Table 1.
Characteristics and laboratory data from four groups of non-diabetic Apoe−/− mice and STZ-induced diabetic Apoe−/− mice treated with or without dapagliflozin.
Table 2.
Characteristics and laboratory data from three groups of diabetic db/db mice with or without ipragliflozin, and wild-type mice that received vehicle.
Fig 1.
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.
Table 3.
Correlation of atherosclerosis or foam cell formation to metabolic parameters
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.