COMT Variants are Associated With Breast and Nipple Pain.

Estimates suggest that only 24.9% of infants born in 2019 were exclusively breastfed before 6 months of age, despite the known health benefits of exclusive breastfeeding. Breast and nipple pain is one of the primary determinants of exclusive breastfeeding. Environmental contributions to breastfeeding success have been reported extensively in the literature, but the contribution(s) of maternal genetics has yet to be discovered. The purpose of the study was to identify an association between pain and lactation-related gene variants with exclusive breastfeeding determinants. We selected 4 genes having single nucleotide polymorphisms (SNPs) with potential functional significance in breastfeeding and pain: prolactin receptor (PRLR), oxytocin receptor (OXTR), catechol-O-methyltransferase (COMT), and milk fat globule epidermal growth factor and factor V/VIII domain containing (MFGE8). We performed a cross-sectional secondary analysis of a longitudinal randomized controlled trial study, Promoting Self-Management of Breast and Nipple Pain with Biomarkers and Technology for Breastfeeding Women (NCT05262920). Breast and nipple pain, perceived insufficient milk, and breastfeeding self-efficacy were examined using total scale scores for the Brief Pain Inventory, Visual Analog Scale, H&H Lactation Scale, and the Breastfeeding Self-efficacy Scale-short form, respectively. Of the candidate genes examined, SNPs within COMT were significantly associated with breastfeeding-related outcomes. Specifically, COMT rs4633 and rs4680 minor allele carriers (T, A) reported higher breast and nipple pain intensity than women homozygous for the major allele (C, G). COMT is the most widely researched "pain gene" and has been linked to cold, postoperative, and postpartum pain. This study is the first to identify a contribution of COMT variants to breast and nipple pain and, as a result, to breastfeeding exclusivity. PERSPECTIVE: Two SNPs in the pain gene COMT are associated with breast and nipple pain. Clinically, a minor allele in COMT rs4633 and rs4680 may increase a woman's rating of moderate breast and nipple pain. TRIAL REGISTRATION: PROMPT was registered in ClinicalTrials.gov (protocol #NCT05262920).

Polyphenols and Lactation: Molecular Evidence to Support the Use of Botanical Galactagogues.

Botanicals and herbal supplements contain a diverse array of polyphenols that may affect mammary gland function and promote galactagogue activity. This scoping review is conducted to identify scientific literature elucidating how polyphenols affect mammary gland biology and cellular mechanisms critical for lactation. A literature search of PubMed and Medline reviews relevant studies in dairy animals, rodent models, and cultured mammary epithelial cells that are published from January 2010 until July 2023, to ascertain effects of polyphenols on mechanisms regulating milk production and composition. The PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Review) strategy is applied and 80 studies on polyphenols and their implications on milk production and composition are included in this review. Limited information delineating effects of polyphenols on the molecular pathways that affect lactation are found, although available information suggests modulation of Stat5 signaling/differentiation, Stat3 signaling/remodeling, mTOR and insulin signaling/energy production, and nuclear factor kappa beta (NFκß) signaling/oxidative stress and inflammation may play roles. A profound lack of mechanistic information underscores the critical need for further research to understand the impact of botanical supplements and polyphenols on milk production and composition in humans to establish maternal nutritional guidelines to support lactation and breastfeeding goals.

Stool titanium dioxide is positively associated with stool alpha-1 antitrypsin and calprotectin in young healthy adults.

