Liraglutide Attenuates Hepatic Oxidative Stress, Inflammation, and Apoptosis in Streptozotocin-Induced Diabetic Mice by Modulating the Wnt/ β-Catenin Signaling Pathway

Abstract

Liraglutide has been extensively applied in the treatment of type 2 diabetes mellitus and also has hepatoprotective effects. However, the role of liraglutide treatment on liver injury in a mouse model of type 1 diabetes mellitus (T1DM) induced by streptozotocin (STZ) and its underlying mechanisms remain to be elucidated. In the present study, diabetes was initiated in experimental animals by single-dose intraperitoneal inoculation of STZ. Forty female C57BL/6J mice were equally assigned into five groups: diabetic group, insulin+diabetic group, liraglutide+diabetic group, insulin+liraglutide+diabetic group, and control group for eight weeks. Diabetic mice exhibited a significantly elevated blood glucose level and decreased body weight, and morphological changes of increased steatosis and apoptosis were observed in the liver compared with the control. Furthermore, a significant increase in the levels of malondialdehyde and inflammatory markers such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin 1β (IL-1β) and the proapoptotic proteins caspase-3 and Bax were observed in the livers of diabetic mice, together with marked increases in antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPX) as well as antiapoptotic protein Bcl-2, all of which were significantly mitigated by treatment with liraglutide, insulin, and their combinations. Interestingly, liraglutide monotherapy showed better efficacy in ameliorating liver injury in T1DM mice than insulin monotherapy, similar to the combined drug therapy. Furthermore, the expression of Wnt/β-catenin signaling pathway-associated molecules was upregulated in the liver of mice treated with liraglutide or insulin. The results of the present study suggested that liraglutide improves T1DM-induced liver injury and may have important implications for the treatment of nonalcoholic fatty liver disease (NAFLD) in patients with T1DM.

Figure 1

Effects of liraglutide on histological alterations in DM mice. (a) HE staining of liver tissue. Scale bar is 100 μm in the images. (b) Hepatic steatosis score. (c) PAS staining of liver tissue. (d) Triglyceride content in the liver. Data are expressed as mean ± SD (n = 4/group); p < 0.05 was defined as statistically significant. DM: saline-treated type 1 diabetes group. DMI: insulin treatment group; DML: liraglutide treatment group; DMIL: insulin+liraglutide treatment group; NC: normal glucose tolerance group; ^∗: compared to NC; #: compared to DM; †: compared to DMI; ‡: compared to DML.

Figure 2

Effects of liraglutide on hepatocyte apoptosis in DM mice. (a) Relative mRNA expression of Bcl-2, Bax, and caspase-3 in liver tissues was assessed by quantitative RT-PCR. (b, c) Representative Western blot images and densitometry measurements of Bcl-2, Bax, and caspase-3 expressions in liver tissues. (d) Representative immunofluorescence images of TUNEL-stained liver tissue sections. (e) Percentage of TUNEL-positive cells. Data are expressed as mean ± SD (n = 4/group); p < 0.05 was defined as statistically significant. Scale bar is 200 μm in the images. DM: saline-treated type 1 diabetes group; DMI: insulin treatment group; DML: liraglutide treatment group; DMIL: insulin+liraglutide treatment group; NC: normal glucose tolerance group; ^∗: compared to NC; #: compared to DM; †: compared to DMI; ‡: compared to DML; Bax: Bcl-2-associated X protein; RT-qPCR: reverse transcription quantitative real-time polymerase chain reaction; TUNEL: terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick-end labeling.

Figure 3

Effects of liraglutide on (a) MDA and hepatic oxidative stress markers (b) SOD and (c) GPX. Data are expressed as mean ± SD (n = 4/group); p < 0.05 was defined as statistically significant. DM: saline-treated type 1 diabetes group; DMI: insulin treatment group; DML: liraglutide treatment group; DMIL: insulin+liraglutide treatment group; NC: normal glucose tolerance group; ^∗: compared to NC; #: compared to DM; †: compared to DMI; ‡: compared to DML.

Figure 4

Effects of liraglutide on relative mRNA expression of (a) TNF, (b) IL-1β, and (c) IL-6. Data are expressed as mean ± SD (n = 4/group); p < 0.05 was defined as statistically significant. DM: saline-treated type 1 diabetes group; DMI: insulin treatment group; DML: liraglutide treatment group; DMIL: insulin+liraglutide treatment group; NC: normal glucose tolerance group; ^∗: compared to NC; #: compared to DM; †: compared to DMI; ‡: compared to DML.

Figure 5

Effects of liraglutide on Wnt/β-catenin pathway activity in DM mice. (a) Relative mRNA expression and quantification of Wnt/β-catenin signaling pathway-related molecules. (b, c) Protein bands and relative protein expression of Wnt/β-catenin signaling pathway-related molecules were detected by Western blot analysis; β-actin was included as a reference for normalization. Data are expressed as mean ± SD (n = 4/group); p < 0.05 was defined as statistically significant. DM: saline-treated type 1 diabetes group; DMI: insulin treatment group; DML: liraglutide treatment group; DMIL: insulin+liraglutide treatment group; NC: normal glucose tolerance group; ^∗: compared to NC; #: compared to DM; †: compared to DMI; ‡: compared to DML.