Amelioration of Hyperglycemia with a Sodium-Glucose Cotransporter 2 Inhibitor Prevents Macrophage-Driven Atherosclerosis through Macrophage Foam Cell Formation Suppression in Type 1 and Type 2 Diabetic Mice

Abstract

Direct associations between hyperglycemia and atherosclerosis remain unclear. We investigated the association between the amelioration of glycemia by sodium-glucose cotransporter 2 inhibitors (SGLT2is) and macrophage-driven atherosclerosis in diabetic mice. We administered dapagliflozin or ipragliflozin (1.0 mg/kg/day) for 4-weeks to apolipoprotein E-null (Apoe-/-) mice, streptozotocin-induced diabetic Apoe-/- mice, and diabetic db/db mice. We then determined aortic atherosclerosis, oxidized low-density lipoprotein (LDL)-induced foam cell formation, and related gene expression in exudate peritoneal macrophages. Dapagliflozin substantially decreased glycated hemoglobin (HbA1c) and glucose tolerance without affecting body weight, blood pressure, plasma insulin, and lipids in diabetic Apoe-/- mice. Aortic atherosclerotic lesions, atheromatous plaque size, and macrophage infiltration in the aortic root increased in diabetic Apoe-/- mice; dapagliflozin attenuated these changes by 33%, 27%, and 20%, respectively. Atherosclerotic lesions or foam cell formation highly correlated with HbA1c. Dapagliflozin did not affect atherosclerosis or plasma parameters in non-diabetic Apoe-/- mice. In db/db mice, foam cell formation increased by 4-fold compared with C57/BL6 mice, whereas ipragliflozin decreased it by 31%. Foam cell formation exhibited a strong correlation with HbA1c. Gene expression of lectin-like ox-LDL receptor-1 and acyl-coenzyme A:cholesterol acyltransferase 1 was upregulated, whereas that of ATP-binding cassette transporter A1 was downregulated in the peritoneal macrophages of both types of diabetic mice. SGLT2i normalized these gene expressions. Our study is the first to demonstrate that SGLT2i exerts anti-atherogenic effects by pure glucose lowering independent of insulin action in diabetic mice through suppressing macrophage foam cell formation, suggesting that foam cell formation is highly sensitive to glycemia ex vivo.

Conflict of interest statement

Competing Interests: This study was financially supported from AstraZeneca and Astellas Pharma Inc. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Oral glucose tolerance test.

Glucose (0.5 g/kg body weight) was administered orally through a gavage tube after 6 h of fasting, and blood glucose levels were measured at the specified time points of 0 (pre-glucose/fasting glucose level), and at 15, 30, 60, and 120 min after administration. Glucose curve after oral glucose loading in non-diabetic and diabetic apolipoprotein E-null mice (Apoe−/−) mice that received vehicle or dapagliflozin (A), and the area under the curve (AUC) (B). Glucose curve after oral glucose loading in diabetic db/db mice that received vehicle or ipragliflozin (C), and the AUC (D). The data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by Tukey test for the comparison of Apoe−/− mice: a, p < 0.05 vs. non-diabetic Apoe−/− mice that received vehicle; b, p < 0.05 vs. non-diabetic Apoe−/− mice that received dapagliflozin; c, p < 0.05 vs. diabetic Apoe−/− mice that received dapagliflozin. Unpaired t-test for the comparison of db/db mice: d, p < 0.05 vs. vehicle. n = 5 per group. †p < 0.01, ‡p < 0.001.

Fig 2. Suppressive effects of dapagliflozine administration against the development of aortic atherosclerotic lesions in non-diabetic and diabetic Apoe −/− mice.

Twenty-two mice at 15 weeks of age were made diabetic with peritoneal injections of STZ (50 mg/kg/day) for 5 consecutive days and twenty mice were treated with saline. The 17-week-old non-diabetic and diabetic Apoe−/− mice were orally given SGLT2i (dapagliflozin) or vehicle for 4 weeks, starting from 17 weeks of age. Representative atherosclerotic lesions in the aortic surface stained with oil red O (a-d) and measured (m). Yellow arrows show notable atherosclerotic lesions. In the aortic root, the atheromatous plaques and monocyte/macrophage accumulations were stained with Oil red O (e-h) or anti-MOMA2 antibody (i-l). Black arrows show notable atheromatous plaques. The severity of atheromatous plaques (n) and degree of monocyte/macrophage accumulation (o) were evaluated. The data are expressed as mean ± SEM. One way ANOVA followed by Tukey test: †p < 0.01, ‡p < 0.001, §p < 0.0001.

