Metformin Improved Oxidized Low-Density Lipoprotein-Impaired Mitochondrial Function and Increased Glucose Uptake Involving Akt-AS160 Pathway in Raw264.7 Macrophages
The incidence of coronary heart disease is approximately doubled in type 2 diabetes, making it the most important cause of morbidity and mortality in diabetic patients. Accelerated atherosclerosis has been observed in diabetic patients, with hyperglycemia and insulin resistance identified as the two most significant risk factors. However, clinical trials and meta-analyses have shown limited benefits of intensive glucose-lowering treatment on all-cause mortality and deaths due to cardiovascular causes. Therefore, pharmacologic interventions targeting other specific mechanisms are warranted to alter the increased risk of atherosclerosis in diabetes.
Macrophages in response to the surrounding microenvironment polarize to different phenotypes. There are two main classes of macrophages: classically activated (M1) pro-inflammatory macrophages and alternatively activated (M2) anti-inflammatory macrophages. Studies have revealed that an imbalance in macrophage polarization is a key pathological factor for a variety of immune-related diseases such as autoimmune diseases, tumors, and atherosclerosis. Both M1 and M2 macrophages have been identified in atherosclerotic plaques of humans and mice, and an increased ratio of M1 over M2 polarization in human atherosclerosis is related to plaque instability. In mouse models of atherosclerosis, approaches to increase M1 polarization accelerate plaque formation, while increasing M2 polarization induces atherosclerosis regression. These findings suggest that manipulation of macrophage polarization might have therapeutic potential in treating atherosclerosis.
Cellular metabolism has a great impact on macrophage polarization and functionality. M1 macrophages are highly glycolytic, while M2 macrophages utilize fatty acid metabolism and mitochondrial oxidative phosphorylation (OXPHOS). Recent studies indicate that disruption of cellular energy metabolism directly alters macrophage M1/M2 fate and inflammatory functions. Shifting macrophage metabolism towards glycolysis drives a pro-inflammatory phenotype, whereas promotion of mitochondrial OXPHOS primes macrophages for alternative activation and inhibits the production of pro-inflammatory cytokines. Therefore, improving macrophage polarization imbalance in atherosclerosis might be achieved by regulating glycolysis and mitochondrial energy metabolism.
Metformin has been used as a glucose-lowering medication in humans for more than 60 years and is now recommended as first-line treatment for type 2 diabetes. Several clinical trials suggest a cardiovascular protective effect by using metformin in individuals with diabetes, while the anti-atherosclerotic mechanisms of metformin remain poorly understood. Metformin has been implicated to improve glucose and lipid metabolism by regulating mitochondrial function and adenosine monophosphate-activated protein kinase (AMPK) activity in insulin-targeted cells, but the metabolic effects on atherosclerotic-related immune cells such as macrophages remain elusive. Therefore, this study aimed to investigate the regulatory role of metformin in macrophage energy metabolism and whether this metabolic regulation mediates phenotypic conversion of macrophages in atherosclerosis.
Murine Raw264.7 macrophages were incubated with oxidized low-density lipoprotein (Ox-LDL) (50 mg/mL) in the presence or absence of metformin (15 mmol/L) for 24 hours. 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.
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).
M1 macrophages have been implicated in the pathogenesis of atherosclerosis and are induced by oxidatively-modified lipids and lipoproteins, the major lipid components in the atherosclerotic environment. Therefore, we examined the regulatory role of metformin, with established cardiovascular protective effects in humans and animals, in macrophage polarization in Ox-LDL-treated Raw264.7 macrophages. We characterized the genetic signature of polarized macrophages by quantifying the transcription of M1 and M2 markers. The real-time PCR results showed that Ox-LDL produced an inflammatory phenotype in macrophages by up-regulating the transcription of M1 markers TNF-α (1.01 ± 0.01 vs. 1.69 ± 0.29, P = 0.030) and IL-6 (1.12 ± 0.17 vs. 4.90 ± 1.66, P = 0.030), as well as down-regulating M2 markers IL-10 (0.96 ± 0.05 vs. 0.76 ± 0.04, P = 0.002). Metformin treatment significantly increased the transcription of M2 markers in Ox-LDL-loaded macrophages (IL-10: 0.76 ± 0.04 vs. 0.94 ± 0.01, P < 0.001; Retnla: 0.67 ± 0.08 vs. 1.78 ± 0.34, P = 0.030), while there were no detectable changes in M1 markers (TNF-α: 1.69 ± 0.29 vs. 1.56 ± 0.22, P = 0.850; IL-6: 4.90 ± 1.66 vs. 4.00 ± 1.34, P = 0.710). These results indicated the presence of an anti-inflammatory phenotype primed by metformin in Ox-LDL-loaded macrophages.
