT) coactivate NF-κB/AP-1 for maximal Il6 gene expression in the presence of palmitate. Furthermore, RNA polymerase II (Pol II) levels were enhanced at the Il6 promoter in cells treated with palmitate or LPS. Notably, palmitate metabolism and TLR4 are involved in IL-6 expression and Il6 promoter H3K9/H3K18 acetylation. Overall, these findings help to explain the increase in IL-6 levels seen in obesity.

The THP-1 cell line was purchased from American Type Culture Collection (Manassas, VA, catalog no. TIB-202) and cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% FBS (Life Technologies), 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 μg/ml Normocin (InvivoGen, San Diego, CA), and penicillin-streptomycin (100 U/ml-100 μg/ml; Life Technologies), and incubated at 37°C in 5% CO2 under humidity. HEK-Blue IL-6 cells (IL-6 activity reporter monocytic cells) were purchased from InvivoGen (San Diego, CA, catalog no. hkb-hil6) and were cultured in DMEM growth medium (Life Technologies) containing 4.5 g/l glucose, 2 mM l-glutamine, 10% FBS, penicillin-streptomycin (100 U/ml-100 μg/ml), 100 μg/ml Normocin, and 1× HEK-Blue Selection to select for cells expressing the secreted alkaline phosphatase (SEAP)-STAT3 reporter, and cells were incubated at 37°C in 5% CO2 under humidity. TLR4 knockout THP1 monocytic cells were purchased from InvivoGen (San Diego, CA, catalog no. thpd-kotlr4) and were cultured and maintained in complete RPMI 1640 medium contained 10 μg/ml blasticidin and 100 μg/ml Zeocin, and cells were incubated at 37°C in 5% CO2 under humidity.

Human peripheral blood (25–30 ml) samples were collected from healthy volunteers with variable body mass index (BMI) and were classified as lean (n = 10, BMI ≤ 25) and obese (n = 16, BMI ≥ 30) in EDTA vacutainer tubes. All participants gave written informed consent and the study was approved by the Ethics Committee of Dasman Diabetes Institute, Kuwait (reference no. 04/07/2010; RA-2010-003). Physical characteristics of the study participants are shown in Supplemental Table I.

PBMCs were isolated by using the Histo-Paque density gradient method as described earlier (23). Monocytes were purified from PBMCs using an indirect magnetic colloid labeling method (monocyte isolation kit II, human; catalog no. 130-091-153; Miltenyi Biotec) in which nonmonocytic cells, including T cells, NK cells, B cells, dendritic cells, and basophils, were magnetically labeled with a mixture of biotin-conjugated Abs against CD3, CD7, CD16, CD19, CD56, CD123, and CD235a (glycophorin A) surface Ags and anti-biotin MicroBeads. Purified CD14+ monocytes were eluted through magnetic column purification, and purity (>90%) was determined by flow cytometry.

THP-1 and primary human monocytes were plated at a concentration of 1 × 106 cells/ml per well in 12-well plates. Cells were stimulated with palmitate (150 μM; Sigma-Aldrich), methyl palmitate (150 μM; Sigma-Aldrich), and LPS (10 ng/ml), alone or in combination, for 24 h at 37°C. Cells were harvested for RNA isolation, and conditioned media were collected and stored at −80°C until used. Secreted IL-6 protein was measured in cell supernatants using a human DuoSet ELISA kit (R&D systems), following the manufacturer’s instructions.

Total cellular RNA was extracted using a RNeasy mini kit (Qiagen, Valencia, CA), and cDNA was synthesized from 1 μg of total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). cDNA (50 ng) was amplified for real-time RT-PCR using inventoried TaqMan gene expression assay products (IL-6, Hs00985639_m1; p300: Hs00914223_m1; and GAPDH, Hs03929097_g1), and TaqMan gene expression master mix in a 7500 real-time PCR system (Applied Biosystems). The mRNA levels were normalized against GAPDH mRNA, and the expression of IL-6 mRNA relative to control was calculated using the 2−ΔΔCt method. Relative mRNA expression was shown as fold expression over the average of control gene expression taken as 1, and data were presented as mean ± SEM.

HEK-Blue IL-6 cells are HEK293 cells that are stably transfected with a reporter construct, expressing the SEAP gene under the control of a promoter inducible by a STAT3 transcription factor. Upon stimulation, IL-6 triggers STAT3 activation and leads to the secretion of SEAP in cell supernatant. HEK-Blue IL-6 cells were stimulated with palmitate (150 μM) and LPS (10 ng/ml), alone or in combination, for 24 h at 37°C, and SEAP levels were detected in conditioned media after incubation for 4 h with QUANTI-Blue medium OD measurement at 650-nm wavelength.

Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP enzymatic ChIP kit (catalog no. 9003, CST) following the manufacturer’s instructions. Briefly, THP1 cells stimulated with palmitate and LPS were crosslinked with formaldehyde and digested with micrococcal nuclease followed by sonication to yield fragments ranging from 200 to 800 bp using a Covaris system. The digested chromatin fragments were subjected to immunoprecipitation using primary Abs specific to the p65 subunit of NF-κB (catalog no. 8242, CST), c-Jun (catalog no. 9165, CST), p300 (catalog no. 61401, Active Motif), RNA Pol II (catalog no. 1066641, Qiagen), H3K9 (catalog no. 9649, CST), H3K18 (catalog no. 13998, CST), histone H3 (positive IP control, catalog no. 4620, CST), and normal rabbit IgG (negative IP control, catalog no. 2729, CST), for overnight at 4°C and incubated with protein G magnetic beads for 2 h at 4°C. We eluted the chromatin from an Ab/protein G magnetic beads complex by incubation at 65°C for 30 min and by magnetic separation. We then reverse crosslinked the chromatin by treating with Proteinase K for 2 h at 65°C and purified DNA from the ChIP fraction using the spin column method. The enrichment of DNA sequences was then detected by real-time quantitative PCR (qPCR) using SYBR Green mix and EpiTect qPCR primers (Supplemental Table II, primer IDs) specific to the transcription factor binding sites spanning the IL-6 gene promotor region.

Gene silencing was performed using the transient transfection method with an Amaxa cell line Nucleofector kit V (Lonza) and Amaxa electroporation system (Lonza), following the manufacturer’s instructions. For transient transfection, THP-1 cells (1 × 106) were resuspended in Nucleofector solution and transfected separately using 30 nM p300 small interfering RNA (siRNA) and scrambled negative control siRNA. After 36 h, transfected cells were treated with palmitate (150 μm) and LPS (10 ng/ml) for 24 h. Cells were harvested for RNA isolation, and conditioned media were collected for assaying IL-6 production in supernatants. RT-PCR was performed to assess the effective suppression of constitutive p300 expression in THP-1 cells transfected with p300 siRNA and scrambled negative-control siRNAs.

THP-1 or primary monocytic cells were incubated for 30 min with lysis buffer containing Tris (62.5 mM [pH 7.5]), 1% Triton X-100, and 10% glycerol. Cell lysates were centrifuged at 14,000 rpm for 10 min, supernatants were collected, and protein was measured using Quick Start Bradford 1× dye reagent and a protein assay kit (Bio-Rad, Hercules, CA). Samples (20 μg) were mixed with loading buffer, heated for 5 min at 95°C, and resolved by 12% SDS-PAGE. Resolved proteins were transferred to an Immun-Blot polyvinylidene difluoride membrane (Bio-Rad) by electroblotting, blocked with 5% nonfat milk in PBS for 1 h, and incubated overnight at 4°C with primary Abs (1:100 dilution) against H3K9ac (catalog no. 9649, CST), H3K14ac (catalog no. 7627, CST), H3K18ac (catalog no. 13998, CST), H3K23ac (catalog no. 14932), H3K27ac (catalog no. 8173, CST), H3K56ac (catalog no. 4243, CST), histone H3 (catalog no. 4620, CST), and IL-6 (catalog no. 233706, Abcam). Blots were washed three times with TBST and incubated for 2 h with HRP-conjugated secondary Ab (Promega, Madison, WI). Immunoreactive bands were developed using an Amersham ECL Plus Western blotting detection system (GE Healthcare, Buckinghamshire, U.K.) and visualized using a Molecular Imager VersaDoc MP imaging system (Bio-Rad).

