Severe depletion of ATP and glucose in perinecrotic zone of advanced plaques.

<p>A. Advanced atherosclerotic plaque, delineation in white of viable intima. Extract shows luminal (left) and perinecrotic zone (right) of viable intima. Note high expression of hexokinase II (HKII), indicative of hypoxia, in perinecrotic zone. B and C. Lower concentrations of ATP (B) and glucose (C) in perinecrotic zone (p<0.05). D and E. No significant difference in glycogen (D) and lactate concentrations (E) between luminal and perinecrotic zone. n = 6, paired t-test.</p

Patient characteristics (n = 6).

<p>Patient characteristics (n = 6).</p

Increased levels of ceramides in ischemic myocardium.

<p>Content of ceramide (A), sphingomyelin (B), phosphatidylcholine (C) and phosphatidylethanolamine (D), n = 7 per group. Results are shown as mean ± SEM, **<i>P</i><0.01 <i>vs.</i> control, ***<i>P</i><0.001 <i>vs.</i> control.</p

Depletion of ATP, glucose and glycogen in advanced human plaques.

<p>A. Energy metabolites were analyzed in intermediate (CCA) and advanced (ICA) segments of human endarterectomies. B. Metabolite concentrations were assessed in the viable part of the intima (delineated), i.e. intimal area minus necrotic core. C, D and E. ATP (C), glucose (D) and glycogen concentrations (E) were lower in advanced segments than in intermediate segments of the same plaque. Note logarithmic scale for ATP and glycogen. F. Lactate concentrations were higher in advanced segments of the plaque. n = 6, Wilcoxon Signed-Rank Test.</p

Increased expression of LDLr and LRP1.

<p><b>(A)</b> RT-QPCR analyses of mRNA expression of LDLr, VLDLr and SR-B1, n = 4 per group. <b>(B)</b> mRNA expression of VLDLr in HL-1 cells incubated in hypoxia for 0.5, 1, 6 and 24 h (n = 3). <b>(C)</b> Representative immunoblots of LDLr and LRP1. (D–E) Quantification of LDLr protein bands <b>(D)</b> and LRP1 protein bands <b>(E)</b> n = 6–7 per group. Results are shown as mean ± SEM, *<i>P</i><0.05 <i>vs.</i> control, **<i>P</i><0.01 <i>vs.</i> control, ***<i>P</i><0.001 <i>vs.</i> control.</p

Increased levels of cholesteryl esters in the pig myocardium.

<p><b>(A)</b> Content of cholesteryl esters, <b>(B)</b> triglycerides and <b>(C)</b> free cholesterol, n = 7 per group. Results are shown as mean ± SEM, ***<i>P</i><0.001 <i>vs.</i> control.</p

GLUT3 knockdown reduces glucose uptake and lipid droplet formation in hypoxic human macrophages.

<p>Human monocyte-derived macrophages were transfected with control siRNA or siRNA against GLUT3 and cultured in medium containing 11 mmol/l glucose for 24 h in hypoxia (1% O₂). (<b>A</b>) Representative immunoblots of GLUT3 and tubulin in hypoxic human macrophages transfected with negative control (NC) or GLUT3 siRNA. (<b>B</b>) Quantification of (<b>A</b>). Data are mean ± SEM from 4 macrophage donors. (<b>C</b>) Knockdown of GLUT3 reduces glucose uptake in hypoxic human macrophages. Data are mean ± SEM from 6 macrophage donors. (<b>D</b>) Knockdown of GLUT3 reduces triglyceride biosynthesis from radiolabeled glucose in hypoxic human macrophages. Data are mean ± SEM from 3 macrophage donors. (<b>E</b>) Micrograph of Oil Red O-stained hypoxic macrophages transfected with control siRNA or GLUT3 siRNA. Scale bar 10 µm. (<b>F</b>) Quantification of (<b>E</b>). Data are mean ± SEM of all cells present in 20 randomly selected pictures from each of 4 macrophage donors. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 vs negative siRNA control.</p

Hypoxia-induced increase in glucose uptake promotes accumulation of cytosolic lipid droplets in human macrophages through increased triglyceride synthesis.

<p>(A,B) Human monocyte-derived macrophages were cultured for 24 h in different glucose concentrations in the absence of exogenous lipids in normoxia (21% O₂) or hypoxia (1% O₂). (A) Micrograph of Oil Red O–stained macrophages exposed to the indicated glucose concentrations in normoxia or hypoxia. Scale bar 10 µm. (B) Quantification of (A). Data are mean ± SEM of all cells present in 20 randomly selected pictures from each of 7 macrophage donors. (C–E) Human monocytes-derived macrophages were incubated in medium containing 11 mmol/l glucose for 24 h in normoxia or hypoxia. (C) Glucose uptake. Data are mean ± SEM from 6 macrophage donors. (D) Triglyceride biosynthesis from radiolabeled glucose. Data are mean ± SEM from 7 macrophage donors. (E) Lactate biosynthesis from radiolabeled glucose. Data are mean ± SEM from 4 macrophage donors*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001 vs normoxia at same glucose concentration; ††<i>P</i><0.01, †††<i>P</i><0.001 vs normoxia at 1 mmol/l glucose.</p

GLUT3 is abundant in macrophages isolated from human atherosclerotic plaques and in hypoxic human macrophages.

<p>(<b>A</b>) Representative immunohistochemical staining of sections of a human atherosclerotic carotid plaque with antibodies against GLUT3, HIF-1á and the macrophage marker CD68. (<b>B</b>) GLUT3 and HIF-1á mRNA expression in CD14⁺ macrophages isolated from 8 human atherosclerotic carotid plaques. (<b>C</b>–<b>F</b>) GLUT3 mRNA and protein levels in human monocyte-derived macrophages cultured in medium containing 11 mmol/l glucose for 24 h in normoxia (21% O₂) or hypoxia (1% O₂). (<b>C</b>) Real-time RT-PCR analyses of GLUT3 mRNA expression. Data are mean ± SEM from 6 macrophage donors. (<b>D</b>) Representative immunoblots of GLUT3 and tubulin in normoxic (N) and hypoxic (H) human macrophages. (<b>E</b>) Quantification of immunoblots. Data are mean ± SEM from 6 macrophage donors. (<b>F</b>) Representative immunoflourescent images of macrophages cultured in normoxia or hypoxia and stained for GLUT3 (green) and nuclei (blue). Scale bar 10 µm. **<i>P</i><0.01, ***<i>P</i><0.001 vs normoxia.</p