Estimation of Titanium Dioxide Intake by Diet and Stool Assessment among US Healthy Adults.

BACKGROUND: Titanium dioxide (TiO2/E171) is used in foods primarily as a whitening agent. Little is known regarding TiO2 exposure in the United States. OBJECTIVES: To quantify stool TiO2 content among US adults and evaluate its association with estimated intake. METHODS: Adults participated in phase 1 [three 24-h dietary recalls (DRs) and stool TiO2 measured from 3 matched samples (n = 52)] and/or phase 2 [tailored FFQ and stool TiO2 measured from 3 samples over 3 mo (n = 61)]. TiO2 in foods was estimated from a database, and concentration in 49 additional foods and 339 stool samples were quantified using inductively coupled plasma mass spectrometry. Associations between dietary and stool TiO2 were assessed by log-linear multivariable regression. USDA food groups (n = 49, servings/d) were related to stool TiO2 by stepwise regression. RESULTS: TiO2 food content varied by brand. Mean TiO2 intake from three 24-h DRs [0.19 ± 0.31 mg/(kg body weight · d)] was lower than from the FFQ [0.30 ± 0.21 mg/(kg body weight · d)]. Dietary TiO2 was not predictive of stool TiO2, in phase 1 or phase 2, 10^(ß) per 10 times higher dietary TiO2: 1.138 [10^(95% CI): 0.635, 2.037, P = 0.66] and 0.628 [10^(95% CI): 0.206, 1.910, P = 0.41], respectively. Food groups related to stool TiO2 were 1) milk desserts, sauces, and gravies [10^(ß) per servings/d: 3.361; 10^(95% CI): 0.312, 36.163; P = 0.002] and 2) yeast breads [10^(ß): 1.430; 10^(95% CI): 0.709, 2.884; P = 0.002] in phase 1 and 1) cream and cream substitutes [10^(ß) = 10.925; 10^(95% CI): 1.952, 61.137; P = 0.01] and 2) milk and milk drinks [10^(ß) = 0.306; 10^(95% CI): 0.086, 1.092, P = 0.07] in phase 2. CONCLUSIONS: Intake of certain foods was associated with higher stool TiO2 content. There is a need for valid estimation of TiO2 intakes via the improvement of a dietary assessment method and a TiO2 food composition database. Future research should assess whether high stool TiO2 content is related to adverse health outcomes.

ZnT2 Is Critical for TLR4-Mediated Cytokine Expression in Colonocytes and Modulates Mucosal Inflammation in Mice.

A wide range of microbial pathogens can enter the gastrointestinal tract, causing mucosal inflammation and infectious colitis and accounting for most cases of acute diarrhea. Severe cases of infectious colitis can persist for weeks, and if untreated, may lead to major complications and death. While the molecular pathogenesis of microbial infections is often well-characterized, host-associated epithelial factors that affect risk and severity of infectious colitis are less well-understood. The current study characterized functions of the zinc (Zn) transporter ZnT2 (SLC30A2) in cultured HT29 colonocytes and determined consequences of ZnT2 deletion in mice on the colonic response to enteric infection with Citrobacter rodentium. ZnT2 in colonocytes transported Zn into vesicles buffering cytoplasmic Zn pools, which was important for Toll-like receptor 4 (TLR4) expression, activation of pathogen-stimulated NF-κß translocation and cytokine expression. Additionally, ZnT2 was critical for lysosome biogenesis and bacterial-induced autophagy, both promoting robust host defense and resolution mechanisms in response to enteric pathogens. These findings reveal that ZnT2 is a novel regulator of mucosal inflammation in colonocytes and is critical to the response to infectious colitis, suggesting that manipulating the function of ZnT2 may offer new therapeutic strategies to treat specific intestinal infections.

Zinc Exposure Promotes Commensal-to-Pathogen Transition in Pseudomonas aeruginosa Leading to Mucosal Inflammation and Illness in Mice.

