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.     

Focus on the supramolecular structure of milk fat in dairy products

Bovine fat is dispersed in raw milk as natural milk fat globules, with an average diameter of 4 microm, which are enveloped in a biological membrane, the milk fat globule membrane (MFGM). However, dairy processes modify the supramolecular structure and the surface composition of milk fat. Thus, milk fat is present in many dairy products under various forms. In this study, we focused on the fact that natural milk fat globules are rarely consumed in their native state, i.e. in fresh raw milk. In most drinking milks, fat globules are homogenised in order to avoid their rising at the surface of the products. Furthermore, fat globules are heat treated to avoid the growth of micro-organisms. As a consequence of the technological process applied, the volume-weighted average diameter of fat globules in drinking milks is in the range 0.2-0.5 microm. Homogenisation of fat globules led to the partial disruption of the MFGM and to the adsorption of milk proteins. Moreover, this study showed that in cheeses, milk fat can be dispersed as (i) fat globules with the MFGM, (ii) aggregates of fat globules, (ii) homogenised fat globules, (iii) free fat and (iv) a combination of different phases and structures. The knowledge of the supramolecular structure of milk fat in dairy products is of primary importance regarding its technological, sensorial and nutritional properties.     

Chemical composition of the membranes of fat globules of cow's milk

The methods of microthin-layer chromatography on plates with silica gel and disc-electrophoresis in PAAG are used to determine the lipid and protein composition of fat globule membranes of cow milk on the first (foremilk) and the tenth day (milk) of lactation as well as of the buttermilk, a by-product of the technological processing of milk. Differences are found in the quantitative content of the basic classes of lipids and proteins in membranes of fat globules produced from foremilk and milk of cows. The membranes of buttermilk and milk fat globules are characterized by the identical qualitative and similar quantitative chemical composition, that permits using buttermilk as easily available and rich source of components from membranes of cow milk fat globules in the first place of phospholipids and sterols.     

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.     

Raman spectroscopy of the milk globule membrane and triglycerides

The acyl chain mobilities of the lipids of bovine milk fat globules and the component triglycerides have been determined by Raman spectroscopy as a function of temperature from -100°C to 80°C. A broad transition is observed centered about 2–6°C. The C-H and C-C stretching bands in the spectra of liquid and crystalline triglycerides are used comparatively to show that the lipids of the milk globule membrane are 30–40% more ordered than the lipids of the intact milk fat globules at 20°C. Synthetic triglyceride melts, quenched rapidly, are used to illustrate the effect of the thermal history of a sample on lipid order as determined spectroscopically.Strong infrared amide I and amide II bands at 1646 and 1543 cm^?1, respectively, indicate that the protein conformation of the globule membrane is not characterized by extensive regions of beta-sheet structure. Raman spectra of the globule triglycerides indicate cis unsaturation of $\text{39 ± 5\%}$ by comparison to triolein and trielaidin.     

Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent.

Detergent solubilized bovine milk fat globule membrane material studied by crossed immunoelectrophoresis combined with histochemical techniques revealed four major protein complexes. All four were found to bind to concanavalin A and three were identified as sialoglycoproteins. Xanthine oxidase activity was associated with the non-sialoglycoprotein precipitate. Immunoabsorption with intact milk fat globules showed an internal location of the xanthine oxidase, whereas the three other main proteins plus Mg2+-ATPase and 5'-nucleotidase were disposed on the outer membrane surface. The major proteins from milk fat globule membrane and membrane material isolated from skim milk showed immunochemical identity.     

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.     

Erratum

Detergent solubilized bovine milk fat globule membrane material studied by crossed immunoelectrophoresis combined with histochemical techniques revealed four major protein complexes. All four were found to bind to concanavalin A and three were identified as sialoglycoproteins. Xanthine oxidase activity was associated with the non-sialoglycoprotein precipitate. Immunoabsorption with intact milk fat globules showed an internal location of the xanthine oxidase, whereas the three other main proteins plus Mg²⁺-ATPase and 5′-nucleotidase were disposed on the outer membrane surface. The major proteins from milk fat globule membrane and membrane material isolated from skim milk showed immunochemical identity.     

