Concentrations (pM) of AEA, PE, OEA, LEA and 2-AG in the medium following TNFα treatment of DU145 cells.

<p>Concentrations (pM) of AEA, PE, OEA, LEA and 2-AG in the medium following TNFα treatment of DU145 cells.</p

Metabolism of 100 nM AEA by DU145 cells.

<p>Panels A show the time courses for the hydrolysis of 100 nM [Et-³H]AEA. Note that the values are per well, and not normalised to the protein content (shown in Panel B). In Panel C, rates of hydrolysis were determined for each experiment from the individual slopes (going through the origin) of the first two time points divided by the protein content. Data are means and 95% confidence intervals, N = 9. For the vehicle and flurbiprofen treated cells, the bootstrapped linear main effects model gave P values of TNFα, 0.19; flurbiprofen, 0.62. The interaction model gave a P value for TNFα x flurbiprofen of 0.80. However, a small effect of flurbiprofen can be masked by the large inter-experimental variation. Expressing the effect of flurbiprofen as % of the corresponding control value gave values of: untreated cells, 90 (81–99.5); TNFα- treated cells 87 (77–96) (means and 95% confidence limits, N = 9). Panels D-E: TLC separation of [Ara-³H]AEA, [³H]arachidonic acid (AA), [³H]PGF_2α and [³H]bimtoprost (Bimat) using ethyl acetate: methanol (90:10 v/v) as solvent system. Panel D shows the complete sampling from a single experiment, and Panel E shows the total recovery for three separate experiments over the R_f range shown. In Panel F, cells were incubated with 100 nM [³H]AEA, labelled in the arachidonoyl part of the molecule for 30 min prior to workup and separation by TLC. Shown are means of individual experiments conducted in triplicate for vehicle (V) and TNFα (T)-treated DU145 cells, and for vehicle and LPS + IFNγ-treated (L/I) RAW264.7 cells.</p

Concentrations (pM) of lipids in the medium following treatment with TNFα and addition of AEA to DU145 cells.

<p>Concentrations (pM) of lipids in the medium following treatment with TNFα and addition of AEA to DU145 cells.</p

Effect of treatment of DU145 cells with TNFα upon mRNA levels of AEA and 2-AG metabolic enzymes.

<p>Effect of treatment of DU145 cells with TNFα upon mRNA levels of AEA and 2-AG metabolic enzymes.</p

Primer sequences used in the present study.

<p>Primer sequences used in the present study.</p

Expression of <i>PTGS2</i> (COX2), <i>NAPEPLD</i>, <i>FAAH</i> and <i>NAAA</i> in DU145 and RAW264.7 cells.

<p>Panels A and B. mRNA levels of <i>PTGS2</i> (A) and <i>NAPEPLD</i> (B) measured in DU145 cell lysates at different times after TNFα treatment. The individual data points summarised in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185011#pone.0185011.t002" target="_blank">Table 2</a> are shown for these two proteins. Panels C and D show mRNA for <i>PTGS2</i>, <i>NAPEPLD</i>, <i>FAAH</i> and <i>NAAA</i> in control (V) and TNFα treated (2 h, T) DU145 cells (Panel B), and in control and LPS + IFNγ-treated (24 h, L/I) RAW264.7 cells (Panel C). Data are for 8–9 separate experiments, undertaken concomitantly with the uptake experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185011#pone.0185011.g002" target="_blank">Fig 2</a>. ***P<0.001, **P<0.01, ^NSP>0.05, exact two-sided permutation tests (complete enumeration) for the comparison shown. Panel E shows a Western blot for COX-2 with unstimulated and stimulated DU145 (TNFα) and RAW264.7 (LPS + IFNγ) cells. Human recombinant COX-2 is included as a positive control.</p

Uptake of 100 nM [Ara-³H]AEA into RAW264.7 and DU145 cells.

<p>Panel A-C show data for control and LPS + IFNγ-treated RAW264.7 cells; Panels D-F for control and TNFα-treated DU145 cells. Panels A, D show the time courses for the accumulation of radiolabel 5, 10, 20 and 30 min after addition of 100 nM of [Ara-³H]AEA. Note that the uptake is per well, and not normalised to the protein content (shown in Panels B and E). In Panel C, rates of uptake were determined for each experiment from the individual slopes of each the time course divided by the protein content. Shown are means and 95% confidence intervals, N = 8. Data was analysed using bootstrapped linear models (for details, see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185011#pone.0185011.ref040" target="_blank">40</a>]). For the main effects model, the P values were: LPS + IFNγ, P<0.0001; flurbiprofen, P = 0.89; URB597, P<0.0001. For the interactions model, the P values of the three bivariate interactions and the trivariate interaction were all ~0.9. The time courses for uptake into DU145 cells shown in Panel D could not be used to obtain robust slope replots. In consequence, the difference in uptake (per unit protein) between TNFα-treated and control cells were determined for each time point. The data is shown in Panel F (means and 95% confidence intervals, N = 6–7). A one-way ANOVA not assuming equal variances gave a P value of 0.018. Note that at the 30 min incubation time point, the confidence limits do not straddle zero.</p