The data obtained were expressed as mean ± SEM, and group means were compared using an unpaired t test and one-way ANOVA. GraphPad Prism software (version 6.05; GraphPad Software, La Jolla, CA) was used for statistical analysis, as well as for graphical representation of the data. All p values ≤0.05 were considered statistically significant.

IL-6 expression is enhanced in various metabolic diseases, and elevated serum levels of IL-6 may be involved in the progression of insulin resistance (2427). Palmitate and LPS, both drivers of inflammation, are elevated along with IL-6 in obesity. We first asked whether palmitate and endotoxin (LPS), in combination, might augment the expression of IL-6 in monocytic cells. To test this, we stimulated both THP1 monocytic cells and primary monocytes with palmitate and LPS, alone or in combination for 24 h, and measured IL-6 expression at the gene and protein levels. In both cellular models, palmitate and LPS were each able to induce a significant amount of Il6 mRNA, however, when the cells were cotreated with both agents, there was a higher than additive effect (Fig. 1A, 1C). Secreted IL-6 protein levels were also induced by both palmitate and LPS, and again a multiplicative effect was noted when the two were administered simultaneously (Fig. 1B, 1D).

FIGURE 1.

Palmitate enhances the ability of LPS to increase IL-6 production by monocytic cells. THP-1 cells were incubated for 24 h with palmitate (150 μM), LPS (10 ng/ml), or the combination, before harvest. (A) Total RNA was extracted, and Il6 mRNA was quantified by quantitative RT-PCR. Relative mRNA expression is expressed as fold change. (B) Secreted IL-6 protein in culture media as determined by ELISA. (C and D) Primary monocytes were isolated from PBMCs of healthy volunteers. Monocytes were incubated with palmitate and/or LPS for 24 h. Il6 mRNA (C) and IL-6–secreted protein (D) were determined. (E) IL-6 reporter cells were stimulated with conditioned media for 24 h. Biological activity of the IL-6 reporter cells (HEK-Blue IL-6 cells) allows the detection of bioactive human IL-6 by monitoring the activation of the STAT3 pathway, as described in Materials and Methods. (F and G) Cells were primed with palmitate for 5 h, washed with RPMI 1640, and then treated with LPS. Cells were primed with LPS for 5 h and treated with palmitate. After 24 h of incubation IL-6 was determined. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001. PA, palmitate.

FIGURE 1.

Palmitate enhances the ability of LPS to increase IL-6 production by monocytic cells. THP-1 cells were incubated for 24 h with palmitate (150 μM), LPS (10 ng/ml), or the combination, before harvest. (A) Total RNA was extracted, and Il6 mRNA was quantified by quantitative RT-PCR. Relative mRNA expression is expressed as fold change. (B) Secreted IL-6 protein in culture media as determined by ELISA. (C and D) Primary monocytes were isolated from PBMCs of healthy volunteers. Monocytes were incubated with palmitate and/or LPS for 24 h. Il6 mRNA (C) and IL-6–secreted protein (D) were determined. (E) IL-6 reporter cells were stimulated with conditioned media for 24 h. Biological activity of the IL-6 reporter cells (HEK-Blue IL-6 cells) allows the detection of bioactive human IL-6 by monitoring the activation of the STAT3 pathway, as described in Materials and Methods. (F and G) Cells were primed with palmitate for 5 h, washed with RPMI 1640, and then treated with LPS. Cells were primed with LPS for 5 h and treated with palmitate. After 24 h of incubation IL-6 was determined. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001. PA, palmitate.

Close modal

To ensure that IL-6 produced by palmitate and/or LPS was biologically active, we added conditioned media from treated monocytic cells to HEK-Blue IL-6R cells, a model in which HEK293 cells are stably transfected with the IL-6R gene along with a reporter expressing SEAP. As expected, JAK-STAT3 activity was strongly induced in the IL-6 reporter cells by the conditioned media from cells cotreated with palmitate and LPS as compared with the cells treated with either agent alone (Fig. 1E). These data indicate that IL-6 produced in response to coadministration of palmitate and LPS is biologically active.

To confirm a priming effect of palmitate, we treated cells with palmitate for 5 h before adding LPS for an additional 24 h. As before, we found that priming with palmitate potentiated LPS-induced IL-6 production. However, priming with LPS for 5 h and then exposing to palmitate did not result in enhancement of IL-6 production (Fig. 1F, 1G).

Because the Il6 promoter is regulated by multiple NF-κB and AP-1 responsive sequences (28, 29) (Fig. 2A), we sought to assess whether these factors mediate the priming effect of palmitate on Il6 expression. To verify direct binding of NF-κB to the Il6 promoter, we performed ChIP followed by real-time qPCR. Quantitative ChIP analysis revealed significant recruitment of NF-κB to several of its known binding sites by LPS and palmitate alone, specifically at the −4350, −364, and +6621 positions relative to the transcription start site of the Il6 promoter (Fig. 2B–D), although only the −4350 site showed any degree of additivity. As expected, LPS was able to activate the generic element, and the addition of palmitate enhanced the activity of LPS.

FIGURE 2.

Targeted NF-κB and AP-1 response elements within the Il6 promoter. (A) Schematic of location of three transcription factor binding sites for NF-κB and one binding site for AP-1 in the Il6 promoter. (BE) Cells were treated with vehicle, palmitate, LPS, or palmitate/LPS for 24 h. Chromatin was immunoprecipitated with anti–NF-κB and anti–c-Jun Abs separately. Levels of NF-κB and AP-1 binding were measured using primer for NF-κB or AP-1 DNA binding sites in the Il6 promoter as shown in (A). Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001. PA, palmitate.

FIGURE 2.

Targeted NF-κB and AP-1 response elements within the Il6 promoter. (A) Schematic of location of three transcription factor binding sites for NF-κB and one binding site for AP-1 in the Il6 promoter. (BE) Cells were treated with vehicle, palmitate, LPS, or palmitate/LPS for 24 h. Chromatin was immunoprecipitated with anti–NF-κB and anti–c-Jun Abs separately. Levels of NF-κB and AP-1 binding were measured using primer for NF-κB or AP-1 DNA binding sites in the Il6 promoter as shown in (A). Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001. PA, palmitate.

Close modal

We next assessed whether AP-1 binding to the Il6 promoter is affected by palmitate and/or LPS, using ChIP-PCR of c-Jun as a proxy. We noted significant recruitment of c-Jun to the −364 position after treatment with palmitate or LPS, but we saw no synergistic induction of c-Jun binding when palmitate was used to prime the LPS response (Fig. 2E).

Taken together, our data suggest that NF-κB and AP-1 contribute to the regulation of Il6 gene expression in response to palmitate and LPS, but also that synergy of these two agents does not result from enhanced binding cooperativity.

We next asked whether the priming effect of palmitate was dependent on HAT activity. Chromatin accessibility is increased by acetylation of histones by HATs such as CBP or p300, which serve as versatile transcriptional coactivators (30, 31). To investigate whether p300 is recruited to the NF-κB and AP-1 binding sites of the Il6 promoter after treatment with palmitate and/or LPS, ChIP assays were performed on cross-linked chromatin using anti-p300 Ab followed by qPCR. Our data reveal an abundant level of p300 enrichment in the aforementioned NF-κB and AP-1 binding sites of the Il6 promotor (Fig. 3A–D). Taken together, our results suggest that p300 interactions with NF-κB/AP-1 can augment the transcriptional activation of Il6 gene expression by chromatin remodeling.

