Metformin improved oxidized low-density lipoprotein-impaired mitochondrial function and increased glucose uptake involving Akt-AS160 pathway in raw264.7 macrophages

Abstract

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.