Milk Fat Globule Secretion Research Articles

Xanthine oxidoreductase mediates membrane docking of milk-fat droplets but is not essential for apocrine lipid secretion.

Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation. XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion. XOR mediates apical membrane reorganization during milk lipid secretion. Loss of XOR delays milk fat globule secretion. XOR loss alters the proteome of milk fat globules. Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane-bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary-specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild-type gland but not in the knockout, suggesting that XOR mediates 'docking' to this membrane. Secreted milk fat globules were isolated from mouse milk of wild-type and XOR MGKO dams, and subjected to LC-MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion.

Comparative proteomics of milk fat globule membrane in different species reveals variations in lactation and nutrition

In present study, 312, 554, 175 and 143 proteins were identified and quantified by label-free quantitative proteomics in human, cow, goat and yak milk fat globule membrane (MFGM), respectively. Fifty proteins involved in vesicle mediate transport and milk fat globule secretion were conserved among species. Moreover, proteins involved in lipid synthesis and secretion (xanthine dehydrogenase/oxidase, stomatin and CD36), showed different expression pattern and the host defense proteins exhibited various profiles within species. Notably, the content and activity of lipid catabolic enzymes were significantly higher in human MFGM, which could be indicative of the superior fat utilization in breast fed infants. Our findings unraveled the significant differences in protein composition of human milk and conventionally used substitutes of it. The in-depth study of lipid metabolic enzymes in human MFGM will probably contribute to the improvement of the fat utilization through modulation of lipid catabolic enzymes in infant formula.

Immunocytochemical Evidence for Golgi Vesicle Involvement in Milk Fat Globule Secretion.

The exact mechanism of secretion of the milk fat globule (MFG) from the mammary secretory cell is still controversial. We have previously suggested close involvement of Golgi vesicles in this process. This paper provides direct immunocytochemical evidence that butyrophilin is present in the Golgi stack and vesicles in ovine and caprine mammary glands. We suggest that it is the butyrophilin in the Golgi vesicle membrane that forms the specific association with the adipophilin on the lipid surface in the cytoplasm. Exocytosis of the associated Golgi vesicle will then initiate the process of MFG secretion. Further exocytosis of associated Golgi vesicles will continue and complete the process. Areas of the plasmalemma that have butyrophilin delivered by previous non-lipid associated Golgi exocytoses may also contribute to the process of forming the milk fat globule membrane (MFGM).

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.

Characterisation of the potential SNARE proteins relevant to milk product release by mouse mammary epithelial cells

Casein micelles and fat globules are essential components of milk and are both secreted at the apical side of mammary epithelial cells during lactation. Milk fat globules are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. SNARE proteins are known to take part in cellular membrane trafficking and in exocytosis events in many cell types and we therefore attempted to identify those relevant to casein secretion. With this aim, we performed a detailed analysis of their expression by RT-PCR in both whole mouse mammary gland and in purified mammary acini at various physiological stages, as well as in the HC11 cell line. The expression of some regulatory proteins involved in SNARE complex formation such as Munc-13, Munc-18 and complexins was also explored. The amount of certain SNAREs appeared to be regulated depending on the physiological stage of the mammary gland. Co-immunoprecipitation experiments indicated that SNAP-23 interacted with syntaxin-6, -7 and -12, as well as with VAMP-3, -4 and -8 in mammary epithelial cells during lactation. Finally, the subcellular localisation of candidate SNAREs in these cells was determined both by indirect immunofluorescence and immunogold labelling. The present work provides important new data concerning SNARE proteins in mammary epithelial cells and points to SNAP-23 as a potential central player for the coupling of casein and milk fat globule secretion during lactation.

