Milk fat globule membrane: the role of its various components in infant health and development

Breastfeeding confers many benefits to the breast-fed infant which are reflected by better short-term and long-term outcomes as compared to formula-fed infants. Many components of breast milk are likely to contribute to these favorable outcomes, and there has recently been focus on the milk fat globule membrane (MFGM). This fraction is a heterogenous mixture of proteins (many of them glycosylated), phospholipids, sphingolipids, gangliosides, choline, sialic acid and cholesterol which is lacking in infant formula as milk fat (which is also low in these components) is replaced by vegetable oils. Many of these components have been shown to have biological effects, and there is considerable evidence from preclinical studies and clinical trials that providing bovine MFGM results in improved outcomes, in particular with regard to infections and neurodevelopment. Since bovine MFGM is commercially available, it is possible to add it to infant formula. There are, however, considerable variations in composition among commercial sources of bovine MFGM, and as it is not known which of the individual components provide the various bioactivities, it becomes important to critically review studies to date and to delineate the mechanisms behind the activities observed. In this review, we critically examine the preclinical and clinical studies on MFGM and its components in relation to resistance to infections, cognitive development, establishment of gut microbiota and infant metabolism, and discuss possible mechanisms of action.     

Monoclonal antibodies recognizing epitopes carried on both glycolipids and glycoproteins of the human milk fat globule membrane.

The molecules of the human milk fat globule membrane (MFGM) which bind four murine monoclonal antibodies (LICR LON M3, M8, M18 and M24) raised against the human MFGM have been identified. By using 'Western' blotting [Burnette (1981) Anal. Biochem. 112, 195-203] it was shown that each antibody reacted with a different set of proteins. M3 and M24 were similar in their pattern of reaction with the membrane proteins, but were quite distinct from M8 and M18, which also differed from each other. Glycopeptides prepared from the MFGM by exhaustive Pronase digestion were able to inhibit partially the binding of M3 and M24, and prevent totally the binding of M8 and M18, to the MFGM in an enzyme-linked immunoabsorbent assay. Oligosaccharides obtained by the deproteination of human milk also completely inhibited the binding of M3, M18 and M24 to the MFGM. However, the binding of M8 was not inhibited by these saccharides, and therefore M8 may not be recognizing a simple carbohydrate determinant. By using an enzyme-linked assay, M8 and M18 were shown not to bind to MFGM glycolipid, whereas M3 and M24 did, and this was confirmed by overlaying thin layer chromatograms of MFGM lipids with these antibodies. Both M3 and M24 showed a similar complex pattern of reaction, binding to more than one glycolipid moiety. By these means all four antibodies have been shown to react with antigens which involve carbohydrate side chains carried on different proteins, and two were also shown to react with such determinants on glycolipids.     

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.     

Qualitative and quantitative profiling of the bovine milk fat globule membrane proteome

Milk is a biological fluid of unique quality and complexity. It has co-evolved with mammals and mankind to nourish offspring and contains macro- and micronutrients for growth and development of the newborn. The milk fat globule membrane (MFGM) represents an important milk fraction, which is rich in bioactive proteins. In order to better understand functionality of milk fractions and, thereby, enhance the benefits of milk products, detailed qualitative and quantitative protein knowledge of fractions such as MFGM is required.We report the qualitative and quantitative profiling of two MFGM-enriched milk fractions, a whey protein concentrate (WPC) and a buttermilk protein concentrate (BMP), as derived from three different analytical workflows. First, an LC-MS/MS-based shotgun approach revealed 244 protein identities in WPC and 133 in BMP, respectively, and provided an extensive characterisation of the protein content in those two fractions. Second, label-free profiling resulted in rapid and efficient semi-quantitative comparison and yielded valuable protein fingerprints. Third, absolute quantification of selected MFGM proteins was achieved by stable isotope dilution (SID)-MS, in combination with multiple reaction monitoring (MRM) detection. In summary, we provide new information on composition, quantity and possible health benefits of two MFGM-enriched milk fractions highly valuable for future nutritional applications.     

