Microlipid droplets in milk secreting mammary epithelial cells: evidence that they originate from endoplasmic reticulum and are precursors of milk lipid globules

Microlipid droplets, structures with diameters less than 0.5 micron, resemble larger cytoplasmic lipid droplets of milk secreting mammary epithelial cells in triacylglycerol core and surface coat composition. Previously, evidence was obtained that microlipid droplets fuse with and support growth of cytoplasmic lipid droplets, which are immediate precursors of large milk lipid globules. Morphological observations suggested that microlipid droplets may also be secreted directly from mammary epithelial cells, yielding the very small lipid globules of milk. The secretion mechanism, which involves envelopment of triacylglycerol droplets in apical plasma membrane, appeared to be the same for microlipid droplets as for larger cytoplasmic lipid droplets. Microlipid droplets appeared to originate by blebbing from cisternae of endoplasmic reticulum. By immunogold cytochemical localization and by immunological identification of electrophoretically separated polypeptides, endoplasmic reticulum, micro- and cytoplasmic lipid droplets, and milk lipid globules had a number of common polypeptides. Kinetics of incorporation of radiolabeled palmitate or glycerol into triacylglycerols and phospholipids were consistent with a possible endoplasmic reticulum origin of microlipid droplets and with the view that microlipid droplets may be secreted directly from the cell or may fuse with cytoplasmic lipid droplets.     

Lipid droplets as dynamic organelles connecting storage and efflux of lipids

Neutral lipids are stored in the cytosol in so-called lipid droplets. These are dynamic organelles with neutral lipids as the core surrounded by a monolayer of amphipathic lipids (phospholipids and cholesterol) and specific proteins (PAT proteins and proteins involved in the turnover of lipids and in the formation and trafficking of the droplets). Lipid droplets are formed at microsomal membranes as primordial droplets with a diameter of 0.1-0.4 microm and increase in size by fusion. In this article, we review the assembly and fusion of lipid droplets, and the processes involved in the secretion of triglycerides. Triglycerides are secreted from cells by two principally different processes. In the mammary gland, lipid droplets interact with specific regions of the plasma membrane and bud off with an envelope consisting of the membrane, to form milk globules. In the liver and intestine, very low-density lipoproteins (VLDL) and chylomicrons are secreted by using the secretory pathway of the cell. Finally, we briefly review the importance of lipid droplets in the development of insulin resistance and atherosclerosis.     

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.     

Secretory membranes of the lactating mammary gland

Summary The lactating mammary gland is one of the most highly differentiated and metabolically active organs in the body. Membranes of the lactating mammary cell have important roles in transmitting from one membrane to another of hormonal information and in milk secretion, which is the final event. During milk secretion, the projection of the surface membrane into the alveolar lumen by enveloping intracellular lipid droplets with the apical plasma membrane is one of the most remarkable aspects of biological membrane action throughout nature.This review focuses on current knowledge about membranes in the lactating mammary gland. (1) Advances in the isolation and properties of membranes, especially the plasma membrane and Golgi-derived secretory vesicles, concerned with milk secretion from the lactating mammary gland are described. (2) Milk serum components are secreted by fusing the membranes of secretory vesicles that condense milk secretions with the plasma membrane in the apical regions. This occurs through the formation of a tubular-shaped projection and vesicular depression in a ball-and-socket configuration, as well as by simple fusion. (3) Intracellular lipid droplets are directly extruded from the mammary epithelial cells by progressive envelopment of the plasma membranes in the apical regions. (4) The balance between the surface volume lost in enveloping lipid droplets and that provided by fusion of the secretory vesicle and other vesicles with the apical plasma membrane is discussed. (5) The membrane surrounding a milk fat globule, which is referred to as the milk fat globule membrane (MFGM), is composed of at least the coating membrane of an intracellular lipid droplet, of the apical plasma membrane and secretory vesicle membrane, and of a coat material. Consequently, MFGM is molecularly different from the plasma membrane in composition. (6) MFGM of bovine milk is structurally composed of an inner coating membrane and outer plasma membrane just after segregation. These two membranes are fused and reorganized through a process of vesiculation and fragmentation to stabilize the fat globules. Hypothetical structural models for MFGM from bovine milk fat globules just after secretion and after rearrangement are proposed.Abbrevations MFGM milk fat globule membrane - HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - INT 2-(p-indophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - Sph sphingomyelin - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PS phosphatidyl serine - PI phosphatidyl inositol - PAS periodic acid-Schiff reagent - CB Coomassie brilliant blue R-250 Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

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.     

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.     

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.     

Nuclear coactivator protein p100 is present in endoplasmic reticulum and lipid droplets of milk secreting cells

We have identified the p100 protein, previously known as a novel cellular coactivator, as a constituent of endoplasmic reticulum and cytosolic lipid droplets from milk secreting cells. Cytosolic lipid droplets of terminally differentiated mammary epithelial cells are secreted as milk lipid globules. However, milk lipid globules did not have detectable amounts of p100 protein. The p100 protein was found also in cytosol from lactating mammary gland, in storage lipid droplets from mouse adipocytes, and in endoplasmic reticulum from liver. Immunofluorescence microscopy of mammary epithelial cells confirmed the presence of p100 in non-nuclear regions of these cells. Partial sequence analysis of tryptic peptides from p100 from cow mammary gland showed extensive homology with the reported sequence of p100 determined from a human cDNA. Antibodies against a peptide synthesized to duplicate a sequence in human p100 recognized a protein of the size of p100 in cow, mouse and rat cell fractions.     