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) is associated gastrointestinal (GI) inflammation and illness; however, factors motivating commensal-to-pathogen transition are unclear. Excessive zinc intake from supplements is common in humans. Due to the fact that zinc exposure enhances P. aeruginosa colonization in vitro, we hypothesized zinc exposure broadly activates virulence mechanisms, leading to inflammation and illness. P. aeruginosa was treated with excess zinc and growth, expression and secretion of key virulence factors, and biofilm production were determined. Effects on invasion, barrier function, and cytotoxicity were evaluated in Caco-2 cells co-cultured with P. aeruginosa pre-treated with zinc. Effects on colonization, mucosal pathology, inflammation, and illness were evaluated in mice infected with P. aeruginosa pre-treated with zinc. We found the expression and secretion of key virulence factors involved in quorum sensing (QS), motility (type IV pili, flagella), biosurfactants (rhamnolipids), toxins (exotoxin A), zinc homeostasis (CzcR), and biofilm production, were all significantly increased. Zinc exposure significantly increased P. aeruginosa invasion, permeability and cytotoxicity in Caco-2 cells, and enhanced colonization, inflammation, mucosal damage, and illness in mice. Excess zinc exposure has broad effects on key virulence mechanisms promoting commensal-to-pathogen transition of P. aeruginosa and illness in mice, suggesting excess zinc intake may have adverse effects on GI health in humans.

A common genetic variant in zinc transporter ZnT2 (Thr288Ser) is present in women with low milk volume and alters lysosome function and cell energetics.

Suboptimal lactation is a common, yet underappreciated cause for early cessation of breastfeeding. Molecular regulation of mammary gland function is critical to the process lactation; however, physiological factors underlying insufficient milk production are poorly understood. The zinc (Zn) transporter ZnT2 is critical for regulation of mammary gland development and maturation during puberty, lactation, and postlactation gland remodeling. Numerous genetic variants in the gene encoding ZnT2 (SLC30A2) are associated with low milk Zn concentration and result in severe Zn deficiency in exclusively breastfed infants. However, the functional impacts of genetic variation in ZnT2 on key mammary epithelial cell functions have not yet been systematically explored at the cellular level. Here we determined a common mutation in SLC30A2/ZnT2 substituting serine for threonine at amino acid 288 (Thr288Ser) was found in 20% of women producing low milk volume (n = 2/10) but was not identified in women producing normal volume. Exploration of cellular consequences in vitro using phosphomimetics showed the serine substitution promoted preferential phosphorylation of ZnT2, driving localization to the lysosome and increasing lysosome biogenesis and acidification. While the substitution did not initiate lysosome-mediated cell death, cellular ATP levels were significantly reduced. Our findings demonstrate the Thr288Ser mutation in SLC30A2/ZnT2 impairs critical functions of mammary epithelial cells and suggest a role for genetic variation in the regulation of milk production and lactation performance.

Increased early enteral zinc intake improves weight gain in hospitalised preterm infants.

AIM: To test the hypothesis that enteral zinc intake is associated with improved preterm infant growth during neonatal intensive care unit (NICU) hospitalisation. METHODS: This prospective cohort study enrolled 105 preterm infants at a tertiary referral centre. Enteral zinc intake was calculated at day of life 14, and growth was measured as change in weight, length and head circumference from birth to discharge. Nonparametric tests assessed the contribution of breast milk vs formula and enteral zinc intake on weight, length and head circumference growth. Partial correlations evaluated the impact of baseline health status and caloric intake on growth. Multiple regression analysis was then completed to determine the unique contribution of zinc intake to weight gain and head circumference growth. RESULTS: Total enteral zinc intake was positively associated with weight gain (r = 0.4, p < 0.01) and head circumference growth (r = 0.3, p < 0.01) during NICU hospitalisation. Further, multiple regression analysis showed higher zinc intake is linked to weight gain during NICU hospitalisation after accounting for postmenstrual age at birth. CONCLUSION: Increased early enteral zinc intake is linked to weight gain during NICU hospitalisation, highlighting the importance of enteral zinc intake in early infant nutrition.

Zinc transporter 2 interacts with vacuolar ATPase and is required for polarization, vesicle acidification, and secretion in mammary epithelial cells.