Mitochondria play a critical role in oxidative metabolism, fuel energy for polarization towards M2 macrophages, and sequential immune responses. Therefore, we investigated the regulatory role of metformin in mitochondrial metabolism in Ox-LDL-treated macrophages. Our results showed that metformin significantly decreased Ox-LDL-up-regulated ROS production (2.50 ± 0.07 vs. 2.15 ± 0.04, P = 0.040), and also increased Ox-LDL-impaired mitochondrial membrane potential (0.67 ± 0.05 vs. 0.86 ± 0.05, P = 0.040), correlating with an elevated cellular ATP production in metformin-treated macrophages (0.026 ± 0.001 vs. 0.035 ± 0.003, P = 0.020). We further tested the effects of metformin on mitochondrial DNA integrity and mitochondrial morphologies, both reported to regulate mitochondrial function. The results indicated that metformin up-regulated Ox-LDL-diminished mitochondrial DNA copy number (mt-ND-4: 0.42 ± 0.16 vs. 1.04 ± 0.24, P = 0.040; mt-ND-1: 0.82 ± 0.42 vs. 1.33 ± 0.40, P = 0.450), and increased the protein expression of fusion-related protein Mfn2 (0.55 ± 0.01 vs. 0.76 ± 0.01, P = 0.002), with little effects on fusion-related protein OPA1 (0.84 ± 0.03 vs. 0.77 ± 0.04, P = 0.110) and fission-related protein Drp-1 (0.77 ± 0.02 vs. 0.82 ± 0.01, P = 0.070). All these results suggested that metformin might promote macrophage oxidative metabolism by improving Ox-LDL-impaired mitochondrial function.
We then explored the characteristics of lipid and glucose metabolism in Ox-LDL-loaded macrophages after treatment with metformin. Our results showed that metformin significantly up-regulated Ox-LDL-impaired p-ACC (0.44 ± 0.02 vs. 0.78 ± 0.03, P = 0.002) and CPT-1b expression (0.61 ± 0.00 vs. 0.95 ± 0.01, P < 0.001), which subsequently mediated lipid synthesis inhibition and fatty acid oxidation, respectively. However, there were no detectable changes of NDUFS3 expression between groups (P = 0.230), the core sub-unit of mitochondrial complex I, which was considered as the main respiratory-chain target of metformin. This indicated that metformin might improve oxidative metabolism independent of mitochondrial respiratory chain complexes regulation. With respect to glucose metabolism, we found that glucose consumption was decreased after Ox-LDL loading (5.77 ± 0.04 vs. 6.39 ± 0.03, P < 0.001), which was reversed by metformin treatment (4.78 ± 0.04 vs. 5.47 ± 0.01, P < 0.001). In contrast, metformin significantly down-regulated Ox-LDL-induced lactic acid production in Raw264.7 macrophages (8.61 ± 0.67 vs. 6.42 ± 0.06, P = 0.002). Moreover, a decreased ratio of lactic output by glucose input in the presence of metformin was observed in Ox-LDL-treated macrophages (1.92 ± 0.15 vs. 1.18 ± 0.01, P < 0.001), implying that metformin mediated-increased glucose flux might enter into oxidative metabolic pathway instead of glycolysis in Ox-LDL-loaded macrophages.
Two paralogue Rab GTPase activating proteins AS160 (also known as TBC1D4) and TBC1D1 have been implicated in the regulation of glut traffic to plasma membrane in insulin-targeted cells through phosphorylation by Akt and AMPK, respectively. However, few studies have evaluated the possible role of AS160 and TBC1D1 in noninsulin-stimulated glucose transport. As we observed the increased membrane translocation of GLUT1, the primary glucose transporter in murine macrophages by metformin treatment, the role of metformin in AS160 and TBC1D1 regulation, as well as the upper-stream kinases Akt and AMPK were examined in Ox-LDL-loaded macrophages. Western blotting analysis showed that in the absence of Ox-LDL, metformin treatment up-regulated AMPK and TBC1D1 phosphorylation (p-AMPK: 0.59 ± 0.02 vs. 0.93 ± 0.13, P = 0.030; p-TBC1D1: 0.63 ± 0.06 vs. 0.96 ± 0.04, P = 0.020), despite little change of Akt and AS160 phosphorylation (p-Akt: 0.82 ± 0.06 vs. 0.60 ± 0.06, P = 0.060; p-AS160: 1.17 ± 0.23 vs. 0.61 ± 0.04, P = 0.080). However, after Ox-LDL loading, metformin improved Ox-LDL-diminished p-Akt, specifically Ser473, and phosphorylation of AS160 (p-Akt: 0.47 ± 0.05 vs. 1.02 ± 0.08, P = 0.040; p-AS160: 0.51 ± 0.04 vs. 1.03 ± 0.03, P = 0.004), while AMPK and TBC1D1 seemed to be unaffected (p-AMPK: 0.68 ± 0.06 vs. 0.50 ± 0.07, P = 0.230; p-TBC1D1: 0.56 ± 0.07 vs. 0.71 ± 0.07, P = 0.180). Altogether, these data implied that metformin mediated-activation of Akt-AS160 phosphorylation, instead of AMPK-TBC1D1, might play an important role in promoting glucose uptake in Ox-LDL-loaded macrophages.
In conclusion, this study shows that metformin regulated macrophage cellular metabolism in a way that is intimately linked to the cell’s inflammatory phenotype. Metabolic reprogramming of Ox-LDL loaded macrophages towards alternatively activated M2 cells by metformin treatment provides a new therapeutic target for the treatment of atherosclerosis.
doi.org/10.1097/CM9.0000000000000333
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