Changes in lipid droplets morphometric features in mammary epithelial cells upon exposure to non-esterified free fatty acids compared with VLDL.

The effects of the macrostructure of long chain fatty acids on the lipid metabolism and biosynthesis of lipid droplets (LD) was studied in mammary epithelial cells (MEC). MEC were exposed to similar compositions and concentrations of fatty acids in the form of either triglycerides (Tg), as part of the very-low-density lipids (VLDL) isolated from lactating cow plasma, or as non-esterified- free fatty acids (FFA). Exposing MEC to FFA resulted in two distinct processes; each independently could increase LD size: an elevation in Tg production and alterations in phospholipid (PL) composition. In particular, the lower PC/PE ratio in the FFA treatment indicated membrane destabilization, which was concomitant with the biosynthesis of larger LD. In addition, 6 fold increase in the cellular concentration of the exogenously added linoleic acid (C18:2) was found in MEC treated with FFA, implying that long chain fatty acids administrated as FFA have higher availability to MEC, enabling greater PL synthesis, more material for the LD envelope, thereby enhancing LD formation. Availability of long chain fatty acids administrated as VLDL-Tg, is dependent on LPL which its activity can be inhibited by the hydrolysis products. Therefore, we used increasing concentrations of albumin, to reduce the allosteric inhibition on LPL by the hydrolysis products. Indeed, a combined treatment of VLDL and albumin, increased LD size and number, similar to the phenotype found in the FFA treatment. These results reveal the role played by the macrostructure of long chain fatty acids in the regulation of LD size in MEC which determine the size of the secreted MFG

Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism.

Milk fat globule size is determined by the size of its precursors-intracellular lipid droplets-and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content

Specific free fatty acids (FFAs) influence amounts and ratio between polar and neutral lipids in mammary epithelial cells.

<p>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 (<i>P</i> < 0.05).</p

Specific free fatty acids (FFA) influence the amount and composition of phospholipid in mammary epithelial cells.

<p>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 (<i>P</i> < 0.05). PI: phosphatidylinositol; PE: phosphotidylethanolamine; PS: phosphatidylserine; PC: phosphotidylcholine; SM: sphingomyelin.</p

Transcription levels of activity markers of mitochondria and phospholipid converting enzyme are modulated by free fatty acids (FFAs).

<p>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 (<i>P</i> < 0.05).</p

Primer Sequences Used for Real-Time PCR Analysis.

<p>Primer Sequences Used for Real-Time PCR Analysis.</p

Intracellular lipid droplet size is altered by the presence of various free fatty acids (FFAs) in the culture medium.

<p>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 (<i>P</i>> 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 (<i>P</i> < 0.05).</p

Triglyceride (Tg) secretion from mammary epithelial cells is altered in the presence of specific free fatty acids (FFAs).

<p>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 (<i>P</i> < 0.05).</p

Different size distribution of lipid droplets in the medium induced by specific free fatty acids (FFAs).

<p>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.</p