FIGURE 3.

p300 is involved in Il6 gene expression by palmitate/LPS. (AD) Monocytic cells were treated with or without palmitate and subsequently stimulated with LPS. ChIP-qPCR was performed on anti-p300 immunoprecipitated chromatin using primers for NF-κB or AP-1 DNA binding sites in the Il6 promoter. Data are expressed as fold enrichment. Results are presented as mean ± SEM from three separate experiments. (E) Monocytic cells were transfected with p300 siRNA or scramble siRNA and transfection efficacy was tested by RT-PCR. (F and G) Cells deficient in p300 were treated as described and IL-6 expression was determined by qPCR and ELISA. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PA, palmitate.

FIGURE 3.

p300 is involved in Il6 gene expression by palmitate/LPS. (AD) Monocytic cells were treated with or without palmitate and subsequently stimulated with LPS. ChIP-qPCR was performed on anti-p300 immunoprecipitated chromatin using primers for NF-κB or AP-1 DNA binding sites in the Il6 promoter. Data are expressed as fold enrichment. Results are presented as mean ± SEM from three separate experiments. (E) Monocytic cells were transfected with p300 siRNA or scramble siRNA and transfection efficacy was tested by RT-PCR. (F and G) Cells deficient in p300 were treated as described and IL-6 expression was determined by qPCR and ELISA. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PA, palmitate.

Close modal

To further confirm the role of p300 in the priming effect of palmitate, monocytic cells were transfected with p300 siRNA and scrambled siRNA control (Fig. 3E). Our data show that p300 deficiency significantly reduced the priming effect of palmitate on Il6 gene expression (Fig. 3F). Consistently, the protein level of IL-6 was significantly downregulated by siRNA-mediated knockdown of p300 (Fig. 3G). Overall, deficiency of p300 eliminated the priming effect of palmitate on LPS-mediated IL-6 production by monocytic cells.

RNA Pol II is the central enzyme that catalyzes DNA-directed mRNA synthesis during the transcription of protein-coding genes (32, 33). Accordingly, we investigated the association of the NF-κB or AP-1 binding sites with RNA Pol II and active transcription of Il6. ChIP of RNA Pol II in palmitate and LPS cotreated cells showed more enrichment of RNA Pol II in the Il6 promotor site compared with RNA Pol II binding in cells treated with palmitate or LPS alone (Fig. 4). These results suggest that the acetylation level mediated by p300 HAT activity facilitates the binding of NF-κB/AP-1 to the Il6 promotor site and becomes more permissive for RNA Pol II to bind to the transcription complex to trigger the Il6 transcription process.

FIGURE 4.

Palmitate/LPS enhances Pol II occupancy on proximal promoter region of Il6. (AD) Monocytic cells were untreated or treated with palmitate and subsequently stimulated with LPS. ChIP-qPCR was performed on anti-RNA Pol II immunoprecipitated chromatin using primers for NF-κB or AP-1 DNA binding sites in the Il6 promoter. Data are expressed as fold enrichment level. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PA, palmitate.

FIGURE 4.

Palmitate/LPS enhances Pol II occupancy on proximal promoter region of Il6. (AD) Monocytic cells were untreated or treated with palmitate and subsequently stimulated with LPS. ChIP-qPCR was performed on anti-RNA Pol II immunoprecipitated chromatin using primers for NF-κB or AP-1 DNA binding sites in the Il6 promoter. Data are expressed as fold enrichment level. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PA, palmitate.

Close modal

Transcription factors activate gene expression in part through the recruitment of HATs, which open chromatin by promoting the acetylation of specific histone residues (34). Conversely, deacetylation of histones by histone deacetylases (HDACs) represses gene expression (35). We next addressed whether palmitate affects the acetylation status of histone H3. We tested the priming effect of palmitate on acetylation at histone H3K4, H3K9, H3K14, H3K18, H3K23, H3K27, and H3K56 (Supplemental Fig. 1). We found that palmitate treatment caused hyperacetylation only at histone H3K9 and H3K18 residues in monocytic cells. However, cells treated with LPS did not display hyperacetylation of histone H3K9 and H3K18 (Fig. 5A–C). These results reflect overall histone acetylation in the cells. To assess how palmitate and LPS affect acetylation at specific regions of the Il6 locus, we used ChIP-PCR, focusing on the same regions at which we previously detected NF-κB and c-Jun binding. The specificity of each ChIP was validated using an IgG control. Palmitate or LPS stimulation of monocytic cells led to an increase in the levels of H3K9ace (Fig. 5D–F) and H3K18ace (Fig. 5G–I) at all three NF-κB binding regions and the single AP-1 binding region examined in the Il6 promoter by quantitative RT-PCR. In comparison, LPS-stimulated H3K9ace and H3K18ace levels were further enhanced in monocytic cells pretreated with palmitate. Taken together, these results indicate that the acetylation of H3K9 and H3K18 is strongly associated with this synergy between palmitate and LPS for Il6 gene transcription.

FIGURE 5.

Palmitate/LPS treatment of the monocytic cells enhances histone modification at the Il6 promoter. (A) Monocytic cells were treated with vehicle, palmitate, LPS, or palmitate+LPS for 5 h. Histone acetylation levels, as detected by Western blot analysis, are shown. (B and C) Quantification of Western blots. Monocytic cells were untreated or treated with palmitate and subsequently stimulated with LPS. (DF) Histone modifications at the Il6 promoter were determined by analyzing chromatin that was immunoprecipitated with anti-acetylated histone H3 lysine 9 (H3K9Ac) or IgG (as a control) Ab. Levels of histone modifications were measured using PCR primers for three NF-κB binding sites. (GI) Histone modifications at the Il6 promoter were determined by analyzing chromatin that was immunoprecipitated with anti-acetylated histone H3 lysine 18 (H3K18Ac) or IgG (as a control) Ab. Levels of histone modifications were measured using PCR primers for three NF-κB DNA binding sites. Differences are expressed as fold enrichment levels. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. PA, palmitate.

FIGURE 5.

Palmitate/LPS treatment of the monocytic cells enhances histone modification at the Il6 promoter. (A) Monocytic cells were treated with vehicle, palmitate, LPS, or palmitate+LPS for 5 h. Histone acetylation levels, as detected by Western blot analysis, are shown. (B and C) Quantification of Western blots. Monocytic cells were untreated or treated with palmitate and subsequently stimulated with LPS. (DF) Histone modifications at the Il6 promoter were determined by analyzing chromatin that was immunoprecipitated with anti-acetylated histone H3 lysine 9 (H3K9Ac) or IgG (as a control) Ab. Levels of histone modifications were measured using PCR primers for three NF-κB binding sites. (GI) Histone modifications at the Il6 promoter were determined by analyzing chromatin that was immunoprecipitated with anti-acetylated histone H3 lysine 18 (H3K18Ac) or IgG (as a control) Ab. Levels of histone modifications were measured using PCR primers for three NF-κB DNA binding sites. Differences are expressed as fold enrichment levels. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. PA, palmitate.

Close modal

We asked whether effects at the level of histone acetylation could account for the priming effect of palmitate on IL-6 production. Consistent with this idea, suppression of HAT activity with anacardic acid did not reduce induction of Il6 gene expression or IL-6 protein secretion by palmitate or LPS alone, but it did have a significant impact on the ability of palmitate to enhance the action of LPS (Fig. 6A, 6B).

FIGURE 6.

