Medium Chain Triglyceride (MCT) Oil Affects the Immunophenotype via Reprogramming of Mitochondrial Respiration in Murine Macrophages (2024)

2.1. Cell Culture and Oil Treatments

The murine macrophage RAW 264.7 cell line was purchased from the Korea Cell Line Bank (KCLB, Seoul, Korea). The cells were seeded on 24-well plates at a concentration of 1 × 10⁵ cells/mL in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic solution (10,000 U/mL penicillin G, 10,000 μg/mL streptomycin, 25 μg/mL amphotericin B). Medium chain fatty acid-triglyceride (MCT) enriched oil was purchased from Nisshin Oillio (Healthy Resetta™, Tokyo, Japan), and canola oil purchased from CJ CheilJedang (Seoul, Korea) served as a control. MCT and control oils were dissolved in DMSO (Daejung Chemicals, Siheung, Korea) and the RAW 264.7 cells were treated for 24 h at 37 °C in a 5% CO₂ incubator. The final concentration of DMSO in the cell culture medium was maintained under 0.01% (v/v). Following oil intervention in medium, macrophages were stimulated by incubating them with 500 ng/mL of TLR ligand approved LPS from Escherichia coli O111:B3 (Sigma-Aldrich, St. Louis, MO, USA) for 12 h [10].

2.2. Animals and Dietary Interventions

C57BL/6 male mice at the age of 4 weeks were purchased from Raon Bio (Yongin, Korea). The care, use, and treatment of all mice in this study were conducted in strict accordance with guidelines from the Institutional Animal Care and Use Committees of Kyung Hee University. Custom-made mouse diets were purchased from Raon Bio (Yongin, Korea), with the control AIN-76A diet modified by replacement of corn oil with 80% (MCT high diet) or 20% MCT oil (MCT low diet) (Table 1). Following 1 week of mouse acclimation by feeding the AIN-76A diet, mice were randomly separated into three groups (n = 7) and fed either MCT high, MCT low, or control AIN-76A diets for 4 weeks ad libitum. After dietary interventions, mice were sacrificed in a CO₂ chamber.

2.3. Bone Marrow-Derived Macrophage (BMDM) Culture and Macrophage Polarization

Mouse mononuclear cells were collected from the femurs and tibias of C57BL/6 mice and treated with NH₄Cl solutions (Stemcell Technology, Canada) on ice for 10 min to lyse the red blood cells. Mature bone marrow-derived macrophages (BMDMs, M0) were obtained from differentiated mononuclear cells stimulated with 10 ng/mL macrophage colony-stimulating factor (M-CSF, Sigma-Aldrich, St. Louis, MO, USA) for 7 days. BMDMs were further cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS at 37 °C in a 5% CO₂ incubator for macrophage polarization as follows: 100 ng/mL LPS with 50 ng/mL interferon (INF)-γ for M1 polarization or 10 ng/mL interleukin (IL)-4 with 10 ng/mL IL-13 for M2 activation were used to treat mature BMDMs for 24 h [11].

2.4. Measurement of Oxygen Consumption Rate

The oxygen consumption rate (OCR) was measured using a Seahorse Bioscience XF extracellular flux analyzer (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions. Oil treated RAW 264.7 cells and polarized BMDMs were seeded in XF cell culture plates with non-buffered DMEM supplemented with 4500 mg/L glucose, 4 mM glutamine, and 1 mM sodium pyruvate. Then, the plate was incubated for 1 h at 37 °C in a non-CO₂ incubator and sequentially treated with 1 μM oligomycin (ATP synthase inhibitor), 0.5 μM carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP, uncoupling agent of mitochondrial respiration), and 0.5 μM rotenone/antimycin A mix (inhibitor of complex I and III of electron transport chain, respectively) for measurements of basal and maximal respiration and ATP production through assessing changes in oxygen consumption rate in real time. Basal and maximal respiration and ATP production were calculated by the following equations:

Basal respiration: Last oxygen consumption rates before oligomycin injection—non-mitochondrial oxygen consumption rates

Maximal respiration: Maximum oxygen consumption rates after FCCP injection—non-mitochondrial oxygen consumption rates

ATP production: basal oxygen consumption rates—minimum oxygen consumption rates after oligomycin injection