High Fat Diet Feeding Impairs Lactogenesis and Butyrophilin Expression in the Mammary Gland of Mice

Overfeeding and obesity negatively impacts milk production in animals, but the underlying mechanisms are unknown. To address this, female C57BL/6J mice were assigned at weaning to a low‐fat control diet (LF, 7% by wt), or a high‐fat diet (HF, 35% by wt) for 8 wks then mated. Dams were killed on pregnancy day 14 (P14) or lactation day 1 (L1) and blood, gonadal fat pads and mammary glands were harvested. HF dams had 2–3 fold heavier mammary glands and fat pads as compared to LF dams. HF dams had 2–4 fold higher serum leptin and 25–35% lower serum adiponectin as compared to LF dams. Mammary whole mounts showed less ductal branching and alveolar buds in HF as compared to LF dams. Epithelial content digitally quantified from stained mammary sections confirmed significantly (~50%) less mammary development in HF. LF and HF litter sizes were similar, but the number of viable pups was lower in HF, and those that lived had little to no milk present in the stomach. Cross‐fostering HF litters onto LF dams rescued pups whereas the reverse was not true. Protein expression of adipophilin and butyrophilin increased in LF mammary gland between P14 and L1, while protein expression of perilipin did not. At L1, butyrophilin expression in HF mammary gland was reduced by at least 50% as compared to LF while adipophilin and perilipin expression remained stable. The results suggest that high fat diets hinder lactogenesis by inhibiting mammary gland development and expression of butyrophilin, a protein important for the onset of milk fat globule secretion. Funded by Mead Johnson Nutrition.

Screening, cloning and sequence analysis ofgBTN1A1gene in mammary gland of Xinong Saanen goat

AbstractButyrophilin 1A1 (BTN1A1), which is a member of the Ig superfamily, is highly expressed in the lactating mammary gland and is secreted into milk in association with lipid droplets, and concentrated in the apical plasma membranes of mammary epithelial cells. Because butyrophilin is expressed only during lactation, it appears to be essential for milk fat globule production. However, there is little information about its properties in the dairy goat. The objective of this study was to confirm whether butyrophilin was related to milk changes in different lactation periods of Xinong Saanen goats. Using suppression subtractive hybridization analysis, we identified a significant increase in butyrophilin mRNA in Xinong Saanen goat mammary gland at middle lactation when compared to that of late lactation. The gBTN1A1 complementary DNA has an open reading frame of 1581 nucleotides encoding a putative protein of 526 amino acids. Amino acids 1–26 were the signal peptide domain. The homologies of nucleotide and peptide sequences of goat butyrophilin with bovine (NM_174508), human (NM_001732) and mouse (AK145168) butyrophilin were 97 and 96%, 88 and 84%, 84 and 70%, respectively. The secondary structure, transmembrane domain and hydrophilicity of goat butyrophilin were similar to the those of the bovine, human and mouse protein. In conclusion, we confirmed that the goat mammary gland mRNA level of gBTN1A1 was responsible for goat milk fat globule secretion.

Composition of Milk Fat from Cows Selected for Milk Fat Globule Size and Offered Either Fresh Pasture or a Corn Silage-Based Diet

The objective of this study was to examine the synthesis and composition of milk produced by dairy cows that secrete either small milk fat globules (SMFG) or large milk fat globules (LMFG), and to study their response to diets known to alter milk composition. Four groups of 3 multiparous dairy cows were assigned to 2 isoenergetic feeding treatments: a corn silage treatment supplemented with soybean meal, and fresh pasture supplemented with cereal concentrate. The 4 groups comprised 2 groups of 3 dairy cows that produced SMFG (3.44μm) and 2 groups of 3 dairy cows that produced LMFG (4.53μm). The SMFG dairy cows produced higher yields of milk, protein, and calcium. Nevertheless, their milk had lower fat and protein contents. Both SMFG and LMFG cows secreted similar amounts of milk fat; therefore, higher globule membrane contents in milk fat were observed in SMFG cows. Higher calcium mineralization of the casein micelles in SMFG cows suggests that it may be possible to improve cheese-making properties even if the lower protein content may lead to lower cheese yields. The SMFG cows secrete milk fat with a higher concentration of monounsaturated fatty acids and a lower concentration of short-chain fatty acids. They also have a higher C18:1/C18:0 ratio than LMFG cows. This suggests that SMFG cows have more significant fatty acid elongation and desaturation. The pasture treatment led to an increase in milk and protein yields because of increased energy intake. It also resulted in lower milk fat yield and fat and protein contents. The pasture treatment led to a decrease in milk fat globule size and, as expected, an increase in monounsaturated and polyunsaturated fatty acid contents. However, it induced a decrease in the protein content, and in calcium mineralization of casein micelles, which suggests that this type of milk would be less suitable for making cheese. This study also shows that there is no correlation between the cows, based on milk fat globule size and diet. These results open up possibilities for improving milk fat quality based on milk fat globule size, and composition. The mechanisms involved in milk fat globule secretion are still to be determined.