Anacardic acid, TSA, and butyrate affect synergistic IL-6 production by palmitate and LPS. Cells were treated with anacardic acid (4 μM) 1 h before vehicle, palmitate, LPS, or palmitate+LPS stimulation for 24 h. Cells and culture supernatant were collected. (A) Total RNA was extracted, and Il6 mRNA was quantified by RT-PCR. (B) Secreted IL-6 protein in culture media was determined by ELISA. (C and D) Cells were treated with TSA (25 nM) for 1 h before stimulation with vehicle, palmitate, LPS, or palmitate+LPS for 24 h. Il6 mRNA and protein were determined. (E) Cells were preincubated with sodium butyrate (2 mM) for 1 h and then stimulated with vehicle, palmitate, LPS, or palmitate+LPS. IL-6 protein was determined. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. **p < 0.01, ***p < 0.001. PA, palmitate.

FIGURE 6.

Anacardic acid, TSA, and butyrate affect synergistic IL-6 production by palmitate and LPS. Cells were treated with anacardic acid (4 μM) 1 h before vehicle, palmitate, LPS, or palmitate+LPS stimulation for 24 h. Cells and culture supernatant were collected. (A) Total RNA was extracted, and Il6 mRNA was quantified by RT-PCR. (B) Secreted IL-6 protein in culture media was determined by ELISA. (C and D) Cells were treated with TSA (25 nM) for 1 h before stimulation with vehicle, palmitate, LPS, or palmitate+LPS for 24 h. Il6 mRNA and protein were determined. (E) Cells were preincubated with sodium butyrate (2 mM) for 1 h and then stimulated with vehicle, palmitate, LPS, or palmitate+LPS. IL-6 protein was determined. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. **p < 0.01, ***p < 0.001. PA, palmitate.

Close modal

Next, we asked whether inhibition of HDACs by trichostatin A (TSA) could substitute for palmitate in potentiating LPS-induced IL-6 production. Preincubation of monocytic cells for 1 h with TSA prior to treatment with LPS significantly increased IL-6 release compared with LPS alone (Fig. 6C, 6D). Similarly, inhibiting HDACs with sodium butyrate substitutes for the priming effect of palmitate (Fig. 6E). In addition, we also tested inhibition of HDAC expression by palmitate and/or LPS and found that expression of HDACs 1, 5, 9, and 10 were significantly inhibited by combined stimulation with palmitate and LPS, but not so by stimulation with palmitate or LPS alone (Supplemental Fig. 2). These data overall indicate that the priming effect of palmitate on IL-6 production is dependent on activity at the level of histone acetylation.

Fatty acids and their metabolites can act directly or indirectly to regulate metabolism and immune function (36). Therefore, we asked whether palmitate needs to be metabolized to exert its synergistic effect with LPS. To test this, THP-1 cells were primed with methyl palmitate, a nonmetabolizable analog of palmitic acid, followed by LPS stimulation. Methyl palmitate priming followed by LPS stimulation did not upregulate IL-6 production compared with palmitate priming (Fig. 7A, 7B), suggesting that activation of palmitate is required for the priming effect. Consistent with this, pretreatment of THP-1 cells with triacsin C, a competitive inhibitor of the acyl-CoA synthetases (ACSLs) that catalyze the coupling of a fatty acid to coenzyme A, significantly reduced the priming effect of palmitate on LPS-induced IL-6 production (Fig. 7C, 7D). To further verify whether the effect of palmitate on LPS-induced IL-6 expression in monocytic cells was ACSL-dependent, we transfected cells with ACSL1 (a dominant isoform in monocytic cells) siRNA, which achieved 60% suppression in ACSL1 gene expression compared with scrambled (control) siRNA (Fig. 7E). Consistent with the pharmacological inhibition data, IL-6 expression was significantly reduced in ACSL1 siRNA-transfected cells compared with control siRNA after palmitate/LPS costimulation (Fig. 7F, 7G).

FIGURE 7.

Palmitate activation is required for the priming effect. THP-1 cells were incubated for 24 h with vehicle, palmitate (150 μM), methyl palmitate (150 μM), LPS (10 ng/ml), or in combination. (A) Total RNA was extracted, and Il6 mRNA was quantified by RT-PCR. Relative mRNA expression is expressed as fold change. (B) Secreted IL-6 protein in culture media as determined by ELISA. (C) IL-6 mRNA expression in THP-1 cells stimulated with vehicle, palmitate, or LPS alone or in combination with LPS in the presence of vehicle (DMSO) or triacsin C (1 μM), etomoxir (5 μM), or myriocin (1 μM) (D) Secreted IL-6 protein in culture media as determined by ELISA. (E) THP-1 monocytic cells were transfected with either control or ACSL1 siRNA and incubated for 36 h. Real-time PCR was done to measure ACSL1 expression. (F and G) ACSL1-deficient THP-1 cells were stimulated with vehicle, palmitate, or LPS alone or in combination for 24 h. IL-6 expression was determined. Data are expressed as mean ± SEM (n = 3) from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PA, palmitate.

FIGURE 7.

Palmitate activation is required for the priming effect. THP-1 cells were incubated for 24 h with vehicle, palmitate (150 μM), methyl palmitate (150 μM), LPS (10 ng/ml), or in combination. (A) Total RNA was extracted, and Il6 mRNA was quantified by RT-PCR. Relative mRNA expression is expressed as fold change. (B) Secreted IL-6 protein in culture media as determined by ELISA. (C) IL-6 mRNA expression in THP-1 cells stimulated with vehicle, palmitate, or LPS alone or in combination with LPS in the presence of vehicle (DMSO) or triacsin C (1 μM), etomoxir (5 μM), or myriocin (1 μM) (D) Secreted IL-6 protein in culture media as determined by ELISA. (E) THP-1 monocytic cells were transfected with either control or ACSL1 siRNA and incubated for 36 h. Real-time PCR was done to measure ACSL1 expression. (F and G) ACSL1-deficient THP-1 cells were stimulated with vehicle, palmitate, or LPS alone or in combination for 24 h. IL-6 expression was determined. Data are expressed as mean ± SEM (n = 3) from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PA, palmitate.

Close modal

Palmitoyl-CoA can have many fates in the cell, but the two best characterized are importation into the mitochondria for β-oxidation or conversion to sphingosine by serine palmitoyltransferase, with subsequent metabolism to ceramides. Pretreatment of THP-1 cells with etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT I), the rate-limiting enzyme for fatty acyl-CoA uptake into the mitochondria, did not suppress IL-6 production in response to palmitate/LPS costimulation (Fig. 7C, 7D), and in fact it enhanced the priming effect. We next turned our attention to the sphingosine pathway, as ceramides have been implicated in inducing inflammatory signaling pathways in numerous cell types (37). THP-1 cells were pretreated with the serine palmitoyltransferase inhibitor myriocin. IL-6 production by palmitate/LPS costimulation was not reduced by myriocin pretreatment (Fig. 7C, 7D) and was actually increased. Altogether, our results indicate that palmitate must be activated to palmitoyl-CoA to exert the priming effect, but the relevant disposition of the palmitoyl-CoA remains unclear and unlikely to involve β-oxidation or sphingosine synthesis. Consistent with the inability of methyl palmitate to prime Il6 expression, the nonmetabolizable analog was also unable to induce hyperacetylation at H3K9 and H3K18, either overall or at the specific regions where we previously detected NF-κB and c-Jun binding (Fig. 8).

FIGURE 8.

Methyl palmitate does not enhance histone modification at the Il6 promoter. (A) Monocytic cells were treated with vehicle, palmitate, or methyl palmitate for 5 h. Histone acetylation levels were detected by Western blot. (B and C) Quantification of Western blots. (DF) Histone modifications at the Il6 promoter were determined by analyzing chromatin immunoprecipitated with anti-acetylated histone 3 lysine 9 (H3K9Ac) or IgG (as a control). Levels of histone modifications were measured using PCR primers for three NF-κB binding sites. (GI) Histone modifications at the Il6 promoter were determined by analyzing chromatin that was immunoprecipitated with anti-acetylated histone 3 lysine 18 (H3K18Ac) or IgG (as a control). Levels of histone modifications were measured using PCR primers for three NF-κB DNA binding sites. Differences are expressed as fold enrichment levels. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. PA, palmitate.