Titanium dioxide (TiO2/E171) is used widely in foods, primarily as a food additive. Animal models have shown that chronic TiO2 exposure may disturb homeostasis of the gastrointestinal tract by increasing gut permeability, inducing gut inflammation, and increasing the likelihood of microbial infection. Adults have a wide range of ingested TiO2,which span two to three orders of magnitude, with a small portion of individuals consuming near gram quantities of TiO2/day. However, research on the health effects of chronic ingestion of TiO2/E171 in humans is limited. We hypothesized that regularly ingested TiO2/E171 is associated with increased gut inflammation and gut permeability in healthy adults. We tested this hypothesis in a cross-sectional design by measuring clinically established stool markers of gut inflammation (calprotectin, lactoferrin) and gut permeability (alpha-1 antitrypsin; A1AT) in 35 healthy adults, and comparing these markers between relatively high and low TiO2 exposure groups. Participants were stratified by TiO2 stool content (high dry stool TiO2 content: 0.95-9.92 µg/mg, n = 20; low content: 0.01-0.04 µg/mg; n = 15). Differences in gut health markers were tested between high and low exposure groups by independent samples t-test or Mann-Whitney U test. Multivariable linear regression was used to assess the association between TiO2 in dry stool and measured stool alpha-1 antitrypsin (A1AT). Participants in the high stool TiO2 group had greater stool A1AT (42.7 ± 21.6 mg/dL; median: 38.3; range: 1.0-49.2 mg/dL), compared to the low TiO2 group (22.8 ± 13.6 mg/dL; median: 20.9; range: 8.7-93.0 mg/dL), P = 0.003. There was also greater stool calprotectin in the high TiO2 group (51.4 ± 48.6 µg/g; median 29.2 µg/g; range: 15.3-199.0 µg/g) than in the low group (47.5 ± 63.3 µg/g; median 18.8 µg/g; range: 1.6-198.1 µg/g), P = 0.04. No clear difference was observed for lactoferrin (high TiO2 group 1.6 ± 2.1 µg/g; median: 0.68 µg/g; range: 0.01-7.7 µg/g, low TiO2 group: 1.3 ± 2.6 µg/g; median: 0.2; range: 0.01-7.6 µg/g) (P = 0.15). A1AT concentration was positively associated with stool TiO2, after adjusting for confounders (ß ± SE: 19.6 ± 7.2; P = 0.01) R2 = 0.38). Community dwelling, healthy adults with the highest TiO2 stool content had higher stool A1AT and calprotectin, compared to those with the lowest TiO2 stool content. Ongoing research is needed to validate these observations in larger groups, and to determine the long-term effects of ingested TiO2 on human gut health, using these and additional health endpoints.

The ins and outs of mammary gland calcium and zinc transport: A brief review.

Milk is an excellent source of all macrominerals and trace elements, which are essential for proper function of a wide variety of vital processes. The concentrations of minerals in milk are influenced by numerous factors, including stage of lactation, time of day, nutritional and health status of the mother, as well as maternal genotype and environmental exposures. Additionally, tight regulation of mineral transport within the secretory mammary epithelial cell itself is critical for the production and secretion of milk. In this brief review, we focus on the current understanding of how the essential divalent cations calcium (Ca) and zinc (Zn) are transported in the mammary gland (MG) with a focus on molecular regulation and the consequence of genotype. A deeper grasp of mechanisms and factors affecting Ca and Zn transport in the MG is important to understanding milk production, mineral output, and MG health to inform intervention design and novel diagnostic and therapeutic strategies in production animals and humans.

Human Milk-Derived Levels of let-7g-5p May Serve as a Diagnostic and Prognostic Marker of Low Milk Supply in Breastfeeding Women.

