Imeglimin Inhibits Macrophage Foam Cell Formation and Atherosclerosis in Streptozotocin-Induced Diabetic ApoE-Deficient Mice

Abstract

Atherosclerotic cardiovascular disease is a major complication of diabetes, whose progression is significantly accelerated by hyperglycemia. Imeglimin, a novel oral antidiabetic agent, has demonstrated efficacy in glucose control; however, its role in diabetes-related cardiovascular complications has not yet been fully explored. This study aimed to investigate the effects of imeglimin on foam cell formation and atherosclerosis in the context of diabetes. THP-1 macrophages were treated with oxidized low-density lipoprotein (LDL) and high glucose to induce foam cell formation in vitro. Additionally, ApoE^-/- mice with streptozotocin-induced diabetes were used to determine the effects of imeglimin in vivo by analyzing metabolic parameters and atherosclerotic plaque formation. Imeglimin inhibited macrophage-derived foam cell formation by promoting the expression of ATP-binding cassette transporters (ABC) A1 and ABCG1 and downregulating the expression of CD36. The effects of imeglimin on ABCG1 and CD36 expression regulation was mediated by AMPK. In diabetic ApoE^-/- mice, imeglimin reduced the atherosclerotic plaque area, decreased fasting glucose and LDL cholesterol levels, and upregulated ABCG1 expression in the liver and aorta. These findings suggest that imeglimin may have a preventive effect on foam cell formation and a therapeutic role in atherosclerosis progression in diabetic conditions.

Keywords: atherosclerosis; cholesterol efflux; foam cell formation; imeglimin; macrophage; type 2 diabetes.

Figure 1

Effect of imeglimin on foam cell formation. THP-1 macrophages were pretreated with imeglimin and then exposed to 100 μg/mL ox-LDL and 30 mM glucose. (A) The formation of foam cells was detected by Oil Red O staining. (B) The lipid droplet area was quantified using Image J. Results are expressed as the means ± SEMs of at least three independent experiments. **** p < 0.0001. HG, high glucose; IME, imeglimin; ox-LDL, oxidized low-density lipoproteins.

Figure 2

Effect of imeglimin on cholesterol efflux. THP-1 macrophages were pretreated with imeglimin and then exposed to 100 μg/mL ox-LDL and 30 mM glucose. Treatment with imeglimin promotes cholesterol efflux in THP-1 macrophage-derived foam cells. Cholesterol efflux was assayed using a fluorometric assay kit. Results are expressed as the means ± SEMs from five independent experiments, each performed in triplicate. ** p < 0.01.

Figure 3

Effects of imeglimin on the expression of ABCA1, ABCG1, and CD36 proteins in ox-LDL/HG-treated THP1 macrophages. THP-1 macrophages were pretreated with imeglimin and then exposed to 100 μg/mL ox-LDL and 30 mM glucose. (A) Western blot analysis showing the expression of ABCA1, ABCG1, and CD36 proteins; (B) Relative protein expression levels of ABCA1, ABCG1, and CD36 (quantified using ImageJ). Results are expressed as the means ± SEMs of at least three independent experiments. * p < 0.05, ** p < 0.01. HG, high glucose; IME, imeglimin; oxLDL, oxidized lowdensity lipoproteins.

Figure 4

Effect of imeglimin on the expression of ABCA1, ABCG1, and CD36 in the presence of the AMPK inhibitor compound C. THP-1-derived macrophages were pretreated with compound C, incubated with 100 μM imeglimin for 24 h, and exposed to 100 μg/mL oxLDL and 30 mM glucose. (A) Western blot analysis showing the expression of total AMPK (tAMPK) and phosphorylated AMPK (p-AMPK) proteins; (B) Relative protein expression levels of t-AMPK and pAMPK (quantified using ImageJ); (C) Western blot analysis showing the expression of ABCA1, ABCG1, and CD36 proteins; (D) Relative protein expression levels of ABCA1, ABCG1, and CD36 (quantified using ImageJ). Results are expressed as the means ± SEMs of at least three independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001. HG, high glucose; IME, imeglimin; ox-LDL, oxidized low-density lipoproteins.

Figure 5

Body weight changes in mice after nine weeks of imeglimin treatment. Diabetic mice show a decrease in body weight over time compared to control ApoE^−/− mice. No significant difference in body weight is observed between diabetic mice from the DMvehicle and DM-imeglimin groups. STZ, streptozotocin.

Figure 6

Effect of imeglimin on atherosclerotic plaque formation in the aortas of diabetic ApoE^−/− mice. (A) Representative images of en face analyses of mice aortas showing atherosclerotic plaques stained with Oil Red O; (B) Quantitative measurement of the en face plaque area (%) observed in mice aortas stained with Oil Red O. Results are expressed as the means ± SEMs of independent experiments (Control: n = 2, DM-vehicle: n = 5, DM-imeglimin: n = 5). * p < 0.05, ** p < 0.01.

Figure 7

Effects of imeglimin on AMPK activity and ABCA1, ABCG1, and CD36 protein expression in diabetic ApoE^-/- mice. (A) Western blot analysis showing the expression of pAMPK, tAMPK, ABCA1, ABCG1, and CD36 in the liver; (B) Relative protein expression levels of pAMPK, tAMPK, ABCA1, ABCG1, and CD36 in the liver (quantified using ImageJ); (C) Western blot analysis showing the expression of pAMPK, tAMPK, ABCA1, ABCG1, and CD36 in the aorta; (D) Relative protein expression levels of pAMPK, tAMPK, ABCA1, ABCG1, and CD36 in the aorta (quantified using ImageJ). Results are expressed as the means ± SEMs of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.