FIGURE 8.

Methyl palmitate does not enhance histone modification at the Il6 promoter. (A) Monocytic cells were treated with vehicle, palmitate, or methyl palmitate for 5 h. Histone acetylation levels were detected by Western blot. (B and C) Quantification of Western blots. (DF) Histone modifications at the Il6 promoter were determined by analyzing chromatin immunoprecipitated with anti-acetylated histone 3 lysine 9 (H3K9Ac) or IgG (as a control). Levels of histone modifications were measured using PCR primers for three NF-κB binding sites. (GI) Histone modifications at the Il6 promoter were determined by analyzing chromatin that was immunoprecipitated with anti-acetylated histone 3 lysine 18 (H3K18Ac) or IgG (as a control). Levels of histone modifications were measured using PCR primers for three NF-κB DNA binding sites. Differences are expressed as fold enrichment levels. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. PA, palmitate.

Close modal

To confirm that TLR4 receptor signaling is involved in the priming effect, we assessed H3K9 and H3K18 hyperacetylation in response to palmitate and methyl palmitate in TLR4 knockout THP-1 cells. Neither analog was able to induce acetylation in the absence of TLR4 signaling (Supplemental Fig. 3A). Furthermore, we noted that TLR4 expression was increased on the surface of monocytes when treated with palmitate, LPS, or costimulation with palmitate and LPS (Supplemental Fig. 4A, 4B). However, no significant change was detected in TNFR1a surface expression following these treatments in monocytic cells (Supplemental Fig. 4C).

Our in vitro data show the involvement of H3K9/H3K18 acetylation in Il6 gene expression and protein production. Next, we asked whether these data were relevant to the clinical setting of obesity. Therefore, we determined the expression of Il6 and acetylation levels of H3K9/H3K18 in monocytes of lean and obese individuals. Our data show that H3K9/H3K18 acetylation and expression of Il6 mRNA and IL-6 protein were elevated in the monocytes of obese compared with lean individuals (Fig. 9). Taken together, our data demonstrate that H3K9/H3K18 hyperacetylation is associated with enhanced IL-6 expression in monocytes of obese individuals.

FIGURE 9.

IL-6 expression along with H3K9 ace and H3K18 ace in monocytes from obese individuals. Monocytic cells were isolated from human peripheral blood samples obtained from lean (n =10) and obese (n = 16) individuals. (A) Il6 mRNA expression was detected by quantitative RT-PCR and represented as fold change over controls. (B) Cell lysates were prepared, and IL-6 protein and H3K9 and H3K18 acetylation was determined by Western blotting. (CE) Quantification of Western blots. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05.

FIGURE 9.

IL-6 expression along with H3K9 ace and H3K18 ace in monocytes from obese individuals. Monocytic cells were isolated from human peripheral blood samples obtained from lean (n =10) and obese (n = 16) individuals. (A) Il6 mRNA expression was detected by quantitative RT-PCR and represented as fold change over controls. (B) Cell lysates were prepared, and IL-6 protein and H3K9 and H3K18 acetylation was determined by Western blotting. (CE) Quantification of Western blots. Data are expressed as mean ± SEM values from three replicates of each experiment, and similar data were obtained from three independent experiments. *p < 0.05.

Close modal

In the current study we demonstrate that the palmitate is able to significantly potentiate the effect of LPS on Il6 gene expression and protein secretion in human monocytic cells via epigenetic mechanisms. Both palmitate and LPS levels are increased in the plasma of obese individuals (4, 6, 7, 38), and we have previously found that palmitate can potentiate the transcription of other inflammatory genes in obesity (11, 39, 40). Saturated free fatty acids and endotoxin (LPS) are well-documented stimulators of monocytes and macrophages and contribute to obesity-related chronic inflammation and insulin resistance (5, 41, 42). Our results show that monocytic cells exposed to palmitate before exposure to LPS enables higher Il6 gene expression and protein production. Analogous to our findings, Bunn et al. (43) showed that palmitate enhances the production of IL-6 by monocytes in the presence of insulin, and Li et al. (44) reported that palmitate and LPS promote IL-6 expression in hepatocytes. However, the mechanistic events occurring in these systems were not elucidated.

Palmitate and LPS both activate two major downstream transcription factors, that is, NF-κB and AP-1 (11, 45), and binding sites for these factors have been identified in the Il6 promoter region (28, 29). Because the Il6 promoter is regulated in part by multiple NF-κB responsive sequences, we sought to assess the role of each NF-κB responsive sequence for the priming effect of palmitate on Il6 expression. Our data suggest that NF-κB and AP-1 contribute to the regulation of Il6 gene expression in response to palmitate and LPS, but we did not detect enhanced binding cooperativity associated with palmitate priming.

Because our results showed that palmitate priming potentiates LPS-induced production of IL-6, we speculated that palmitate may induce epigenetic modifications that may enhance the LPS-induced transcription of the Il6 gene. Multiple studies have demonstrated that histone acetylation is associated with gene expression and active transcription (46, 47). p300 is broadly implicated in controlling H3K acetylation and thereby plays a significant role as a coactivator of transcription factors, including NF-κB and AP-1 (48). Our ChIP-qPCR data showed that p300 is targeted to the Il6 promoter in cells after exposure to palmitate, LPS, or a combination of the two. Furthermore, disruption of p300 by gene knockdown confirmed its critical role in Il6 transcription in the cells exposed to palmitate and LPS.

Histone acetylation promotes transcription by weakening electrostatic interactions between DNA and histones and between adjacent nucleosomes, which makes DNA more accessible to transcription factors and RNA Pol II (49). Pol II recruitment to the promoter is a hallmark of transcriptional induction (50). In our study we found that priming of the palmitate enhanced the LPS-induced association of Pol II to the Il6 promoter corresponding with IL-6 production. Preinitiation complexes containing RNA Pol II are present on the promoters of several genes along with epigenetic histone acetylated marks (33, 51). Because acetylation is involved in the priming effect of palmitate for the production of IL-6, we next addressed the possibility that palmitate alone or in combination could contribute to the acetylation status of histone H3. Previous studies have shown that palmitate can co-operate with other stimuli to promote inflammation (11, 52), but exact epigenetic mechanisms were not explored. We found that palmitate priming caused hyperacetylation at histone H3K9 and H3K18 residues in monocytic cells. However, cells treated with LPS alone did not display hyperacetylation of histone H3K9 and H3K18. Next, we identified the mechanistic role of H3K9/H3K18 modifications in regulating transcriptional activation of the Il6 gene in palmitate-, LPS-, or palmitate/LPS-treated cells. LPS stimulation of palmitate pre-exposed cells led to changes in Il6 promoter histone acetylation as seen by an increase in the levels of transcriptionally permissive H3K9/H3K18. These data show that H3K9/H3K18 acetylation plays a significant role in the regulation of Il6 gene expression under the influence of palmitate and LPS. Similar modulation of H3K9 and H3K18 acetylation was reported to be a regulatory feature of other actively transcribed inflammatory genes such as Tnfa and Ccl2 in the setting of obesity (53), although those studies did not clarify the role of any specific fatty acid in acetylation. Our findings indicate that acetylated histone H3K9/H3K18 is enriched at the Il6 gene promoter and highly correlates with gene expression in the presence of palmitate.

