Search

Human 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.

Cells in human milk are an untapped source, as potential “liquid breast biopsies”, of material for investigating lactation physiology in a non-invasive manner. We used single cell RNA sequencing (scRNA-seq) to identify milk-derived mammary epithelial cells (MECs) and their transcriptional signatures in women with diet-controlled gestational diabetes (GDM) with normal lactation. Methodology is described for coordinating milk collections with single cell capture and library preparation via cryopreservation, in addition to scRNA-seq data processing and analyses of MEC transcriptional signatures. We comprehensively characterized 3,740 cells from milk samples from two mothers at two weeks postpartum. Most cells (>90%) were luminal MECs (luMECs) expressing lactalbumin alpha and casein beta and positive for keratin 8 and keratin 18. Few cells were keratin 14(+) basal MECs and a small immune cell population was present (

It is estimated that well-nourished women secrete over 6 kg of fat into milk during a 6-month period of breastfeeding, which makes the lactating breast one of the most lipogenic organs in the human body. In addition to being a primary source of energy for neonates, milk lipids participate in the delivery of micronutrients, including fat-soluble vitamins and essential fatty acids required for infant development. The lipid content of human milk averages 3.4% w/v, which is about the same as that of major dairy species, such as cattle and goats, but significantly less than the approximately 50% found in aquatic mammals. The precise composition of lipids in the milk of humans and other mammals is variable and influenced by diet, genetics, and lactation stage, among other factors. Improvements in bioanalytical methods for lipids and proteins have led to a greater and more detailed understanding of the composition and complexity of human breast milk lipids and their role in neonatal nutrition and human health.

Key points Wild-type mice and mice with hepatocyte-specific or whole-body deletions of perilipin-2 (Plin2) were used to define hepatocyte and extra-hepatocyte effects of altered cellular lipid storage on obesity and non-alcoholic fatty liver disease (NAFLD) pathophysiology in a Western-diet (WD) model of these disorders. Extra-hepatocyte actions of Plin2 are responsible for obesity, adipose inflammation and glucose clearance abnormalities in WD-fed mice. Hepatocyte and extra-hepatic actions of Plin2 mediate fatty liver formation in WD-fed mice through distinct mechanisms. Hepatocyte-specific actions of Plin2 are primary mediators of immune cell infiltration and fibrotic injury in livers of obese mice. Abstract Non-alcoholic fatty liver disease (NAFLD) is an obesity- and insulin resistance-related metabolic disorder with progressive pathology. Perilipin-2 (Plin2), a ubiquitously expressed cytoplasmic lipid droplet scaffolding protein, is hypothesized to contribute to NAFLD in humans and rodent models through effects on cellular lipid metabolism. In this study, we delineate hepatocyte-specific and extra-hepatocyte Plin2 mechanisms regulating the effects of obesity and insulin resistance on NAFLD pathophysiology in mice fed an obesogenic Western-style diet (WD). Total Plin2 deletion (Plin2-Null) fully protected WD-fed mice from obesity, insulin resistance, adipose inflammation, steatohepatitis (NASH) and liver fibrosis found in WT animals. Hepatocyte-specific Plin2 deletion (Plin2-HepKO) largely protected against NASH and fibrosis and partially protected against steatosis in WD-fed animals, but it did not protect against obesity, insulin resistance, or adipose inflammation. Significantly, total or hepatocyte-specific Plin2 deletion impaired WD-induced monocyte recruitment and pro-inflammatory macrophage polarization found in livers of WT mice. Analyses of the molecular and cellular processes mediating steatosis, inflammation and fibrosis identified differences in total and hepatocyte-specific actions of Plin2 on the mechanisms promoting NAFLD pathophysiology. Our results demonstrate that hepatocyte-specific actions of Plin2 are central to the initiation and pathological progression of NAFLD in obese and insulin-resistant mice through effects on immune cell recruitment and fibrogenesis. Conversely, extra-hepatocyte Plin2 actions promote NAFLD pathophysiology through effects on obesity, inflammation and insulin resistance. Our findings provide new insight into hepatocyte and extra-hepatocyte mechanisms underlying NAFLD development and progression.