2.5. Assessments of Macrophage Activation Markers

The surface marker expressions of RAW 264.7 cells and BMDMs were determined by flow cytometric analysis following staining with specific antibodies conjugated with fluorescences. Briefly, LPS treated RAW 264.7 cells and differentiated BMDMs were washed with cold PBS with 2% FBS followed by incubation with anti-mouse CD16/CD32 mAbs (Fc block, eBioscience, San Diego, CA, USA) for 15 min at 4 °C to prohibit non-specific antibody binding. Without removing Fc block, fluorescence-conjugated mAbs, i.e., fluorescein isothiocyanate-conjugated anti-mouse CD80 (aCD80-FITC, eBioscience, San Diego, CA, USA), phycoerythrin-conjugated anti-mouse CD86 (aCD86-PE), or allophycocyanin-conjugated anti-mouse MHC-II (aMHC-II-APC, eBioscience, San Diego, CA, USA) were used to treat RAW 264.7 cells 10 min at 4 °C. Similarly, BMDMs were stained by fluorescein isothiocyanate-conjugated anti-mouse F4/80 (aF4/80-FITC), phycoerythrin-conjugated anti-mouse CD206 (aCD206-PE, eBioscience, San Diego, CA, USA), peridinin-chlorophyll proteins-cyanin5.5-conjugated anti-mouse CD11b (aCD11b-PerCP-Cy5.5, eBioscience, San Diego, CA, USA), or allophycocyanin-conjugated anti-mouse CD11c (aCD11c-APC, eBioscience, San Diego, CA, USA). Following surface staining with specific antibodies, cells were washed twice with ice-cold PBS and resuspended in 100 μL PBS, then analyzed by a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). F4/80⁺CD11b⁺ population BMDMs were gated and designated as macrophages. The relative expressions of activation markers, i.e., CD80, CD86, and MHC-II for RAW 264.7 cells and F4/80, CD206, CD11b, and CD11c for BMDMs were determined by mean fluorescence intensity (MFI) as quantified by FlowJo^® software (BD Biosciences, San Jose, CA, USA).

2.6. Cytokine Quantification

Following stimulation of RAW 264.7 cells with LPS or polarization of BMDMs, pro-inflammatory (interleukin (IL)-6 and tumor necrosis factor (TNF)-α) or anti-inflammatory cytokines (IL-10) in the culture medium were quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA). In brief, 96-well plates were coated with capture antibodies overnight at 4 °C. Then, excess antibodies were washed with assay diluent. Cell culture supernatants and each cytokine standard were aliquoted into each well at designated concentrations. After incubating at room temperature and washing with the provided buffer, the captured cytokines were incubated with biotinylated detection antibodies and streptavidin-horse radish peroxidase conjugates (sAv-HRP) at room temperature. Following a series of washing steps, substrate solution was added for enzymatic reactions, and a stop solution (1M H₃PO₄) was applied. After the final reaction steps, the absorbance was measured with a microplate reader (Bio-Rad, Hercules, CA, USA) at a 450-nm wavelength.

2.7. Statistical Analysis

Data are expressed as mean ± standard error of the mean (SEM). Statistical significance was analyzed by one-way analysis of variance (ANOVA), followed by a post-hoc Tukey’s multiple comparison test using Prism software (GraphPad Software, La Jolla, CA, USA). Significant differences were indicated with different letters in the set of data (n = 3–7), with significance identified at p < 0.05. Non significance was denoted as N.S.

2.8. Ethics Statement

The animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of Kyung Hee University (approval ID: KHUASP(GC)-17-030).

3.1. MCT Oil Up-Regulates Mitochondrial Respiration in Macrophages

In an in vitro model using a RAW 264.7 murine macrophage cell-line, canola oil, known for its anti-oxidant and anti-inflammatory effects which inhibit the production of nitric oxide and prostaglandin E₂ [12], served as a control. Cytotoxicity of canola or MCT oil emulsified in DMSO, as well as the vehicle itself, was determined by MTT assay. Neither oil affected cell viability up to 100 μg/mL (data not shown), and the oil concentration was set to 10 μg/mL thereafter through the study. In non-stimulated conditions, OCR of mitochondria were not more strongly affected by MCT oil compared to canola oil and non-oil-treated control (Figure 1A). However, following an onset of inflammatory cues by LPS treatment, there was a significant increase of mitochondrial respiration in MCT treated cells (Figure 1B, circle), compared to canola (square) or non-oil-treated (triangle) controls. Throughout the OCR measurements, oligomycin, FCCP, and rotenone/antimycin A were sequentially injected to specifically calculate the cellular oxygen needed for basal respiration, maximal respiration, and ATP production [13]. As quantitatively assessed, MCT oil significantly up-regulated OCR for basal respiration (153.58 ± 4.57 pmoles/min/10E5 cells), maximal respiration (153 ± 7.15 pmoles/min/10E5 cells), and ATP production (79.62 ± 0.26 pmoles/min/10E5 cells), while canola (93.03 ± 3.88 pmoles/min/10E5 cells for basal respiration, 97.91 ± 4.28 pmoles/min/10E5 cells for maximal respiration, and 57.97 ± 4.75 pmoles/min/10E5 cells for ATP production) and non-oil-treated (83.29 ± 0.63 pmoles/min/10E5 cells for basal respiration, 86.78 ± 1.65 pmoles/min/10E5 cells for maximal respiration and 54.48 ± 0.79 pmoles/min/10E5 cells for ATP production) controls exhibited no differences (Figure 1C).

