Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells

Abstract

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O2-dependent shuttle of oxidizable fatty acid substrates. MB's impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively.

Fig 1. MB-dependent fatty acid profiles.

Fatty acid profiles (C12-C20) of total lipids of the mouse milk (A), MDA-MB468 breast cancer cells cultured under normoxia (Nx, B) and severe hypoxia (0.2% O₂, C). In all graphs, MBwt as grey bars vs MBko black. FA levels are depicted as “% of FA / total FA pool”. Students t-test was used for statistics (n = 4–8); mean ± SD. *p≤0.05, **p≤0.01, ***p≤0.001. The axis is labelled FFA.

Fig 2. Impact of MB on indices of saturated and monounsaturated fatty acids.

FA compositions of the mouse milk and MDA-MB468 cells were presented as the percent of major MUFAs (C16 and C18, n9 and n7 species; A and F, respectively) or SFAs (C12, C14, C16, C18, C20; B and G, respectively) among total FAs amount, and as the MUFA vs. SFA ratio (C and H, respectively). The delta-9 desaturation index was measured as oleate (C18:1n9) vs. stearate (C18:0) ratio (D and I, respectively) or palmitoleate (C16:1n7) vs. palmitate (C16:0) ratio (E and J respectively). MDA-MB468 cells were exposed to four O₂ tensions for 72 hrs: Nx, 5% and 1% O₂ (oxygenated MB), 0.2% O₂ (deoxygenated MB). In all graphs, MBwt as grey bars versus MBko black. Students t-test was used for statistics (n = 4–8); mean ± SD. *p≤0.05, **p≤0.01, ***p≤0.001.

Fig 3. MB’s control of the limited oxidation of fatty acids.

Limited oxidation was measured as C14:0 vs. C16:0 ratio or C16:1n9 vs. C18:1n9 ratio in mouse milk (A, B) and MDA-MD468 cells (C, D). In all graphs, MBwt as grey bars versus MBko black. MDA-MB468 cells were exposed to Nx, 5% O₂, 1% O₂ (oxygenated MB) or 0.2% O₂ (deoxygenated MB). Students t-test was used for statistics (n = 4–8); mean ± SD. **p≤0.005, ***p≤0.001.

Fig 4. MB-based trafficking of fatty acids.

MB in MCF7 (A) and MDA-MB468 (B) cells controls the transport of a fluorogenic palmitate derivative (BODIPY-FL C16) between cytoplasm and vesicular depots in an O₂-dependent fashion. Cells were incubated with BODIPY (green) for 30 min before exposure for 72 hrs to normoxia (Nx, air, oxygenated MB) or severe hypoxia (Hx, 0.2% O₂, deoxygenated MB). The nuclei of MCF7 cells were stained with DAPI (blue). The figures shown are the representative of 6 independent experiments. Scale bar: 10 μm. Magnification: 63x. (C) MBwt and MBko MDA-MB468 cells incubated under normoxia (Nx) or severe hypoxia (Hx) were co-stained with BODIPY and a lysosome marker (Cytopainter red). The figures shown are representative of 3 independent experiments. Scale bar: 10 μm. Magnification: 63x. MBko (Nx) cells were stained by DAPI for nuclei. (D) Lysosomal (Cytopainter red) staining in MDA-MB468 cells was analysed by FACS measurement with the signal intensity expressed as geometric mean (X-GMean). Students t-test was used for statistics (n = 5); mean ± SD. *p≤0.05.

Fig 5. Lipid droplet homeostasis and MB.

MB regulates lipid droplet homeostasis in an O₂-dependent fashion. MCF7 (A) and MDA-MB468 (B) cells were incubated with BODIPY-FL C16 (green) for 30 min before exposure for 72 hrs to normoxia (Nx, oxygenated MB) or severe hypoxia (Hx, 0.2% O₂, deoxygenated MB). Cells were then fixed and co-stained with Nile red for lipid droplets and DAPI (blue) for nuclei. The figures shown are representative of 3 independent incubations. Scale bar: 20 μm. Magnification: 63x. Small inserts represent selected areas in higher magnification.

Fig 6. Model.

Schematic diagram of MB’s role as an O₂-dependent shuttle of FAs in mammary epithelial cells. Under normoxia and moderate hypoxia (left panel), oxygenated MB (MBO₂) binds C16/C18 FAs and promotes cytoplasmic solubility and FA oxidation in association with smaller lipid droplets. Under severe hypoxia (right), MB unloads O₂ and FA and deposits FAs to lipid droplets of larger sizes, potentially to prevent ROS poisoning, lipotoxicity and to store FA substrates for energy production upon reoxygenation.