Ultrastructural analysis of milk fat globule membranes of colostrum and human milk

Lacteous fat globules with their membranes from human milk and calostrum were studied by scanning and transmission electron microscopy. The first appear as spheroidal structures with some irregularities on the surface. Under the transmission electron microscope these irregularities are composed of islets from a material morphologically similar to cytoplasm, and with structures that resemble a fragment of rugose endoplasmic reticulum. The membranes in specimens fixed immediately after secretion are tri-layered, similar in appearance to those of a single membrane, but in samples fixed between two and four hours after secretion the details are unclear. Through freeze-etching, the laminar aspect of the fat globules is observed.     

Fat absorptive processes in the intestine of the Antarctic fish Notothenia coriiceps (Richardson, 1844)

Absorption of lipids by the enterocytes of Notothenia coriiceps, an omnivorous Antarctic and subAntarctic fish, was studied by light and electron microscopy. The lipids are absorbed by the anterior and middle intestine segments. They appear as fat droplets that measure from 0.5 to 7 μm of diameter and which accumulate in the apical cytoplasm within the first 24 h and seem to be the main fat storage form in the enterocytes. Fat is also observed as lipid particles with 60–300 nm inside the rough endoplasmic reticulum and cytoplasmic smooth vesicles. The epithelial intercellular space and the mucosal inner lamina contain lipid particles, which probably are the fat transport form. Our observations show that an intense lipid absorptive process takes place in N. coriiceps digestive system, due to the great extension of the intestine involved and due to the great lipid accumulation found in the epithelial compartment.     

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.     

A Test of Current Models for the Mechanism of Milk‐Lipid Droplet Secretion

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin‐2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N‐linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN‐enhanced green fluorescent protein was highly mobile in areas of mouse milk‐lipid droplets that had not undergone post‐secretion changes, and endogenous mouse BTN comprised only 0.5–0.7% (w/w) of the total protein, i.e. over 50‐fold less than in the milk‐lipid droplets of cow and other species. These data are incompatible with models of milk‐lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk .     

Milk lipid globule precursor release from endoplasmic reticulum reconstituted in a cell-free system.

Lipid droplet precursors of milk lipid globules are believed to be derived from elements of endoplasmic reticulum in milk-secreting mammary epithelial cells. Endoplasmic reticulum isolated from mammary gland was able to generate small droplets morphologically resembling microlipid droplet precursors of milk lipid globules. Droplet generation was time and temperature dependent and required a cytosol fraction of Mr greater than 10,000. Droplet generation was enhanced by, but did not require, addition of nucleoside triphosphates, fatty acids, coenzyme A, glycerol-3-phosphate, and dithiothreitol. Microlipid droplets generated in this cell-free system were enriched in triacylglycerols and resembled microlipid droplets formed within mammary epithelial cells in polar lipid and polypeptide composition. Endoplasmic reticulum immobilized onto nitrocellulose retained activity in generation of putative microlipid droplets, and this immobilization method provided a facile means for separation of the donor from the generated products.     

Antibodies to the major insoluble milk fat globule membrane-associated protein: specific location in apical regions of lactating epithelial cells

Milk lipid globules of various species are surrounded by a membrane structure that is separated from the triglyceride core of the globule by a densely staining fuzzy coat layer of 10- to 50-nm thickness. This internal coat structure remains attached to the membrane during isolation and extraction with low- and high-salt buffers, is insoluble in nondenaturing detergents, and is enriched in an acidic glycoprotein (butyrophilin) with an apparent Mr of 67,000. Guinea pig antibodies against this protein, which show cross-reaction with the corresponding protein in some (goat) but not other (human, rat) species, have been used for localization of butyrophilin on frozen sections of various tissues from cow by immunofluorescence and electron microscopy. Significant reaction is found only in milk-secreting epithelial cells and not in other cell types of mammary gland and various epithelial tissues. In milk-secreting cells, the staining is restricted to the apical cell surface, including budding milk lipid globules, and to the periphery of the milk lipid globules contained in the alveolar lumina. These findings indicate that butyrophilin, which is constitutively secreted by surface budding in coordination with milk lipid production, is located at the apical surface and is not detected at basolateral surfaces, in endoplasmic reticulum, and in Golgi apparatus. This protein structure represents an example of a cell type-specific cytoskeletal component in a cell apex. It is suggested that this antigen provides a specific marker for the apical surface of milk- secreting cells and that butyrophilin is involved in the vectorial discharge of milk lipid globules.     

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.     

