The role of exocytosis in the apocrine secretion of milk lipid globules in mouse mammary gland during lactogenesis.

Functional relations between exocytotic vesicle membranes, plasmalemma and milk fat globule membranes (MFGM) were studied during the final stages of mouse mammary gland differentiation, in the gland during full lactation and in the postpartum gland in which the synthesis of secretory products was partly inhibited by application of 2-Br-alpha-ergocryptine. Analysis of ultrathin sections, freeze-fracture replicas, scanning electron microscopy and application of a cytochemical marker filipin showed that the apocrine secretion of lipid globules was closely related to the exocytosis of milk proteins. During the last days of gestation the secretion of lipid globules resulted from many exocytotic events of the secretory vesicles that accumulated and fused around the cytoplasmic lipid droplets. Seldom the lipid droplet protruded partly into the gland lumen and a part of its surface became covered with the apical plasmalemma. Although apical plasmalemma became more important in the formation of MFGM in the postpartum period, we could still confirm a direct contribution of secretory vesicle membranes to the final detachment of the lipid globule. The application of 2-Br-alpha-ergocryptine hindered the apocrine secretion of the lipid globules and a situation similar to the situation in the prepartum gland was observed.     

Phosphoproteomics of the goat milk fat globule membrane: New insights into lipid droplet secretion from the mammary epithelial cell

Mechanisms of milk lipid secretion are highly controversial. Analyzing the fine protein composition of the “milk fat globule membrane” (MFGM), the triple‐layered membrane surrounding milk lipid droplets (LDs) can provide mechanistic clues to better understand LD biosynthesis and secretion pathways in mammary epithelial cells (MECs). We therefore combined a high‐sensitive Q‐Exactive LC‐MS/MS analysis of MFGM‐derived peptides to the use of an in‐house database intended to improve protein identification in the goat species. Using this approach, we performed the identification of 442 functional groups of proteins in the MFGM from goat milk. To get a more dynamic view of intracellular mechanisms driving LD dynamics in the MECs, we decided to investigate for the first time whether MFGM proteins were phosphorylated. MFGM proteins were sequentially digested by lysine‐C and trypsin proteases and the resulting peptides were fractionated by a strong cation exchange chromatography. Titanium beads were used to enrich phosphopeptides from strong cation exchange chromatography eluted fractions. This approach lets us pinpoint 271 sites of phosphorylation on 124 unique goat MFGM proteins. Enriched GO terms associated with phosphorylated MFGM proteins were protein transport and actin cytoskeleton organization. Gained data are discussed with regard to lipid secretory mechanisms in the MECs. All MS data have been deposited in the ProteomeXchange with identifier PXD001039 ( http://proteomecentral.proteomexchange.org/dataset/PXD001039 ).     

The distribution of MUC1, an apical membrane glycoprotein, in mammary epithelial cells at the resolution of the electron microscope: implications for the mechanism of milk secretion

The distribution of the glycoprotein, mucin 1 (MUC1), was determined in lactating guinea-pig mammary tissue at the resolution of the electron microscope. MUC1 was detected on the apical plasma membrane of secretory epithelial cells, the surface of secreted milk-fat globules, the limiting membranes of secretory vesicles containing casein micelles and in small vesicles and tubules in the apical cytoplasm. Some of the small MUC1-containing vesicles were associated with the surfaces of secretory vesicles and fat droplets in the cytoplasm. MUC1 was detected in much lower amounts on basal and lateral plasma membranes. By quantitative immunocytochemistry, the ratio of MUC1 on apical membranes and milk-fat globules to that on secretory vesicle membranes was estimated to be 9.2:1 (density of colloidal gold particles/microm membrane length). The ratio of MUC1 on apical membranes compared with basal/lateral membranes was approximately 99:1. The data are consistent with a mechanism for milk-fat secretion in which lipid globules acquire an envelope of membrane from the apical surface and possibly from small vesicles containing MUC1 in the cytoplasm. During established lactation, secretory vesicle membrane does not appear to contribute substantially to the milk-fat globule membrane, or to give rise in toto to the apical plasma membrane.     

