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 woundhealing 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. Although it is recognized that full-term pregnancy at an early age reduces the lifetime risk of developing breast cancer, women of all ages have a transient increase in breast cancer risk with a recent pregnancy.1-4 Breast cancers diagnosed up to five years out from a completed pregnancy have been referred to as pregnancy-associated or PABC. 5,6 Several studies have shown that PABC frequently metastasizes, resulting in poor prognosis for the patient.6 -8 Epidemiological data identify women whose breast cancer is diagnosed postpartum, rather than during pregnancy, as having the worst outcomes.7-14 Further, when breast cancer patients were matched for known prognostic indicators, the postpartum window proved to be an independent factor for metastasis, whereas a diagnosis during pregnancy did not. 11,14,15 We have proposed the involution-hypothesis to account for the high metastatic oc-

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.

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.

regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium.

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.

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.

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.

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.