Liraglutide attenuates hepatic iron levels and ferroptosis in db/db mice

Abstract

Liver pathological changes are as high as 21%-78% in diabetic patients, and treatment options are lacking. Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor that is widely used in the clinic and is approved to treat obesity and diabetes. However, the specific protection mechanism needs to be clarified. In the present study, db/db mice were used to simulate Type 2 diabetes mellitus (T2DM), and they were intraperitoneally injected daily with liraglutide (200 μg/kg/d) for 5 weeks. Hepatic function, pathologic changes, oxidative stress, iron levels, and ferroptosis were evaluated. First, liraglutide decreased serum AST and ALT levels, and suppressed liver fibrosis in db/db mice. Second, liraglutide inhibited the ROS production by upregulating SOD, GSH-PX, and GSH activity as well as by downregulating MDA, 4-HNE, and NOX4 expression in db/db mice. Furthermore, liraglutide attenuated iron deposition by decreasing TfR1 expression and increasing FPN1 expression. At the same time, liraglutide decreased ferroptosis by elevating the expression of SLC7A11 and the Nrf2/HO-1/GPX4 signaling pathway in the livers of db/db mice. In addition, liraglutide decreased the high level of labile iron pools (LIPs) and intracellular lipid ROS induced by high glucose in vitro. Therefore, we speculated that liraglutide played a crucial role in reducing iron accumulation, oxidative damage and ferroptosis in db/db mice.

Keywords: Liver; ferroptosis; high glucose; iron overload; liraglutide; oxidative stress.

Graphical abstract

Figure 1.

LIRA improve the disorders of glycometabolism in db/db mice. (a) The blood glucose of mice from 9^th to the last day of 14^th week. (The data are shown as the means ± SEM. n = 6. *p < 0.05, **p < 0.01 vs. db/m group. ^##p < 0.01 vs. db/db group 9^th week. ^$p < 0.05, ^$$p < 0.01 vs. LIRA group 9^th week. ^&p < 0.05, ^&&p < 0.01 vs. db/db group.). (b) The HOMA-insulin resistance (HOMA-IR) of db/m, db/db and LIRA groups. (c) The intraperitoneal glucose tolerance test of mice with 120 min. (d) The area under of the curve as shown in panel C. The data are shown as the means ± SEM. n = 6. ***p < 0.001 vs. db/m group. ^##p < 0.01 vs. db/db group.

Figure 2.

The liver dysfunction and fibrosis in db/db mice. (a) The liver H&E staining of db/m, db/db and LIRA groups (n = 3). The black arrow represents the normal hepatocytes (db/m group) or ballooning hepatocytes (db/db and LIRA groups). The yellow arrow represents the adipocytes. (b) The serum ALT and AST content (n = 4–5). (c) Sirius red staining of mice liver (n = 3). (d) The immunohistochemical staining of TGF-β protein (n = 3). (e) The mean density of TGF-β protein as shown in panel D. (f-g) The expression and statistics of Collagen I and Collagen III proteins in liver tissue (n = 6). The results are presented as the mean ± SEM. *p < 0 05, **p < 0 01 vs. db/m group. ^#p < 0 05, ^##p < 0 01 vs. db/db group.

Figure 3.

The oxidative stress levels in the liver tissue of db/db mice. (a-b) The expression and statistics of NOX4 and 4-HNE in liver tissue (n = 6). (c-d) The total SOD and GSH-PX activity (n = 5–6). (e-f) The GSH and MDA content (n = 5–6). The results are presented as the mean ± SEM. *p < 0 05, **p < 0 01 vs. db/m group. ^#p < 0 05, ^##p < 0 01 vs. db/db group.

Figure 4.

The iron levels and iron-related transportproteins in the liver tissue of db/db mice. (a) The Perls’ staining of liver tissue (n = 3). (b) The mean density of Fe content as shown in panel A. (c-d) The expression and statistics of TfR1, DMT1 and FPN1 proteins (n = 6). The results are presented as the mean ± SEM. *p < 0 05, **p < 0 01 vs. db/m group. ^#p < 0 05, ^##p < 0 01 vs. db/db group.

Figure 5.

The ferroptosis related signaling pathway in the liver tissue of db/db mice. (a) The ultrastructure of mitochondria observed by TEM (n = 3). The black arrow represents the mitochondrial outer membrane. The red arrow represents regional focal fatty infiltration. (b-c) The expression and statistics of GPX4 and SLC7A11 proteins (n = 6). (d-e) The expression and statistics of Nrf2 and HO-1 proteins (n = 6). The results are presented as the mean ± SEM. *p < 0 05, **p < 0 01 vs. db/m group. ^#p < 0 05, ^##p < 0 01 vs. db/db group.

Figure 6.

Liraglutide inhibited the ferroptosis in HepG2 treated with high glucose. (a-b) The cell viability of HepG2 cells treated with high glucose with 0, 25, 50, 75, 100 mM for 24 h and 48 h (n = 6, *p < 0 05, **p < 0 01 vs. Con group). (c) The cell viability of HepG2 cells treated high glucose (HG, 75 mM), Erastin (200 nM), liraglutide (LIRA, 100 nM), Fer-1 (2 μM), DFO (200 μM). (d-f) The expression and statistics of GPX4, SLC7A11 and TfR1 proteins (n = 3). (g) The fluorescence absorption spectrum of BODIPY 581/591. (h) The folds changes of fluorescence absorption as shown in panel G. (i) The folds changes of fluorescence absorption of Calcein-AM with different groups (n = 6). The results are presented as the mean ± SEM. *p < 0 05, **p < 0 01 vs. Con group. ^#p < 0 05, ^##p < 0 01 vs. HG group.

Figure 7.

The schematic description of ferroptosis involved in liver of db/db mice. The diabetic mice owned a high glycemic index in whole body, accompanied with hepatic fibrosis and iron overload. The high level of TfR1 and lower level of FPN1 contributed to elevated LIP in liver. The excessive Fe²⁺ could aggravate lipid ROS generation by Fenton action. The unbalance system X_c⁻ could lead to glutathione exchange was inhibited, GSH synthesis was decreased, GPX4 was declined, resulting in ferroptosis. Nrf2 could promote HO-1 expression to elevated GPX4, against as lipid ROS injury. The liraglutide could decrease the overload iron level in liver by adjusting the TfR1 and FPN1 expression. The liraglutide could improve system X_c⁻ and Nrf2 /HO-1 pathway to against to ferroptosis in liver of db/db mice.