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.

Key points Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation. XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion. XOR mediates apical membrane reorganization during milk lipid secretion. Loss of XOR delays milk fat globule secretion. XOR loss alters the proteome of milk fat globules. Abstract Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane-bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary-specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild-type gland but not in the knockout, suggesting that XOR mediates ‘docking’ to this membrane. Secreted milk fat globules were isolated from mouse milk of wild-type and XOR MGKO dams, and subjected to LC-MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion.

Background/Objectives The current obesity epidemic has spurred exploration of the developmental origin of adult heath and disease. A mother’s dietary choices and health can affect both the early wellbeing and lifelong disease-risk of the offspring. Subjects/Methods To determine if changes in the mother’s diet and adiposity have long-term effects on the baby’s metabolism, independently from a prenatal insult, we utilized a mouse model of diet-induced-obesity and cross-fostering. All pups were born to lean dams fed a low fat diet but were fostered onto lean or obese dams fed a high fat diet. This study design allowed us to discern the effects of a poor diet from those of mother’s adiposity and metabolism. The weaned offspring were placed on a high fat diet to test their metabolic function. Results In this feeding challenge, all male (but not female) offspring developed metabolic dysfunction. We saw increased weight gain in the pups nursed on an obesity-resistant dam fed a high fat diet, and increased pathogenesis including liver steatosis and adipose tissue inflammation, when compared to pups nursed on either obesity-prone dams on a high fat diet or lean dams on a low fat diet. Conclusion Exposure to maternal over-nutrition, through the milk, is sufficient to shape offspring health outcomes in a sex- and organ-specific manner, and milk from a mother who is obesity-prone may partially protect the offspring from the insult of a poor diet.

The lactating mammary epithelial cell exists in a highly structured epithelial monolayer, interacting with adjacent epithelial cells through tight junctions, adherens and gap junctions and to myoepithelial cells through hemidesmosomes. Within the cell the endoplasmic reticulum, Golgi and secretory vesicles mediate protein and lactose synthesis and secretion; cytoplasmic lipid droplets coalesce at the plasma membrane to create the secreted milk fat globule. A large variety of transporters bring substrates, ions, and water to appropriate compartments within the cell.

Insulin is known to be an important regulator of milk secretion in the lactating mammary gland. Here we examine the role of insulin signaling in mammary development in pregnancy using a mouse with a floxed insulin receptor (IR) crossed with a mouse expressing Cre specifically in the mammary gland. In the mammary glands of these IRfl/flCre+mice, expression of IR is significantly diminished throughout development. Glands from these mice had 50% fewer alveoli at midpregnancy; casein and lipid droplets were diminished by 60 and 75%, respectively, indicating a role for IR both in alveolar development and differentiation. In an acinar preparation from mammary epithelial cells (MEC) isolated from pregnant mice, insulin stimulated lumen formation, mammary cell size, acinar size, acinar casein content, and the formation of lipid droplets with a Kmof ∼1.7 nM. IGF-I and IGF-II had no effect at concentrations below 50 nM, and a function blocking antibody to the IGF type 1 receptor did not alter the response to insulin. We conclude that insulin interacting with IR is essential for mammary differentiation during murine pregnancy. Using array analysis, we then examined the expression of genes up- or downregulated >1.5-fold in the IRfl/flCre+MECs, finding significant downregulation of differentiation specific genes and upregulation of cell cycle and extracellular matrix genes. We conclude that insulin fosters differentiation and may inhibit cell proliferation in the mammary gland of the midpregnant mouse.