Low milk supply (LMS) is associated with early breastfeeding cessation; however, the biological underpinnings in the mammary gland are not understood. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally downregulate gene expression, and we hypothesized the profile of miRNAs secreted into milk reflects lactation performance. Longitudinal changes in milk miRNAs were measured using RNAseq in women with LMS (n = 47) and adequate milk supply (AMS; n = 123). Relationships between milk miRNAs, milk supply, breastfeeding outcomes, and infant weight gain were assessed, and interactions between milk miRNAs, maternal diet, smoking status, and BMI were determined. Women with LMS had lower milk volume (p = 0.003), were more likely to have ceased breast feeding by 24 wks (p = 0.0003) and had infants with a lower mean weight-for-length z-score (p = 0.013). Milk production was significantly associated with milk levels of miR-16-5p (R = -0.14, adj p = 0.044), miR-22-3p (R = 0.13, adj p = 0.044), and let-7g-5p (R = 0.12, adj p = 0.046). Early milk levels of let-7g-5p were significantly higher in mothers with LMS (adj p = 0.0025), displayed an interaction between lactation stage and milk supply (p < 0.001), and were negatively related to fruit intake (p = 0.015). Putative targets of let-7g-5p include genes important to hormone signaling, RNA regulation, ion transport, and the extracellular matrix, and down-regulation of two targets (PRLR and IGF2BP1/IMP1) was confirmed in mammary cells overexpressing let-7g-5p in vitro. Our data provide evidence that milk-derived miRNAs reflect lactation performance in women and warrant further investigation to assess their utility for predicting LMS risk and early breastfeeding cessation.

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.

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.

Loss-of-function SLC30A2 mutants are associated with gut dysbiosis and alterations in intestinal gene expression in preterm infants.

Loss of Paneth cell (PC) function is implicated in intestinal dysbiosis, mucosal inflammation, and numerous intestinal disorders, including necrotizing enterocolitis (NEC). Studies in mouse models show that zinc transporter ZnT2 (SLC30A2) is critical for PC function, playing a role in granule formation, secretion, and antimicrobial activity; however, no studies have investigated whether loss of ZnT2 function is associated with dysbiosis, mucosal inflammation, or intestinal dysfunction in humans. SLC30A2 was sequenced in healthy preterm infants (26-37 wks; n = 75), and structural analysis and functional assays determined the impact of mutations. In human stool samples, 16S rRNA sequencing and RNAseq of bacterial and human transcripts were performed. Three ZnT2 variants were common (>5%) in this population: H346Q, f = 19%; L293R, f = 7%; and a previously identified compound substitution in Exon7, f = 16%). H346Q had no effect on ZnT2 function or beta-diversity. Exon7 impaired zinc transport and was associated with a fractured gut microbiome. Analysis of microbial pathways suggested diverse effects on nutrient metabolism, glycan biosynthesis and metabolism, and drug resistance, which were associated with increased expression of host genes involved in tissue remodeling. L293R caused profound ZnT2 dysfunction and was associated with overt gut dysbiosis. Microbial pathway analysis suggested effects on nucleotide, amino acid and vitamin metabolism, which were associated with the increased expression of host genes involved in inflammation and immune response. In addition, L293R was associated with reduced weight gain in the early postnatal period. This implicates ZnT2 as a novel modulator of mucosal homeostasis in humans and suggests that genetic variants in ZnT2 may affect the risk of mucosal inflammation and intestinal disease.

Early-life formula feeding is associated with infant gut microbiota alterations and an increased antibiotic resistance load.

BACKGROUND: Infants are at a high risk of acquiring fatal infections, and their treatment relies on functioning antibiotics. Antibiotic resistance genes (ARGs) are present in high numbers in antibiotic-naive infants' gut microbiomes, and infant mortality caused by resistant infections is high. The role of antibiotics in shaping the infant resistome has been studied, but there is limited knowledge on other factors that affect the antibiotic resistance burden of the infant gut. OBJECTIVES: Our objectives were to determine the impact of early exposure to formula on the ARG load in neonates and infants born either preterm or full term. Our hypotheses were that diet causes a selective pressure that influences the microbial community of the infant gut, and formula exposure would increase the abundance of taxa that carry ARGs. METHODS: Cross-sectionally sampled gut metagenomes of 46 neonates were used to build a generalized linear model to determine the impact of diet on ARG loads in neonates. The model was cross-validated using neonate metagenomes gathered from public databases using our custom statistical pipeline for cross-validation. RESULTS: Formula-fed neonates had higher relative abundances of opportunistic pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Klebsiella oxytoca, and Clostridioides difficile. The relative abundance of ARGs carried by gut bacteria was 69% higher in the formula-receiving group (fold change, 1.69; 95% CI: 1.12-2.55; P = 0.013; n = 180) compared to exclusively human milk-fed infants. The formula-fed infants also had significantly less typical infant bacteria, such as Bifidobacteria, that have potential health benefits. CONCLUSIONS: The novel finding that formula exposure is correlated with a higher neonatal ARG burden lays the foundation that clinicians should consider feeding mode in addition to antibiotic use during the first months of life to minimize the proliferation of antibiotic-resistant gut bacteria in infants.

