Milk Fat Content and DGAT1 Genotype Determine Lipid Composition of the Milk Fat Globule Membrane

During secretion of milk fat globules, triacylglycerol (TAG) droplets are enveloped by a phospholipid (PL) trilayer. Globule size has been found to be related to polar lipid composition and fat content, and milk fat content and fatty acid composition have been associated with the diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism; however, the association between the DGAT1 polymorphism and fat globule size and polar lipid composition has not been studied. The ratio between polar and neutral lipids as well as the composition of the polar lipids in milk has industrial as well as nutritional and health implications. Understanding phenotypic and genotypic factors influencing these parameters could contribute to improving milk lipid composition for dairy products. The focus of the present study was to determine the effect of both fat content and DGAT1 polymorphism on PL/TAG ratio, as a marker for milk fat globule size, and detailed PL composition. Milk samples were selected from 200 cows such that there were equal numbers of samples for the different fat contents as well as per DGAT1 genotype. Samples were analyzed for neutral and polar lipid concentration and composition. PL/TAG ratio was significantly associated with both fat content and DGAT1 genotype. Phosphatidylinositol and phosphatidylserine concentrations were associated with fat content*DGAT1 genotype with a stronger association for the AA than the KK genotype. Sphingomyelin concentration tended to interact with fat content*DGAT1 genotype. Phosphatidylethanolamine (PE) concentration showed a biphasic response to fat content, suggesting that multiple biological processes influence its concentration. These results provide a new direction for controlling polar lipid concentration and composition in milk through selective breeding of cows

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 (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

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

Deleterious effect of n-3 polyunsaturated fatty acids in non-alcoholic steatohepatitis in the fat-1 mouse model

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of pathologies, ranging from hepatocellular steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. It has been suggested that fish oil containing n-3 polyunsaturated fatty acids (n-3 PUFA) induce beneficial effects in NAFLD. However, n-3 PUFA are sensitive to peroxidation that generate free radicals and reactive aldehydes. We aimed at determining whether changing the tissue ratio of n-3 to n-6 PUFA may be beneficial or alternatively harmful to the etiology of NAFLD. The transgenic Fat-1 mouse model was used to determine whether n-3 PUFA positively or negatively affect the development of NAFLD. fat-1mice express the fat-1 gene of Caenorhabditis elegans, which encodes an n-3 fatty-acid desaturase that converts n-6 to n-3 fatty acids. Wild-type C57BL/6 mice served as the control group. Both groups of mice were fed methionine and choline deficient (MCD) diet, which induces NASH within 4 weeks. The study shows that NASH developed faster and was more severe in mice from the fat-1 group when compared to control C57BL/6 mice. This was due to enhanced lipid peroxidation of PUFA in the liver of the fat-1 mice as compared to the control group. Results of our mice study suggest that supplementing the diet of individuals who develop or have fatty livers with n-3 PUFA should be carefully considered and if recommended adequate antioxidants should be added to the diet in order to reduce such risk

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