Open in a separate window

Figure 1

Medium chain triglyceride (MCT) exacerbates mitochondrial oxygen consumption in RAW 264.7 cells. The oxygen consumption rate (OCR) was measured as a marker of oxidative phosphorylation (OXPHOS) using a Seahorse Extracellular Flux analyzer. Samples were initially treated with oils under non-stimulated (A) and lipopolysaccharide (LPS)-stimulated conditions (B). During extracellular flux analysis, cells were sequentially treated with oligomycin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and rotenone with antimycin A to assess OXPHOS phenotype based on OCR levels. Based on OCR data (B), the basal respiration, maximal respiration, and ATP production were calculated and are indicated as OCR in pmoles/min (C). Data are presented as mean ± SEM (n = 4), and significant differences are indicated with different letters (p < 0.05) within each basal respiration, maximal respiration, and ATP production column.

As an in vivo model of MCT-induced regulation of energy metabolism in macrophages, M1/M2 polarized BMDMs isolated from C57BL/6 mice following dietary interventions were examined to assess the mitochondrial consumption of oxygen. The defined AIN-76A diet was used as a control normal diet, while either 20% (MCT low) or 80% (MCT high) corn oil was replaced with MCT oil. The body weight gain exhibited no difference among the diet groups indicating the isocaloric diet uptake in mice (Supplementary Figure S1). In non-polarized (M0) BMDMs, MCT oil-fed groups exhibited significantly up-regulated mitochondrial oxygen consumption compared to the normal AIN-76A diet-fed group (Figure 2A). In accordance to the RAW 264.7 cellular model, dietary provision of MCT resulted in dose-dependent increase of OCR. In detail, MCT high-fed mice exhibited significantly increased OCR for basal respiration (12.47 ± 4.7 pmoles/min/10E4 cells), maximal respiration (11 ± 3.87 pmoles/min/10E4 cells), and ATP production (10.26 ± 3.72 pmoles/min/10E4 cells) in M0 BMDMs compared to normal diet-fed mice (2.54 ± 0.28 pmoles/min/10E4 cells for basal respiration, 1.12 ± 0.45 pmoles/min/10E4 cells for maximal respiration, and 2.86 ± 0.27 pmoles/min/10E4 cells for ATP production). The MCT low-fed group exhibited OCR (6.07 ± 1.72 pmoles/min/10E4 cells for basal respiration, 4.5 ± 1.2 pmoles/min/10E4 cells for maximal respiration, and 5.37 ± 0.93 pmoles/min/10E4 cells for ATP production) that was intermediate between MCT high and AIN-76A control groups (Figure 2B).

Open in a separate window

Figure 2

MCT upregulates OXPHOS in non-polarized and M2 polarized BMDMs. To assess OCR in non-polarized (M0) and polarized (M1 and M2) conditions, bone marrow-derived macrophages were collected from C57BL/6 mice fed with custom diets (AIN-76A, MCT Low, MCT High) and treated with macrophage colony-stimulating factor (M-CSF). Following stimulation with LPS/IFN-γ for M1 polarization or interleukin (IL)-4/IL-13 for M2 polarization, cells were transferred to Seahorse microplates for assessing OCR. OCR was assessed using a Seahorse Extracellular Flux analyzer in non-polarized (A), M1 (C), and M2 (E) polarized conditions. The mitochondrial respiration parameters (basal respiration, maximal respiration, and ATP production) were calculated and are presented as pmoles/min in each non-polarized (B), M1 (D), and M2 (F) polarized BMDMs. Data are indicated as mean ± SEM (n = 4), and significant differences are indicated with different letters (p < 0.05, N.S. means no significant difference) within each basal respiration, maximal respiration, and ATP production column (B, D, and F).