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.

Macrolets: Outsized Extracellular Vesicles Released from Lipopolysaccharide-Stimulated Macrophages that Trap and Kill Escherichia coli.

Macrophages release a variety of extracellular vesicles (EVs). Here we describe a previously unreported class of EVs that are released from macrophages in response to Escherichia coli endotoxin, lipopolysaccharide (LPS), that we have named "macrolets" since they are extruded as large "droplets" released from macrophages. Morphologically, macrolets are anuclear, bounded by a single lipid membrane and structurally dependent on an actin cytoskeleton. Macrolets are enriched in tetraspanins and separable on this basis from their parent macrophages. Macrolets are distinguished from classic exosomes by their larger size (10-30 µm), discoid shape, and the presence of organelles. Macrolets are rich in both interleukin 6 (IL-6) and interleukin 6 receptor (IL-6R),and are capable of trapping and killing E. coli in association with production of reactive oxygen species. Our observations offer insights into the mechanisms by which macrophage activities may be amplified in sites of infection, inflammation, and healing.

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.

Milk-derived miRNA profiles elucidate molecular pathways that underlie breast dysfunction in women with common genetic variants in SLC30A2.

Studies in humans and pre-clinical animal models show milk-derived miRNAs reflect mammary gland function during lactation. The zinc transporter SLC30A2/ZnT2 plays a critical role in mammary gland function; ZnT2-null mice have profound defects in mammary epithelial cell (MEC) polarity and secretion, resulting in sub-optimal lactation. Non-synonymous genetic variation in SLC30A2 is common in humans, and several common ZnT2 variants are associated with changes in milk components that suggest breast dysfunction in women. To identify novel mechanisms through which dysfunction might occur, milk-derived miRNA profiles were characterized in women harboring three common genetic variants in SLC30A2 (D103E, T288S, and Exon 7). Expression of ten miRNAs differed between genotypes, and contributed to distinct spatial separation. Studies in breast milk and cultured MECs confirmed expression of ZnT2 variants alters abundance of protein levels of several predicted mRNA targets critical for breast function (PRLR, VAMP7, and SOX4). Moreover, bioinformatic analysis identified two novel gene networks that may underlie normal MEC function. Thus, we propose that genetic variation in genes critical for normal breast function such as SLC30A2 has important implications for lactation performance in women, and that milk-derived miRNAs can be used to identify novel mechanisms and for diagnostic potential.

The Role of Genetic Variant rs13266634 in SLC30A8/ZnT8 in Postoperative Hyperglycemia After Major Abdominal Surgery.