Fig 3. Foam cell formation in exudate peritoneal macrophages obtained from non-diabetic and diabetic Apoe −/− mice (A), and diabetic db/db mice (B).

Four days after an intraperitoneal injection of thioglycolate, the exudated peritoneal cells were isolated from the treated non-diabetic and diabetic Apoe−/− mice at 21 weeks of age (Fig 3A), or from the diabetic db/db mice at 13 weeks of age (Fig 3B). Adherent macrophages were incubated for 18 hours with the RPMI-1640 medium containing 10μg/mL oxidized low-density lipoprotein (LDL) in the presence of 0.1 mmoL [3H]olate that was conjugated with bovine serum albumin. The cellular lipids were extracted and the radioactivity of the cholesterol [3H]olate was determined with thin-layer chromatography. Foam cell formation was expressed as cholesteryl ester (CE) accumulation. The values show mean ± SEM. One-way ANOVA followed by Tukey test: ‡p < 0.001, §p < 0.0001.

Fig 4. Correlation of atherosclerosis to glycemic control or foam cell formation in non-diabetic and diabetic Apoe −/− mice.

Fig 4A shows the correlation between atherosclerosis and HbA1c in non-diabetic and diabetic Apoe−/− mice. Diabetic mice that received vehicle (n = 15); r = 0.56, p < 0.01. Diabetic mice that received dapagliflozin (n = 7); r = 0.94, p < 0.005. Combined diabetic mice (n = 22); r = 0.77, p < 0.0001. Fig 4B shows the correlation between foam cell formation and atherosclerosis in non-diabetic and diabetic Apoe−/− mice. Diabetic mice that received vehicle (n = 10); r = 0.91, p < 0.0005. Diabetic mice that received dapagliflozin (n = 5); r = 0.98, p < 0.005. Diabetic mice combined (n = 15); r = 0.95, p < 0.0001. r values indicate Pearson correlation coefficients. Pearson’s correlation test, p < 0.05.

Fig 5. Correlation between foam cell formation and glycemic control in diabetic Apoe −/− mice (A) or diabetic db/db mice (B).

Fig 5A shows the correlation between foam cell formation and HbA1c in non-diabetic and diabetic Apoe−/− mice. Diabetic mice that received vehicle (n = 10); r = 0.77, p < 0.01. Diabetic mice that received dapagliflozin (n = 5); r = 0.98, p < 0.005. Combined diabetic mice (n = 15); r = 0.91, p < 0.0001. Fig 5B shows the correlation between foam cell formation and HbA1c in diabetic db/db mice and wild-type (C57/BL6) mice. Diabetic mice that received vehicle (n = 18); r = 0.71, p < 0.001. Diabetic mice that received ipragliflozin (n = 13); r = 0.87, p < 0.0001. Combined diabetic mice (n = 31); r = 0.88, p < 0.0001. r values indicate Pearson correlation coefficients. Pearson’s correlation test, p < 0.05.

Fig 6. Changes in gene expression related to foam cell formation in the macrophages obtained from Apoe −/− mice.

Gene expressions were measured in the peritoneal macrophages obtained from non-diabetic Apoe−/− mice (n = 7), diabetic Apoe−/− mice that received vehicle (n = 6), and those that received dapagliflozin (n = 5). Gene expressions of (A) lectin-like ox-LDL receptor-1 (Lox-1), (B) CD36, (C) acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), (D) ATP-binding cassette transporter A1 (ABCA1), and (E) ATP-binding cassette sub-family G member1 (ABCG1) and the association with GAPDH were analyzed by real-time RT-PCR before the addition of ox-LDL. The values show mean ± SEM. One-way ANOVA followed by Tukey test for the comparison of Apoe−/− mice: *p < 0.05, †p < 0.01.

Fig 7. Changes in gene expression related to foam cell formation in the macrophages obtained from db/db mice and wild-type mice (C57/BL6).

Gene expressions were measured in the peritoneal macrophages obtained from wild-type (C57/BL6) mice (n = 3), diabetic db/db mice that received vehicle (n = 12), and those that received ipragliflozin (n = 6). Gene expressions of (A) Lox-1, (B) CD36, (C) ACAT1, (D) ABCA1, and (E) ABCG1 in association with GAPDH were analyzed by real-time RT-PCR before the addition of ox-LDL. The values show mean ± SEM. One-way ANOVA followed by Tukey test: *p < 0.05, †p < 0.01.