Purification and properties of the major sialoglycoprotein of the milk fat globule membrane

The major periodate-Schiff positive component (glycoprotein-2) of bovine milk fat globule membranes (MFGM) has been purified by extraction of washed cream with chloroform/methanol followed by chromatography on Sephadex G-200 in sodium dodecyl sulfate. The glycoprotein is > 95% pure by polyacrylamide electrophoresis in dodecyl sulfate and shows the same prominent component at gel percentages of from 5 to 12.5. The molecular weight obtained by extrapolation of the apparent molecular weights on these gels to higher gel percentages was 70,000. An apparent molecular weight of 105,000 was obtained by gel filtration in 1% dodecyl sulfate on Sepharose 4B. The glycoprotein contains 50% carbohydrate by weight, with sialic acid (30.5%), N-acetylglucosamine (22.3%), galactose (15.9%), N-acetylgalactosamine (14.0%), mannose (11.1%), and fucose (5.8%) being the major monosaccharides. Leucine, glutamic acid, and glycine are the major amino acids. Affinity chromatography of deoxycholate-solubilized MFGM indicates that glycoprotein-2 is not the major concanavalin A receptor of these membranes.     

Adipocyte hypertrophy is associated with lysosomal permeability both in vivo and in vitro: role in adipose tissue inflammation

Obesity in both humans and rodents is characterized by adipocyte hypertrophy and the presence of death adipocytes surrounded by macrophages forming crown-like structures. However, the biochemical pathways involved in triggering adipocyte death as well as the role of death adipocytes in adipose tissue remodeling and macrophage infiltration remain poorly understood. We now show that induction of adipocyte hypertrophy by incubation of mature adipocytes with saturated fatty acids results in lysosomal destabilization and cathepsin B (ctsb), a key lysosomal cysteine protease, activation and redistribution into the cytosol. ctsb activation was required for the lysosomal permeabilization, and its inhibition protected cells against mitochondrial dysfunction. With the use of a dietary murine model of obesity, ctsb activation was detected in adipose tissue of these mice. This is an early event during weight gain that correlates with the presence of death adipocytes, and precedes macrophage infiltration of adipose tissue. Moreover, ctsb-deficient mice showed decreased lysosomal permeabilization in adipocytes and were protected against adipocyte cell death and macrophage infiltration to adipose tissue independent of body weight. These data strongly suggest that ctsb activation and lysosomal permeabilization in adipocytes are key initial events that contribute to the adipocyte cell death and macrophage infiltration into adipose tissue associated with obesity. Inhibition of ctsb activation may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.     

Variability of milk fat globule membrane protein gene between cattle and riverine buffalo.

A study on butyrophilin (BTN) gene was conducted to detect variability at nucleotide level between cattle and buffalo. Hae III PCR-RFLP was carried out in crossbred cattle and it revealed polymorphism at this locus. Three genotypes namely, AA, BB and AB and two alleles were observed with frequencies 0.78, 0.17, 0.04 and 0.87, 0.13, respectively. The sequences of different cattle, buffalo and sheep breeds have been reported in the EMBL gene bank with accession numbers: AY491468 to AY491475. The nucleotides, which have been substituted from allele A to B, were found to be C to G (71st nucleotide), C to T (86th nucleotide), A to T (217th nucleotide), G to A (258th nucleotide), A to C (371st nucleotide) and C to T (377th nucleotide). The nucleotide substitution at 71st, 86th and 377th position of the fragment were expected to be a silent mutation where as nucleotide changes at 217th, 258th and 371st positions were expected to be substituted by lysine with arginine, valine with isoleucine and leucine with proline in allele B. The differences of nucleotides and amino acids between cattle, buffalo and sheep breeds have been revealed and on the basis of nucleotide as well as protein variability the phylogenetic diagram have been developed indicating closeness between cattle and buffalo.     

