Metformin alleviates hepatic iron overload and ferroptosis through AMPK-ferroportin pathway in HFD-induced NAFLD

Abstract

Metformin prevents progression of non-alcoholic fatty liver disease (NAFLD). However, the potential mechanism is not entirely understood. Ferroptosis, a recently recognized nonapoptotic form of regulated cell death, has been reported to be involved in the pathogenesis of NAFLD. Here, we investigated the effects of metformin on ferroptosis and its potential mechanism in NAFLD. We found that metformin prevented the progression of NAFLD, and alleviated hepatic iron overload (HIO), ferroptosis and upregulated ferroportin (FPN) expression in vivo and in vitro. Mechanically, metformin reduced the lysosomal degradation pathway of FPN through activation AMPK, thus upregulated the expression of FPN protein, alleviated HIO and ferroptosis, and prevented progression of NAFLD. These findings discover a mechanism of metformin, suggesting that targeting FPN may have the therapeutic potential for treating NAFLD and related disorders.

Keywords: Biochemistry; Biological sciences; Cell biology; Pharmacology; Physiology.

Graphical abstract

Figure 1

Metformin prevented the progression of NAFLD in HFD rats (A and B) Body weight and liver weight of rats fed with normal diet (ND) or high-fat diet (HFD) without or with metformin (150 mg/kg or 300 mg/kg) intragastrically for 8 weeks. Data are represented as mean ± SEM (n = 6 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (C and D) Serum ALT and AST of rats fed with normal diet (ND) or high-fat diet (HFD) without or with metformin (150 mg/kg or 300 mg/kg) intragastrically for 8 weeks. Data are represented as mean ± SEM (n = 6 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (E) Liver histopathology assessed by H&E staining (magnification, 200×), scale bar: 100 μm. (F) NAFLD active score. Data are represented as mean ± SEM (n = 6 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, ^∗∗∗p < 0.001. (G) Liver fibrosis determined by Masson staining (magnification, 200×), scale bar: 100 μm. (H) Liver fibrosis score. Data are represented as mean ± SEM (n = 6 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗∗p < 0.001. NC, normal control; HFD-C, high fat diet control; Met, metformin; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Figure 2

Metformin ameliorated hepatic iron overload and ferroptosis in HFD-fed rats (A and B) Serum ferritin and hepatic iron content of rats fed with ND or HFD without or with metformin (150 mg/kg or 300 mg/kg) intragastrically for 8 weeks. Data are represented as mean ± SEM (n = 5–6 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05, ^∗∗∗p < 0.001. (C) Hepatic iron deposition assessed by Prussian blue staining (magnification, 200×), scale bar: 100 μm. (D) Pearson correlation analysis between hepatic iron and serum IL-6. (E) Spearman correlation analysis between hepatic iron and NAS. (F) Spearman correlation analysis between hepatic iron and liver fibrosis score. (G) MDA concentrations in liver tissue in rats fed with ND or HFD without or with metformin (150 mg/kg or 300 mg/kg) intragastrically for 8 weeks. Data are represented as mean ± SEM (n = 5–6 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05. (H) qPCR analysis of liver GPX4 in rats fed with ND or HFD without or with metformin (150 mg/kg or 300 mg/kg) intragastrically for 8 weeks. Data are represented as mean ± SEM (n = 5–6 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05. (I) Western blotting of hepatic GPX4, FTH, and FPN in rats fed with ND or HFD without or with metformin (150 mg/kg or 300 mg/kg) intragastrically for 8 weeks, GAPDH was used as the loading control. (J) Representative immunofluorescence staining image of hepatic GPX4 of rats fed with ND or HFD without or with metformin (150 mg/kg or 300 mg/kg) intragastrically for 8 weeks (magnification, 200×), scale bar: 100 μm. IL-6, interleukin-6; NAFLD, nonalcoholic fatty liver disease; MDA, malondialdehyde; FPN, ferroportin; FTH, ferritin heavy chain; GPX4, glutathione peroxidase 4; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