Preventing obesity requires a precise balance between deposition into and mobilization from fat stores, but regulatory mechanisms are incompletely understood. Drosophila Split ends (Spen) is the founding member of a conserved family of RNA-binding proteins involved in transcriptional regulation and frequently mutated in human cancers. We find that manipulating Spen expression alters larval fat levels in a cell-autonomous manner. Spen-depleted larvae had defects in energy liberation from stores, including starvation sensitivity and major changes in the levels of metabolic enzymes and metabolites, particularly those involved in β-oxidation. Spenito, a small Spen family member, counteracted Spen function in fat regulation. Finally, mouse Spen and Spenito transcript levels scaled directly with body fat in vivo, suggesting a conserved role in fat liberation and catabolism. This study demonstrates that Spen is a key regulator of energy balance and provides a molecular context to understand the metabolic defects that arise from Spen dysfunction.

Recent pregnancy correlates with decreased survival for breast cancer patients compared with non–pregnancy-associated breast cancer. We hypothesize that postpartum mammary involution induces metastasis through wound-healing programs known to promote cancer. It is unknown whether alternatively activated M2 macrophages, immune cells important in wound-healing and experimental tumorigenesis that also predict poor prognosis for breast cancer patients, are recruited to the normal involuting gland. Macrophage markers CD68, CSF-1R, and F4/80 were examined across the pregnancy and involution cycle in rodent and human mammary tissues. Quantitative immunohistochemistry revealed up to an eightfold increase in macrophage number during involution, which returned to nulliparous levels with full regression. The involution macrophages exhibit an M2 phenotype as determined by high arginase-1 and low inducible nitric oxide synthase staining in rodent tissue, and by mannose receptor expression in human breast tissue. M2 cytokines IL-4 and IL-13 also peaked during involution. Extracellular matrix (ECM) isolated from involuting rat mammary glands was chemotactic for macrophages compared with nulliparous mammary ECM. Fibrillar collagen levels and proteolysis increased dramatically during involution, and denatured collagen I acted as a strong chemoattractant for macrophages in cell culture, suggesting proteolyzed fibrillar collagen as a candidate ECM mediator of macrophage recruitment. M2 macrophages, IL-4, IL-13, fibrillar collagen accumulation, and proteolysis of collagen are all components of tumor promotional microenvironments, and thus may mediate promotion of breast cancers arising in the postpartum setting.

That milk secretion is not the final differentiated state of the mammary alveolar cells is a relatively new concept. Recent work has suggested that secreting, mammary epithelial cells (MECs) have another function to perform before they undergo cell death in the involuting mammary gland. That is, they help in the final clearance and breakdown of their neighboring cells (and likely residual milk as well.) They become, for a short time, amateur phagocytes, or efferocytes, and then are believed to die and be cleared themselves. Although relatively little study has been made of this change in the functional state of the MEC, nevertheless we may speculate from the involution literature, and extend findings from other systems of apoptotic cell clearance, on some of the mechanisms involved. And with the finding that involution may represent a unique susceptibility window for the progression of metastatic breast cancer, we may suggest areas for future research along these lines as well.

Following the cessation of lactation, the mammary gland undergoes a physiologic process of tissue remodeling called involution in which glandular structures are lost, leaving an adipose tissue compartment that takes up a much larger proportion of the tissue. A quantitative morphometric analysis was undertaken to determine the mechanisms for clearance of the epithelial cells during this process. The involution process was set in motion by removal of pups from 14-day lactating C57BL/6 mice. Within hours, milk-secreting epithelial cells were shed into the glandular lumen. These cells became apoptotic, exhibiting exposure of phosphatidylserine residues on their surfaces, activation of effector caspase-3, staining for caspase-cleaved keratin 18, loss of internal organellar structure, and nuclear breakdown, but minimal blebbing or generation of apoptotic bodies. Clearance of residual milk and the shed epithelial cells was rapid, with most of the removal occurring in the first 72 h. Intact apoptotic epithelial cells were engulfed in large numbers by residual viable epithelial cells into spacious efferosomes. This process led to essentially complete involution within 4 days, at which point estrous cycling recommenced. Macrophages and other inflammatory cells did not contribute to the clearance of either residual milk or apoptotic cells, which appeared to be due entirely to the epithelium itself.

