Fig 2: Application of a cell-based model to assess the metformin and iron chelation treatment on iron misregulation and glucose dyshomeostasis(A) Schematic of cell stimulations. Cells were grown for 48 h in either low glucose (1 g/L) or high glucose (10 g/L) high iron media (additional 10 μM). Cells were then treated with either metformin (2 mM) (green octagon) or deferoxamine (25 μM) (purple diamond), an iron chelator.(B–F) Representative western blot of cell stimulations for transferrin receptor (TFRC) (B), ferritin heavy chain (FTH1) (C), fatty acid synthase (FAS) (D), sterol regulatory binding protein 1 (SREBP-1) (E), and glucose transporter 2 (GLUT2) (F).(G–K) Densitometry analysis of western blot for FAS (G), precursor form of SREBP-1 (∼125 kDa) (H), GLUT2 (I), TFR1 (J), and FTH1 (K) in stimulated cells (n = 3). Loading controls in panels B, C, and D are from the same membranes as are loading controls in panels E and F as these proteins were detected via sequential probing of a single gel. Full uncropped blots with molecular weight markers for all western blot panels presented in this figure are provided in Figure S10. Data displayed as Mean ± SEM. α-tubulin was utilized as a loading control for all western blot analyses. One-tailed t test was used for all statistical analysis. ∗p-value <0.05, ∗∗p-value <0.01, ∗∗∗p-value <0.001, ∗∗∗∗p-value <0.0001.

Fig 3: (A) Ferrous iron (Fe2+) contents and transferrin receptor (TFRC) levels, (B) glutathione peroxidase 4 (GPX4) activities, reduced glutathione (GSH), and 4-hydroxynonenal (4-HNE) levels in lungs of mice at two time points after cecal ligation and puncture (CLP). Calcitriol treatment reduced Fe2+, TFRC, and 4-HNE levels, and enhanced GSH levels and GPX4 activities in ovariectomized mice with sepsis. OB, mice with a sham-ovariectomy operation and a high-fat diet (HFD) (n = 8); OVSS, mice with an ovariectomy, an HFD, and then cecal ligation and puncture (CLP) was performed, and saline was injected after CLP, sacrificed on either 24 h (OVSS-24) or 72 h (OVSS-72) (n = 8 at each time point); and OVSD, mice with an ovariectomy, an HFD, and CLP, and calcitriol was injected after CLP, sacrificed on either 24 h (OVSD-24) or 72 h (OVSD-72) (n = 8 at each time point). Values are expressed as the mean ± SEM. Comparisons among experimental groups were analyzed by a two-way ANOVA followed by the Bonferroni post hoc test. +Significantly differs from the OVSS group on 72 h. ⁣∗Significantly differs from the OVSS group at the same time point (p < 0.05).

Fig 4: HGD induces hepatic iron overload(A) Inductively coupled plasma mass spectrometry (ICP-MS) analysis of liver tissue of HGD and vehicle mice. Samples were normalized by tissue weight. (n = 6) (B) Real-time quantitative PCR of iron regulators in the liver of HGD and vehicle mice. Gene expression analysis was performed for divalent metal transporter 1 (DMT1), ferroportin (FPN1), transferrin receptor 1 (TFRC), ferritin heavy chain (FHC), ferritin light chain (FLC), hepcidin (HAMP), iron-responsive element-binding protein 1 (IRP1), and iron-responsive element-binding protein 2 (IRP2). (n = 6) (C) Representative western blot of transferrin receptor (TFRC) in the liver of HGD and vehicle mice.(D) Representative western blot of ferritin heavy chain 1 (FTH1) in the liver of HGD and vehicle mice.(E)Representative western blot of divalent metal transporter 1 (DMT1) in the liver of HGD and vehicle mice.(F) Representative western blot of ferroportin (FPN1) in the liver of HGD and vehicle mice.(G) Densitometry analysis of TFRC western blot for HGD and vehicle mice (n = 6).(H) Densitometry analysis of FTH1 western blot for HGD and vehicle mice (n = 6).(I) Densitometry analysis of DMT1 western blot for HGD and vehicle mice (n = 6).(J) Densitometry analysis of FPN1 western blot for HGD and vehicle mice (n = 6). Full uncropped blots with molecular weight markers for all western blot panels presented in this figure are provided in Figure S9. Data displayed as Mean ± SEM. α-tubulin was utilized as a loading control for all western blot analysis. One-tailed t test was used for all statistical analyses. ∗p-value <0.05, ∗∗p-value <0.01, ∗∗∗p-value <0.001, ∗∗∗∗p-value <0.0001.