Following M1 polarization of M0 BMDM by LPS stimulation, metabolic phenotype was partially affected by MCT intervention compared to the normal diet-fed group (Figure 2C). In detail, OCR in MCT high (9.35 ± 1.57 pmoles/min/10E4 cells for basal respiration, 5.84 ± 0.41 pmoles/10E4 for maximal respiration, and 7.38 ± 0.89 pmoles/min/10E4 cells for ATP production) and MCT low (6.13 ± 1.83 pmoles/min/10E4 cells for basal respiration, 4.08 ± 1.06 pmoles/min/10E4 cells for maximal respiration, and 4.78 ± 1.06 pmoles/min/10E4 cells for ATP production) groups did not significantly differ from the AIN-76A control (3.67 ± 0.47 pmoles/min/10E4 cells for basal respiration, 2.01 ± 0.34 pmoles/min/10E4 cells for maximal respiration, and 4.78 ± 1.06 pmoles/min/10E4 cells for ATP production). However, following dissected quantification of OCR by appropriate drug treatments, maximal respiration of LPS-stimulated (M1) BMDMs in the MCT high-fed group was significantly elevated compared to the normal diet group. Even though the difference in OCR was not statistically significant for basal respiration and ATP production, similar trends to those for maximal respiration were observed (Figure 2D).

Alternatively, M0 BMDMs were polarized to the M2 phenotype by the addition of IL-4 in culture medium. OCRs for MCT oil-fed groups were significantly elevated compared to normal diet-fed controls (Figure 2E). In detail, the MCT high-fed group exhibited significant increments of basal respiration, maximal respiration, and ATP production in M2 BMDMs (15.17 ± 4.03, 11.13 ± 3.62, and 11.97 ± 2.89 pmoles/min/10E4 cells, respectively) compared to the AIN-76A fed control group (3.33 ± 0.54, 0.94 ± 0.25, and 3.47 ± 0.67 pmoles/min/10E4 cells, respectively). Similar to OCR quantification in M0, OCR in the MCT low-fed group (6.6 ± 1.85 pmoles/min/10E4 cells for basal respiration, 4.25 ± 1.54 pmoles/min/10E4 cells for maximal respiration, and 5.67 ± 1.17 pmoles/min/10E4 cells for ATP production) was intermediate between those of MCT and normal diet-fed controls (Figure 2F).

3.2. MCT Oil Downregulates Activation Marker Expression on Macrophage Surfaces

Given that the mitochondrial respiration of macrophages was modulated by MCT oil through up-regulation of oxygen consumption in vitro (RAW 264.7 cells) and ex vivo (murine BMDMs), we examined whether the regulation of mitochondrial respiration by MCT oil affects the expressions of activation markers on macrophage surfaces. In a cellular model using RAW 264.7 cells, costimulatory molecules, i.e., expression of CD80 (B7-1), CD86 (B7-2), and MHC-II, were determined by specific staining with fluorescence-conjugated antibodies followed by flow cytometry. Canola oil treatment significantly upregulated (p < 0.05) CD80 expression in RAW 264.7 cells (25,455 ± 2963 MFI) compared to non-oil-treated controls (16,115 ± 1485 MFI). However, MCT oil-treated cells exhibited comparable expression of CD80 (17,205 ± 253.7 MFI) to non-oil-treated controls (Figure 3A). In addition, MCT oil significantly suppressed the expressions of CD86 (2399 ± 51.46 MFI) and MHC-II molecules (4376 ± 392.5 MFI) compared to non-oil-treated controls (2705 ± 52.93 MFI for CD86 expression and 6490 ± 567.5 MFI for MHC-II expression), while canola oil did not affect the expressions of either CD86 (2652 ± 52.93 MFI) or MHC-II (5726 ± 167.1 MFI) (Figure 3B,C). These results indicate that MCT oil has a potent anti-inflammatory effect on LPS-stimulated M1-like condition in macrophages.

Open in a separate window

Figure 3

MCT induces macrophages to have anti-inflammatory phenotypes by modulating the expression of surface markers. CD80 (A), CD86 (B), and MHC-II (C) expressions of RAW 264.7 cells were evaluated following oil treatment and/or LPS stimulations (n = 3). CD11c (D) was evaluated for the M1 marker and CD206 (E) for the M2 marker in BMDMs from mice fed with custom diets following LPS and IL-4 stimulation, respectively (n = 7). Cells were stained with antibodies specific to surface molecules and analyzed by a flow cytometer. Values are indicated as mean ± SEM, and statistical significance is indicated with different letters (p < 0.05).