We also found that inhibition of histone acetylation blocks the priming effect of palmitate for IL-6 production. Suppression of histone acetylation via anacardic acid inhibited the priming effect of palmitate on IL-6 production. Increased acetylation by HDAC inhibitors (TSA or butyrate) substitutes the palmitate priming effect in this synergy. Our results are in line with previous studies showing that inhibition of HAT by TSA reduces LPS-induced IL-8 production (54). Taken together, our data showed that treatment of monocytic cells with LPS in the presence of palmitate significantly increases the levels of H3K9/H3K18 acetylation, p300-HAT, as well as Pol II and NF-κB/AP-1 recruitment at Il6 promoter regions, all corresponding with the increased Il6 gene expression/production.

Because fatty acid metabolism is involved in induction of inflammatory responses (44, 52, 55), we tested whether palmitate metabolism plays a role in the priming effect of palmitate. We used genetic and pharmacological methods to show that palmitate must be activated to palmitoyl-CoA to see the priming effect. Furthermore, our siRNA studies suggest a specific role for ACSL1. Bunn et al. (43) showed that increased fatty acid flux through the glycerolipid biosynthesis pathway was involved in proinflammatory cytokine expression in monocytes. Unexpectedly, we found that neither inhibition of β-oxidation nor repression of de novo ceramide biosynthesis was able to block the priming effect of palmitate, and in fact enhanced it. One explanation for the enhancement is that blocking downstream metabolism through these pathways increases the pool of palmitoyl-CoA available for use by other metabolic pathways that can stimulate IL-6 expression. One such fate might be esterification in the triglyceride synthesis pathway, the intermediates of which have been implicated in promoting IL-6 expression in a variety of cells (56, 57). Consistent with our findings in monocytic cells, Staiger et al. (58) showed that neither β-oxidation of fatty acids nor ceramide biosynthesis was involved in IL-6 production by palmitate in endothelial cells. However, Schwartz et al. (59) demonstrated that palmitate metabolism to ceramide was critical to the amplification of LPS-induced inflammation in human monocytes. Li et al. (44) reported that LPS and palmitate stimulate de novo ceramide synthesis, which plays an essential role in the synergistic stimulation of IL-6 gene expression by LPS and palmitate in hepatocytes. Thus, the specific mechanisms of this synergy may be context-dependent. Palmitate is believed to operate through TLR4-dependent pathways (60, 61), which our data further corroborate.

Several lines of evidence show that obese humans and mice have high levels of palmitate, LPS, and IL-6 (21, 58, 62, 63). Our in vitro studies showed that LPS and palmitate interaction enhances IL-6 production through acetylation of H3K9 and H3K18. To identify the clinical relevance of this study, we asked whether the levels of acetylation of H3K9/H3K18 were increased along with IL-6 expression in monocytes isolated from obese individuals. Our data also point to a profound relationship among these markers in obese individuals which highlights its significance in metabolic inflammation and human health and disease.

In summary, our findings support a model in which increased levels of palmitate in obesity create a setting for LPS to potentiate IL-6 production through H3K9ace/H3K18ace, p300, Pol II, and the NF-κB/AP-1 axis, representing a possible contribution of palmitate to metabolic inflammation.

This work was supported by Kuwait Foundation for the Advancement of Sciences Grant RA AM 2020-017 (to R.A.) and National Institutes of Health Grant DK R01102170 (to E.D.R.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • ACSL

