Organellar Contacts of Milk Lipid Droplets

Milk-secreting epithelial cells of the mammary gland are functionally specialized for the synthesis and secretion of large quantities of neutral lipids, a major macronutrient in milk from most mammals. Milk lipid synthesis and secretion are hormonally regulated and secretion occurs by a unique apocrine mechanism. Neutral lipids are synthesized and packaged into perilipin-2 (PLIN2) coated cytoplasmic lipid droplets within specialized cisternal domains of rough endoplasmic reticulum (ER). Continued lipid synthesis by ER membrane enzymes and lipid droplet fusion contribute to the large size of these cytoplasmic lipid droplets (5–15 μm in diameter). Lipid droplets are directionally trafficked within the epithelial cell to the apical plasma membrane. Upon contact, a molecular docking complex assembles to tether the droplet to the plasma membrane and facilitate its membrane envelopment. This docking complex consists of the transmembrane protein, butyrophilin, the cytoplasmic housekeeping protein, xanthine dehydrogenase/oxidoreductase, the lipid droplet coat proteins, PLIN2, and cell death-inducing DFFA-like effector A. Interactions of mitochondria, Golgi, and secretory vesicles with docked lipid droplets have also been reported and may supply membrane phospholipids, energy, or scaffold cytoskeleton for apocrine secretion of the lipid droplet. Final secretion of lipid droplets into the milk occurs in response to oxytocin-stimulated contraction of myoepithelial cells that surround milk-secreting epithelial cells. The mechanistic details of lipid droplet release are unknown at this time. The final secreted milk fat globule consists of a triglyceride core coated with a phospholipid monolayer and various coat proteins, fully encased in a membrane bilayer

Organellar Contacts of Milk Lipid Droplets

Milk-secreting epithelial cells of the mammary gland are functionally specialized for the synthesis and secretion of large quantities of neutral lipids, a major macronutrient in milk from most mammals. Milk lipid synthesis and secretion are hormonally regulated and secretion occurs by a unique apocrine mechanism. Neutral lipids are synthesized and packaged into perilipin-2 (PLIN2) coated cytoplasmic lipid droplets within specialized cisternal domains of rough endoplasmic reticulum (ER). Continued lipid synthesis by ER membrane enzymes and lipid droplet fusion contribute to the large size of these cytoplasmic lipid droplets (5–15 μm in diameter). Lipid droplets are directionally trafficked within the epithelial cell to the apical plasma membrane. Upon contact, a molecular docking complex assembles to tether the droplet to the plasma membrane and facilitate its membrane envelopment. This docking complex consists of the transmembrane protein, butyrophilin, the cytoplasmic housekeeping protein, xanthine dehydrogenase/oxidoreductase, the lipid droplet coat proteins, PLIN2, and cell death-inducing DFFA-like effector A. Interactions of mitochondria, Golgi, and secretory vesicles with docked lipid droplets have also been reported and may supply membrane phospholipids, energy, or scaffold cytoskeleton for apocrine secretion of the lipid droplet. Final secretion of lipid droplets into the milk occurs in response to oxytocin-stimulated contraction of myoepithelial cells that surround milk-secreting epithelial cells. The mechanistic details of lipid droplet release are unknown at this time. The final secreted milk fat globule consists of a triglyceride core coated with a phospholipid monolayer and various coat proteins, fully encased in a membrane bilayer

Comparative proteomic analysis of human milk fat globules and paired membranes and mouse milk fat globules identifies core cellular systems contributing to mammary lipid trafficking and secretion

