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.

Endogenous myoglobin expression in mouse models of mammary carcinoma reduces hypoxia and metastasis in PyMT mice

“…Towards a possible function of this ectopically expressed MB protein, it should be noted that the globin occurs exclusively within luminal cells (as opposed to myoepithelial cells) in healthy and malignantly transformed mammary epithelia of ´mice and men´ 3 , thus suggesting MB to also function as a fatty acid carrier in vivo. This O 2 -dependent shuttling of C16 saturated and C18 monounsaturated fatty acids by MB was already implied in earlier in vitro studies 8 – 11 and further substantiated in a recent study from our group 12 . Beyond the mammary gland, we also demonstrated that the presence of MB in the brown adipose tissue (BAT) of mice appears to link oxygen and lipid-based thermogenic metabolism by shifting the lipid droplet (LD) equilibrium towards higher counts of smaller droplets (i.e., towards a browning phenotype) 13 .…”

“…Our earlier observation in brown adipose tissue correlated loss of MB with bigger lipid droplets, even though the overall lipid content didn’t differ 13 . Moreover, in the mammary epithelial cells, we also showed that MB plays a role in the trafficking of fatty acids in an O 2 -dependent manner 12 . Our current results suggest that the loss of MB in the WapCre;Trp53 flox /MB tumors, which associates with a higher load of entrapped fat in the cells, could either be explained by an increased invasive behavior of the tumor to the pre-existing mammary adipose tissue, or by the fact that the tumor cells themselves are accumulating more lipid droplets, or that cancer stem cells increasingly differentiate into adipocytes, a mechanism has been demonstrated to be induced in tumor cells undergoing EMT 31 .…”

“…After washing in TBST buffer, the horseradish peroxidase (HRP) conjugated secondary antibodies were diluted 1:5000 in blocking buffer (donkey-anti-rabbit, NA934V, Amersham/ goat-anti-mouse, sc-2031, Santa Cruz Biotechnology) and added to the membranes, which were placed on a shaker for 1 h. Super Signal West Femto Maximum Sensitivity Substrate (ThermoFisher, 34095) was used to detect of the bands in a luminescent image analyzer LAS-3000 (Fujifilm). Antibodies have been previously validated by us 12 , 13 , 16 . We could not provide the full-length membranes for some blots because the membranes were cut before hybridization with the primary antibody.…”

Autocatalytic Mechanism in the Anaerobic Reduction of Metmyoglobin by Sulfide Species

“…The nonspecific binding of polysulfides has been observed in the reaction of human methemoglobin with sulfide . Previous reports on the binding of nonpolar species to myoglobin have been presented; for example the nonspecific binding of fatty acids to myoglobin is considered to participate in their trafficking and metabolism. − It should be considered that it cannot be ruled out that polysulfides may be below the limit of detection by cold cyanolysis under the reaction conditions selected.…”

Divergent Reactivity of Hemoglobin Isoforms α2Aβ2 and α2Dβ2 from Meleagris gallopavo in Native and Fatty Acid-Modified States

“…Previous works have outlined the ability of FFA, particularly medium-chain (C 16 –C 18 ) fatty acids, to bind the heme protein, oxymyoglobin (Mb). , However, this work made little mention of the influence of FFA binding on the Mb redox status. Recently, it was reported that linoleic acid (LA) can interact with turkey met-hemolysate, producing a bis-histidyl ferric (Bis-His) Hb species, more commonly known as hemichrome , which strongly inhibited lipid oxidation compared to native met-hemolysate .…”