The molecular mechanisms controlling the onset of copious milk secretion are only now beginning to be elucidated. We have known for nearly four decades that progesterone suppresses milk secretion during pregnancy, and that the fall in progesterone near parturition is necessary for secretory activation. Similarly, we've known for 15 years that transforming growth factor beta (TGFbeta) also suppresses milk secretion. Yet no formal link between the two has ever been established. This work aims to review the evidence for and against a link between progesterone and TGFbeta, raise unanswered questions, and to propose further lines of research.

Clearance of apoptotic cells is critical to tissue homeostasis and resolution of inflammatory lesions. Macrophages are known to remove dying cells and release anti-inflammatory mediators in response; however, many cells traditionally thought of as poor phagocytes can mediate this function as well. In the lactating mammary gland following weaning, alveolar epithelial cell death is massive, yet the gland involutes rapidly, attaining its prepregnancy state in a matter of days. We found histologic evidence of apoptotic cell phagocytosis by viable mammary epithelial cells (MEC) in the involuting mouse mammary gland. Cultured MEC were able to engulf apoptotic cells in vitro, utilizing many of the same receptors used by macrophages, including the phosphatidylserine receptor (PSR), CD36, the vitronectin receptor alpha(v)beta3, and CD91. In addition, MEC, like macrophages, produced TGFbeta in response to stimulation of the PSR by apoptotic cells or the anti-PSR ab 217G8E9, and downregulated endotoxin-stimulated proinflammatory cytokine production. These data support the hypothesis that amateur phagocytes play a significant role in apoptotic cell clearance and its regulation of inflammation.

Murine milk contains 18 mg ml−1 serum albumin, a concentration equal to that in the serum of the lactating mouse. We examined cellular transport using in vivo methods in the mouse. At steady state the specific activity of 125I-albumin injected into the blood stream was equal in plasma and whey, confirming that milk albumin is extra-mammary in origin. Fluorescent albumin crossed the gland from basolateral surface to lumen via cytoplasmic vesicles, but was not transported in the apical to basal direction. Albumin was segregated from transferrin at the basal surface of the epithelial cells and did not colocalize with either caveolin-1 or -2. Vesicular transport was not disrupted by filipin providing additional evidence that, unlike the vascular endothelium, caveoli are not involved. Cytoplasmic albumin was localized to vesicles containing IgA and transport was disrupted by agents that interfere with clathrin-mediated endocytosis. Together, these findings provide evidence that albumin is transported across the mammary epithelium by the same pathway as immunoglobulin. The possibility that the massive transfer of albumin into mouse milk is mediated by fluid phase transport is considered.

Clearance of cellular corpses is a critical feature of apoptosis in vivo during development, tissue homeostasis, and resolution of inflammation. As the professional phagocytes of the body, macrophages play a key role in this process. By recognizing emerging signals using several different receptors, macrophages engulf apoptotic cells swiftly and efficiently. In addition, the binding of apoptotic cells profoundly down-regulates the ability of the macrophage to produce inflammatory mediators by inducing the release of antiinflammatory mediators. Finally, macrophages may actually induce cell death in specific cells during embryogenesis. Abnormalities of apoptotic cell clearance may contribute to the pathogenesis of chronic inflammatory diseases, including those of autoimmune etiology. It is also possible that certain malignant tumor cells co-opt the mechanisms for apoptotic cell clearance to avoid immune surveillance by subverting macrophage and dendritic cell responses.

The processes by which the involuting mammary gland clears residual milk and milk fat, as well as apoptotic cells, have gone largely unstudied in the modern literature. Here we review the evidence for and against the involvement of professional phagocytes of hematopoetic lineage in this process. Additionally we present evidence that mammary epithelial cells themselves are capable of phagocytosis and may be responsible for the majority of apoptotic cell and residual milk clearance during murine involution. In this scheme these cells regulate their cytokine production in response to apoptotic cells in a manner similar to other cells, including macrophages. The ensuing model describes a process of involution that actively suppresses an inflammatory response in the gland, allowing for effective tissue remodeling and damage prevention.

