Milk Lipids: A Complex Nutrient Delivery SystemJ. Bruce German
The evolutionary origin of lactation and the composition, structures, and functions of milk’s biopolymers illustrates that the Darwinian pressure on lactation selected for biopolymers with considerable structural complexity and discrete functions within the digestive system . For example, complex sugar polymers, oligosaccharides, possess unique properties in guiding the growth of intestinal bacteria that are not possible by feeding their simple sugars; proteins exhibit enzymatic activities towards other milk components rendering those components both more digestible but also releasing biologically active products. To date, however, the most complex structure in mammalian milk, the fat globule, has not been effectively examined beyond its simple composition. The globules of milk are heterogeneous in size, composition and function, and new research tools and models are beginning to understand the mechanisms that control the assembly of globules in the mammary gland and the disassembly within the infant.
Milk globules represent a unique biological particle class composed of a triglyceride core bounded by a phospholipid monolayer, assembled in the endoplasmic reticulum, and a complete bilayer structure enrobed around the globule by the mammary epithelial plasma membrane during globule secretion [2, 3]. The size, diversity, and composition of milk fat globules changes during lactation and as a function of genetics, diet, and mammary gland metabolism . The size of fat globules is directed in part by the availability of specific precursor lipids . Phosphatidyl choline and phosphatidyl ethanolamine compete for occupancy of the globule surface, and their distinct physical properties guide the fusion events that ultimately determine the size of globules as they form and are secreted into milk. The proportion of these two complex phospholipids is thus a vital determinant of globule assembly. Lipid trafficking within the mammary epithelial cells is determined in part by available fatty acids and by mitochondria. These research breakthroughs not only provide a mechanistic understanding for these processes but offer the possibility to guide globule size, composition, and function with exogenous treatment .
Fat globules undergo complex disruption during digestion within the intestine of the infant. The importance of the digestion process has been implied by the discoveries of endogenous lipase enzymes within the milk itself that guide lipid digestion even in the absence of digestion capacity within early infants. Complex lipids interact with the aqueous phase to produce a diverse tableau of possible structures that exhibit considerable organization within the nanometer and micrometer length scales. The biological membrane is just one example of a three-dimensional lipid phase that forms spontaneously depending on the concentration and composition of lipids. As the lipid composition of fat globules changes within the intestine due to changes in the molecular structure of complex lipids resulting from hydrolysis, new three-dimensional phases/ structures can form spontaneously. However, these phases are dynamic and change spontaneously as a function of concentration. Dynamic lipid structures have been ostensibly impossible to follow scientifically due to their ephemeral nature. High-intensity, coherent X-rays are formed by synchrotron accelerators. It is thus possible to introduce complex lipid mixtures into these beams and follow the structures formed in real time. Recent studies on the phase changes during lipolysis have demonstrated that the ensemble of complex lipids of mammalian milks forms distinct cubic structures in real time . The cubic phase is of particular interest since it is a three-dimensional bicontinuous phase capable of dissolving and transporting both water-soluble and lipid-soluble components in all directions. The cubic phase presence has been linked to successful absorption of a variety of fat-soluble nutrients, yet it has not been possible previously to determine if milk lipids form cubic phases. Thus, the lipid globule constitutes the precursors for a complex, higher-order, structured delivery system that self-assembles within the infant’s intestine, facilitating absorption by the infant.
The composition of mammalian milk lipids is a perplexingly sensitive process. Unlike the other biopolymer classes (proteins, glycans, polynucleotides), lipid composition is significantly altered by the composition of fatty acids within the maternal diet. As a result of the widespread observations of the environmental influences on milk composition, considerable research has focused on this aspect. Much less interest has focused on the fatty acids that are synthesized by the mammary gland itself. Some fatty acids are relatively constant in milk across species, and new research is beginning to reveal functions of these fatty acids beyond simple fuel provision. The most abundant single fatty acid in milk, palmitic acid, has been identified as a potent ligand for the PGC-1ß transcription coactivator within the liver . This nuclear regulator orchestrates the transcription of the complex machinery necessary to synthesize and assemble very-low-density lipoproteins within the liver, thus guiding not only liver lipid secretion but whole-body energy metabolism. The fatty acid derivative of desaturation of palmitic acid, palmitoleic acid, has been identified as possessing an alternative signaling function. Characterized as the first of a class of lipid signaling lipids “lipokines,” palmitoleic acid controls hepatic gluconeogenesis, lipid uptake in the muscle, and potentially food intake systemically . The presence of these fatty acids within milk suggests that various aspects of fuel metabolism within the infant are under direct control of the composition of milk’s constitutive fatty acids.