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.     

Ultrastructure of the milk fat globule membrane with and without triglyceride

Summary The primary milk fat globule membrane (MFGM) around freshly secreted milk fat globules consists of a unit membrane separated from the triglyceride core by a dense material. This dense material may widen to include cytoplasmic organelles or may form small blebs. Preincubation and fixation of the globules at temperatures between 4° C and 60° C has no effect on the width or appearance of the dense material. Isolated MFGM profiles show structures identical to those found on intact globules. The dense material on the isolated MFGM profiles is unaffected by extractions which remove essentially all the triglyceride present in the pellets of MFGM.The structure of the primary MFGM is therefore independent of any triglyceride content and the earlier suggestions that the dark material represented a triglyceride layer of high melting point adsorped during cooling of the globules after milking are not supported by the work described in this paper.     

Release of remnant plasma membrane from milk fat globules by Triton X-100

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material ($\text{50 000 × g}$, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-released material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20–22°C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.     

Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins

Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as environmental factors. It is therefore important to study the qualitative and quantitative differences of bovine milk samples. Proteins in milk can be present in casein micelles, in the serum (the water soluble fraction) or in fat globules. These fat globules have a double membrane layer with proteins being bound to or being incapsulated in the membrane layer. The identification and molecular composition of the milk proteins have gained increased interest in recent years. Proteomic techniques make it now possible to identify up to many thousands of proteins in one sample, however quantification of proteins is as yet not straightforward. We analyzed the proteins of the milk fat globule membrane using dimethyl labeling methods combined with a filter-aided sample preparation protocol. Using these methods, it is now possible to quantitatively study the detailed protein composition of many milk samples in a short period of time.     

Studies on the structure of milk fat globule membrane.

Milk fat globule membrane was solubilized with sodium dodecyl sulfate and mercaptoethanol and the membrane proteins were separated by SDS-polyacrylamide gel electrophoresis. The membrane preparations contained three major size classes of polypeptide of 155,000, 62,500 and 43,500 daltons. At least five glycopeptides were separated of which two stained intensely with periodic acid-Schiff reagent, but poorly with coomassie blue. Trypsin hydrolysis of whole cream and isolated milk fat globule membrane revealed major differences in the rates of protein hydrolysis. Many of the membrane proteins of whole cream resisted proteolysis compared with the same proteins in the isolated membrane. Two glycopeptides were resistant to trypsin digestion in either preparation. Treatment of whole cream with neuraminidase led to the release of at least 70% of the protein-bound sialic acid. Whole cream and isolated membrane samples were iodinated with 125I in the presence of lactoperoxidase and hydrogen peroxide. The membrane proteins were significantly more accessible to lactoperoxidase-125I i in isolated membrane compared with the proteins of whole cream. Polypeptides of molecular weight 43,500 and approximately 48,000 daltons were predominantly labelled in whole cream and could be eluted from the fat globules with magnesium chloride (1.5m). The results strongly suggest that the proteins of milk fat globule membrane are asymmetrically arranged in the membrane and that most of the protein-bound sialic acid is present on the external surface of milk fat globules.     

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.     

Galactose transfer in the membranes of human milk fat globules (author's transl)]

Galactosyltransferase which catalyzes the transfer from UDP-galactose to either endogeneous glycoproteins, free N-acetylglucosamine or N-acetylglucosaminyl residues in the carbohydrate portion of glycoproteins, or to glucose when alpha-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.     