Metformin ameliorated iron overload and ferroptosis in PA-treated WRL68 cells (A) Cell viability assessed with a CCK-8 assay after 24 h or 48 h of pretreatment with different concentrations of PA (0.1 mM–0.6 mM). Data are represented as mean ± SEM (n = 4 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05, vs. control group; ^#p < 0.05, vs. PA 0.1 mM group; ^$p < 0.05, vs. PA 0.2 mM group; ^&p < 0.05, vs. PA 0.3 mM group. (B) Cell survival assessed by CCK-8 after 24 h of pretreatment with different concentrations of metformin (0.01 mM–25 mM) and PA (0.4 mM) for 24 h. Data are represented as mean ± SEM (n = 5 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05, vs. control group; ^#p < 0.05, vs. PA 0.4 mM group. (C–F) Intracellular levels of TG, MDA, GSH, and T-SOD in WRL68 cells treated with PA (0.4 mM) without or with metformin (0.5 mM, 1.0 mM, and 2.5 mM) or AICAR (1.0 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^#p < 0.05, ^##p < 0.01, vs. PA 0.4 mM group; ^&&p < 0.01, vs. PA + Metformin 0.5 mM group. (G) Distribution of iron between cells and supernatants in WRL68 cells treated with PA (0.4 mM), FAC (0.1 mM) without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^$$p < 0.05, vs. PA + FAC group. (H) Representative images of Oil Red O staining of WRL68 cells treated with PA (0.4 mM) in the absence or presence of Fer-1 (4 μM) or metformin (2.5 mM) for 24 h (magnification, 200×), scale bar: 100 μm. (I) Western blotting of AMPKα, P-AMPKα, and GPX4 after 24 h of PA (0.4 mM) treatment in the absence or presence of Fer-1 (4 μM) or metformin (2.5 mM) in WRL68 cells. GAPDH was used as the loading control. (J) Cell viability assessed by CCK-8 after 24 h of pretreatment with erastin (20 μM) without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, ^∗∗∗p < 0.001. (K–M) Intracellular levels of MDA, GSH and T-SOD in WRL68 cells treated with erastin (20 μM) without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^$$p < 0.01, vs. erastin group. (N) Analysis of total iron, Fe²⁺and Fe³⁺ in pellets of WRL68 cells after erastin (20 μM) treatment for 24 h without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^##p < 0.01, vs. erastin group. (O) Total iron, Fe²⁺ and Fe³⁺ in supernatants of WRL68 cells after erastin (20 μM) treatment for 24 h without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group. PA, palmitic acid; Met, metformin; TG, triglyceride; MDA, malondialdehyde; GSH, glutathione; T-SOD, total superoxide dismutase; Fer-1, ferrostatin-1.

Figure 4

Effects of metformin on iron overload in vitro NAFLD model (A) Concentrations of total iron, Fe²⁺and Fe³⁺ in pellets of WRL68 cells treated with PA (0.4 mM), and FAC (0.1 mM) without or with metformin (2.5 mM) or AICAR (1.0 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^$p < 0.05, ^$$p < 0.01, vs. PA + FAC group. (B) Concentrations of total iron, Fe²⁺ and Fe³⁺ in supernatants of WRL68 cells treated with PA (0.4 mM), and FAC (0.1 mM) without or with metformin (2.5 mM) or AICAR (1.0 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05, ^∗∗p < 0.01, vs. control group; ^$p < 0.05, ^$$p < 0.01, vs. PA + FAC group; ^&&p < 0.01, vs. PA+FAC+Metformin group. (C and D) Intracellular levels of TG and TC in WRL68 cells treated with FAC (0.1 mM, 0.2 mM) without or with PA (0.4 mM) in the absence or presence of metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^##p < 0.01, vs. FAC 0.2 mM group; ^$$p < 0.01, vs. PA + FAC 0.1 mM group; ^&&p < 0.01, vs. PA + FAC 0.2 mM group. (E–G) Intracellular levels of MDA, GSH and T-SOD in WRL68 cells treated with FAC (0.1 mM, 0.2 mM) without or with PA (0.4 mM) in the absence or presence of metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^$$p < 0.01, vs. PA + FAC group; ^&&p < 0.01, vs. PA+FAC+Metformin group. TG, triglyceride; TC, total cholesterol; FAC, ferric ammonium citrate; MDA, malondialdehyde; GSH, glutathione; T-SOD, total superoxide dismutase.

