fLMF chemotaxis assays with human macrophages.

<p>Control (Ctrl) and IL-4 (10 ng/ml), IL-13 (10 ng/ml), IFNγ (50 ng/ml), LPS (100 ng/ml), and 3M-002 (3 µM) stimulated macrophages were allowed to migrate towards the chemotactic factor fLMF (+). Medium without chemotactic factor (−) was used as control for basal migration in each experiment. Graphs show the mean migration index compared to each individual control (n = 3) and error bars display the standard deviation (±SD). *p<0.05; **p<0.01.</p

FPR1 cell surface expression on human neutrophils, monocytes, and macrophages.

<p>FACS analysis was performed to investigate cell surface expression of FPR1. A) Neutrophils. B) Monocytes. C) 9 day old macrophages. Autofluorescence of the cells is shown in light grey, the isotype control in grey and cells labeled with FPR1 Ab in black. D) Quantitative representation of the FPR1 median fluorescence intensity (antibody MFI minus isotype MFI) NG: neutrophils (n = 3). MO: monocytes (n = 5). MA: 9 day old macrophages (n = 9).</p

Regulation of FPR1 mRNA expression in human macrophages.

<p>A) Relative mRNA expression of FPR1 after stimulation of human macrophages with different stimuli for 24 hours. IL-3 (20 ng/ml), IL-4 (10 ng/ml), IL-13 (10 ng/ml), IL-1β (5 ng/ml), IL-6 (10 ng/ml), INFγ (50 ng/ml), TNFα (1 ng/ml), CpG (100 ng/ml), LPS (100 ng/ml), Poly I:C (1 ng/ml), 3M-001 (3 µM), 3M-002 (3 µM). B) Time-course experiment of FPR1 mRNA expression in macrophages stimulated with IL-4 (10 ng/ml) (dotted) and IL-13 (10 ng/ml) (white) for 24, 48 and 72 hours. The values are normalized for GAPDH mRNA expression and are presented relative to non-stimulated control macrophages (black). Bars display the mean and the standard deviation (±SD) of three independent experiments. *p<0.05, **p<0.01.</p

Regulation of FPR1 cell surface expression in human macrophages.

<p>FACS analysis was performed to investigate cell surface expression of FPR1 upon treatment with the indicated stimuli. A) FPR1 protein expression after stimulation of human macrophages for 48 hours with stimuli which were shown to regulate FPR1 mRNA expression (IL-4 (10 ng/ml), IL-13 (10 ng/ml), INFγ (50 ng/ml), LPS (100 ng/ml), 3M-002 (3 µM)). B) Time-course experiment of FPR1 protein expression in controls macrophages (black), or macrophages stimulated with IL-4 (10 ng/ml) (dotted) and IL-13 (10 ng/ml) (white) for 24, 48 and 72 hours. Values are presented relative to unstimulated macrophages. Bars display the mean and the standard deviation (±SD) of three independent experiments. *p<0.05, **p<0.01.</p

Resting perfusion map acquired from a single participant, shown in radiological orientation (scale: 0–90 ml/min/100ml).

<p>Resting perfusion map acquired from a single participant, shown in radiological orientation (scale: 0–90 ml/min/100ml).</p

Women show higher perfusion than men and DHEAS correlates negatively with perfusion.

<p>a) Sex difference in whole brain grey matter perfusion: perfusion is higher in women (<i>M</i> = 35.97 ml/min/100 ml, <i>SD</i> = 5.37) than in men (<i>M</i> = 30.47 ml/min/100 ml, <i>SD</i> = 5.91, <i>p</i> = .006). Single dots represent the subjects' individual values. The horizontal line within the boxes indicate medians, the edges of the boxes are the 25^th and 75^th percentiles, and the whiskers represent 1.5 times the interquartile range. b) Sex difference (women > men) in regional perfusion: women show higher regional perfusion than men (<i>p</i> = .004, FWE-corrected). c) Simple regression analysis with whole brain perfusion values as the dependent variable and DHEAS as the only predictor: a significant model was found (<i>p</i> = .007, adjusted <i>R</i>^<i>2</i> = .180) with a standardised β = -.452 for DHEAS. d) DHEAS effects in men and women: DHEAS correlates negatively with regional perfusion in both sexes (<i>p</i> = .004, FWE-corrected). Colour bar in a) and c) denotes a non-parametric <i>t</i> score, given by <i>a1</i>/[standard error(<i>a1</i>)], see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135827#sec002" target="_blank">methods</a>. Images are shown in neurological orientation. Slices are at MNI z-coordinates -45, -30, -15, 0, 15, 30, 45, 60, 75 (from top left to bottom right).</p

Mechanisms underlying loss of protective ROS signaling in early type-2 diabetic hearts.