To investigate the role of low-density lipoprotein (LDL) in the delivery of cholesterol to the mammary gland during pregnancy and lactation, we examined the distribution of radioactivity from 125I-tyramine cellobiose-LDL injected into the tail vein of female mice at various stages of the reproductive cycle. Changes in the proportion of isotope taken up by the mammary gland largely reflected the increased weight of the gland in pregnancy and lactation. In addition, during lactation, radioactivity was found in the milk and was associated with a protein of the molecular weight of apoB-100. Quantitatively similar results were obtained with mice homozygous for disruption of the LDL receptor gene (LDLR null). Analysis of endogenous lipoproteins showed that the milk lipoprotein particles were denser than the corresponding serum lipoproteins and largely depleted of triglyceride and cholesterol. Using fluorescence microscopy we visualize the sorting of apoB protein from the LDL lipid phase at the basal surface of the mammary epithelial cell of both wild-type and LDLR-null mice. Our findings provide evidence that the mammary epithelium of the lactating mouse is able to take up LDL from the plasma by a non-LDLR-mediated process. An apoB-containing particle from which the cholesterol has been removed is transferred into milk.—Monks, J., P. U. Huey, L. Hanson, R. H. Eckel, M. C. Neville, and S. Gavigan. A lipoprotein-containing particle is transferred from the serum across the mammary epithelium into the milk of lactating mice. J. Lipid Res. 2001. 42: 686–696.

1. The concentration of iron in mouse milk is approximately 3 times that of the serum. Although there is clear evidence for the presence of the transferrin receptor in the rodent mammary gland, the precise mechanisms of iron transfer into milk are not known. 2. Milk iron was linearly related to the serum iron:transferrin ratio in lactating mice whose serum iron ranged from 8 to 66 microM. 3. Increasing the iron binding capacity of the milk by 340 microM by targeting the lactoferrin transgene to the mammary gland did not alter the relation between milk iron and the serum iron:transferrin ratio. 4. The steady-state distribution ratio of 125I-transferrin between plasma and milk was about 0.2, indicating that transcytosed transferrin contributed a maximum of 6% of the milk iron. 5. Fluorescently labelled transferrin incubated with the in situ gland localized mainly near the basal surface of the mammary alveolar cells. 6. These experiments provide evidence that the initial and rate-limiting step in the transfer of iron into milk is binding to a basal transferrin receptor. 7. A theoretical model of the relation between milk and serum iron suggests that the affinity of apotransferrin for the basal recycling system may be higher than observed in many other cell types.

The mammary fat pad is essential for development of the mammary epithelium, providing signals that mediate ductal morphogenesis and, probably, alveolar differentiation. The "cleared" fat pad is often used as a transplantation site. Considering the crucial role of the fat pad, its properties have received relatively little attention from researchers in the field. Some of the questions whose investigation is pertinent to understanding both normal mammary development and carcinogenesis are outlined in this commentary in the spirit of stimulating enquiry into this important subject. It is clear from a brief perusal of the available literature that until studies are specifically designed to clearly differentiate between functional effects of the fibrous and the adipose stroma, more substantive information about their differential effects on mammary development and tumorigenesis will not be forthcoming.

Perilipin-1 (Plin1), a prominent cytoplasmic lipid droplet (CLD) binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT) induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps.

Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands.

The lactating mammary gland synthesizes large amounts of triglyceride from fatty acids derived from the blood and from de novo lipogenesis. The latter is significantly increased at parturition and decreased when additional dietary fatty acids become available. To begin to understand the molecular regulation of de novo lipogenesis, we tested the hypothesis that the transcription factor sterol regulatory element binding factor (SREBF)-1c is a primary regulator of this system. Expression of Srebf1c mRNA and six of its known target genes increased ≥2.5-fold at parturition. However, Srebf1c-null mice showed only minor deficiencies in lipid synthesis during lactation, possibly due to compensation by Srebf1a expression. To abrogate the function of both isoforms of Srebf1, we bred mice to obtain a mammary epithelial cell-specific deletion of SREBF cleavage-activating protein (SCAP), the SREBF escort protein. These dams showed a significant lactation deficiency, and expression of mRNA for fatty acid synthase ( Fasn), insulin-induced gene 1 ( Insig1), mitochondrial citrate transporter ( Slc25a1), and stearoyl-CoA desaturase 2 ( Scd2) was reduced threefold or more; however, the mRNA levels of acetyl-CoA carboxylase-1α ( Acaca) and ATP citrate lyase ( Acly) were unchanged. Furthermore, a 46% fat diet significantly decreased de novo fatty acid synthesis and reduced the protein levels of ACACA, ACLY, and FASN significantly, with no change in their mRNA levels. These data lead us to conclude that two modes of regulation exist to control fatty acid synthesis in the mammary gland of the lactating mouse: the well-known SREBF1 system and a novel mechanism that acts at the posttranscriptional level in the presence of SCAP deletion and high-fat feeding to alter enzyme protein.