Figure 5

Metformin upregulates the expression of FPN by reducing its lysosomal ubiquitination degradation in an AMPK-dependent manner (A) Metformin upregulates FPN mRNA expression in PA-treated WRL68 cells. Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were determined by one-way ANOVA analysis. ^∗∗∗p < 0.001. (B) Metformin upregulates FPN protein expression and downregulates FTH protein expression in PA-treated WRL68 cells. (C) WRL68 cells were transfected with siRNA-NC or siRNA-FPNs (siRNA-FPN1, siRNA-FPN2, and siRNA-FPN3) for 48 h, and the relative mRNA expression of FPN was determined by RT-PCR. Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were determined by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^$$p < 0.01, vs. SiRNA-NC group; ^&&p < 0.01, vs. SiRNA-FPN2 group. (D) WRL68 cells were transfected with siRNA-FPN3 or siRNA-NC for 48 h. FPN knockdown was confirmed through western blotting. (E–G) Intracellular total iron, Fe²⁺ and Fe³⁺ levels. WRL68 cells were transfected with siRNA-FPN or siRNA-NC for 48 h, then treated with PA (0.4 mM) without or with metformin (2.5 mM) for 24 h, and intracellular total iron, Fe²⁺ and Fe³⁺ levels in WRL68 cells were measured. Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were determined by one-way ANOVA analysis. ^∗p < 0.05, ^∗∗p < 0.01, vs. control+siRNA-NC group; ^#p < 0.05, ^##p < 0.01, vs. PA+siRNA-NC group; ^$p < 0.05, vs. siRNA-FPN group; ^&p < 0.05, vs. PA+siRNA-FPN group. (H) PA-treated WRL68 cells were treated without or with metformin (2.5 mM), were then incubated with 100 μM CQ (a lysosome inhibitor) for 2 h, and the expression of FPN protein was assessed by western blotting. (I) PA-treated WRL68 cells were treated without or with metformin (2.5 mM), were then incubated with 10 μM MG132 (a proteasome inhibitor) for 8 h, and the expression of FPN protein was assessed by western blotting. (J) Ubiquitin/FPN co-immunoprecipitation of cells extracts of WRL68 cells transfected with GFP-tagged human overexpression FPN plasmid. WRL68 cells were divided into four groups: (1) GFP-FPN+CQ group; (2) GFP-FPN+CQ + PA group; (3) GFP-FPN+CQ + PA + Met group, and (4) GFP-FPN+CQ + PA + AICAR group. Cells were then lysed and immunoprecipitated with anti-FPN antibody or IgG as negative control, and immunocomplexes were immunoblotted by anti-ubiquitin or anti-FPN antibody (output). Also, the expressions of FPN in cell lysates before immunoprecipitation were assessed (input). (K) Short hairpin RNAs (shRNA) for transient silencing of AMPKα gene (sh-AMPKα) or non-targeting control shRNA (sh-NC) were constructed. The one with the highest silencing efficiency (sh-AMPKα-2) was selected for the experiment. WRL68 cells were transfected with GFP-tagged human overexpression FPN plasmid. The cells were then divided into four groups: (1) GFP-FPN+CQ+sh-NC;( 2) GFP-FPN+CQ + PA+sh-NC; (3) GFP-FPN +CQ + PA+Met+ sh-NC; and (4) GFP-FPN+CQ + PA+Met+sh-AMPKα-2. Ubiquitin/FPN was assessed by co-immunoprecipitation. PA, palmitic acid; FPN, ferroportin; FTH, ferritin heavy chain; GPX4, glutathione peroxidase 4; CQ, chloroquine.

Figure 6

Metformin ameliorated iron overload through AMPK/FPN pathway in PA+FAC-induced WRL68 cells (A) WRL68 cells were transfected with GFP-tagged human overexpression FPN plasmid. The protein expressions of FPN were confirmed by western blotting. (B–D) Intracellular total iron, Fe²⁺ and Fe³⁺ levels. WRL68 cells were transfected with GFP-tagged human overexpression FPN plasmid or empty vector plasmid, then cells were treated with PA (0.4 mM), FAC (0.1 mM) without or with metformin (2.5 mM) or Compound C (15 μM), and intracellular total iron, Fe²⁺ and Fe³⁺ levels in WRL68 cells were measured. Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were determined by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^##p < 0.01, vs. PA+FAC+empty vector group; ^&&p < 0.01, vs. PA+FAC+empty vector +Met +Compound C group. PA, palmitic acid; FPN, ferroportin; FAC, ferric ammonium citrate.