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Lipoprotein lipase stimulates the binding and uptake of moderately oxidized low-density lipoprotein by J774 macrophages.

ABSTRACT: Lipoprotein lipase (LPL) stimulates the uptake of low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) in different cell types, including macrophages, through bridging of LPL between lipoproteins and extracellular heparan sulphate proteoglycans (HSPG). Because macrophages produce LPL and because modified lipoproteins are present in the arterial wall in vivo, we wondered whether LPL also enhances the uptake of oxidized LDL by J774 macrophages. LDL samples with different degrees of oxidation, as evaluated by relative electrophoretic mobility (REM) as compared with native LDL are used as well as native and acetylated LDL. Addition of 5 microg/ml LPL to the J774 cell culture medium stimulated the binding of both native LDL and moderately oxidized LDL (REM < 3.5) 50-100-fold, and their uptake was stimulated approx. 20-fold. The LPL-mediated binding of native LDL and moderately oxidized LDL was dose-dependent. Preincubation of the cells with heparinase (2.4 units/ml) inhibited the stimulatory effect of LPL, indicating that this LPL-mediated stimulation was due to bridging between the lipoproteins and HSPG. The binding to J774 macrophages of severely oxidized LDL (REM=4.3) was stimulated less than 3-fold by LPL, whereas its uptake was not stimulated significantly. The binding and uptake of acetylated LDL (AcLDL) were not stimulated by LPL, although the LPL-molecule itself does bind to AcLDL. Measurements of the cellular lipid content showed that addition of LPL also stimulated the accumulation in the cells of cholesteryl ester derived from both native LDL and moderately oxidized LDL in a dose-dependent manner. We conclude that our results present experimental evidence for the hypothesis that LPL serves as an atherogenic component in the vessel wall.

SUBMITTER: Hendriks WL

PROVIDER: S-EPMC1217086 | biostudies-other | 1996 Mar

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Macrophage accumulation in the vascular wall is a hallmark of atherosclerosis. Studies showed that shifting of oxidized lipids-induced inflammatory macrophages towards an anti-inflammatory phenotype by promoting oxidative metabolism attenuated atherosclerosis progression. Therefore, this study aimed to investigate whether metformin, which has ameliorated atherosclerosis in animal models and clinical trials, modulated oxidized low-density lipoprotein (Ox-LDL) induced inflammatory status in macrophages by regulating cellular oxidative metabolism. METHODS:Murine raw264.7 macrophages were incubated with Ox-LDL (50 μg/mL) in the presence or absence of metformin (15 μmol/L) for 24 h. Real-time polymerase chain reaction was used to quantify the transcription of classically activated (M1) pro-inflammatory and alternatively activated (M2) anti-inflammatory markers and mitochondrial DNA copy numbers. Cellular reactive oxygen species (ROS) production and mitochondrial membrane potential were detected by immunofluorescence. Cellular adenosine triphosphate (ATP) synthesis, glucose uptake, and lactic acid production were measured by commercial kit and normalized to cellular lysates. Western blotting analysis was performed to detect the expression of mitochondrial fusion/fission related proteins, enzymes mediating lipid metabolism and signaling pathway of glucose transport. Differences between groups were analyzed using one-way analysis of variance. RESULTS:Metformin improved Ox-LDL-impaired anti-inflammatory phenotype in raw264.7 macrophages as shown by up-regulated transcription of anti-inflammatory markers including interleukin 10 (0.76 ± 0.04 vs. 0.94 ± 0.01, P = 0.003) and Resistin-like molecule alpha (0.67 ± 0.08 vs. 1.78 ± 0.34, P = 0.030). Conversely, Ox-LDL-diminished phosphorylation of Akt was up-regulated by metformin treatment (0.47 ± 0.05 vs. 1.02 ± 0.08, P = 0.040), associated with an improvement of mitochondrial function, characterized by decreased ROS generation (2.50 ± 0.07 vs. 2.15 ± 0.04, P = 0.040), increased lipid oxidation, and elevated cellular ATP production (0.026 ± 0.001 vs. 0.035 ± 0.003, P = 0.020). Moreover, metformin-mediated Akt activation increased Akt substrate of 160 kDa (AS160) phosphorylation (0.51 ± 0.04 vs. 1.03 ± 0.03, P = 0.0041), promoted membrane translocation of glucose transporter 1, and increased glucose influx into the cells (4.78 ± 0.04 vs. 5.47 ± 0.01, P < 0.001). CONCLUSION:This study suggested that targeting macrophage metabolism with new or existing drugs had therapeutic potential for the prevention and treatment of diabetes-accelerated atherosclerosis.

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Lipoprotein lipase stimulates the binding and uptake of moderately oxidized low-density lipoprotein by J774 macrophages.

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