Aspect ultrastructural de la lipogénèse dans le tissu adipeux brun

Summary In interscapular brown fat of the rat, appropriately processed so as to maintain membrane structures intact, lipid droplets are in fact liposomes, i.e., lipid filled vacuoles surrounded by a membrane that is related to the smooth endoplasmic reticulum. Thus the endoplasmic reticulum appears as an important component of brown fat cells. — After complete lipid depletion has been achieved by 7 days of fasting, feeding with glucose results in sudden and very conspicuous increase of smooth endoplasmic reticulum; dilatations of the perinuclear cistern and pinocytotic activity at the periphery of the cell contribute to this increase. Simultaneously the cytoplasmic matrix is heavily loaded with glycogen in particulate form. Lipogenesis, as far as can be appreciated by different degrees in electron density, takes place inside the vesicles of the endoplasmic reticulum; increase of such small lipid vacuoles then leads to reconstitution of liposomes; return to the normal aspect is completed 12 hours after the beginning of feeding. — During the phase of glycogen overloading and resulting lipogenesis, glycogen particles may be found in intercellular and pericapillary spaces; the significance of this finding is discussed. — The fundamental difference between both types of fat cells seems to be concerned with the site of lipogenesis: this takes place in the cytoplasmic matrix of white fat cells, so that lipid droplets aggregate without any limiting membrane, whereas in brown fat cells lipogenesis occurs inside cavities of the endoplasmic reticulum and lipids remain permanently enclosed in membranes. This process appears similar to what may be observed occasionally in liver and normally in adrenal cortex, and this might presumably lead to a physico-chemical understanding of the particular aspect of lipogenesis in brown fat, as compared to that in common white fat.

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Travail dédié au Professeur W. Bargmann, en témoignage d'admiration, à l'occasion de son 60e anniversaire.     

Distribution of terminal transferases of acylglycerol synthesis in cell fractions from lactating mammary gland

Cytoplasmic lipid droplets and microlipid droplets, intracellular precursors of milk lipid globules, had little ability to incorporate radioactivity from glycerol 3-phosphate or palmitoyl-CoA into triacylglycerols. The limited incorporation of these precursors by micro- and cytoplasmic lipid droplets from rat and cow mammary gland was into phospholipids primarily. Acyltransferases catalyzing incorporation of glycerol 3-phosphate into acylglycerols were concentrated in a relatively high buoyant density class of rough microsomes. Palmitoyl-CoA-sn-1,2-diacylglycerol acyltransferase activity was distributed heterogeneously among fractions obtained by equilibrium density gradient fractionation of mammary homogenates. Observations suggest that terminal steps of acylglycerol synthesis are localized primarily in rough endoplasmic reticulum of milk secreting mammary epithelial cells. There appears to be a heterogeneous distribution of acyltransferases along the reticular network.     

Butyrophilin of milk lipid globule membrane contains N-linked carbohydrates and cross-links with xanthine oxidase

1. N-glycanase, but not O-glycanase, released carbohydrates from butyrophilin of rat and cow milk lipid globule membranes. 2. 1-Deoxynojirimycin, and inhibitor of glucosidases I and II of the glycoprotein processing pathway, increased the amount or extent of glycosylation of butyrophilin in rat milk lipid globules. 3. Butyrophilin and xanthine oxidase of milk lipid globule membrane had a nearest neighbor relationship, as demonstrated through specific crosslinking of these proteins. 4. From these results it is suggested that butyrophilin has asparagine-linked oligosaccharides which bypass the processing apparatus of endoplasmic reticulum and Golgi apparatus. Butyrophilin may be responsible for anchoring xanthine oxidase to the inner (cytoplasmic) face of milk lipid globule membrane.     

An Updated Perspective on the Dual-Track Model of Enterocyte Fat Metabolism

The small intestinal epithelium has classically been envisioned as a conduit for nutrient absorption, but appreciation is growing for a larger and more dynamic role for enterocytes in lipid metabolism. Considerable gaps remain in our knowledge of this physiology, but it appears that the enterocyte’s structural polarization dictates its behavior in fat partitioning, treating fat differently based on its absorption across the apical versus the basolateral membrane. In this review, we synthesize existing data and thought on this dual-track model of enterocyte fat metabolism through the lens of human integrative physiology. The apical track includes the canonical pathway of dietary lipid absorption across the apical brush-border membrane, leading to packaging and secretion of those lipids as chylomicrons. However, this track also reserves a portion of dietary lipid within cytoplasmic lipid droplets for later uses, including the “second-meal effect,” which remains poorly understood. At the same time, the enterocyte takes up circulating fats across the basolateral membrane by mechanisms that may include receptor-mediated import of triglyceride-rich lipoproteins or their remnants, local hydrolysis and internalization of free fatty acids, or enterocyte de novo lipogenesis using basolaterally absorbed substrates. The ultimate destinations of basolateral-track fat may include fatty acid oxidation, structural lipid synthesis, storage in cytoplasmic lipid droplets, or ultimate resecretion, although the regulation and purposes of this basolateral track remain mysterious. We propose that the enterocyte integrates lipid flux along both of these tracks in order to calibrate its overall program of lipid metabolism.