Dulaglutide exerts beneficial anti atherosclerotic effects in ApoE knockout mice with diabetes: the earlier, the better

Abstract

There has been no report about the mechanism for anti-atherosclerotic effects of dulaglutide (Dula) and/or about the difference of its effectiveness between in an early and a late phase of diabetes. To address such questions, streptozotocin (STZ) was intraperitoneally injected to ApoE knockout mice at 8 weeks of age. Either Dula or vehicle was administered to STZ-induced diabetic ApoE knockout mice from 10 to 18 weeks of age as an early intervention group and from 18 to 26 weeks as a late intervention group. Next, non-diabetic ApoE knockout mice without STZ injection were subcutaneously injected with either Dula or vehicle. In an early intervention group, atherosclerotic lesion in aortic arch and Mac-2 and CD68-positive areas in aortic root were significantly smaller in Dula group. In abdominal aorta, expression levels of some villain factors were lower in Dula group. In a late intervention group, there were no immunohistological differences in aortic root and expression levels of various factors between two groups. Furthermore, even in non-diabetic ApoE knockout mice, expression levels of inflammatory and macrophage markers were reduced by treatment with Dula. Taken together, Dula exerts more beneficial anti-atherosclerotic effects in an early phase of diabetes rather than in a late phase.

Figure 1

Blood glucose levels, body weights and food intake in an early and a late intervention group in STZ-induced ApoE knockout mice. STZ-induced diabetic ApoE knockout mice were subcutaneously injected either Dula (0.6 mg/kg × 2/week) or vehicle in an early and a late intervention group. (a) Blood glucose levels, (b) body weights and (c) food intake in an early intervention group. Dula (black circle) (n = 9), control (white circle) (n = 9). (d) Blood glucose levels, (e) body weights and (f) food intake in a late intervention group. Dula (n = 8), control (n = 10). Values are the mean ± SEM of data obtained from each group. #p < 0.05 (Early Cont vs Early Dula), §p < 0.05 (Late Cont vs Late Dula).

Figure 2

Atherosclerotic lesion in aortic arch and Masson trichrome staining of aortic root in ApoE knockout mice. (a) Representative Sudan-IV staining pictures in aortic arch. (b) Sudan-IV positive area (% lesion area) (c) Representative Masson trichrome staining pictures in aortic root. (d) Masson trichrome staining (% lesion area). (e) Necrotic core in aortic root (% lesion area). Non-DM Cont (n = 7), non-DM Dula (n = 6), Early Cont (n = 5–6), Early Dula (n = 5 ~ 6), Late Cont (n = 6), Late Dula (n = 6). Values are the mean ± SEM of data obtained from each group. #p < 0.05 (Early Cont vs Early Dula), §p < 0.05 (Late Cont vs Late Dula).

Figure 3

CD68 and Mac2 staining of aortic root in early and late intervention groups in STZ-induced ApoE knockout mice. (a) Representative CD68 staining pictures, (b) CD68 positive area (% lesion area), (c) Representative Mac2 staining pictures, (d) Mac2 positive area (% lesion area). Early Cont (n = 5–6), Early Dula (n = 5–6), Late Cont (n = 6), Late Dula (n = 6). #p < 0.05 (Early Cont vs Early Dula).

Figure 4

mRNA expression levels of Glp-1r and inflammatory and coagulation markers in abdominal aorta. (a) Glp-1r mRNA expression levels, (b–g) mRNA expression levels of inflammatory and coagulation markers expressed as fold change relative to non-diabetic control group. Non-diabetic Cont (n = 8), Non-diabetic Dula (n = 8), Early diabetic Cont (n = 8), Early diabetic Dula (n = 6), Late diabetic Cont (n = 8), Late diabetic Dula (n = 7). Values are the mean ± SEM of data obtained from each group. #p < 0.05 (Early Cont vs Early Dula).

Figure 5

mRNA expression levels related to cell adhesion molecules and plaque stability in abdominal aorta. (a, b) mRNA expression levels related to cell adhesion molecules such as Vcam-1 and Icam. (c–g) mRNA expression levels related to plaque stability such as Mmp-2, Mmp-9, Mmp-3, Timp-1 and Timp2 expressed as fold change relative to non-diabetic control group. Non-diabetic Cont (n = 8), Non-diabetic Dula (n = 8), Early diabetic Cont (n = 8), Early diabetic Dula (n = 6), Late diabetic Cont (n = 8), Late diabetic Dula (n = 7). Values are the mean ± SEM of data obtained from each group. *p < 0.05 (non-DM Cont vs non-DM Dula), #p < 0.05 (Early Cont vs Early Dula).

Figure 6

mRNA expression levels of macrophage markers and M2-like macrophage markers in abdominal aorta. (a, b) mRNA expression levels of macrophage markers such as F4/80 and Cd68. (c–g) mRNA expression levels M2-like macrophage markers such as Il-10, Cd206, Fizz1, Arg-1 and Ym-1 expressed as fold change relative to control group. Dula (n = 7), Control (n = 8). *p < 0.05 (non-DM cont vs non-DM Dula), #p < 0.05 (Early Cont vs Early Dula), §p < 0.05 (Late Cont vs Late Dula).