Anti-CD3 antibodies induce T cells from unprimed animals to secrete IL-4 both in vitro and in vivo

Recently, functional heterogeneity among Th cells has been recognized. Based on pattern of lymphokine secretion, two mutually exclusive subsets of CD4+ cells have been defined and designated Th1 (secreting IL-2 and IFN-gamma) and Th2 (secreting IL-4 and IL-5). Identification of these subsets was mostly based on the study of long term cultured T cell lines and clones, and little is known about the Th heterogeneity in vivo. In particular, it has been suggested that IL-4 producing cells cannot be detected in vivo or in primary stimulations in vitro unless responder cells had been previously primed. Our data however, indicate that anti-CD3 mediated stimulation can induce T cells isolated from unprimed animals to IL-4 production. An assay system based on the ability of IL-4 to increase Ia expression of B cells present in the environment of activated T cells was found to be more sensitive than detection of secreted IL-4 in the supernatant by conventional bioassays and was used to study IL-4 production by unprimed lymphocytes polyclonally stimulated in vivo and in vitro by anti-CD3 mAb. The results obtained indicate that CD4+ CD8- T cells able to produce IL-4 upon receptor-specific stimulation exist in the preimmune pool of adult animals. Remarkably, these cells can also be stimulated in vivo by treating animals with anti-CD3 mAb, as indicated by the in vivo induction of IL-4 specific mRNA and hyper-Ia expression on B cells. These results indicate that the inability to detect IL-4 in primary cultures is not due to different activation requirements of Th2 cells but may simply result from their lower frequency in unprimed animals.     

Identification and partial characterization of the xanthine oxidase transitions of the milk fat globule membrane

Differential scanning calorimetry of bovine milk fat globule membranes (MFGM) yields five to eight transitions, depending on the conditions employed during isolation and assay of the membranes. Transitions A, B, and C were shown in a previous publication to derive from lipid melting, while transition D was found to stem from the unfolding of a structural protein termed butyrophilin [K. C. Appell, T. W. Kennan, and P. S. Low (1982) Biochim. Biophys. Acta 690, 243-250]. In this report we present evidence that the E1, E2, and F endotherms derive from the major MFGM protein, xanthine oxidase. Support for this contention derives from (i) thermal gel analysis; (ii) thermal inactivation analysis; (iii) comparison of the calorimetric properties of endotherms I, II, and III of purified xanthine oxidase with transitions E1, E2, and F of MFGM; (iv) comparison of the properties of a peculiar exotherm in scans of both the purified enzyme and MFGM; and (v) examination of the effects of specific ligands, reducing agents, and pH on both the xanthine oxidase and MFGM transition. The existence of three independent endotherms (I, II, and III) in purified xanthine oxidase demonstrates that the enzyme is composed of multiple independent domains. The interconversion of transitions I (E1) and II (E2) with a change in the redox conditions of the medium implies that these two transitions may be manifestations of the interconvertible dehydrogenase and oxidase forms of the enzyme, respectively. The relative independence of the I/II transitions from transition III further shows that only slight interaction between the major domains of xanthine oxidase exists.     

The yeast phospholipid N-methyltransferases catalyzing the synthesis of phosphatidylcholine preferentially convert di-C16:1 substrates both in vivo and in vitro

Phosphatidylcholine (PC) is an important and abundant structural component of the membranes of eukaryotic cells. In the yeast Saccharomyces cerevisiae, the primary route for the biosynthesis of PC consists of three consecutive methylation steps of phosphatidylethanolamine (PE) catalyzed by the phospholipid N-methyltransferases Cho2p and Opi3p. To investigate how these biosynthetic enzymes contribute to the composition of the PC species profile, the precursor-product relationships between PE and newly synthesized PC were determined at the level of the molecular species by using electrospray ionization tandem mass spectrometry and stable isotope labeling. In vivo labeling of yeast cells for 10 min with [methyl-D3]methionine revealed the preferential methylation of di-C16:1 PE over a range of PE species compositions. A similar preferential conversion of di-C16:1 PE to PC was found in vitro upon incubating isolated microsomes with S-adenosyl[methyl-D3]methionine. Yeast opi3 and cho2 deletion strains were used to distinguish between the substrate selectivities of Cho2p and Opi3p, respectively. Both biosynthetic enzymes were found to participate in the speciesselective methylation with Cho2p contributing the most. The combined results indicate that the selective methylation of PE species by the methyltransferases plays an important role in shaping the steady-state profile of PC molecular species in yeast.     