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).     

Proteomics of the milk fat globule membrane from Camelus dromedarius

Camel milk has been widely characterized with regards to casein and whey proteins. However, in camelids, almost nothing is known about the milk fat globule membrane (MFGM), the membrane surrounding fat globules in milk. The purpose of this study was thus to identify MFGM proteins from Camelus dromedarius milk. Major MFGM proteins (namely, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already evidenced in cow milk were identified in camel milk using MS. In addition, a 1D‐LC‐MS/MS approach led us to identify 322 functional groups of proteins associated with the camel MFGM. Dromedary MFGM proteins were then classified into functional categories using DAVID (the Database for Annotation, Visualization, and Integrated Discovery) bioinformatics resources. More than 50% of MFGM proteins from camel milk were found to be integral membrane proteins (mostly belonging to the plasma membrane), or proteins associated to the membrane. Enriched GO terms associated with MFGM proteins from camel milk were protein transport (p‐value = 1.73 × 10^?14), translation (p‐value = 1.08 × 10^?11), lipid biosynthetic process (p‐value = 6.72 × 10^?10), hexose metabolic process (p‐value = 1.89 × 10^?04), and actin cytoskeleton organization (p‐value = 2.72 × 10^?04). These findings will help to contribute to a better characterization of camel milk. Identified MFGM proteins from camel milk may also provide new insight into lipid droplet formation in the mammary epithelial cell.     

Characterization of a secretory vesicle-rich fraction from lactating bovine mammary gland.

Fractions enriched in secretory vesicles were obtained from lactating bovine mammary tissue by a straightforward procedure involving gentle homogenization and centrifugation in isotonic milk salt solution containing Ficoll. Secretory vesicle-rich fractions could also be obtained from lactating rat mammary gland by this procedure. With rats, yields of vesicles were substantially increased by administration of colchicine or thioglucose to animals several hours before sacrifice. Isolated fractions were enriched in lactose and consisted predominantly of 0.2–1.2 μm diameter vesicles, many of which contained casein micelles. Enzymatic, compositional and morphological examination revealed vesicle preparations to be largely free of contamination by rough endoplasmic reticulum, mitochondria, nuclei, peroxisomes and lysosomes. Specific activity of several marker enzymes of the secretory vesicle fraction were similar to, or intermediate between, Golgi apparatus and milk lipid globule membranes. Amounts of cholesterol and gangliosides in vesicle fractions approached levels found in plasma membranes. In distribution of major phospholipids, secretory vesicles were intermediate between Golgi apparatus and milk lipid globule membranes. The pattern of polypeptides of secretory vesicle membrane was qualitatively similar to that of Golgi apparatus membranes. While there were similarities between these polypeptide patterns and that of lipid globule membranes, the latter contained relatively more of certain polypeptides, particularly the internal coat-associated polypeptides of the globule membrane. These observations are discussed in relation to the endomembrane hypothesis and the origin of the membrane of milk lipid globules.     

Redox constituents in milk fat globule membranes and rough endoplasmic reticulum from lactating mammary gland