Alternatively, BMDMs isolated from normal diet-fed mice were induced to transform to either M1 or M2 phenotypes by the addition of LPS or IL-4 in culture medium, respectively. Following differentiation, the expressions of CD11c (M1 marker) and CD206 (M2 marker) were assessed by specific antibody staining and flow cytometry. Following LPS stimulation, cells exhibited significantly elevated expressions of CD11c molecules (LPS 26,335 ± 1109 vs. non-LPS 8995 ± 1153 MFI). Cells from MCT high-fed mice in which 80% of total dietary lipid (corn oil) was substituted for MCT tended to suppress the expression of CD11c (21,508 ± 1201 MFI) but the difference was not significant. Of particular interest, cells from MCT low-fed mice, in which MCT replaced 20% of total dietary lipid (corn oil), exhibited significantly down-regulated expression of CD11c (19,981 ± 2132 MFI) compared to AIN-76A normal diet-fed controls (26,335 ± 1109 MFI) (Figure 3D). In IL-4-treated conditions inducing M2 polarization, increase of CD206 expression by IL-4 stimulation in the AIN-76-A diet fed group (76,703 ± 8136 MFI) was significantly augmented by dietary interventions with high levels of MCT (MCT High, 107,127 ± 6089 MFI). Moderate doses of MCT in the MCT low diet group resulted in expression of CD206 (82,656 ± 6323 MFI) that was intermediate between those of AIN-76A and MCT high diet-fed mice, but the difference was not significant (Figure 3E).

Taken together, these results indicate that MCT oil suppresses inflammatory surface markers while elevating anti-inflammatory activation markers of murine macrophages both in vitro and ex vivo.

3.3. MCT Oil Induces an Anti-Inflammatory M2-Like Phenotype in Macrophages

Having found that MCT oil significantly increases M2 surface markers while suppressing M1 surface markers, it was further examined whether the secretion of inflammatory and anti-inflammatory cytokines in macrophages was affected by MCT oil. RAW 264.7 macrophages incubated with MCT oil showed significantly suppressed inflammatory cytokine secretion in LPS-induced conditions. MCT oil significantly down-regulated the production of IL-6 (24.48 ± 1.38 ng/mL) compared to non-oil-treated controls (36.45 ± 2.26 ng/mL). Canola oil, known for its anti-inflammatory and anti-oxidant effects [14], also suppressed the secretion of IL-6 (26.92 ± 2.03 ng/mL) (Figure 4A). MCT oil resulted in similar reductions in TNF-α secretion (17.89 ± 0.67 ng/mL) compared to non-oil-treated controls (28.35 ± 1.69 ng/mL), while canola oil treatment resulted in no significant differences from the control (22.11 ± 2.38 ng/mL) (Figure 4B).

Open in a separate window

Figure 4

MCT inhibits the production of pro-inflammatory cytokines while enhancing anti-inflammatory cytokine secretion. IL-6 (A) and TNF-α (B) production of RAW 264.7 macrophages treated with conventional oil samples under LPS-stimulated conditions (n = 3). Pro-inflammatory cytokine, IL-6 (C), and anti-inflammatory cytokine, IL-10 (D) secretion by mice BMDMs under non-polarized (M0), M1, and M2 polarized conditions (n = 7). The quantification of cytokines in culture supernatants was determined by ELISA. Data are indicated as mean ± SEM, and statistical significance is presented with different letters (p < 0.05, N.S. means no significant difference, N.D. means not detected) within each M0, M1, and M2 column (C,D).

To further assess the dietary effects of MCT on cytokine production, BMDMs (M0) isolated from either AIN-76A control, MCT low, or MCT high diet fed mice were polarized to M1 (LPS) or M2 (IL-4) phenotypes. There was no significant difference in inflammatory cytokine IL-6 production between different diet-fed groups under non-polarized (M0) and LPS-treated (M1) conditions. Following M2 differentiation by IL-4, the secretions of IL-6 was not detected in any diet-fed BMDMs (Figure 4C). In contrast, MCT significantly increased the anti-inflammatory cytokine, IL-10, in all subsets of murine BMDMs ex vivo. The MCT high-fed group exhibited significantly up-regulated IL-10 secretion (1246 ± 194.6 pg/mL) of BMDMs compared to normal diet-fed (318.1 ± 238.3 pg/mL) and MCT low-fed (266 ± 173.9 pg/mL) groups in non-polarized (M0) status. In inflammatory LPS-treated (M1) conditions, IL-10 was also significantly elevated in MCT high-fed mice (1209 ± 245.7 pg/mL) compared to MCT low-fed (351.3 ± 139.6 pg/mL) and normal diet-fed (176.5 ± 148.6 pg/mL) controls. Consistent with previous results, MCT-high fed BMDMs secreted significantly more IL-10 (2135 ± 159.7 pg/mL) compared to other diet-fed groups (Figure 4D). These data suggest that MCT induces M0 and M1 macrophages to transform into M2-like macrophages and enhances anti-inflammatory properties of all subsets of macrophages.

