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Lipid Droplet Fusion in Mammary Epithelial Cells is Regulated by Phosphatidylethanolamine Metabolism

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Abstract

Mammary epithelial cells (MEC) secrete fat in the form of milk fat globules (MFG) which are found in milk in diverse sizes. MFG originate from intracellular lipid droplets, and the mechanism underlying their size regulation is still elusive. Two main mechanisms have been suggested to control lipid droplet size. The first is a well-documented pathway, which involves regulation of cellular triglyceride content. The second is the fusion pathway, which is less-documented, especially in mammalian cells, and its importance in the regulation of droplet size is still unclear. Using biochemical and molecular inhibitors, we provide evidence that in MEC, lipid droplet size is determined by fusion, independent of cellular triglyceride content. The extent of fusion is determined by the cell membrane’s phospholipid composition. In particular, increasing phosphatidylethanolamine (PE) content enhances fusion between lipid droplets and hence increases lipid droplet size. We further identified the underlying biochemical mechanism that controls this content as the mitochondrial enzyme phosphatidylserine decarboxylase; siRNA knockdown of this enzyme reduced the number of large lipid droplets threefold. Further, inhibition of phosphatidylserine transfer to the mitochondria, where its conversion to PE occurs, diminished the large lipid droplet phenotype in these cells. These results reveal, for the first time to our knowledge in mammalian cells and specifically in mammary epithelium, the missing biochemical link between the metabolism of cellular complex lipids and lipid-droplet fusion, which ultimately defines lipid droplet size.

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Acknowledgements

The authors would like to acknowledge Dr. Sergei Grigoryan for his assistance in confocal microscopy imaging. This research was partially supported by the Nutrigenomics Center of the Hebrew University of Jerusalem, Israel, and by the Israeli Dairy Board grant #8200327.

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Authors and Affiliations

  1. The Animal Science Department, The Robert H Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, PO Box 12, Rehovot, 76100, Israel

    Bat-Chen Cohen, Chen Raz & Nurit Argov-Argaman

  2. Department of Ruminant Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel

    Avi Shamay

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Lipid droplet fusion. Representative fusion event of two lipid droplets, observed in MEC treated with 100 μM palmitic acid + 10 μM DZA and stained for their lipid droplets with Nile red. Optical sections were recorded with time-lapse imaging and reconstructed as 3D images from deconvolved images. Arrows indicate a pair of fusing lipid droplets. (AVI 78 KB)

10911_2017_9386_MOESM2_ESM.tif

DZA addition has no effect on live cell number compared to palmitic acid alone. Number of live cells determined by Trypan blue staining was similar in MEC treated with 10 μM DZA+100 μM palmitic acid relative to palmitic acid alone, both for 24 h. Data are presented as mean ± SD. (TIF 29 KB)

10911_2017_9386_MOESM3_ESM.tif

Time and dose response for NaN3 + NaF and oleic acid treatments. a Addition of 5 mM NaN3 + 20 mM NaF to medium containing 100 μM oleic acid eliminated MEC presenting large lipid droplets (>2.5 μm) from culture. Distribution of MEC phenotype, distinguished by the presence or absence of large lipid droplets after 24 h of treatment, was determined by chi-square test (P ≤ 0.05). b Phospholipid composition (weight %) of MEC treated as in (a) shows lower PE and higher sphingomyelin percent in oleic acid + NaN3 + NaF treatment relative to oleic acid alone. Data are presented as mean ± SD (*P ≤ 0.05). c, d Effects of NaN3 + NaF + oleic acid concentration and duration of treatment on live cell number. c 5 mM NaN3 + 20 mM NaF in the presence of 100 μM oleic acid for 24 h decreased the number of cells relative to treatment with oleic acid alone. d 2.5 mM NaN3 + 10 mM NaF in the presence of 360 μM oleic acid did not change live cell number relative to oleic acid alone after 2 and 4 h of treatment. Number of live cells was determined by Trypan blue staining. e MEC treated with 360 μM oleic acid + 2.5 mM NaN3 + 10 mM NaF presented smaller droplets relative to oleic acid alone after 4 h of treatment. Neutral lipids were stained with Nile red (red) and nuclei were stained with DAPI (blue). Scale bars, 20 μm. Data are presented as mean ± SD (*P ≤ 0.05). (TIF 912 KB)

Relatively slow movement of lipid droplets in MEC treated with oleic acid + NaN3 + NaF. Time-lapse imaging of Nile red-stained lipid droplets in MEC treated with 360 μM oleic acid + 2.5 mM NaN3 + 10 mM NaF reveals slower movement relative to oleic acid alone (see Online Resource <link rid="Sec22">5</link>). (AVI 199 KB)

Lipid droplet movement in MEC treated with oleic acid. Time-lapse imaging of Nile red-stained lipid droplets in MEC treated with 360 μM oleic acid reveals rapid movement and noticeable fusion between lipid droplets. (AVI 286 KB)

10911_2017_9386_MOESM6_ESM.tif

Differential responsiveness of MEC cultures to similar oleic acid concentration. Different batches of MEC primary culture were treated with 100 μM free oleic acid for 24h. Culture #1 showed a much greater number of large lipid droplets than culture #2. The largest droplet measured in each culture (2 replicates, 30 cells per replicate) was 5.0 μm and 3.7 1 μm in culture #1 and culture #2 respectively. The average diameter of the 3 largest lipid droplets was 3.4±0.7 μm and 2.8±0.3 in culture #1 and culture #2 respectively. Neutral lipids were stained with Nile red (red) and nuclei were stained with DAPI (blue). Scale bars, 20 μm. (TIF 1919 KB)

HPLC–ELSD solvent gradient method used for quantifying triglycerides and phospholipids. (PDF 87 KB)

10911_2017_9386_MOESM8_ESM.pdf

PSD mRNA sequence and sequence amplified by real-time PCR. mRNA coding sequence of bovine PSD (NCBI reference sequence: NM_001024475.1, gi|66792777:137-1387) with forward and reverse primers marked in bold and underlined, and PCR product marked with light gray. PCR product sequence was verified by sequencing. (PDF 32 KB)

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Cohen, BC., Raz, C., Shamay, A. et al. Lipid Droplet Fusion in Mammary Epithelial Cells is Regulated by Phosphatidylethanolamine Metabolism. J Mammary Gland Biol Neoplasia 22, 235–249 (2017). https://doi.org/10.1007/s10911-017-9386-7

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