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.