<p><u>Panel A</u>: increase in isoform 2 of complex IV (COX) subunit IV (COX IV-2) in cardiac mitochondria from fructose-fed (ff) rats as compared to healthy rats. <u>Panel B</u>: uncoupling protein 3 (UCP 3) is increased in cardiac mitochondria of fructose-fed (ff) rats as compared with healthy rats. COX, cytochrome c oxidase. <u>Panel C</u>: leak respiration (normalized to citrate synthase (CS) activity) with pyruvate as measured by polarography in permeabilized cardiac fibers from fructose-fed rats collected at reperfusion. <u>Panel D</u>: relationship between hydrogen peroxide (H₂O₂) emission capacity determined by Amplex Red assay and leak respiration (normalized to citrate synthase activity) with pyruvate measured by polarography in permeabilized cardiac fibers from healthy (reproduced from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104971#pone.0104971-Zaugg1" target="_blank">[19]</a>) and fructose-fed (ff) rats collected at reperfusion. ff-IR(3 min), untreated hearts exposed to 15 min of ischemia and 3 min of reperfusion; ff-IR/IL(3 min), hearts exposed to 15 min of ischemia and 3 min of reperfusion with 1% Intralipid® at the onset of reperfusion; Arrows illustrate the opposing response to Intralipid® treatment in healthy vs. early diabetic hearts (see manuscript for details). **, significantly increased from healthy. Data are mean ± SD. N = 4–5 hearts in each group.</p

Mitochondrial fatty acid uptake and complex IV inhibition by palmitoylcarnitine in early type-2 diabetic hearts.

<p><u>Panel A</u>: carnitine palmitoyltransferase 1 and 2 (CPT1 and CPT2, respectively) activity at the onset of reperfusion in healthy (h-IR/IL(3 min)) and early diabetic (ff-IR/IL(3 min) hearts treated with 1% Intralipid®. *, significantly different from healthy hearts. N = 10 hearts in each group. <u>Panel B</u>: concentration-dependent inhibition of complex IV by palmitoylcarnitine (C16∶0c) in permeabilized cardiac fibers of healthy (reproduced from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104971#pone.0104971-Zaugg1" target="_blank">[19]</a>) and fructose-fed (ff) rats. Complex IV inhibition is given as relative decrease in oxygen consumption. IC₅₀, concentration of palmitoylcarnitine that reduces the respiration rate by 50%. N = 5–6 hearts per group. Data are or mean ± SD (panels A) or mean ± SEM (panel B).</p

Mitochondrial respiration in saponin-skinned cardiac fibers harvested at the end of the ischemia-reperfusion protocols.

<p>Mitochondrial respiration was measured in the presence of glucose-derived (pyruvate/malate) or fat-derived substrates (palmitoylcarnitine/malate). The measured oxygen consumption (normalized to citrate synthase activity) is expressed as nmol O₂ s⁻¹/CS. Data are presented as mean (SD). *, significantly increased compared to all other groups;#, significantly increased compared to ff-IR/IL.</p><p>ff-IR, hearts from fructose-fed rats exposed to ischemia-reperfusion (IR) without treatment (N = 10); ff-IR/SEV, hearts from fructose-fed rats exposed to IR with sevoflurane (2 vol.-%) conditioning (N = 10); ff-IR/IL, hearts from fructose-fed rats exposed to IR with Intralipid (1%) treatment at the onset of reperfusion (N = 6).</p

Complex IV inhibition by metabolites of Intralipid® fatty acid constituents.

<p>Panel A: Concentration-dependent inhibition of complex IV by palmitoyl(C16∶0c)-, oleoyl(C18∶1c)-, and linoleoylcarnitine (C18∶2c) in permeabilized cardiac fibers. Complex IV inhibition is given as relative decrease in oxygen consumption. Panel B and C: Concentration-dependent hydrogen peroxide (H₂O₂) emission capacity determined by Amplex Red assay in permeabilized cardiac fibers exposed to increasing concentrations of palmitoyl- and linoleoylcarnitine. *, significantly different from untreated. Data are mean (SEM). N = 6 hearts in each group/concentration.</p