Table 1.
HPLC Solvent Gradient Method Used for Quantifying Triglycerides, Cholesterol and Phospholipids Using Liquid Chromatography Equipped with ELSD Detector.
Table 2.
Primer Sequences Used for Real-Time PCR Analysis.
Fig 1.
Specific free fatty acids (FFAs) influence amounts and ratio between polar and neutral lipids in mammary epithelial cells.
Mammary epithelial cells were treated with 100 μM FFA (capric, palmitic or oleic acid) or with FFA-free medium (control) for 24 h; lipids were then extracted and analyzed by HPLC-ELSD. (A) Total lipid amount. (B) Triglyceride amount. (C) Total phospholipid amount. (D) Triglyceride-to-phospholipid ratio. All data are presented as mean ± SEM. Different letters indicate significant differences between treatment groups (P < 0.05).
Fig 2.
Specific free fatty acids (FFA) influence the amount and composition of phospholipid in mammary epithelial cells.
Mammary epithelial cells were treated with 100 μM FFA (capric, palmitic or oleic acid) or with FFA-free medium (control) for 24 h; lipids were extracted and analyzed by HPLC-ELSD. (A) Membrane lipid amounts. (B) Phospholipid weight %. The percent of the amount of an individual phospholipid out of the summed phospholipids amounts. (C) Weight ratio between phosphatidylcholine and phosphatidylethanolamine. All data are presented as mean ± SEM. Different letters indicate significant differences between treatment groups (P < 0.05). PI: phosphatidylinositol; PE: phosphotidylethanolamine; PS: phosphatidylserine; PC: phosphotidylcholine; SM: sphingomyelin.
Fig 3.
Transcription levels of activity markers of mitochondria and phospholipid converting enzyme are modulated by free fatty acids (FFAs).
Mammary epithelial cells were treated with 100 μM FFA (capric, palmitic or oleic acid) or with FFA-free medium (control) for 2 h; RNA was extracted and gene-expression levels of (A) PGC-1α, (B) PGC-1β, (C) NDUFAF3 and (D) PEMT were analyzed by real-time PCR. All data are presented as mean ± SEM of the expression level of the assayed gene normalized to the geometric mean of two housekeeping genes. Different letters indicate significant differences between treatment groups (P < 0.05).
Fig 4.
Specific free fatty acids (FFAs) in culture medium alter mitochondrial quantity in mammary epithelial cells.
Mammary epithelial cells were treated with 100 μM FFA (palmitic or oleic acid) or with FFA-free medium (control) for 24 h, and mitochondria were stained with Mitotracker deep red FM. Mitochondrial amount was determined by quantification of the fluorescence intensity of Mitotracker deep red staining and presented as fold change compared to the control. Data are presented as mean ± SEM. Different letters indicate significant differences between treatment groups (P < 0.05).
Fig 5.
Intracellular lipid droplet size is altered by the presence of various free fatty acids (FFAs) in the culture medium.
After cultivating mammary epithelial cells with 100 μM FFA (palmitic or oleic acid) or with FFA-free medium (control) for 24 h, lipid droplets were stained with Nile red. (A) Representative images showing the cellular phenotype according to the presence and size of cytoplasmic lipid droplets. Cells were categorized into three groups according to their lipid droplet phenotype: without lipid droplets, with small lipid droplets, or with large lipid droplets. Scale bar = 10 μm. (B) Distribution of mammary epithelial cells with different lipid droplet phenotypes was analyzed by chi-square test (P> 0.05). (C) Number of lipid droplets, by size categories. (D) Maximal lipid droplet diameter. In C and D, data are presented as mean ± SEM and different letters indicate significant differences between treatment groups (P < 0.05).
Fig 6.
Different size distribution of lipid droplets in the medium induced by specific free fatty acids (FFAs).
After cultivating mammary epithelial cells with 100 μM FFA (palmitic or oleic acid) or with FFA-free medium (control) for 24 h, medium was collected and lipid droplets were stained with Nile red. Representative images of lipid droplets in the medium collected from palmitate and oleate treatments (A and B, respectively). Droplets were measured and divided into three size groups: 0 > X < 3, 3 > X < 5, and ≤5 μm. Size distribution of lipid droplets in the medium was compared by chi-square test (C). Scale bar = 20 μm.
Fig 7.
Triglyceride (Tg) secretion from mammary epithelial cells is altered in the presence of specific free fatty acids (FFAs).
Mammary epithelial cells were treated with 100 μM FFA (palmitic or oleic acid) or with FFA-free medium (control) for 24 h, then the medium was collected and Tg content was determined. Data are presented as mean ± SEM. Different letters indicate significant differences between treatment groups (P < 0.05).
Fig 8.
Suggested mechanism for free fatty acids effect on intracellular and secreted lipid droplets in mammary gland epithelial cells.
The presence of free palmitic or oleic acid in the culture medium increased Tg amount in mammary epithelial cells. In addition oleic acid induced changes in the PGC-1 coactivators transcription pattern which increased mitochondria number. Consequently, higher conversion rates of PS to PE occurred and resulted in altered membrane composition. The higher PE content in the membrane induced fusion of intracelllular lipid droplets which results in larger lipid droplets in the cytoplasm. The higher PE also increased plasma membrane curvature and hence increased the secretion rates of the large lipid droplets which resulted in higher Tg concentration in cultrue medium after incubation with oleic acid. In the presence of palmitic acid (left hand side of the illustration) in the culture medium Tg content was also elevated and also proffered incorporation of palmitic acid into PC increased its content in the cellular membranes. PC induced membrane stability which inhibited the fusion rates of intracellular lipid droplets and resulted in small intracelluar and secreted lipid droplets. This suggested mechanism may explain the reason for the association between the size of the milk fat globules and the membrane phospholipid composition of mammary epithelial cells. C16:0- palmitic acid. C18:1- oleic acid. PGC1- PPAR gamma coactivator 1.PS- Phosphatidylserine. PE- Phosphatidylethanolamine. PC- Phosphatidylcholine. Tg- Triglyceride. Orange arrows- pathways induced by oleate. Yellow arrows- pathways induced by palmitate. Blue arrows- pathways induced by both oleate and palmitate. Membrane composition: Green- phoaphatidylinositol. Red- Phosphatidylchole. Pink- Phosphatidylserine. Yellow- Phosphatidylethanolamine. Blue- Sphingomyelin.