    acyl-CoA synthetase

  •  
  • BMI

    body mass index

  •  
  • ChIP

    chromatin immunoprecipitation

  •  
  • HAT

    histone acetyltransferase

  •  
  • HDAC

    histone deacetylase

  •  
  • Pol II

    polymerase II

  •  
  • qPCR

    quantitative PCR

  •  
  • SEAP

    secreted alkaline phosphatase

  •  
  • siRNA

    small interfering RNA

  •  
  • TSA

    trichostatin A

1.
Xu
H.
,
G. T.
Barnes
,
Q.
Yang
,
G.
Tan
,
D.
Yang
,
C. J.
Chou
,
J.
Sole
,
A.
Nichols
,
J. S.
Ross
,
L. A.
Tartaglia
,
H.
Chen
.
2003
.
Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.
J. Clin. Invest.
112
:
1821
1830
.
2.
Weisberg
S. P.
,
D.
McCann
,
M.
Desai
,
M.
Rosenbaum
,
R. L.
Leibel
,
A. W.
Ferrante
Jr
.
2003
.
Obesity is associated with macrophage accumulation in adipose tissue.
J. Clin. Invest.
112
:
1796
1808
.
3.
Reid
B. N.
,
G. P.
Ables
,
O. A.
Otlivanchik
,
G.
Schoiswohl
,
R.
Zechner
,
W. S.
Blaner
,
I. J.
Goldberg
,
R. F.
Schwabe
,
S. C.
Chua
Jr.
,
L. S.
Huang
.
2008
.
Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis.
J. Biol. Chem.
283
:
13087
13099
.
4.
Quehenberger
O.
,
A. M.
Armando
,
A. H.
Brown
,
S. B.
Milne
,
D. S.
Myers
,
A. H.
Merrill
,
S.
Bandyopadhyay
,
K. N.
Jones
,
S.
Kelly
,
R. L.
Shaner
, et al
2010
.
Lipidomics reveals a remarkable diversity of lipids in human plasma.
J. Lipid Res.
51
:
3299
3305
.
5.
Cani
P. D.
,
J.
Amar
,
M. A.
Iglesias
,
M.
Poggi
,
C.
Knauf
,
D.
Bastelica
,
A. M.
Neyrinck
,
F.
Fava
,
K. M.
Tuohy
,
C.
Chabo
, et al
2007
.
Metabolic endotoxemia initiates obesity and insulin resistance.
Diabetes
56
:
1761
1772
.
6.
Creely
S. J.
,
P. G.
McTernan
,
C. M.
Kusminski
,
M.
Fisher
,
N. F.
Da Silva
,
M.
Khanolkar
,
M.
Evans
,
A. L.
Harte
,
S.
Kumar
.
2007
.
Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes.
Am. J. Physiol. Endocrinol. Metab.
292
:
E740
E747
.
7.
Monte
S. V.
,
J. A.
Caruana
,
H.
Ghanim
,
C. L.
Sia
,
K.
Korzeniewski
,
J. J.
Schentag
,
P.
Dandona
.
2012
.
Reduction in endotoxemia, oxidative and inflammatory stress, and insulin resistance after Roux-en-Y gastric bypass surgery in patients with morbid obesity and type 2 diabetes mellitus.
Surgery
151
:
587
593
.
8.
Ghoshal
S.
,
J.
Witta
,
J.
Zhong
,
W.
de Villiers
,
E.
Eckhardt
.
2009
.
Chylomicrons promote intestinal absorption of lipopolysaccharides.
J. Lipid Res.
50
:
90
97
.
9.
Cani
P. D.
,
S.
Possemiers
,
T.
Van de Wiele
,
Y.
Guiot
,
A.
Everard
,
O.
Rottier
,
L.
Geurts
,
D.
Naslain
,
A.
Neyrinck
,
D. M.
Lambert
, et al
2009
.
Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability.
Gut
58
:
1091
1103
.
10.
Schäffler
A.
,
J.
Schölmerich
.
2010
.
Innate immunity and adipose tissue biology.
Trends Immunol.
31
:
228
235
.
11.
Ahmad
R.
,
A.
Al-Roub
,
S.
Kochumon
,
N.
Akther
,
R.
Thomas
,
M.
Kumari
,
M. S.
Koshy
,
A.
Tiss
,
Y. A.
Hannun
,
J.
Tuomilehto
, et al
2018
.
The synergy between palmitate and TNF-α for CCL2 production is dependent on the TRIF/IRF3 pathway: implications for metabolic inflammation.
J. Immunol.
200
:
3599
3611
.
12.
Kumari
M.
,
X.
Wang
,
L.
Lantier
,
A.
Lyubetskaya
,
J.
Eguchi
,
S.
Kang
,
D.
Tenen
,
H. C.
Roh
,
X.
Kong
,
L.
Kazak
, et al
2016
.
IRF3 promotes adipose inflammation and insulin resistance and represses browning.
J. Clin. Invest.
126
:
2839
2854
.
13.
Kado
S.
,
T.
Nagase
,
N.
Nagata
.
1999
.
Circulating levels of interleukin-6, its soluble receptor and interleukin-6/interleukin-6 receptor complexes in patients with type 2 diabetes mellitus.
Acta Diabetol.
36
:
67
72
.
14.
Scheller
J.
,
A.
Chalaris
,
D.
Schmidt-Arras
,
S.
Rose-John
.
2011
.
The pro- and anti-inflammatory properties of the cytokine interleukin-6.
Biochim. Biophys. Acta
1813
:
878
888
.
15.
Wieckowska
A.
,
B. G.
Papouchado
,
Z.
Li
,
R.
Lopez
,
N. N.
Zein
,
A. E.
Feldstein
.
2008
.
Increased hepatic and circulating interleukin-6 levels in human nonalcoholic steatohepatitis.
Am. J. Gastroenterol.
103
:
1372
1379
.
16.
Hennigar
S. R.
,
J. P.
McClung
,
S. M.
Pasiakos
.
2017
.
Nutritional interventions and the IL-6 response to exercise.
FASEB J.
31
:
3719
3728
.
17.
Eder
K.
,
N.
Baffy
,
A.
Falus
,
A. K.
Fulop
.
2009
.
The major inflammatory mediator interleukin-6 and obesity.
Inflamm. Res.
58
:
727
736
.
18.
Sindhu
S.
,
R.
Thomas
,
P.
Shihab
,
D.
Sriraman
,
K.
Behbehani
,
R.
Ahmad
.
2015
.
Obesity is a positive modulator of IL-6R and IL-6 expression in the subcutaneous adipose tissue: significance for metabolic inflammation.
PLoS One
10
:
e0133494
.
19.
Mohamed-Ali
V.
,
S.
Goodrick
,
A.
Rawesh
,
D. R.
Katz
,
J. M.
Miles
,
J. S.
Yudkin
,
S.
Klein
,
S. W.
Coppack
.
1997
.
Subcutaneous adipose tissue releases interleukin-6, but not tumor necrosis factor-α, in vivo.
J. Clin. Endocrinol. Metab.
82
:
4196
4200
.
20.
Park
E. J.
,
J. H.
Lee
,
G.-Y.
Yu
,
G.
He
,
S. R.
Ali
,
R. G.
Holzer
,
C. H.
Osterreicher
,
H.
Takahashi
,
M.
Karin
.
2010
.
Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression.
Cell
140
:
197
208
.
21.
Bastard
J. P.
,
C.
Jardel
,
E.
Bruckert
,
P.
Blondy
,
J.
Capeau
,
M.
Laville
,
H.
Vidal
,
B.
Hainque
.
2000
.
Elevated levels of interleukin 6 are reduced in serum and subcutaneous adipose tissue of obese women after weight loss.
J. Clin. Endocrinol. Metab.
85
:
3338
3342
.
22.
Cottam
D. R.
,
S. G.
Mattar
,
E.
Barinas-Mitchell
,
G.
Eid
,
L.
Kuller
,
D. E.
Kelley
,
P. R.
Schauer
.
2004
.
The chronic inflammatory hypothesis for the morbidity associated with morbid obesity: implications and effects of weight loss.
Obes. Surg.
14
:
589
600
.
23.
Ahmad
R.
,
S.
El Bassam
,
P.
Cordeiro
,
J.
Menezes
.
2008
.
Requirement of TLR2-mediated signaling for the induction of IL-15 gene expression in human monocytic cells by HSV-1.
Blood
112
:
2360
2368
.
24.
Senn
J. J.
,
P. J.
Klover
,
I. A.
Nowak
,
R. A.
Mooney
.
2002
.
Interleukin-6 induces cellular insulin resistance in hepatocytes.
Diabetes
51
:
3391
3399
.
25.
Rotter
V.
,
I.
Nagaev
,
U.
Smith
.
2003
.
Interleukin-6 (IL-6) induces insulin resistance in 3T3-L1 adipocytes and is, like IL-8 and tumor necrosis factor-α, overexpressed in human fat cells from insulin-resistant subjects.
J. Biol. Chem.
278
:
45777
45784
.
26.
Kern
P. A.
,
S.
Ranganathan
,
C.
Li
,
L.
Wood
,
G.
Ranganathan
.
2001
.
Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance.
Am. J. Physiol. Endocrinol. Metab.
280
:
E745
E751
.
27.
Rieusset
J.
,
K.
Bouzakri
,
E.
Chevillotte
,
N.
Ricard
,
D.
Jacquet
,
J. P.
Bastard
,
M.
Laville
,
H.
Vidal
.
2004
.
Suppressor of cytokine signaling 3 expression and insulin resistance in skeletal muscle of obese and type 2 diabetic patients.
Diabetes
53
:
2232
2241
.
28.
Libermann
T. A.
,
D.
Baltimore
.
1990
.
Activation of interleukin-6 gene expression through the NF-kappa B transcription factor.
Mol. Cell. Biol.
10
:
2327
2334
.
29.
Xiao
W.
,
D. R.
Hodge
,
L.
Wang
,
X.
Yang
,
X.
Zhang
,
W. L.
Farrar
.
2004
.
NF-kappaB activates IL-6 expression through cooperation with c-Jun and IL6-AP1 site, but is independent of its IL6-NFkappaB regulatory site in autocrine human multiple myeloma cells.