This growing body of evidence argues for a broader view of milk composition that includes the complex structures of large biopolymers, their structures as ensembles, their distinct activities within the milk as it is digested, and the influence of this structural dimension across the nanometer and micrometer length scales on the health value of milk within the entire diet.
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The evolution of lactation and the composition, structures, and functions of milk’s bio- polymers illustrates that the Darwinian pressure on lactation selected for gene products with considerable structural complexity and diverse functions within the digestive system. For example, complex sugar polymers – oligosaccharides – possess unique proper- ties in guiding the growth of intestinal bacteria that are not possible by feeding their simple sugars. The proteins of milk are diverse with some exhibiting enzymatic activities towards other milk components rendering those components both more digestible but also releasing biologically active products. Thus, research into milk’s biopolymers has been most enlightening when milk was investigated for the formation and disassembly of its structures and for the functions within the infant. To date however, the most complex structure in mammalian milk, the fat globule, has not been effectively examined beyond its simple composition. The globules of milk are heterogeneous in size, composition, and function. With new research tools, scientists are beginning to understand the mechanisms that control the assembly of globules in the mammary gland and the disassembly within the infant.
Milk fat has been largely ignored by diet and health research apart from linking fat to levels of cholesterol in blood. As a result, milk fat has been viewed as a net damaging contribution to human diets and even for infants. Such a negative value is not justified by more broadly based biological perspectives. Computational analysis of the evolution of the repertoire of mammalian genes responsible for lactation produced an unexpected conclusion. The most conserved sequence identities within the lactation subset of genes through mammalian evolution are the genes responsible for lipid secretion . From a purely evolutionary conservation perspective, such results position milk as a lipid delivery system. Nonetheless, it is not clear from disease protection or long-term performance what lipids are providing to infants. Similarly, it is not evident when genetic, environmental or nutritional factors in maternal mammary gland compromise the synthesis of milk lipids. This lack of mechanistic understanding of milk synthesis and globule assembly is an important barrier to policy for dietary intakes for mothers. Without a mechanistic understanding of functions of milk lipids, it is not possible to develop methods that are capable of diagnosing functional impairments in infants due to insufficiencies or excesses in particular fatty acids, complex lipids, or even metabolic cofactors. The diversity of lipid molecules in milk implies a diversity of functions, yet scientists are still focusing almost exclusively on fatty acids. Lipids are far more complex, and in that complexity resides the assembly of cellular membranes, components of dispersed particles, epithelial barriers in skin, surfactant lipids in lungs and vesicles within and without cellular compartments. In addition to diverse structural roles, lipids provide an array of substrates for metabolic fuel, and they provide a precursor pool for a range of signaling molecules. Recent developments in biochemistry, gene annotation, and biophysics are enabling a renewed focus on the synthesis of milk lipids within the mammary gland. As structures are identified, they are being assigned novel functions within the infant. Saturated and monounsaturated fatty acids are becoming recognized as metabolic signal molecules acting systemically. The complex composition of human milk lipids directs a spontaneous self-assembly process during digestion creating structures at the nanometer length scale that enhance absorption of a wide range of nutrients.