Butyrophilin controls milk fat globule secretion

The molecular mechanism underlying milk fat globule secretion in mammary epithelial cells ostensibly involves the formation of complexes between plasma membrane butyrophilin and cytosolic xanthine oxidoreductase. These complexes bind adipophilin in the phospholipid monolayer of milk secretory granules, the precursors of milk fat globules, enveloping the nascent fat globules in a layer of plasma membrane and pinching them off the cell. However, using freeze-fracture immunocytochemistry, we find these proteins in locations other than those previously inferred. Significantly, butyrophilin in the residual plasma membrane of the fat globule envelope is concentrated in a network of ridges that are tightly apposed to the monolayer derived from the secretory granule, and the ridges coincide with butyrophilin labeling in the globule monolayer. Therefore, we propose that milk fat globule secretion is controlled by interactions between plasma membrane butyrophilin and butyrophilin in the secretory granule phospholipid monolayer rather than binding of butyrophilin-xanthine oxidoreductase complexes to secretory granule adipophilin.

Milk xanthine oxidase: Properties and physiological roles

Xanthine oxidoreductase (XOR) is a complex molybdoflavoenzyme that occurs as a major protein component of the milk fat globule membrane surrounding lipid droplets in milk. The enzyme has broad substrate specificity and is capable of reducing oxygen to generate the reactive oxygen species (ROS), superoxide and hydrogen peroxide. It can also reduce nitrite, yielding reactive nitrogen species (RNS), such as nitric oxide and peroxynitrite. By virtue of its capacity to generate ROS and RNS, milk XOR may play an antimicrobial defensive role in the neonatal gut, complementing endogenous enzyme of the intestinal epithelium. XOR is also involved in secretion of milk fat globules in a process dependent on the enzyme protein rather than on its enzymic activity, which is known to vary greatly with time after parturition and also between species. Some potential hazards and benefits of ingestion of cows milk XOR are briefly discussed.

Physiological Roles of Xanthine Oxidoreductase

Xanthine oxidoreductase (XOR) is a major protein component of the milk fat globule membrane (MFGM) surrounding fat droplets in milk and its enzymology is well characterised. The enzyme is widely distributed in mammalian tissues and is generally accepted to be a key enzyme of purine catabolism. It catalyses the oxidation of a wide range of substrates and can pass electrons to molecular oxygen, generating reactive oxygen species (ROS); similar reduction of nitrite yields reactive nitrogen species (RNS). While XOR has been implicated in ischemia‐reperfusion injury, its involvement in normal physiological processes has been less studied. It is argued here that XOR‐derived ROS and RNS play a role in innate immunity, specifically in the inflammatory response and in anti‐microbial defense of the gastrointestinal tract. XOR‐derived species could also be involved in signalling. Additionally, XOR is likely to play a part in metabolism of xenobiotics and has recently been shown to mediate the secretion of milk fat globules. The human enzyme has only relatively recently been characterized. The enzyme purified from breast milk shows very low enzymatic activity, and it is suggested that human XOR has evolved so as to be regulated by an exceptional range of pre‐ and posttranslational mechanisms.

Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo

In the present study we provide evidence that the cytodifferentiation of primary mouse mammary epithelial cells within the alveolar-like structures formed after culture on a reconstituted basement membrane resembles development in vivo during late pregnancy and early lactation. During the first two days in culture on a basement membrane gel in the presence of lactogenic hormones, epithelial cells isolated from mid-pregnant mice are disorganized and central lumina are largely absent. Levels of mRNA for the milk proteins, beta-casein and transferrin, are dramatically reduced. By the second or third day in culture, cytoplasmic polarization becomes evident and prominent apical junctional complexes are formed. Synthesis of both mRNA and milk protein is reinitiated at this time. By day 4, well-defined lumina appear, and abundant synthesis and secretion of casein and lipid is observed. A striking feature of this differentiation in culture is the specific localization of milk protein gene expression (beta-casein mRNA) to luminal epithelial cells in the alveolar-like structures. At the ultrastructural level, increased milk protein synthesis and secretion are paralleled by a fourfold increase in rough ER that resembles the dramatic increase in the ER observed in vivo following parturition. One indication of tissue-specific differentiation observed in later cultures (days 4-11) is the synthesis and secretion of abundant casein micelles. A second characteristic of lactating mammary epithelial cells in vivo that has not previously been observed in culture is the secretion of milk fat globules. Taken together, these observations indicate that mammary epithelial cells plated onto a reconstituted basement membrane differentiate to the lactating phenotype in culture.