1. West A.P., Shadel G.S., Ghosh S. Mitochondria in innate immune responses. Nat. Rev. Immunol. 2011;11:389–402. doi:10.1038/nri2975. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

2. Mills E.L., O’Neill L.A. Reprogramming mitochondrial metabolism in macrophages as an anti-inflammatory signal. Eur. J. Immunol. 2016;46:13–21. doi:10.1002/eji.201445427. [PubMed] [CrossRef] [Google Scholar]

3. Geeraerts X., Bolli E., Fendt S.M.S.-M., Van Ginderachter J.A.A. Macrophage Metabolism as Therapeutic Target for Cancer, Atherosclerosis, and Obesity. Front. Immunol. 2017;8:289. doi:10.3389/fimmu.2017.00289. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

4. Pike L.S., Wu M. Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate. PLoS ONE. 2014;9:e109916. [PMC free article] [PubMed] [Google Scholar]

5. Huang S.-C., Everts B., Ivanova Y., O’Sullivan D., Nascimento M., Smith A.M.M., Beatty W., Love-Gregory L., Lam W.Y.Y., O’Neill C.M.M., et al. Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages. Nat. Immunol. 2014;15:846–855. doi:10.1038/ni.2956. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

6. Qu Q., Zeng F., Liu X., Wang Q.J., Deng F. Fatty acid oxidation and carnitine palmitoyltransferase I: Emerging therapeutic targets in cancer. Cell Death Dis. 2016;7:e2226. doi:10.1038/cddis.2016.132. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

7. Nagao K., Yanagita T. Medium-chain fatty acids: Functional lipids for the prevention and treatment of the metabolic syndrome. Pharm. Res. 2010;61:208–212. doi:10.1016/j.phrs.2009.11.007. [PubMed] [CrossRef] [Google Scholar]

8. Marten B., Pfeuffer M., Schrezenmeir J. Medium-chain triglycerides. Int. Dairy J. 2006;16:1374–1382. doi:10.1016/j.idairyj.2006.06.015. [CrossRef] [Google Scholar]

9. Yu S., Choi J.H., Kim H.J., Park S.H., Go G., Kim W. In Vitro Evidence of Anti-Inflammatory and Anti-Obesity Effects of Medium-Chain Fatty Acid-Diacylglycerols. J. Microbiol. Biotechnol. 2017;27:1617–1627. doi:10.4014/jmb.1703.03071. [PubMed] [CrossRef] [Google Scholar]

10. Kwon Y., Yu S., Choi G.S., Kim J.H., Baik M., Su S.T., Kim W. Puffing of Rehmannia glutinosa enhances anti-oxidant capacity and down-regulates IL-6 production in RAW 264.7 cells. Food Sci. Biotechnol. 2019;28:1235–1240. doi:10.1007/s10068-019-00566-z. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

11. Jablonski K.A., Amici S.A., Webb L.M., Ruiz-Rosado J.D.D., Popovich P.G., Partida-Sanchez S., Guerau-De-arellano M. Novel markers to delineate murine M1 and M2 macrophages. PLoS ONE. 2015;10:e0145342. doi:10.1371/journal.pone.0145342. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

12. Loganes C., Ballali S., Minto C. Main Properties of Canola Oil Components: A Descriptive Review of Current Knowledge. Open Agric. J. 2016;10:69–74. doi:10.2174/1874331501610010069. [CrossRef] [Google Scholar]

13. Kalyanaraman B., Cheng G., Hardy M., Ouari O., Lopez M., Joseph J., Zielonka J., Dwinell M.B. A review of the basics of mitochondrial bioenergetics, metabolism, and related signaling pathways in cancer cells: Therapeutic targeting of tumor mitochondria with lipophilic cationic compounds. Redox Biol. 2018;14:316–327. doi:10.1016/j.redox.2017.09.020. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

14. Ren J., Chung S.H. Anti-inflammatory effect of α-linolenic acid and its mode of action through the inhibition of nitric oxide production and inducible nitric oxide synthase gene expression via NF-κB and mitogen-activated protein kinase pathways. J. Agric. Food Chem. 2007;55:5073–5080. doi:10.1021/jf0702693. [PubMed] [CrossRef] [Google Scholar]