Additional file 1: Table S1. of High-fat diet feeding differentially affects the development of inflammation in the central nervous system

Composition of the diets used in the diet-induced obesity model. Table S2. High-fat diet-induced changes in phospholipid and lysophospholipid cortical levels. Table S3. High-fat diet-induced changes in phospholipid and lysophospholipid cerebellar levels. Table S4. High-fat diet-induced changes in lipid levels. (PDF 354 kb

Additional file 1: of Oxysterol levels and metabolism in the course of neuroinflammation: insights from in vitro and in vivo models

Figure S1. Activation of BV2 cells and oxysterol levels in control BV2 cells. (A) 2.5 × 105 cells were incubated with vehicle (CTL) or LPS (100 ng/mL) for the indicated time points. mRNA was extracted and RT-qPCR performed for IL-1β, IL-6, and TNF-α. The data are expressed as the mean ± S.E.M in percentage of the respective CTL. ***p < 0.001, **p < 0.01, and *p < 0.05 vs CTL. (B) Oxysterols were quantified in BV2 cells incubated without LPS. The data are expressed as the mean ± S.E.M in pmol/10*106 cells. (C) 2.5 × 105 cells were incubated with vehicle (CTL) or 10 U/mL of IL-4 for the indicated time points. mRNA was extracted and RT-qPCR performed for Arg1 and CD206. The data are expressed as the mean ± S.E.M in percentage of the respective CTL. Figure S2. LPS-induced activation of primary co-culture of astrocytes and microglia and oxysterol levels in control cells. (A) 2.5 × 105 cells were incubated with LPS (100 ng/mL) or vehicle (CTL) for 8 h. mRNA was extracted and RT-qPCR performed for IL-1β, IL-6, and TNF-⍺. The data are expressed as mean ± S.E.M in percentage of CTL set at 100. ****p < 0.0001 and ***p < 0.001 vs CTL. (B) Oxysterols were quantified in co-culture of primary microglia and astrocytes incubated without LPS. The data are expressed as the mean ± S.E.M in pmol/10 × 106 cells. Figure S3. mRNA expression of pro-inflammatory markers in (A) the brain and (B) spinal cord of mice with LPS-induced inflammation in comparison to control mice. Mice (seven per group) were treated with LPS (300 μg/kg) or vehicle (CTL) and sacrificed after 4 or 8 h. mRNA was extracted and RT-qPCR was performed for IL-6 and TNF-α. The data are expressed as the mean ± S.E.M in percentage of CTL set at 100. ***p < 0.001 vs CTL. Figure S4. Oxysterol levels in CTL mice. Oxysterols were analyzed in seven control mice, in the brain (A), the spinal cord (B), and the liver (C). The data are expressed as the mean ± S.E.M in pmol/g of tissue. Figure S5. Effect of LPS-induced inflammation on oxysterol levels in the liver in comparison to CTL mice. Seven mice per group were treated with LPS (300 μg/kg) or vehicle (CTL) and sacrificed after 4 or 8 h. The data are expressed as mean ± S.E.M in percentage of CTL. **p < 0.01 and *p < 0.05 vs CTL. (PDF 103 kb

A Mechanistic Study on Nanoparticle-Mediated Glucagon-Like Peptide‑1 (GLP-1) Secretion from Enteroendocrine L Cells

L cells have attracted particular interest because of the pleiotropic effects of their secreted peptides (i.e., glucagon-like peptide (GLP) 1 and 2, peptide YY (PYY)). L cells express different G-protein-coupled receptors (GPCRs) that can be activated by endogenous ligands found in the gut lumen. We herein hypothesized that lipid-based nanoparticles could mimic endogenous ligands and thus activate GLP-1 secretion in type 2 diabetes mellitus treatment. To assess this hypothesis, lipid-based nanoparticles (nanostructured lipid carriers (NLC), lipid nanocapsules (LNC), and liposomes) and PLGA nanoparticles were added to the L cells and GLP-1 secretion was quantified. Among these nanoparticles, only NLC resulted effective at inducing GLP-1 secretion in both murine and human L cells <i>in vitro</i>. The mRNA expression of proglucagon showed that this effect was due to an increased GLP-1 secretion and not to an increased GLP-1 synthesis. The mechanism by which NLC triggered GLP-1 secretion by L cells revealed an extracellular interaction of NLC, exerting a physiological GLP-1 secretion. We herein demonstrate that nanomedicine can be used to induce GLP-1 secretion from murine and human L cells