Influence of colchicine on lactating mammary gland of the cow and rat with special reference to the exocytosis and to the milk fat globule secretion.

Abstract A study of the influence of colchicine on milk secretory cells was coducted in rats and cows, to ascertain the role of microtubules in the process of exocytosis and fat globule secretion. It was demonstrated with the aid of light microscopy, transmission electronmicroscopy and scanning electronmicroscopy that colchicin treatment apparently interferes with the fusion of secretory vesicles to the surface membrane of secretory cells and therefore interupts exocytosis. The significance of these findings are discussed. Summary A study of the influence of colchicine on the secretion of the milk fat globules and of the exocytosis process has been made. Besides, the effects of this drug on the ultrastructure of the lactating mammary cell was analyzed. Colchicine induced similar changes in both cow and rat mammary glands, characterized by a progressive accumulation of secretory vesicles and lipid droplets, the loss of the polarity of the endoplasmic reticulum and the translational movement of the secretory vesicles and of the milk fat globules. The interference on microtubles assembly appears to be the main cause of the inhibition of the milk fat globules secretionl. It is probable that the secretory vesicles lose their capacity to fuse with the apical membrance of the secretory cell in a characteristic exocytosis process. Zusammenfassung Der Einfluß von Colchizin auf die laktierende Milchdrüse von Kuh und Ratte mit besonderer Beachtung der Exocytose und der Sekretion von Milchfettkügelchen Untersucht wurde der Einfluß von Colchizin auf die Sekretion von Milchfettkügelchen und auf den Prozeßder exocytose. Daneben wurde die Wirkung dieser Droge auf die Ultrastruktur der laktierenden Milchdrüsenzellen beachtet. Colchizin rief sowohl bei der Milchdrüse der Kuh als auch bei jener der RAtteähnliche Veränderungen hervor, die durch eine fortschreitende Ansammulung von sekretorischen Vesikeln und Fetttröpfchen, Verlust der polarität des endoplasmatischen Retikulums und Zerreißen der Mikrotubuli charakterisiert waren. Diese Untersuchungsergebnisse weisen darauf hin, daß Colchizin an den Übertragungsmechafnismen der sekretorischen Vesikel und der Milchfettkügelchen angreift. Die Störung der Gesamtheit der Mikrotubuli scheint der Hauptgrund fü die Behinderung der Sekretion von Milchfettkügelchen zu sein. Es ist wahrscheinlich, daß die sekretorischen Vesikel ihre Fáhigkeit verlieren, mit der apikalen Membran der sekretorischen Zelle in jenem Charakteristischen Vorgang der Expcytose zusammenzufließen. Résumé Influence de la colchicine sur la glande mammaire en lactation de la Vache et de la Ratte, avec référence particulière à l'exocyose et à la sécrétion des golbules gras du lait On a étudié l'influence de la colchincine sur la sécrétion des globules gras du lait et sur le processus de l'exocytose; on a analysé, en outre, les effets de cette drogue sur l'ultrastructure de la cellule mammaire en lactation. La colchicine induit des cangements similaires dans les glands mammaires, ches la Vache comme chez la Ratte: ceux-ci se caractérisent par une accumulation progressive de vésicules de sécrétion et de gouttelettes lipidiques, par la pette de polarité du réticulum endoplasmique et par la urpture des microtubules. Ces résultats démontrent que la colchicine affecte le mécanisme de transport des vésicules de sécrétion et des globules gras du lait. La pertubation de l'appareil microtubulaire semble être la cause principale de l'inhibition de la sécrétion des globules gras du lait. Il est probable que les vésicules sécrétoires perdent leur capacitéà fusinner avec la membrane apicale des cellules séscétrices selon le processus caractéristique de l'exocytose. Resumen La influencia de la colquicina sobre la glándula mamaria lactante de la vaca y de la rata, con referencia especial a la exocitosis y la secreción de los golóbulos de grasa en la leche Se investigó la influencia de la colquicina sobre la secreción delos fgloóulos de grasa en la leche y sobre la exocitosis. Además, se analizó el efecto de esta droga sobre la ultraestructura de la célula mamaria lactante. Los cambios inducidos por la colguicina en las glándulas mamarias fueron los mismos en la vaca y en la rata, caracterizados por una acumulactón progresiva de vesículas seretoras y gotitas de lípidos, la pérdida de la polaridad del retículo endoplésmico y la ruptura de los mocrotúbulos. Estos resltados indicaron que la colquicina afecta los movimientos de transporte de las vesículas secretoras y de los glóbulos grasos de la leche. La interferencia en la organización de los microtúbulos parece ser la causa pricipal para la inhibición de la secreción de los glóbulos de grasa de la leche. Por razones similares, probablemente también las vesículas fsecretoras pierden su capacided de fusionarse con la membrana apical de la célula secretora, como sucede en el proceso típico de la exocitosis.