Polyunsaturated phosphatidylcholine and lipolysis: metabolic interactions in vitro and in vivo.

Polyunsaturated phosphatidylcholine (EPL) is known to have a number of effects on lipid metabolism. We tested the drug on rat adipose tissue in vitro: stimulated lipolysis was inhibited without any effect on cyclic AMP level. Administration of EPL in rats enhanced the basal lipolysis and inhibited the hormone stimulated process. In fasting rats, the high plasma FFA level was further increased by EPL, while no modification on total esterified fatty acids, glucose and cholesterol contents was found. These effects could be due to the activity of EPL on various enzymes, for instance lipoprotein lipase, or to a general effect on membranes.     

Active component of Fatsia japonica enhances the transduction efficiency of Tat-SOD fusion protein both in vitro and in vivo

It has been reported that Tat-SOD can be directly transduced into mammalian cells and skin and acts as a potential therapeutic protein in various diseases. To isolate the compound that can enhance the transduction efficiency of Tat-SOD, we screened a number of natural products. 3-O-[beta-D-Glucopyranosyl(1-->4)-alpha-L-arabinopyranosyl]- hederagenin (OGAH) was identified as an active component of Fatsia japonica and is known as triterpenoid glycosides (hederagenin saponins). OGAH enhanced the transduction efficiencies of Tat-SOD into HeLa cells and mice skin. The enzymatic activities in the presence of OGAH were markedly increased in vitro and in vivo when compared with the controls. Although the mechanism is not fully understood, we suggest that OGAH, the active component of Fatsia japonica, might change the conformation of the membrane structure and it may be useful as an ingredient in antiaging cosmetics or as a stimulator of therapeutic proteins that can be used in various disorders related to reactive oxygen species (ROS).     

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.     

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.     

Griseofulvin interacts with microtubules both in vivo and in vitro

Immunofluorescence microscopy using monospecific tubulin antibody shows that in vivo griseofulvin interferes with the expression of both cytoplasmic and spindle microtubules in tissue culture cells in a concentration-dependent manner. In mouse 3T3 cells cytoplasmic microtubules are destroyed at a griseofulvin concentration of 5 × 10^?5m. At this concentration no increase of the mitotic index is observed but the cells are arrested in interphase, probably due to the destruction of cytoplasmic microtubules. Lowering the drug concentration to 10^?5m allows 3T3 cells to accumulate in c-mitotic (“colchicin-mitotic”) arrest. In HeLa cells the display of spindle microtubules observed in drug-arrested cells appears similar to that seen in normal metaphase cells only at lower griseofulvin concentrations. Higher drug concentrations induce c-mitotic arrest accompanied by an increasing loss of typical metaphase tubulin structures.In vitro polymerization experiments with brain tubulin using both light-scattering and electron microscopy show that in the presence of griseofulvin tubulin can aggregate rapidly in the cold. This behaviour is not found in the absence of the drug. Thus both in vivo and in vitro experiments show that griseofulvin, like other c-mitotic drugs, acts at the level of tubulin polymerization and that its effects are concentration dependent.     

Dihydrotanshinone I inhibits angiogenesis both in vitro and in vivo

Dihydrotanshinone I （DI）, a naturally occurring compound extracted from Salvia miltiorrhiza Bunge, has been reported to have cytotoxicity to a variety of tumor cells. In this study, we investigated its anti-angiogenic capacity in human umbilical vein endothelial cells. DI induced a potent cytotoxicity to human umbilical vein endothelial cells, with an IC50 value of approximately 1.28 μg/ml.At 0.25-1μg/ml, DI dose-dependently suppressed human umbilical vein endothelial cell migration, invasion, and tube formation detected by wound healing, Transwell invasion and Matrigel tube formation assays, respectively. Moreover, DI showed significant in vivo anti-angiogenic activity in chick embryo chorioailantoic membrane assay. DI induced a 61.1% inhibitory rate of microvessel density at 0.2 μg/egg. Taken together, our results showed that DI could inhibit angio-genesis through suppressing endothelial cell proliferation, migration, invasion and tube formation, indicating that DI has a potential to be developed as a novel anti-angiogenic agent.