An important feature of the mammary gland is its ability to undergo profound morphological, physiological, and intracellular changes to establish and maintain secretory function. During this process, key polarity proteins and receptors are recruited to the surface of mammary epithelial cells (MECs), and the vesicle transport system develops and matures. However, the intracellular mechanisms responsible for the development of secretory function in these cells are unclear. The vesicular zinc (Zn2+) transporter ZnT2 is critical for appropriate mammary gland architecture, and ZnT2 deletion is associated with cytoplasmic Zn2+ accumulation, loss of secretory function and lactation failure. The underlying mechanisms are important to understand as numerous mutations and non-synonymous genetic variation in ZnT2 have been detected in women that result in severe Zn2+ deficiency in exclusively breastfed infants. Here we found that ZnT2 deletion in lactating mice and cultured MECs resulted in Zn2+-mediated degradation of phosphatase and tensin homolog (PTEN), which impaired intercellular junction formation, prolactin receptor trafficking, and alveolar lumen development. Moreover, ZnT2 directly interacted with vacuolar H+-ATPase (V-ATPase), and ZnT2 deletion impaired vesicle biogenesis, acidification, trafficking, and secretion. In summary, our findings indicate that ZnT2 and V-ATPase interact and that this interaction critically mediates polarity establishment, alveolar development, and secretory function in the lactating mammary gland. Our observations implicate disruption in ZnT2 function as a modifier of secretory capacity and lactation performance.

ZnT2 is critical for lysosome acidification and biogenesis during mammary gland involution.

Mammary gland involution, a tightly regulated process of tissue remodeling by which a lactating mammary gland reverts to the prepregnant state, is characterized by the most profound example of regulated epithelial cell death in normal tissue. Defects in the execution of involution are associated with lactation failure and breast cancer. Initiation of mammary gland involution requires upregulation of lysosome biogenesis and acidification to activate lysosome-mediated cell death; however, specific mediators of this initial phase of involution are not well described. Zinc transporter 2 [ZnT2 ( SLC30A2)] has been implicated in lysosome biogenesis and lysosome-mediated cell death during involution; however, the direct role of ZnT2 in this process has not been elucidated. Here we showed that ZnT2-null mice had impaired alveolar regression and reduced activation of the involution marker phosphorylated Stat3, indicating insufficient initiation of mammary gland remodeling during involution. Moreover, we found that the loss of ZnT2 inhibited assembly of the proton transporter vacuolar ATPase on lysosomes, thereby decreasing lysosome abundance and size. Studies in cultured mammary epithelial cells revealed that while the involution signal TNFα promoted lysosome biogenesis and acidification, attenuation of ZnT2 impaired the lysosome response to this involution signal, which was not a consequence of cytoplasmic Zn accumulation. Our findings establish ZnT2 as a novel regulator of vacuolar ATPase assembly, driving lysosome biogenesis, acidification, and tissue remodeling during the initiation of mammary gland involution.

Immune and metabolic responses in early and late sepsis during mild dietary zinc restriction.

BACKGROUND: Mild dietary zinc (Zn) deficiency is widespread in human populations, but its influence on recovery after acute illness is poorly understood. In a mouse model of abdominal sepsis (cecal ligation puncture), systemic immune responses and liver metabolism were monitored in early (24 h) and late (5 d) phases, under control conditions and during mild dietary Zn restriction. METHODS: Mice were fed diets adequate or marginally deficient (ZM) in Zn (30 versus 10 mg zinc/kg diet) for 4 wk, before undergoing laparotomy alone (nonseptic control) or cecal ligation puncture (septic). RESULTS: Among nonseptic mice, the ZM state was not associated with differences in inflammation or metabolic responses. Among septic mice, mortality did not differ between the zinc adequate and ZM groups. In the early phase, the ZM state amplified increases in plasma interleukin (IL) 6, tumor necrosis factor alpha, and IL-10, while dampening the interferon gamma response. In the late phase, subtle but significant ZM-associated increases were observed in plasma IL-5 and interferon gamma levels and hepatic protein synthesis, the latter of which appeared to be mammalian target of rapamycin independent and was associated with increased hepatic tumor necrosis factor alpha messenger RNA content. CONCLUSIONS: Without increasing mortality, the ZM state is associated with a more disordered acute systemic inflammatory response and persistence or enhancement of acute phase responses within the liver parenchyma.

Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation.

The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women.

Biological underpinnings of breastfeeding challenges: the role of genetics, diet, and environment on lactation physiology.

