Molecular Mechanism Of Metformin In Modulating Foam Cell Phenotype

Background and objectiveAtherosclerotic cardiovascular disease is the main cause of morbidity and mortality in diabetic patients.Macrophage-derived foam cell formation is the hallmark of atherosclerosis.Studies have shown that diabetes-related metabolic factors such as hyperglycemia,dyslipidemia and increased oxidative stress,disrupt cholesterol homeostasis in foam cells,resulting in increased intracellular lipid accumulation,exacerbating local inflammatory responses,and promoting the development of atherosclerosis.However,cardiovascular outcome in diabetic patients after modulation of conventional risk factors proved to be disappointing.Clinical trials have demonstrated that intensive glycemic control didn’t affect diabetic-related macrovascular end point.Drugs designated to elevate decreased HDL-C concentrations in diabetic patients proved to have little cardiovascular benefit.Therefore,it has been the focus of research to explore specific diabetes-accelerated atherosclerosis mechanism for treatment of vascular complications.A body of evidence has pointed out diabetes promoted foam cell formation and atherosclerosis progression by impairing HDL-mediated reverse cholesterol through downregulation of the expression of cholesterol efflux receptors ABCA-1 and ABCG-1.As studies showed that pharmaceutical or genetic approaches to enhance cholesterol efflux by upregulating cholesterol transporters reduced lipid deposition in foam cells and attenuated progression of atherosclerosis,reverse cholesterol transport seemed to be a plausible therapeutic target for diabetic atherosclerosis.Metformin is the first-line treatment of type 2 diabetes.Studies have shown that it not only improves insulin resistance by promoting glucose and lipid metabolism,but also plays an important role in anti-ageing and treatment for cancer.Metformin exerted its beneficial effects partly by AMPK activation.Clinical trials and meta-analysis both implied that metformin reduces the risk of cardiovascular events in diabetic patients with less adverse effects compared to insulin and other hypoglycemic agents.Studies exploring molecular mechanism of metformin in attenuating atherosclerosis have found that it improved vascular endothelial cell dysfunction and inhibited vascular smooth muscle cell inflammation.However,there are few reports about the effect of metformin on foam cell formation,especially in the terms of reverse cholesterol transport process.In this study,we ought to investigate the effects of metformin on Ox-LDL-induced foam cell phenotype including cholesterol transport,inflammatory status and cellular metabolism under hyperglycemia,thus revealing the new mechanism of metformin in the prevention and treatment of diabetic cardiovascular complications.Methods and results1.In vitro foam cell model was established by incubating macrophage cellline raw264.7 with 50μg/ml Ox-LDL for 48 h.During the last 24h incubation metformin or PBS was added.Oil red O staining and fluorescent staining by BIODIPY493/503 were used to detect intracellular lipid droplets.Cholesterol quantification kit was used to detect intracellular cholesterol contents.Western blot and qPCR were used to detect the translational and transcriptional levels of cholesterol influx and efflux related proteins.Cholesterol uptake was evaluated with NBD-cholesterol by flow cytometry.Fluorescent microplate reader detected cholesterol efflux to ApoA-I and HDL with NBD-cholesterol,respectively.The results showed that metformin significantly reduced intracellular lipid droplet formation and decreased total cholesterol especially cholesterol-ester content,implying that metformin intervention ameliorated foam cell formation.Metformin treatment significantly increased HDL-mediated cholesterol efflux by upregulating the translation and transcription of ABCG-1,one of the ABC cholesterol transporters,without affecting cholesterol uptake and scavenger receptors responsible for cholesterol intake.2.Foam cell model was established as above.ELISA was used to detect the secretion of inflammatory cytokines into the medium after metformin treatment.qPCR was used to detect the transcription level of inflammation and polarization-related genes The results showed that Ox-LDL primed macrophage towards an inflammatory phenotype.Metformin significantly up-regulated Ox-LDL-impaired secretion and transcription of anti-inflammatory factor IL-10,alleviating the inflammatory status of foam cells.3.Foam cell model was established as above.Glucose detection kit was used to detect the cellular glucose consumption after metformin treatment.The lactic acid detection kit was to quantify the lactic acid level in the culture medium.ATP detection kit was used to detect cellular ATP production.With fluorescent probe DCFH-DA and JC-1,intracellular ROS and mitochondrial membrane potential were detected by flow cytometry,respectively.Western blot was used to detect the changes of enzymes involved in lipid metabolism and mitochondrial fission/fusion related proteins.The results showed that metformin increased the glucose consumption of foam cells,reduced lactate acid secretion and increased ATP production.Metformin also downregulated lipid synthesis and improved cellular oxidative metabolism by increasing phosphorylation of ACC and CPT-lb expression.Moreover,metformin treatment reduced cellular oxidative stress,upregulated Ox-LDL-impaired mitochondrial membrane potential and promoted mitochondrial fission by decreasing the expression of mitofusion related protein Mfn2Conclusions1.Metformin increased cholesterol efflux to HDL by upregulating the expression of reverse cholesterol transporter ABCG-1,resulting in reduced intracellular cholesterol deposition,contributing to decreased foam cell formation2.Metformin shifted the foam cell away from inflammatory phenotype by increasing the secretion and transcript of anti-inflammatory cytokine IL-10.3.Metformin reduced Ox-LDL-induced intracellular oxidative stress,upregulated Ox-LDL-impaired mitochondrial membrane potential,promoted mitochondrial fission and improved the oxidative metabolism of foam macrophages cells.