Assembling Lipid Particles in the Mammary EpitheliaThe assembly of complex lipids into highly organized bilayer membrane structures is the central organizational and energetic paradigm of biology. However, lipids provide a variety of other structural assemblies to higher organisms in particular as transport. The milk fat globule is one of biology’s more prodigious lipid transport systems. The particles themselves are highly disperse with sizes spanning three orders of magnitude and vary with stage of lactation, health of the mother, etc., with the average diameter varying between 1 and 10 μm . The fat globule is a core of triacylglycerides assembled within the endoplasmic reticulum (ER) of the mammary epithelial cell from fatty acids of dietary, adipose depot, and lipogenic origins. This core of triacylglyceride is coated by a single monolayer of phospholipids derived from the ER membrane . The milk fat globule is unique in containing also an intact bilayer membrane, which enrobes the globule as it exits the epithelial cell. This membrane as a result is com- posed of the lipids and proteins of the epithelial cell plasma membrane, including significant quantities of cholesterol, phosphatidylcholine, and sphingomyelin [4, 5]. Research has documented that these complex lipids have effects on composition and function of tissues literally from intestine to brain [6–8]. Yet one of the most important determinants of both composition and structure, globule size, is poorly understood. New research methods and models are mak- ing it possible to interrogate the more dynamic and complex lipid structures that are integral to the structures and functions of milk lipids.
The basic steps of globule assembly in the mammary epithelia are described . Triglyceride synthesis by enzymes within the ER gives rise to an inert lipid phase of triglycerides bounded by a monolayer of ER phospholipids that is released into the cytoplasm as a spherical lipid particle. What happens after their formation is both the most unique property of milk fat globules and their least understood. Globules migrating towards the apical membrane fuse into larger discrete particles, and then the membrane and particle surfaces become enriched in key proteins notably xanthine oxidase and butyrophilin. At this stage, the particles’ outer surface interacts with the inner face of the plasma membrane, and the entire particle with surrounding plasma membrane is excreted as an intact milkfat globule.
The most important determinants of globule size are the flux of triglycerides into the nascent particle within the ER, their release into the cytosol and the subsequent fusion (or not) of these particles prior to attachment with the plasma membrane and secretion. Triglyceride flux is driven by the substrate fatty acid availability and the net energy charge within the cell. Breakthrough new research demonstrates that these same determinants are responsible for particle fusion . Central to these breakthroughs is the realization that specific fatty acids are more than simple substrates to triglyceride formation, they influence the metabolic pathways and biophysical events that guide particle formation, composition, and fusion (Fig. 1). Lipid synthesis is metabolically controlled in part by the PGC1 family of transcription coactivators that drive the expression of the concert of genes necessary to assemble mitochondria and synthesize lipid particles . PGC1 is upregulated in the mammary epithelia by palmitic and oleic acids yet asymmetrically. Palmitic acid enhanced triglyceride and phosphatidyl choline concentration. Oleic acid enhanced triglyceride production and PGC1 activation and mitochondrial biosynthesis with a net increase in phosphatidyl ethanolamine concentration. This difference in phospholipid composition has an important effect on particle fusion events. As a membrane constituent, phosphatidyl ethanolamine is fusagenic. Thus, with these insightful breakthroughs in the basic mechanisms of particle fusion and the metabolic production of fusagenic phospholipids, guiding globule size distribution is becoming an accessible mammary gland outcome.
Fig. 1. Assembly of lipid globules within the mammary gland. Fatty acids with distinct metabolic activities and their consequences to biophysical events are indicated. C16:0, palmitic acid; C18:1, oleic acid; PGC1, PPARγ coactivator 1; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; Tg, triglyceride. Modified from Co- hen et al. .
Milk Fat Globule Disassembly
Fig. 2. Milk lipid structures: macroscopic, microscopic, and diffraction patterns during digestion.
Lipids as Metabolic Signals
The dossier of lipid molecules documented to exhibit a signaling function and trigger and/or regulate cellular processes continues to grow. Many of these follow classic pathways in which protein receptors bind to different lipids transducing cellular events downstream into cellular processes [18, 19]. The oxylipins are lipid signals of local stress. The essential n-6-polyunsaturated fatty acids and in particular arachidonic acid have been demonstrated to provide these signaling functions .