15. Liu Y.-C., Zou X.-B., Chai Y.-F., Yao Y.-M. Macrophage polarization in inflammatory diseases. Int. J. Biol. Sci. 2014;10:520–529. doi:10.7150/ijbs.8879. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

16. Kim L., Kim J.Y. Chondroprotective effect of curcumin and lecithin complex in human chondrocytes stimulated by IL-1β via an anti-inflammatory mechanism. Food Sci. Biotechnol. 2019;28:547–553. doi:10.1007/s10068-018-0470-6. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

17. Shapouri-Moghaddam A., Mohammadian S., Vazini H., Taghadosi M., Esmaeili S.A., Mardani F., Seifi B., Mohammadi A., Afshari J.T., Sahebkar A. Macrophage plasticity, polarization, and function in health and disease. J. Cell. Physiol. 2018;233:6425–6440. doi:10.1002/jcp.26429. [PubMed] [CrossRef] [Google Scholar]

18. Zhou X., Li W., Wang S., Zhang P., Wang Q., Xiao J., Zhang C., Zheng X., Xu X., Xue S., et al. YAP Aggravates Inflammatory Bowel Disease by Regulating M1/M2 Macrophage Polarization and Gut Microbial Homeostasis. Cell Rep. 2019;27:1176–1189. doi:10.1016/j.celrep.2019.03.028. [PubMed] [CrossRef] [Google Scholar]

19. Kelly B., O’Neill L.A.J. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res. 2015;25:771–784. doi:10.1038/cr.2015.68. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

20. Zhang Q., Wang H., Mao C., Sun M., Dominah G., Chen L., Zhuang Z. Fatty acid oxidation contributes to IL-1β secretion in M2 macrophages and promotes macrophage-mediated tumor cell migration. Mol. Immunol. 2018;94:27–35. doi:10.1016/j.molimm.2017.12.011. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

21. Van den Bossche J., Baardman J., Otto N.A., van der Velden S., Neele A.E., van den Berg S.M., Luque-Martin R., Chen H.J., Boshuizen M.C.S., Ahmed M., et al. Mitochondrial Dysfunction Prevents Repolarization of Inflammatory Macrophages. Cell Rep. 2016;17:684–696. doi:10.1016/j.celrep.2016.09.008. [PubMed] [CrossRef] [Google Scholar]

22. Han M.S., Jung D.Y., Morel C., Lakhani S.A., Kim J.K., Flavell R.A., Davis R.J. JNK expression by macrophages promotes obesity-induced insulin resistance and inflammation. Science. 2013;339:218–222. doi:10.1126/science.1227568. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

23. Kim H.-J., Yoon H.-J., Kim S.-Y., Yoon Y.-R. A Medium-Chain Fatty Acid, Capric Acid, Inhibits RANKL-Induced Osteoclast Differentiation via the Suppression of NF-κB Signaling and Blocks Cytoskeletal Organization and Survival in Mature Osteoclasts. Mol. Cells. 2014;37:598–604. doi:10.14348/molcells.2014.0153. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

24. Martinez F.O., Sica A., Mantovani A., Locati M. Macrophage activation and polarization. Front. Biosci. 2008;13:453–461. doi:10.2741/2692. [PubMed] [CrossRef] [Google Scholar]

25. Zhang M.-Z., Wang X., Wang Y., Niu A., Wang S., Zou C., Harris R.C. IL-4/IL-13–mediated polarization of renal macrophages/dendritic cells to an M2a phenotype is essential for recovery from acute kidney injury. Kidney Int. 2017;91:375–386. doi:10.1016/j.kint.2016.08.020. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

26. Raschke W.C., Baird S., Ralph P., Nakoinz I. Functional macrophage cell lines transformed by abelson leukemia virus. Cell. 1978;15:261–267. doi:10.1016/0092-8674(78)90101-0. [PubMed] [CrossRef] [Google Scholar]

27. Wu W.H.H., Lin B.Y.Y., Kuo Y.H.H., Huang C. Triglycerides constituted of short and medium chain fatty acids and dicarboxylic acids in Momordica charantia, as well as capric acid, inhibit PGE2 production in RAW264.7 macrophages. Food Chem. 2009;117:306–311. doi:10.1016/j.foodchem.2009.04.004. [CrossRef] [Google Scholar]

28. Bertevello P.L.L., De Nardi L., Torrinhas R.S.S., Logullo A.F.F., Waitzberg D.L.L. Partial Replacement of omega-6 Fatty Acids with Medium-Chain Triglycerides, but Not Olive Oil, Improves Colon Cytokine Response and Damage in Experimental Colitis. J. Parenter. Enter. Nutr. 2012;36:442–448. doi:10.1177/0148607111421788. [PubMed] [CrossRef] [Google Scholar]