Following the cessation of lactation, the mammary gland undergoes a physiologic process of tissue remodeling called involution in which glandular structures are lost, leaving an adipose tissue compartment that takes up a much larger proportion of the tissue. A quantitative morphometric analysis was undertaken to determine the mechanisms for clearance of the epithelial cells during this process. The involution process was set in motion by removal of pups from 14-day lactating C57BL/6 mice. Within hours, milk-secreting epithelial cells were shed into the glandular lumen. These cells became apoptotic, exhibiting exposure of phosphatidylserine residues on their surfaces, activation of effector caspase-3, staining for caspase-cleaved keratin 18, loss of internal organellar structure, and nuclear breakdown, but minimal blebbing or generation of apoptotic bodies. Clearance of residual milk and the shed epithelial cells was rapid, with most of the removal occurring in the first 72 h. Intact apoptotic epithelial cells were engulfed in large numbers by residual viable epithelial cells into spacious efferosomes. This process led to essentially complete involution within 4 days, at which point estrous cycling recommenced. Macrophages and other inflammatory cells did not contribute to the clearance of either residual milk or apoptotic cells, which appeared to be due entirely to the epithelium itself.

The processes by which the involuting mammary gland clears residual milk and milk fat, as well as apoptotic cells, have gone largely unstudied in the modern literature. Here we review the evidence for and against the involvement of professional phagocytes of hematopoietic lineage in this process. Additionally we present evidence that mammary epithelial cells themselves are capable of phagocytosis and may be responsible for the majority of apoptotic cell and residual milk clearance during murine involution. In this scheme these cells regulate their cytokine production in response to apoptotic cells in a manner similar to other cells, including macrophages. The ensuing model describes a process of involution that actively suppresses an inflammatory response in the gland, allowing for effective tissue remodeling and damage prevention.

Utilizing a novel protocol to study transport of substances into mouse milk in situ, we have shown that many “fluid-phase” markers are taken up by mammary epithelial cells and deposited in milk. Since the tight junctions are closed and impermeable even to small molecules, extra-alveolar substances (those not synthesized by the alveolar cells) must be transported into the milk by the epithelial cells themselves. The markers we have used include dextran, lucifer yellow dye, horseradish peroxidase, and albumin. Using these markers and immunostaining for endogenous proteins, we have visualized transcytotic vesicles involved in transporting these markers to milk.

Lactoferrin, one of four major protein components of human milk, has long been thought to protect the breast and the infant against infection through its bacteriostatic and bacteriocidal activity1. However, it has also been reported to stimulate growth of the infant intestine2 and to modulate the activity of immune cells3. Lactoferrin shows both tissue-specific and species-specific regulation. In the mouse uterus, for example, it is clear that estrogen is a major regulator of lactoferrin secretion4. In humans and cows lactoferrin appears to be present in milk at higher levels during the onset of lactation5,6 and to decrease during established lactation. However, the actual rate of secretion is highly species-dependent. Lactoferrin secretion is especially low during established lactation in ruminants; correspondingly cows’ milk has less than 0.1 mg/ml lactoferrin5,7. In both human and cow mammary secretions lactoferrin concentrations are high at involution and in milk from the post-mastitic gland, a phenomenon that has been best documented in the cow5,8.