Milk fat globule membranes (MFGM) and rough endoplasmic reticulum (RER) membranes were isolated from milk and lactating mammary gland from the cow and were characterized by biochemical and electron microscope methods in terms of gross composition (proteins, phospholipids, neutral lipids, cholesterol, RNA, and DNA) and purity. Both fractions contained significant amounts of a b-type cytochrome with several properties similar to those of cytochrome b5 from liver, as well as a rotenone- insensitive NADH- and NADPH-cytochrome c reductase. The b-type cytochrome content in the apical plasma membrane-derived MFGM was of the same order of magnitude as it was in RER membranes. It was characterized by a high resistance to extraction by low- and high-salt concentrations and nonionic detergents. MFGM contained much more flavin and much higher activities of xanthine oxidase than the RER membranes. The same redox components were found in MFGM and mammary RER from women, rats, mice, and goats, but in absolute contents great differences between the species were noted. The cytochromes described here differed from liver cytochrome b5 in some spectral properties. The alpha-band of the reduced hepatic cytochrome b5 is asymmetric with a maximum at 555 nm that is split into two distinct peaks at low temperatures. The alpha-band of the b-type cytochromes from MFGM and mammary RER appears as one symmetrical peak at about 560 nm that is not split at low temperatures. When treated with cyanide, MFGM and mammary microsomes showed difference spectra of a reduced b-type cytochrome. Under the same conditions, liver microsomes gave a completely different spectrum. These findings demonstrate the presence of a b-type cytochrome and associated redox enzymes in MFGM, i.e., a derivative of the apical cell surface membrane that is regularly used for envelopment of the milk fat globule during secretion.     

Regulation and functional relevance of milk fat globules and their components in the mammary gland

Mammary gland and epithelial cells are unique to mammals and are under the control of lactogenic hormones such as prolactin. Recent findings indicated that major components of milk fat globule membrane (MFGM) are under the control of lactogenic hormones, and that the major components butyrophilin and xanthine oxidoreductase are indispensable for milk fat secretion. Further, prolactin signaling is negatively controlled by two highly related protein tyrosine phosphatases, PTP1B and TC-PTP. Milk fat globule EGF factor 8 (MFG-E8) is one of the major components of MFGM and is upregulated during lactation. MFG-E8 is further upregulated in the involuting mammary gland. MFG-E8 on exosome-like membrane vesicles in the milk recovered from post-weaning but not lactating mammary glands exhibits higher binding activity to phosphatidylserine and apoptotic mammary epithelial cells, and serves as a link between apoptotic mammary epithelial cells and phagocytes. Recent reports using MFG-E8 deficient mice support the view that MFG-E8 is indispensable for eliminating apoptotic mammary epithelial cells during involution.     

Bovine milk exosome proteome

Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology.     

Freeze-fracture observations of the lactating rat mammary gland. Membrane events during milk fat secretion

Membrane events during milk fat secretion were analyzed by freeze-fracture of the rat mammary gland. Two modes of milk fat secretion were observed: extrusion of fat droplets surrounded by a portion of the apical plasma membrane of the alveolar epithelial cells and, less frequently, release into the alveolar lumen of fat droplets contained in intracytoplasmic vacuoles. The extrusion process consists of two asynchronous events: clearing of membrane particles (probably including integral membrane proteins) and bulging of the apical plasma membrane. Most fat droplets are extruded with a bilayer membrane envelope (milk fat globule membrane) partially devoid of particles. The segregation of membrane particles may represent the onset of a process of structural degradation of the milk fat globule membrane.     

Intracellular origin and secretion of milk fat globules

The cream or fat fraction of milk consists of fat droplets composed primarily of triacylglycerols that are surrounded by cellular membranes. In this review we discuss what is known about how these droplets are formed in and secreted by mammary epithelial cells during lactation. This secretion mechanism, which appears to be unique, is unlike the exocytotic mechanism used by other cell types to secrete lipids. Milk fat globules originate as small, triacylglycerol-rich, droplets that are formed on or in endoplasmic reticulum membranes. These droplets are released from endoplasmic reticulum into the cytosol as microlipid droplets coated by proteins and polar lipids. Microlipid droplets can fuse with each other to form larger cytoplasmic lipid droplets. Droplets of all sizes appear to be unidirectionally transported to apical cell regions by as yet unknown mechanisms that may involve cytoskeletal elements. These lipid droplets appear to be secreted from the cell in which they were formed by being progressively enveloped in differentiated regions of apical plasma membrane. While plasma membrane envelopment appears to be the primary mechanism by which lipid droplets are released from the cell, a mechanism involving exocytosis of lipid droplets from cytoplasmic vacuoles also has been described. As discussed herein, while we have a general overview of the steps leading to the fat globules of milk, virtually nothing is known about the molecular mechanisms involved in milk fat globule formation, intracellular transit, and secretion.     