29. Kono H., Fujii H., Ogiku M., Tsuchiya M., Ishii K., Hara M. Enteral diets enriched with medium-chain triglycerides and N-3 fatty acids prevent chemically induced experimental colitis in rats. Transl. Res. 2010;156:282–291. doi:10.1016/j.trsl.2010.07.012. [PubMed] [CrossRef] [Google Scholar]

30. Druml W., Fishcer M., Pidlich J., Lenz K. Fat elimination in chronic hepatic failure: Long-chain vs. medium-chain triglycerides. Am. J. Clin. Nutr. 1995;61:812–817. doi:10.1093/ajcn/61.4.812. [PubMed] [CrossRef] [Google Scholar]

31. Kadochi Y., Mori S., Fujiwara-Tani R., Luo Y., Nishiguchi Y., Kishi S., Fujii K., Ohmori H., Kuniyasu H. Remodeling of energy metabolism by a ketone body and medium-chain fatty acid suppressed the proliferation of CT26 mouse colon cancer cells. Oncol. Lett. 2017;14:673–680. doi:10.3892/ol.2017.6195. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

32. Shinohara H., Ogawa A., Kasai M., AOYAMA T. Effect of Randomly Interesterified Triacylglycerols Containing Medium- and Long-Chain Fatty Acids on Energy Expenditure and Hepatic Fatty Acid Metabolism in Rats. Biosci. Biotechnol. Biochem. 2005;69:1811–1818. doi:10.1271/bbb.69.1811. [PubMed] [CrossRef] [Google Scholar]

33. Schönfeld P., Wojtczak L. Short- and medium-chain fatty acids in energy metabolism: The cellular perspective. J. Lipid Res. 2016;57:943–954. doi:10.1194/jlr.R067629. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

34. Dayrit F.M. Lauric acid is a medium-chain fatty acid, coconut oil is a medium-chain triglyceride. Philipp. J. Sci. 2014;143:157–166. [Google Scholar]

35. Nomura M.M., Liu J., Rovira I.I.I., Gonzalez-Hurtado E., Lee J., Wolfgang M.J.J., Finkel T. Fatty acid oxidation in macrophage polarization. Nat. Immunol. 2016;17:216–217. doi:10.1038/ni.3366. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

36. Johnson A.R., Qin Y., Cozzo A.J., Freemerman A.J., Huang M.J., Zhao L., Sampey B.P., Milner J.J., Beck M.A., Damania B., et al. Metabolic reprogramming through fatty acid transport protein 1 (FATP1) regulates macrophage inflammatory potential and adipose inflammation. Mol. Metab. 2016;5:506–526. doi:10.1016/j.molmet.2016.04.005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

37. Huang S.C.C., Smith A.M., Everts B., Colonna M., Pearce E.L.E.J., Schilling J.D., Pearce E.L.E.J. Metabolic Reprogramming Mediated by the mTORC2-IRF4 Signaling Axis Is Essential for Macrophage Alternative Activation. Immunity. 2016;45:817–830. doi:10.1016/j.immuni.2016.09.016. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

38. Wanten G.J.A., Naber A.H.J., Kruimel J.W., Tool A.T.J., Roos D., Jansen J.B.M.J. Influence of structurally different lipid emulsions on human neutrophil oxygen radical production. Eur. J. Clin. Investig. 1999;29:357–363. doi:10.1046/j.1365-2362.1999.00486.x. [PubMed] [CrossRef] [Google Scholar]

39. Wanten G.J.A., Geijtenbeek T.B.H., Raymakers R.A.P., van Kooyk Y., Roos D., Jansen J.B.M.J., Naber A.H.J. Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil β2 integrin expression, adhesion, and degranulation. J. Parenter. Enter. Nutr. 2000;24:228–233. doi:10.1177/0148607100024004228. [PubMed] [CrossRef] [Google Scholar]

40. Wanten G.J.A., Roos D., Naber A.H.J. Effects of structurally different lipid emulsions on human neutrophil migration. Clin. Nutr. 2000;19:327–331. doi:10.1054/clnu.2000.0113. [PubMed] [CrossRef] [Google Scholar]

41. Wanten G.J., Curfs J.H., Meis J.F., Naber A.H. Phagocytosis and killing of Candida albicans by human neutrophils after exposure to structurally different lipid emulsions. J. Parenter. Enter. Nutr. 2001;25:9–13. doi:10.1177/014860710102500109. [PubMed] [CrossRef] [Google Scholar]