Cancer Biol. Ther.
3
:
1007
1017
.
30.
Chan
H. M.
,
N. B.
La Thangue
.
2001
.
p300/CBP proteins: HATs for transcriptional bridges and scaffolds.
J. Cell Sci.
114
:
2363
2373
.
31.
Dal Piaz
F.
,
A.
Tosco
,
D.
Eletto
,
A. L.
Piccinelli
,
O.
Moltedo
,
S.
Franceschelli
,
G.
Sbardella
,
P.
Remondelli
,
L.
Rastrelli
,
L.
Vesci
, et al
2010
.
The identification of a novel natural activator of p300 histone acetyltranferase provides new insights into the modulation mechanism of this enzyme.
ChemBioChem
11
:
818
827
.
32.
Orphanides
G.
,
T.
Lagrange
,
D.
Reinberg
.
1996
.
The general transcription factors of RNA polymerase II.
Genes Dev.
10
:
2657
2683
.
33.
Guenther
M. G.
,
S. S.
Levine
,
L. A.
Boyer
,
R.
Jaenisch
,
R. A.
Young
.
2007
.
A chromatin landmark and transcription initiation at most promoters in human cells.
Cell
130
:
77
88
.
34.
Sterner
D. E.
,
S. L.
Berger
.
2000
.
Acetylation of histones and transcription-related factors.
Microbiol. Mol. Biol. Rev.
64
:
435
459
.
35.
Chen
H. P.
,
Y. T.
Zhao
,
T. C.
Zhao
.
2015
.
Histone deacetylases and mechanisms of regulation of gene expression.
Crit. Rev. Oncog.
20
:
35
47
.
36.
Cullberg
K. B.
,
J. Ø.
Larsen
,
S. B.
Pedersen
,
B.
Richelsen
.
2014
.
Effects of LPS and dietary free fatty acids on MCP-1 in 3T3-L1 adipocytes and macrophages in vitro.
Nutr. Diabetes
4
:
e113
.
37.
Gill
J. M.
,
N.
Sattar
.
2009
.
Ceramides: a new player in the inflammation-insulin resistance paradigm?
Diabetologia
52
:
2475
2477
.
38.
Boden
G.
2008
.
Obesity and free fatty acids.
Endocrinol. Metab. Clin. North Am.
37
:
635
646
,
viii
ix
.
39.
Hasan
A.
,
N.
Akhter
,
A.
Al-Roub
,
R.
Thomas
,
S.
Kochumon
,
A.
Wilson
,
M.
Koshy
,
E.
Al-Ozairi
,
F.
Al-Mulla
,
R.
Ahmad
.
2019
.
TNF-α in combination with palmitate enhances IL-8 production via the MyD88-independent TLR4 signaling pathway: potential relevance to metabolic inflammation.
Int. J. Mol. Sci.
20
:
4112
.
40.
Sindhu
S.
,
N.
Akhter
,
A.
Wilson
,
R.
Thomas
,
H.
Arefanian
,
A.
Al Madhoun
,
F.
Al-Mulla
,
R.
Ahmad
.
2020
.
MIP-1α expression induced by co-stimulation of human monocytic cells with palmitate and TNF-α involves the TLR4-IRF3 pathway and is amplified by oxidative stress.
Cells
9
:
1799
.
41.
Boden
G.
,
X.
Chen
,
J.
Ruiz
,
J. V.
White
,
L.
Rossetti
.
1994
.
Mechanisms of fatty acid-induced inhibition of glucose uptake.
J. Clin. Invest.
93
:
2438
2446
.
42.
Liang
H.
,
S. E.
Hussey
,
A.
Sanchez-Avila
,
P.
Tantiwong
,
N.
Musi
.
2013
.
Effect of lipopolysaccharide on inflammation and insulin action in human muscle.
PLoS One
8
:
e63983
.
43.
Bunn
R. C.
,
G. E.
Cockrell
,
Y.
Ou
,
K. M.
Thrailkill
,
C. K.
Lumpkin
Jr.
,
J. L.
Fowlkes
.
2010
.
Palmitate and insulin synergistically induce IL-6 expression in human monocytes.
Cardiovasc. Diabetol.
9
:
73
.
44.
Li
Y.
,
Z.
Lu
,
J. H.
Ru
,
M. F.
Lopes-Virella
,
T. J.
Lyons
,
Y.
Huang
.
2018
.
Saturated fatty acid combined with lipopolysaccharide stimulates a strong inflammatory response in hepatocytes in vivo and in vitro.
Am. J. Physiol. Endocrinol. Metab.
315
:
E745
E757
.
45.
Rogero
M. M.
,
P. C.
Calder
.
2018
.
Obesity, inflammation, Toll-like receptor 4 and fatty acids.
Nutrients
10
:
432
.
46.
Grunstein
M.
1997
.
Histone acetylation in chromatin structure and transcription.
Nature
389
:
349
352
.
47.
Struhl
K.
1998
.
Histone acetylation and transcriptional regulatory mechanisms.
Genes Dev.
12
:
599
606
.
48.
Iyer
N. G.
,
H.
Özdag
,
C.
Caldas
.
2004
.
p300/CBP and cancer.
Oncogene
23
:
4225
4231
.
49.
Peleg
S.
,
C.
Feller
,
A. G.
Ladurner
,
A.
Imhof
.
2016
.
The metabolic impact on histone acetylation and transcription in ageing.
Trends Biochem. Sci.
41
:
700
711
.
50.
Adelman
K.
,
M. A.
Kennedy
,
S.
Nechaev
,
D. A.
Gilchrist
,
G. W.
Muse
,
Y.
Chinenov
,
I.
Rogatsky
.
2009
.
Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling.
Proc. Natl. Acad. Sci. USA
106
:
18207
18212
.
51.
Kim
T. H.
,
L. O.
Barrera
,
M.
Zheng
,
C.
Qu
,
M. A.
Singer
,
T. A.
Richmond
,
Y.
Wu
,
R. D.
Green
,
B.
Ren
.
2005
.
A high-resolution map of active promoters in the human genome.
Nature
436
:
876
880
.
52.
Korbecki
J.
,
K.
Bajdak-Rusinek
.
2019
.
The effect of palmitic acid on inflammatory response in macrophages: an overview of molecular mechanisms.
Inflamm. Res.
68
:
915
932
.
53.
Mikula
M.
,
A.
Majewska
,
J. K.
Ledwon
,
A.
Dzwonek
,
J.
Ostrowski
.
2014
.
Obesity increases histone H3 lysine 9 and 18 acetylation at Tnfa and Ccl2 genes in mouse liver.
Int. J. Mol. Med.
34
:
1647
1654
.
54.
Tsaprouni
L. G.
,
K.
Ito
,
I. M.
Adcock
,
N.
Punchard
.
2007
.
Suppression of lipopolysaccharide- and tumour necrosis factor-α-induced interleukin (IL)-8 expression by glucocorticoids involves changes in IL-8 promoter acetylation.
Clin. Exp. Immunol.
150
:
151
157
.
55.
Al-Rashed
F.
,
Z.
Ahmad
,
M. A.
Iskandar
,
J.
Tuomilehto
,
F.
Al-Mulla
,
R.
Ahmad
.
2019
.
TNF-α induces a pro-inflammatory phenotypic shift in monocytes through ACSL1: relevance to metabolic inflammation.
Cell. Physiol. Biochem.
52
:
397
407
.
56.
Coleman
R. A.
,
T. M.
Lewin
,
D. M.
Muoio
.
2000
.
Physiological and nutritional regulation of enzymes of triacylglycerol synthesis.
Annu. Rev. Nutr.
20
:
77
103
.
57.
Schenk
S.
,
M.
Saberi
,
J. M.
Olefsky
.
2008
.
Insulin sensitivity: modulation by nutrients and inflammation.
J. Clin. Invest.
118
:
2992
3002
.
58.
Staiger
H.
,
K.
Staiger
,
N.
Stefan
,
H. G.
Wahl
,
F.
Machicao
,
M.
Kellerer
,
H. U.
Häring
.
2004
.
Palmitate-induced interleukin-6 expression in human coronary artery endothelial cells.
Diabetes
53
:
3209
3216
.
59.
Schwartz
E. A.
,
W. Y.
Zhang
,
S. K.
Karnik
,
S.
Borwege
,
V. R.
Anand
,
P. S.
Laine
,
Y.
Su
,
P. D.
Reaven
.
2010
.
Nutrient modification of the innate immune response: a novel mechanism by which saturated fatty acids greatly amplify monocyte inflammation.
Arterioscler. Thromb. Vasc. Biol.
30
:
802
808
.
60.
Holland
W. L.
,
B. T.
Bikman
,
L. P.
Wang
,
G.
Yuguang
,
K. M.
Sargent
,
S.
Bulchand
,
T. A.
Knotts
,
G.
Shui
,
D. J.
Clegg
,
M. R.
Wenk
, et al
2011
.
Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice.
J. Clin. Invest.
121
:
1858
1870
.
61.
Lee
J. Y.
,
K. H.
Sohn
,
S. H.
Rhee
,
D.
Hwang
.
2001
.
Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4.
J. Biol. Chem.
276
:
16683
16689
.
62.
Starr
M. E.
,
M.
Saito
,
B. M.
Evers
,
H.
Saito
.
2015
.
Age-associated increase in cytokine production during systemic inflammation—II: the role of IL-1β in age-dependent IL-6 upregulation in adipose tissue.
J. Gerontol. A Biol. Sci. Med. Sci.
70
:
1508
1515
.
63.
Jové
M.
,
A.
Planavila
,
J. C.
Laguna
,
M.
Vázquez-Carrera
.
2005
.
Palmitate-induced interleukin 6 production is mediated by protein kinase C and nuclear-factor κB activation and leads to glucose transporter 4 down-regulation in skeletal muscle cells.
Endocrinology
146
:
3087
3095
.

The authors have no financial conflicts of interest.

Supplementary data