Characterisation of the surface of bovine milk fat globule membrane using microelectrophoresis

The nature of the ionogenic groups on the surface of the milk fat globule membrane was studied by microelectrophoresis of intact fat globules after chemical and enzymic modification. The changes in pH-mobility curves effected by formaldehyde and 2,4-dinitrofluorobenzene showed that the membrane surface contained amine groups. These were identified as arising from lysine and arginine by chromatography of their dinitrophenyl derivatives. The contribution of N-acetylneuraminic acid and phosphate to the surface charge was demonstrated by their specific removal by neuraminidase and phospholipase C, respectively. After removal of N-acetylneuraminic acid and phosphate, anionogenic effects remained which were attributed to protein carboxyl groups. These groups could be partially esterified using diazomethane. The effect of sodium dodecyl sulphate and of ionic strength on electrophoretic mobility indicated that the surface contains little neutral lipid and is predominantly ionogenic. The results obtained concerning the nature of the surface of the milk fat globule membrane support the hypothesis that the milk fat globule membrane originates from the plasmalemma of the mammary alveolar cell.     

Native fat globules of different sizes selected from raw milk: thermal and structural behavior

The aim of this study was to characterize differences in the thermal and structural behavior between different sized native milk fat globules. A novel microfiltration process permits the selection of native small fat globules (SFG, 1-3 microm) and large fat globules (LFG, >5 microm) in raw milk, that were analyzed by X-ray diffraction (XRD) coupled to differential scanning calorimetry (DSC). There were no major differences in triglyceride crystalline structures between SFG and LFG, after eliminating thermal history and the influence of cooling rates. The three main 3L and 2L crystalline structures appearing under slow cooling existed regardless of globule size. The supercooling increased for the SFG, mainly due to heterogeneous nucleation in winter milk, and also to compositional variations in spring milk. Differences appeared regarding stabilized crystalline forms at 20 degrees C and subsequent cooling: the SFG contained less 2L triglyceride structures than the LFG. These results can be important in dairy manufactures using tempering periods.     

Glycosphingolipids from bovine milk and milk fat globule membranes: a comparative study. Adhesion to enterotoxigenic Escherichia coli strains

Several components of milk fat globule membranes (MFGMs) have been reported to display beneficial health properties and some of them have been implicated in the defense of newborns against pathogens. These observations prompted us to determine the glycosphingolipid content of MFGMs and their interaction with pathogens. A comparative study with whole milk components was also carried out. Milk fat globules and MFGMs were isolated from milk. Gangliosides and neutral glycosphingolipids were obtained from MFGMs and whole milk and their fatty acid contents were determined by gas chromatography-mass spectrometry (GC-MS). MFGMs and whole milk showed similar ganglioside and neutral glycosphingolipid contents, with whole milk having more GM3 and glucosylceramide and less GD3, O-acetyl GD3, O-acetyl GT3, and lactosylceramide. The fatty acid content of gangliosides from both sources showed a similar composition. However, the neutral glycosphingolipid fatty acid content seemed to be quite different. Whole milk had fewer very-long-chain fatty acids (18.1% vs. 46.4% in MFGMs) and more medium-chain and unsaturated C18:1 and C18:2 fatty acids. Milk fat globules, MFGMs, lactosylceramide, and gangliosides GM3 and GD3 were observed to bind enterotoxigenic Escherichia coli strains. Furthermore, bacterial hemagglutination was inhibited by MFGMs and glycosphingolipids.     

Glycosyl transferases in mouse and human milk fat globule membranes

Summary Membranes isolated from mouse and human milk fat globules were found to contain the enzymes responsible for the synthesis of dolichol monophosphate mannose and dolichol monophosphate glucose as well as those involved in the transference of the glycosyl residues from the two dolichol derivatives to dolichol diphosphate oligosaccharides. The levels of most of the enzymes were comparable to those found in mouse mammary gland microsomes. The presence of enzymes involved in protein glycosylation via dolichol derivatives in the milk fat globule membrane provides evidence in favor of an outward flow of membrane components from the rough endoplasmic reticulum, where these enzymes are active in vivo, towards the cell surface.     

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

Galactose transfer in the membranes of human milk fat globulesGalactosyltransferase 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.