Lactation is a dynamic process that has evolved to produce a complex biological fluid that provides nutritive and nonnutritive factors to the nursing offspring. It has long been assumed that once lactation is successfully initiated, the primary factor regulating milk production is infant demand. Thus, most interventions have focused on improving breastfeeding education and early lactation support. However, in addition to infant demand, increasing evidence from studies conducted in experimental animal models, production animals, and breastfeeding women suggests that a diverse array of maternal factors may also affect milk production and composition. In this review, we provide an overview of our current understanding of the role of maternal genetics and modifiable factors, such as diet and environmental exposures, on reproductive endocrinology, lactation physiology, and the ability to successfully produce milk. To identify factors that may affect lactation in women, we highlight some information gleaned from studies in experimental animal models and production animals. Finally, we highlight the gaps in current knowledge and provide commentary on future research opportunities aimed at improving lactation outcomes in breastfeeding women to improve the health of mothers and their infants.

Subtype-specific accumulation of intracellular zinc pools is associated with the malignant phenotype in breast cancer.

BACKGROUND: Zinc (Zn) hyper-accumulates in breast tumors and malignant cell lines compared to normal mammary epithelium. The mechanisms responsible for Zn accumulation and the consequence of Zn dysregulation are poorly understood. METHODS: Microarrays were performed to assess differences in the expression of Zn transporters and metallothioneins (MTs) in human breast tumors and breast cancer cell lines. Real-time PCR and immunoblotting were employed to profile Zn transporter expression in representative luminal (T47D), basal (MDA-MB-231), and non-malignant (MCF10A) cell lines. Zn distribution in human tumors was assessed by X-ray fluorescence imaging. Zn distribution and content in cell lines was measured using FluoZin-3 imaging, and quantification and atomic absorption spectroscopy. Functional consequences of ZnT2 over-expression in MDA-MB-231 cells including invasion, proliferation, and cell cycle were measured using Boyden chambers, MTT assays, and flow cytometry, respectively. RESULTS: Gene expression profiling of human breast tumors and breast cancer cell lines identified subtype-specific dysregulation in the Zn transporting network. X-ray fluorescence imaging of breast tumor tissues revealed Zn hyper-accumulation at the margins of Luminal breast tumors while Zn was more evenly distributed within Basal tumors. While both T47D and MDA-MB-231 cells hyper-accumulated Zn relative to MCF10A cells, T47D cells accumulated 2.5-fold more Zn compared to MDA-MB-231 cells. FluoZin-3 imaging indicated that Zn was sequestered into numerous large vesicles in T47D cells, but was retained in the cytoplasm and found in fewer and larger, amorphous sub-cellular compartments in MDA-MB-231 cells. The differences in Zn localization mirrored the relative abundance of the Zn transporter ZnT2; T47D cells over-expressed ZnT2, whereas MDA-MB-231 cells did not express ZnT2 protein due to proteasomal degradation. To determine the functional relevance of the lack of ZnT2 in MDA-MB-231cells, cells were transfected to express ZnT2. ZnT2 over-expression led to Zn vesicularization, shifts in cell cycle, enhanced apoptosis, and reduced proliferation and invasion. CONCLUSIONS: This comprehensive analysis of the Zn transporting network in malignant breast tumors and cell lines illustrates that distinct subtype-specific dysregulation of Zn management may underlie phenotypic characteristics of breast cancers such as grade, invasiveness, metastatic potential, and response to therapy.

Zn2+ efflux through lysosomal exocytosis prevents Zn2+-induced toxicity.

Zn(2+) is an essential micronutrient and an important ionic signal whose excess, as well as scarcity, is detrimental to cells. Free cytoplasmic Zn(2+) is controlled by a network of Zn(2+) transporters and chelating proteins. Recently, lysosomes became the focus of studies in Zn(2+) transport, as they were shown to play a role in Zn(2+)-induced toxicity by serving as Zn(2+) sinks that absorb Zn(2+) from the cytoplasm. Here, we investigated the impact of the lysosomal Zn(2+) sink on the net cellular Zn(2+) distribution and its role in cell death. We found that lysosomes played a cytoprotective role during exposure to extracellular Zn(2+). Such a role required lysosomal acidification and exocytosis. Specifically, we found that the inhibition of lysosomal acidification using Bafilomycin A1 (Baf) led to a redistribution of Zn(2+) pools and increased apoptosis. Additionally, the inhibition of lysosomal exocytosis through knockdown (KD) of the lysosomal SNARE proteins VAMP7 and synaptotagmin VII (SYT7) suppressed Zn(2+) secretion and VAMP7 KD cells had increased apoptosis. These data show that lysosomes play a central role in Zn(2+) handling, suggesting that there is a new Zn(2+) detoxification pathway.