Lipids signal to a wide range of biological processes. Their interaction with fuel metabolism and transport may be of particular importance to the development of metabolic regulation within infants. Although the majority of scientific research has focused on the essential fatty acids that must be obtained within the maternal diet, actions of the fatty acids made within the mammary gland are emerging as important as signaling molecules. Palmitic acid is the end product of fatty acid synthesis and is ubiquitous throughout biology. Palmitic acid is: a constituent fatty acid in cellular membranes; a fuel, producing 106 moles of ATP energy via beta oxidation; is acylated to various membrane proteins; and is a major component of storage lipids.
Now to this list must be added signaling. Puigserver and Spiegelman  discovered the functions controlled by a protein complex that they termed the PPAR gene transcription coactivator (PGC-1α). This transcription factor co-activator assembles protein regulatory units in the nucleus to control mitochondrial biogenesis. This laboratory made an equally important realization that when exposed to high levels of palmitic acid, liver cells both in vivo and in vitro actively turned on a closely related protein, PGC-1β . PGC-1β simultaneously controlled fatty acid synthesis, triglyceride assembly, phospholipid synthesis and cholesterol biosynthesis. Their work identified the molecular switch linking dietary saturated fat and serum cholesterol as PGC-1β, and its most potent ligand is palmitic acid. Thus, palmitic acid is the signal activating PGC-1β and its cellular functions. By this transcription mechanism, the liver regulates the net production of very-low-density lipoproteins (VLDL) and their molecular constituents. Palmitic acid acts as a hepatic signal to stimulate VLDL synthesis and assembly. This specific signaling action in the liver and to VLDL production may be important both in infancy and throughout life.
Palmitoleic acid (16:1n7) is formed by the desaturation of palmitic acid by the stearoyl CoA desaturase enzyme. In practice, palmitoleic acid is produced when the enzymes of fatty acid synthesis and stearoyl CoA desaturase are more active than the elongase enzyme converting palmitic to stearic acid. This is typically true during active lipogenesis in humans and other animals. Cao et al.  identified and made a bold prediction characterizing palmitoleic acid as a novel lipokine, or lipid hormone involved in regulating the insulin-sensitizing effects of diet. Their studies are a demonstration of palmitoleate as a lipid signal, and a unifying model of metabolic products acting to control whole body fuel metabolism.
These are examples of signaling systems based on non-essential fatty acids regulating cellular, tissue, and whole body functions. Each of these lipid signals is conspicuously present in human milk. In view of these potentially immediate benefits of dietary lipids in infant health, it seems prudent to consider the complexity of lipids within human breast milk as an important asset that pursuing more simplified compositions puts at risk.
Lipids in milk are highly diverse, and their retention throughout evolution is a compelling story of biological structure and function. Though the research is not complete, principles can be taken forward in considering optimal dietary lipid compositions and structures for infancy. The biosynthesis of fatty acids and complex lipids within the mammary gland is a relatively variable series of processes depending on genetics, diet, maternal physiology, and metabolic status. This variability poses both a challenge and an opportunity. Understanding how globules are synthesized and assembled has proven to be a daunting task. Nonetheless, as the basic mechanistic principles are emerging, opportunities are also emerging to guide these processes towards more healthy, protective, and functional lipid systems. Similarly, the diversity of composition is revealing a diversity of functions within the mammary gland and the infant. The roles of lipid molecules in signaling are proving to be astonishingly diverse. Whereas lipid signaling was once thought to be a simple response to stress via the oxygenation of polyunsaturated fatty acids, a host of lipid molecules are now implicated in normal metabolism, physiology, immunology, and even cognition and behavior. Finally, lipids are a unique class of biomolecules because of the structures that they make within and outside cells. The evolution of mammalian milk has retained the unique fat globule structure across marsupials and all mammals. Finally, new biophysical tools are beginning to assign specific properties to the structural dimension of dietary lipids. Once again, biology is teaching chemistry and pharmacology how best to guide complex processes to desired outcomes.
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