BIOCHEMICAL AND MORPHOLOGICAL COMPARISON OF PLASMA MEMBRANE AND MILK FAT GLOBULE MEMBRANE FROM BOVINE MAMMARY GLAND

Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids. The sphingomyelin content of the phospholipid fraction of both membranes was higher than in these fractions from other cell components, β-carotene, a constituent characteristic of milk fat, was present in the lipid fraction of the plasma membrane. Cholesterol esters of plasma membrane were similar in fatty acid composition to those of milk fat globule membranes. Disc electrophoresis of either membrane preparation on polyacrylamide gels revealed a single major protein component characteristic of plasma membrane from other sources. Distinct morphological differences between plasma membrane and milk fat globule membranes were observed in both thin sections and in negatively stained material. Plasma membrane was vesicular in appearance while milk fat globule membranes had a platelike aspect. These observations are consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents.     

Milk lipid and protein traffic in mammary epithelial cells: joint and independent pathways

In mammary epithelial cells, milk lipids and proteins are synthesised in the same compartment, the endoplasmic reticulum. Lipids, carried through the cytoplasm, associate with the apical membrane which then pinches off and releases the lipid globule. Proteins, carried through membrane compartments are released in the lumen after fusion of secretory vesicles with the apical membrane. These processes assure a relatively constant composition of milk but it is not known whether lipid and protein secretion are linked. The protein composition of the milk fat globule membrane and the stimulatory effects of prolactin and oxytocin on lipid and protein secretion suggest that these processes are coupled and co-regulated. However, it is possible to observe a dissociation between the formation and the secretion of the two constituents, during differentiation and in various experimental conditions, and this suggests that coupling is not strictly required.     

Antiserum to the milk fat globule membrane. Preparation and capacity to suppress milk secretion

A procedure is described for preparing rabbit antiserum to goat milk fat globule membrane. This membrane is derived from the secretory surface of the lactating cell. Immunoelectrophoresis and enzyme-linked immunosorbent assay showed that antibody development reached maximal levels in about 6–8 weeks. Infusion of 5–10 ml of this antiserum into the lactating mammary gland of goats via the teat canal depressed milk yields temporarily on the infused side to 60–80% of normal. Ordinary serum from rabbit, goat or human did not evoke such a response and rabbit complement was not essential for the effect. Fractionation showed that the globulin fraction of the antiserum contained the milk-suppressing principle. Milk from the antiserum-infused side of the udder showed extensive and tenacious clumping of fat globules on standing 12–24 h. The inhibition of milk flow by antibodies to the secretory membrane resembles a previously observed inhibition following infusion of concanavalin A or its succinyl derivative. Binding of antibodies or lectins which recognize specific surface protein components of the lactating cell appears to be involved in the suppression mechanism. The possible relevance of our findings to autoimmune suppression of exocytosis is noted.     

Milk fat globule membranes devoid of intramembranous particles

When isolated milk fat globule membranes from bovine, human, and murine (rat) milk were examined by freeze-fracturing most of the membrane faces were devoid of membrane-intercalated particles whereas a minor portion showed relatively few particles, either in clusters or in apparent random distribution. A reduced particle density was also noted in membranes of intra-alveolar milk fat globules of cows and rats, in contrast to high particle densities in the apical plasma membrane of lactating epithelial cells. The observations suggest that certain membrane constituents recognized as intramembranous particles either are displaced from the region of the apical surface of the mammary epithelial cell which is involved in milk fat globule budding or are dislocated and rearranged during the budding process.     