CONTEXT: Following major surgery, postoperative hyperglycemia (POHG) is associated with suboptimal outcomes among patients with diabetes and nondiabetic patients. A specific genetic variant, rs13266634 (c.973C>T; p.ARG325TRP) in zinc transporter SLC30A8/ZnT8, is associated with protection against type 2 diabetes (T2D), suggesting it may be actionable for predicting and preventing POHG. OBJECTIVE: To determine independent and mediated influences of a genetic variant on POHG in patients undergoing a model major operation, complex ventral hernia repair (cVHR). PATIENTS AND DESIGN: For 110 patients (mean body mass index, 34.9 ± 5.8; T2D history, 28%) undergoing cVHR at a tertiary referral center (January 2012 to March 2017), multivariable regression was used to correlate the rs13266634 variant to preoperative clinical, laboratory, and imaging-based indices of liver steatosis and central abdominal adiposity to POHG. Causal mediation analysis (CMA) was used to determine direct and mediated contributions of SLC30A8/ZnT8 status to POHG. RESULTS: Variant rs13266634 was present in 61 patients (55.4%). In univariate models, when compared with patients with homozygous wild-type genotype (C/C, n = 49), rs13266634 was associated with significantly lower risks of POHG (OR, 0.30; 95% CI, 0.14 to 0.67; P = 0.0038). Multivariable regression indicated that the association was independent (OR, 0.39; 95% CI, 0.15 to 0.97; P = 0.040). Additionally, CMA suggested that rs13266634 protects against POHG directly and indirectly through its influence on liver steatosis and central adiposity. CONCLUSIONS: In medically complex patients undergoing major operations, the rs13266634 variant protects against POHG and its associated outcomes, through independent and mediated contributions. In C/C patients undergoing major operations, SLC30A8/ZnT8 may prove useful to stratify the risk of POHG and potentially as a therapeutic target.

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.

Excess Dietary Zinc Intake in Neonatal Mice Causes Oxidative Stress and Alters Intestinal Host-Microbe Interactions.

SCOPE: Greater than 68% of young infants are exposed to dietary zinc (Zn) levels that are higher than the Tolerable Upper Intake Limit. However, the consequences of excess dietary Zn during early life on intestinal function and host-microbe interactions are unknown. METHODS AND RESULTS: Neonatal mice are gavaged with 100 Zn µg d-1 from postnatal day (PN) 2 through PN10 and indices of intestinal function and host-microbe interactions are compared to unsupplemented mice. Excess dietary Zn causes oxidative stress, increases goblet cell number and mucus production, and are associated with increased intestinal permeability and systemic inflammation. Over 900 genes are differentially expressed; 413 genes display a fold-change >1.60. The Gene Ontology Biological processes most significantly affected include biological adhesion, the immune system, metabolic processes, and response to stimulus. Key genes most highly and significantly upregulated include ALDH2, MT1, TMEM6, CDK20, and COX62b, while CALU, ST3GAL4, CRTC2, SLC28A2, and COMMA1 are downregulated. These changes are associated with a microbiome enriched in pathogenic taxa including Pseudomonadales and Campylobacter, and greater expression of bacterial stress response genes. CONCLUSION: Excess dietary Zn may have unforeseen influences on epithelial signaling pathways, barrier function, and luminal ecology in the intestine that may have long-term consequences on intestinal health.

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.

A genetic variant in SLC30A2 causes breast dysfunction during lactation by inducing ER stress, oxidative stress and epithelial barrier defects.

SLC30A2 encodes a zinc (Zn) transporter (ZnT2) that imports Zn into vesicles in highly-specialized secretory cells. Numerous mutations and non-synonymous variants in ZnT2 have been reported in humans and in breastfeeding women; ZnT2 variants are associated with abnormally low milk Zn levels and can lead to severe infantile Zn deficiency. However, ZnT2-null mice have profound defects in mammary epithelial cell (MEC) polarity and vesicle secretion, indicating that normal ZnT2 function is critical for MEC function. Here we report that women who harbor a common ZnT2 variant (T288S) present with elevated levels of several oxidative and endoplasmic reticulum (ER) stress markers in their breast milk. Functional studies in vitro suggest that substitution of threonine for serine at amino acid 288 leads to hyperphosphorylation retaining ZnT2 in the ER and lysosomes, increasing ER and lysosomal Zn accumulation, ER stress, the generation of reactive oxygen species, and STAT3 activation. These changes were associated with decreased abundance of zona occludens-1 and increased tight junction permeability. This study confirms that ZnT2 is important for normal breast function in women during lactation, and suggests that women who harbor defective variants in ZnT2 may be at-risk for poor lactation performance.

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.

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.