Effects of colchicine on ultrastructure of the lactating mammary cell: Membrane involvement and stress on the Golgi apparatus

The effects of colchicine on ultrastructure of the lactating mammary cell in the rat and goat were studied by electron microscopy. Changes in tissue of the rat were examined over time (1, 2 and 4 h). The goat gland was evaluated by comparing ultrastructure of tissue at the time of maximum milk flow suppression induced by the drug with that of untreated tissue. Colchicine produced notable changes in the tissue of both species: 1) the secretion of lipid droplets and Golgi vesicle contents (exocytosis) was inhibited and the droplets and vesicles became randomly distributed throughout the cell, 2) the Golgi apparatus was significantly reduced in size, 3) casein and lipid continued to be synthesized as evidenced by greater numbers of secretory vesicles and increased sizes of casein micelles and lipid droplets, 4) secretory vesicles showed a propensity to cluster around lipid droplets, 5) isolated microtubules were found occasionally in the control tissue, ordinarily in the vicinity of the Golgi apparatus, but rarely in the colchicine-treated tissue. These observations indicate that colchicine has two effects leading to suppression of exocytosis in the mammary cell: one involves early interference with capacity of secretory vesicle membranes to fuse and a further effect, related to higher concentrations of colchicine, causes intracellular disorganization and loss of polarity. Microtubules were not seen as directly involved in the mechanisms of exocytosis. The secretion of milk fat globules is coupled to exocytosis and thereby is also inhibited by colchicine.

The supression of milk fat globule secretion by colchicine: an effect coupled to inhibition of exocytosis

The effects of colchicine on release of milk lipids from mammary tissue were evaluated by biochemical analysis of milk and morphological study of the tissue following intramammary infusions of the alkaloid into lactating goats. Colchicine produces a reversible drop in milk yield. As the flow of milk resumes, 36–48 h after infusion, the fat content of the milk increases, phospholipid per g of total globule lipid falls, mean size of milk fat globules increased and diameters of fat droplets (presecretory milk fat globules) within lactating cells approximately double. These observations are consistent with the conclusion that colchicine suppresses milk fat globule secretion but that globules continue to grow in size within cells during the suppression period. These findings indicate that secretion of milk fat globules and the skim milk phase are coupled.

Transfert du galactose dans les membranes des globules lipidiques du lait humain

Galactose transfer in the membranes of human milk fat globules Galactosyltransferase which catalyzes the transfer from UDP-galactose to either endogeneous glycoproteins, free N- actylglucosamine or N- acetylglucosaminyl residues in the carbohydrate portion of glycoproteins, or to glucose when α-lactalbumin is added, occurs in human milk fat globule membranes. Various treatments (washing of membranes, freezing and thawing) did not affect this activity. In the presence of Triton X-100, the enzyme shows appreciable latency. This detergent was then used to solubilize the enzyme and to study its main characteristics. A competition and a heat stability experiment show that only one enzyme acts on two substrates (free N- acetylglucosamine or desialyzed and degalactosylated fetuin). UDP-galactose hydrolase activities were very low compared to those of the bovine milk fat globule membranes. Other characteristic enzymes of Golgi vesicles were found in human milk fat globules membranes. It is of interest to find out whether this is the result of contamination with cytoplasmic particles or whether it reflects the participation of Golgi vesicles in human milk fat globule secretion.