A proteomic characterization of water buffalo milk fractions describing PTM of major species and the identification of minor components involved in nutrient delivery and defense against pathogens

Water buffalo has been studied in relation to the exclusive use of its milk for the manufacture of high-quality dairy products. Buffalo milk presents physicochemical features different from that of other ruminant species, such as a higher content of fatty acids and proteins. We report here a detailed proteomic analysis of buffalo skim milk, whey and milk fat globule membrane fractions. Notwithstanding the poor information available on buffalo genome, identification of protein isoforms corresponding to 72 genes was achieved by a combined approach based on 2-DE/MALDI-TOF PMF and 1-DE/muLC-ESI-IT-MS-MS. Major protein components, i.e. alpha(Sl)-, alpha(S2)-, beta-, kappa-caseins, alpha-lactalbumin and beta-lactoglobulin, were characterized for PTM, providing a scientific basis to coagulation/cheese making processes used in dairy productions. Minor proteins detected emphasized the multiple functions of milk, which besides affording nutrition to the newborn through its major components, also promotes development and digestive tract protection in the neonate, and ensures optimal mammary gland function in the mother. Defense against pathogens is guaranteed by an arsenal of antimicrobial/immunomodulatory proteins, which are directly released in milk or occur on the surface of secreted milk-lipid droplets. Proteins associated with cell signaling or membrane/protein trafficking functions were also identified, providing putative insights into major secretory pathways in mammary epithelial cells.     

Plasma membrane fragments in bovine and caprine skim milks

By either differential or linear gradient ultracentrifugation of bovine or caprine skim milks it was possible to obtain fractions which contained 45–75% of the lipid phosphorus and unesterified cholesterol of the skim milk. Electron microscopy of these fractions revealed the presence of numerous membrane-bound vesicles, microvilli and membrane fragments. Assay of the fractions for certain membrane-bound enzymes; i.e. 5′-nucleotide pyrophosphatase, alkaline phosphatase and ATPases, established the presence of all but the latter in the membrane-rich fractions. The distributions of the enzymes in the various fractions were correlated with their lipid phosphorus and cholesterol contents.Compositions of the phospholipids from skim milk membranes, milk fat globule membranes and the plasma membrane of the lactating mammary cell were observed to be similar and unique for having a relatively high level (20–25%) of sphingomyelin. By virtue of secretory processes, all of these membranes appear to be interrelated with each other and with Golgi vesicle membranes. It is concluded that the membrane material in the skim milk originates primarily from plasma membrane of the lactating cell. The possibiltiy that Golgi vesicle membranes form a substantial part of this material is not precluded by the results of this study.Separation of bovine skim milk on a Sepharose 4B gel column demonstrated that virtually all of the 5′-nucleotidase and lipid phosphorus are recovered together in the void volume of the column. Considering the particle size discriminating characteristics of this gel, the skim milk membrane material appears to be constituted of relatively large structures rather than of discrete subunits.     

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.     

RETRACTED ARTICLE: High Fat Feeding of Lactating Mice Causing a Drastic Reduction in Fat and Energy Content in Milk without Affecting the Apparent Growth of Their Pups and the Production of Major Milk Fat Globule Membrane Components MFG-E8 and Butyrophilin

Lactating mice were fed either a low fat or a high fat diet. Milk samples were collected and the composition was examined. Triglyceride and free fatty acid contents were greatly reduced in the milks of high fat diet group, while protein and lactose contents were almost the same between both diet groups. Although the energy content of each component was also lower in milk of high fat diet group, there was apparently no significant difference in the growth of the pups raised by either diet group. This discrepancy might be in part explained by a hypothesis that the pups might monitor calorie content in milk and keep suckling until the energy intake reaches their satisfaction. Moreover, nearly the same amounts of major milk fat globule membrane proteins MFG-E8 and butyrophilin were shown to be present in the milks from both diet groups and gene expression of both proteins in the mammary glands were also indistinguishable, suggesting that production of major MFGM components is not simply related to fat production and secretion.     

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

Summary 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.Supported in part by grant HL 03622 of the U.S. Public Health Service