Fig. 5: Carbon monoxide (CO) interferes with low-density lipoproteins (LDL) and glucose uptake.

a, b Analyses of the effect to LDL uptake by CO-RM. 3T3L1 cells were incubated with 10 μmol l⁻¹ CO-RM or control RuCl₃ for 2 h, then treated with 5 mg l⁻¹ Alexa Fluor 488-acetylated LDL. a Images of 3T3L1 cells treated with or without CO-RM and stained with Alexa Fluor 488-acetylated LDL (green) and DAPI (blue) are shown (scale bar: 10 μm). b Flow-cytometry analysis of the fluorescence intensities of 3T3L1 cells after incubation with Alexa Fluor 488-acetylated LDL for 60 min. The graph shows the mean of fluorescence intensities (per 10,000 cells) (n = 4–5). c Analyses of regulation of the LDL-R or VLDL-R localization by CO-RM. Plasma membrane fraction or whole-cell lysate of 3T3L1 cells with or without CO-RM were analyzed by western blotting using antibodies against LDL-R, VLDL-R, or GAPDH. d Analysis of the effect of 2-DG uptake by CO-RM. After treatment of 0.5 μmol l⁻¹ insulin for 18 min with or without 10 μmol l⁻¹ CO-RM for 2 h, 3T3L1 cells were incubated with 1 μmol l⁻¹ 2-DG for 20 min, and the 2-DG uptake was measured. The graph shows relative fold change by normalizing with 2-DG uptake of cells without CO-RM (n = 4). e Analyses of regulation of the GLUT4 translocation by CO-RM. Plasma membrane fraction of 3T3L1 cells (with or without treatment with CO-RM) was incubated with or without 0.5 µmol l⁻¹ insulin for 2 h. Protein expressions in the plasma membrane or whole-cell lysate were detected by western blotting using antibodies against either GLUT4, progesterone receptor membrane-associated component 1 (PGRMC1), pAkt, Akt, Na-K ATPase α1, or GAPDH. f 3T3L1 cells were transiently transfected with the shRNA-resistant expression vector of wild-type haem oxygenase 1 (HO-1) (WT) or the H25A mutant (H25A). The protein expressions were analyzed by western blotting using antibodies against HO-1 or GAPDH. g Analysis of the effect of HO-1 to 2-DG uptake. 2-DG uptake was measured in 3T3L1 cells (control or PGRMC1 KD) expressing HO-1 WT or H25A mutant. All data represent as mean ± S.E. Statistical analysis was performed using ANOVA with Tukey’s T test. ^*P < 0.05.