ZnT4 (SLC30A4)-null ("lethal milk") mice have defects in mammary gland secretion and hallmarks of precocious involution during lactation.

During lactation, highly specialized secretory mammary epithelial cells (MECs) produce and secrete huge quantities of nutrients and nonnutritive factors into breast milk. The zinc (Zn) transporter ZnT4 (SLC30A4) transports Zn into the trans-Golgi apparatus for lactose synthesis, and across the apical cell membrane for efflux from MECs into milk. This is consistent with observations in "lethal milk" (lm/lm) mice, which have a truncation mutation in SLC30A4, and present with not only low milk Zn concentration, but also smaller mammary glands, decreased milk volume, and lactation failure by lactation day 2. However, the molecular underpinnings of these defects are not understood. Here, we used lactating C57BL/6J(lm/lm) (ZnT4-null) mice to explore the consequences of a ZnT4-null phenotype on mammary gland function during early lactation. Lactating C57BL/6J(lm/lm) mice had significantly fewer, smaller, and collapsed alveoli comprising swollen, lipid-filled MECs during early lactation. These defects were associated with decreased Akt expression and STAT5 activation, indicative of defects in MEC secretion. In addition, increased expression of ZnT2, TNF-α, and cleaved e-cadherin concomitant with increased activation of STAT3 implicated the loss of ZnT4 in precocious activation of involution. Collectively, our study indicates that the loss of ZnT4 has profound consequences on MEC secretion and may promote tissue remodeling in the mammary gland during early lactation.

Molecular regulation of lactation: The complex and requisite roles for zinc.

Lactation provides many health benefits to the nursing infant and breastfeeding mother. In order to successfully breastfeed, the mammary gland must expand and differentiate to activate numerous processes that regulate milk production and secretion. This involves a complex series of molecular, biochemical and cellular events driven largely by lactogenic hormones. Recent advances implicate zinc as a critical modulator of mammary gland function. Here, we provide an overview of our current understanding of the role and regulation of zinc in promoting proliferation, differentiation and secretion in the mammary gland during lactation, and highlight critical gaps in knowledge.

Dietary Iron and Colorectal Cancer Risk: A Review of Human Population Studies.

Iron is an essential micronutrient that is involved in many redox processes and serves as an integral component in various physiological functions. However, excess iron can cause tissue damage through its pro-oxidative effects, potentiating the development of many diseases such as cancer through the generation of reactive oxidative species. The two major forms of iron in the diet are heme and nonheme iron, both of which are found in several different foods. In addition to natural food sources, intake of nonheme iron may also come from fortified foods or in supplement form. This review summarizes the results of human population studies that have examined the role of dietary iron (heme and nonheme), heme iron alone, and iron from supplements in colorectal carcinogenesis.

Oral omega-3 fatty acids promote resolution in chemical peritonitis.