Introduction: Human milk delivers critical nutritional and immunological support to human infants. Milk fat globules (MFGs) and their associated membranes (MFGMs) contain the majority of milk lipids and many bioactive components that contribute to neonatal development and health, yet their compositions have not been fully defined, and the mechanisms responsible for formation of these structures remain incompletely understood.Methods: In this study, we used untargeted mass spectrometry to quantitatively profile the protein compositions of freshly obtained MFGs and their paired, physically separated MFGM fractions from 13 human milk samples. We also quantitatively profiled the MFG protein compositions of 9 pooled milk samples from 18 lactating mouse dams.Results: We identified 2,453 proteins and 2,795 proteins in the majority of human MFG and MFGM samples, respectively, and 1,577 proteins in mouse MFGs. Using paired analyses of protein abundance in MFGMs compared to MFGs (MFGM-MFG; 1% FDR), we identified 699 proteins that were more highly abundant in MFGMs (MFGM-enriched), and 201 proteins that were less abundant in MFGMs (cytoplasmic). MFGM-enriched proteins comprised membrane systems (apical plasma membrane and multiple vesicular membranes) hypothesized to be responsible for lipid and protein secretion and components of membrane transport and signaling systems. Cytoplasmic proteins included ribosomal and proteasomal systems. Comparing abundance between human and mouse MFGs, we found a positive correlation (R2 = 0.44, p < 0.0001) in the relative abundances of 1,279 proteins that were found in common across species.Discussion: Comparative pathway enrichment analyses between human and mouse samples reveal similarities in membrane trafficking and signaling pathways involved in milk fat secretion and identify potentially novel immunological components of MFGs. Our results advance knowledge of the composition and relative quantities of proteins in human and mouse MFGs in greater detail, provide a quantitative profile of specifically enriched human MFGM proteins, and identify core cellular systems involved in milk lipid secretion

Comparative Proteomic Analysis of Paired Human Milk Fat Globules and Membranes and Mouse Milk Fat Globules Identifies Core Cellular Systems Contributing to Mammary Lipid Trafficking and Secretion

AbstractHuman milk delivers critical nutritional and immunological support to the infant. The milk fat globule and its membrane contain many bioactive components, yet the mechanism of milk fat secretion and how milk fat globule (MFG) components are regulated are poorly defined. In this study, we perform quantitative proteomic profiling of milk fat globules from human and mouse milk, as well as from isolated membranes physically disrupted from human milk fat globules.Using paired analyses of the human samples we report membrane enrichment of the proteins involved in docking/tethering the lipid droplet to the membrane as well as minor components involved in the signaling pathway for secretion. Comparing abundance between human and mouse milk fat globules we find that 8 of 12 major milk fat globule proteins are shared between the two species. Comparative pathway enrichment analyses between human and mouse samples reveal similarities in shared membrane trafficking and signaling pathways involved in milk fat secretion. Our results advance knowledge of the composition and relative quantities of proteins in human and mouse milk fat globules in greater detail, provide a quantitative profile of specifically enriched human milk fat globule membrane proteins, and identify core cellular systems involved in milk lipid secretion.</jats:p

Contribution of Xanthine Oxidoreductase to Mammary Epithelial and Breast Cancer Cell Differentiation In Part Modulates Inhibitor of Differentiation-1

Abstract Loss of xanthine oxidoreductase (XOR) has been linked to aggressive breast cancer in vivo and to breast cancer cell aggressiveness in vitro. In the present study, we hypothesized that the contribution of XOR to the development of the normal mammary gland may underlie its capacity to modulate breast cancer. We contrasted in vitro and in vivo developmental systems by differentiation marker and microarray analyses. Human breast cancer microarray was used for clinical outcome studies. The role of XOR in differentiation and proliferation was examined in human breast cancer cells and in a mouse xenograft model. Our data show that XOR was required for functional differentiation of mammary epithelial cells both in vitro and in vivo. Poor XOR expression was observed in a mouse ErbB2 breast cancer model, and pharmacologic inhibition of XOR increased breast cancer tumor burden in mouse xenograft. mRNA microarray analysis of human breast cancer revealed that low XOR expression was significantly associated with time to tumor relapse. The opposing expression of XOR and inhibitor of differentiation-1 (Id1) during HC11 differentiation and mammary gland development suggested a potential functional relationship. While overexpression of Id1 inhibited HC11 differentiation and XOR expression, XOR itself modulated expression of Id1 in differentiating HC11 cells. Overexpression of XOR both inhibited Id1-induced proliferation and -stimulated differentiation of Heregulin-β1–treated human breast cancer cells. These results show that XOR is an important functional component of differentiation whose diminished expression contributes to breast cancer aggressiveness, and they support XOR as both a breast cancer biomarker and a target for pharmacologic activation in therapeutic management of aggressive breast cancer. Mol Cancer Res; 9(9); 1242–54. ©2011 AACR.</jats:p