BACKGROUND: Recent studies suggest that purified omega-3 fatty acids may attenuate acute inflammation and hasten the transition to healing. In this study, we tested the hypothesis that pretreatment with omega-3-rich fish oil (FO) would promote resolution of peritoneal inflammation through production of specific lipid mediators. METHODS: C57/BL6 mice were given a daily 200-µL oral gavage of saline (CTL) or FO (1.0-1.5 g/kg/d docosahexaenoic acid and 1.3-2.0 g/kg/d eicosapentaenoic acid) for 7 d before chemical peritonitis was induced with thioglycollate. Peritoneal lavage fluid was collected before induction and at days 2 and 4 after peritonitis onset. Prostaglandin E2 (PGE2), Leukotriene B4 (LTB4), Resolvin D1 (RvD1), and the composition of immune cell populations were examined in peritoneal lavage exudates. Cells harvested from the peritoneum were assessed for macrophage differentiation markers, phagocytosis, and lipopolysaccharide-induced cytokine secretion profiles (interleukin [IL]-6, IL-10, IL-1ß, TNFα). RESULTS: The ratio of RvD1 to pro-inflammatory PGE2 and LTB4 was increased in the peritoneal cavity of FO-supplemented animals. FO induced a decrease in the number of monocytes in the lavage fluid, with no change in the number of macrophages, neutrophils, or lymphocytes. Macrophage phagocytosis and M1/M2 messenger RNA markers were unchanged by FO with the exception of decreased PPARγ expression. FO increased ex vivo TNFα secretion after stimulation with lipopolysaccharide. CONCLUSIONS: Our findings provide evidence that nutraceutically relevant doses of FO supplements given before and during chemical peritonitis shift the balance of lipid mediators towards a proresolution, anti-inflammatory state without drastically altering the number or phenotype of local innate immune cell populations.

Exome Sequencing of SLC30A2 Identifies Novel Loss- and Gain-of-Function Variants Associated with Breast Cell Dysfunction.

The zinc (Zn) transporter ZnT2 (SLC30A2) is expressed in specialized secretory cells including breast, pancreas and prostate, and imports Zn into mitochondria and vesicles. Mutations in SLC30A2 substantially reduce milk Zn concentration ([Zn]) and cause severe Zn deficiency in exclusively breastfed infants. Recent studies show that ZnT2-null mice have low milk [Zn], in addition to profound defects in mammary gland function during lactation. Here, we used breast milk [Zn] to identify novel non-synonymous ZnT2 variants in a population of lactating women. We also asked whether specific variants induce disturbances in intracellular Zn management or cause cellular dysfunction in mammary epithelial cells. Healthy, breastfeeding women were stratified into quartiles by milk [Zn] and exonic sequencing of SLC30A2 was performed. We found that 36% of women tested carried non-synonymous ZnT2 variants, all of whom had milk Zn levels that were distinctly above or below those in women without variants. We identified 12 novel heterozygous variants. Two variants (D(103)E and T(288)S) were identified with high frequency (9 and 16%, respectively) and expression of T(288)S was associated with a known hallmark of breast dysfunction (elevated milk sodium/potassium ratio). Select variants (A(28)D, K(66)N, Q(71)H, D(103)E, A(105)P, Q(137)H, T(288)S and T(312)K) were characterized in vitro. Compared with wild-type ZnT2, these variants were inappropriately localized, and most resulted in either 'loss-of-function' or 'gain-of-function', and altered sub-cellular Zn pools, Zn secretion, and cell cycle check-points. Our study indicates that SLC30A2 variants are common in this population, dysregulate Zn management and can lead to breast cell dysfunction. This suggests that genetic variation in ZnT2 could be an important modifier of infant growth/development and reproductive health/disease. Importantly, milk [Zn] level may serve as a bio-reporter of breast function during lactation.

Prolactin (PRL)-stimulated ubiquitination of ZnT2 mediates a transient increase in zinc secretion followed by ZnT2 degradation in mammary epithelial cells.

The zinc transporter ZnT2 imports zinc into secretory vesicles and regulates zinc export from the mammary epithelial cell. Mutations in ZnT2 substantially impair zinc secretion into milk. The lactogenic hormone prolactin (PRL) transcriptionally increases ZnT2 expression through the Jak2/STAT5 signaling pathway, increasing zinc accumulation in secretory vesicles and zinc secretion. Herein, we report that PRL post-translationally stimulated ZnT2 ubiquitination, which altered ZnT2 trafficking and augmented vesicular zinc accumulation and secretion from mammary epithelial cells in a transient manner. Ubiquitination then down-regulated zinc secretion by stimulating degradation of ZnT2. Mutagenesis of two N-terminal lysine residues (K4R and K6R) inhibited ZnT2 ubiquitination, vesicular zinc accumulation and secretion, and protein degradation. These findings establish that PRL post-translationally regulates ZnT2-mediated zinc secretion in a multifactorial manner, first by enhancing zinc accumulation in vesicles to transiently enhance zinc secretion and then by activating ubiquitin-dependent ZnT2 degradation. This provides insight into novel mechanisms through which ZnT2 and zinc transport is tightly regulated in mammary epithelial cells.

TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes.

Mammary epithelial cells undergo widespread lysosomal-mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor-α (TNFα), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study is to determine how TNFα retargets ZnT2 to lysosomes. We tested the hypothesis that TNFα signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C-terminus of ZnT2, allowing adaptor protein complex-3 (AP-3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNFα redistributed wild-type (WT) ZnT2 from late endosomes (Pearson's coefficient = 0.202 ± 0.05 and 0.097 ± 0.03; P<0.05) to lysosomes (0.292 ± 0.03 and 0.649 ± 0.03; P<0.0001), which increased lysosomal Zn (P<0.0001) and activated LCD (P<0.0001) compared to untreated cells. Mutation of the dileucine motif (L294V) eliminated the ability of TNFα to redistribute ZnT2 from late endosomes to lysosomes, increase lysosomal Zn, or activate LCD. Moreover, TNFα increased (P<0.05) AP-3 binding to wt ZnT2 but not to L294V immunoprecipitates. Finally, using phospho- and dephospho-mimetics of predicted phosphorylation sites (T281, T288, and S296), we found that dephosphorylated S296 was required to target ZnT2 to accumulate Zn in lysosomes and activate LCD. Our findings suggest that women with variation in the C-terminus of ZnT2 may be at risk for inadequate involution and breast disease due the inability to traffic ZnT2 to lysosomes.

Obesity-Induced Inflammation Is Associated with Alterations in Subcellular Zinc Pools and Premature Mammary Gland Involution in Lactating Mice.

BACKGROUND: Lactation failure is common in overweight and obese women; however, the precise mechanism remains unknown. OBJECTIVE: We tested the hypothesis that obesity-induced inflammation in the mammary gland (MG) redistributes subcellular zinc pools to promote cell death of mammary epithelial cells (MECs) and premature involution. METHODS: Female DBA/2J mice were fed a high-fat (obese; 45% kcal from fat, n = 60) or control diet (lean; 10% kcal from fat, n = 50) for 5 wk and bred. MG cytokines and macrophage infiltration were determined by reverse transcriptase-polymerase chain reaction and F4/80 staining, respectively. Zinc concentration was analyzed by atomic absorption spectroscopy, and zinc transporters and markers of endoplasmic reticulum (ER) stress, autophagy, and involution were measured by immunoblot. To confirm effects of inflammation, tumor necrosis factor-α (TNF) or vehicle was injected into adjacent MGs of lean lactating C57BL/6 mice (n = 5) and cultured MECs (HC11 cells) were treated with TNF in vitro. RESULTS: Seventy-seven percent of obese mice failed to lactate (lean: 39%; P < 0.001). Obese mice capable of lactating had greater macrophage infiltration (obese: 135 ± 40.4 macrophages/mm(2); lean: 63.8 ± 8.9 macrophages/mm(2); P < 0.001) and elevated TNF expression (P < 0.05), concurrent with lower zrt- irt-like protein 7 abundance (P < 0.05) and higher ER zinc concentration (obese: 0.36 ± 0.004 µg Zn/mg protein; lean: 0.30 ± 0.02 µg Zn/mg protein; P < 0.05) compared with lean mice. Heat shock protein 5 (HSPA5) expression (P < 0.05) was suppressed in the MG of obese mice, which was consistent with HSPA5 suppression in TNF-injected MGs (P < 0.01) and MECs treated with TNF in vitro (P < 0.01). Moreover, obesity increased lysosomal activity (P < 0.05) and autophagy in the MG, which corresponded to increased zinc transporter 2 abundance and lysosomal zinc concentration compared with lean mice (obese: 0.20 ± 0.02 µg Zn/mg protein; lean: 0.14 ± 0.01 µg Zn/mg protein; P < 0.05). Importantly, MGs of obese mice exhibited markers of apoptosis (P = 0.05) and involution (P < 0.01), which were not observed in lean mice. CONCLUSIONS: Diet-induced obesity created a proinflammatory MG microenvironment in mice, which was associated with zinc-mediated ER stress and autophagy and the activation of premature involution.